Download Page 1 of 200 - Gamma Delta Conference 2016

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Sponsors
we are very greatful to our sponsors:
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A warm welcome …
…… to London for the 7
th
International Gamma Delta T cell Conference. The
conference arrives at a time of widening and intensifying interest in gamma delta T
cells, spanning their biology through to the cells’ clinical application. Although time
will be short, the Conference will span this spectrum of interests, adopting the by-now
customary approach of focusing on talks selected from the abstracts. Whether your
interests are in skin or gut; mouse or human; IL-17, KGF or Interferon; infectious
disease or cancer, we anticipate that the Conference will provide an unrivalled means
to catch up with the latest developments.
A basic vulnerability of the gamma delta T cell field is our collective inability to
describe in molecular terms the natural history of a gamma delta T cell response from
its initiation through to resolution. This unmet need will serve as a sub-text to the
Conference, challenging us to ask how much closer we are to defining gamma delta
T cell biology, even in a single system, and thereby to predict the outcomes of the
cells’ application in the clinic. So, welcome to London; we have our work cut out!
Adrian Hayday
on behalf of the organising committee
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Index
Sponsors.........................................................................................2
GeneralInformation.......................................................................5
Maps..............................................................................................9
Programme...................................................................................17
Abstracts......................................................................................18
WhatdoγδTcellsrecognize?CD1,haptens,novelinsights..............................................18
WhatdoγδTcellsrecognize?TherolesofButyrophilin/Butyrophilin-likemoleculesinγδ
biology.............................................................................................................................27
Whatfactorsdictateγδbiology?Development,regulationandsignallingpathways.........42
WheredoγδTcellsfunctionandhowdotheygetthere?Tissues/tissuehomeostasis/
migration..........................................................................................................................71
γδTcellfunctioninhealthandmedicine:1.infectiousdisease.........................................86
γδTcellfunctioninhealthandmedicine:2.autoimmunedisease/inflammation/
transplantation...............................................................................................................106
γδTcellfunctioninhealthandmedicine:3.cancer........................................................125
γδTcellfunctioninhealthandmedicine:4.γδTcellsinimmunotherapy......................148
PosterSessionA.........................................................................172
PosterSessionB..........................................................................173
PosterSessionC..........................................................................174
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General Information
Organising committee:
All members of the organising committee will be at the conference.
Additionally other members of the Hayday lab will be able to help you with
issues regarding the conference.
Adrian Hayday:
Anett Jandke:
Natalie Roberts:
Oliver Nussbaumer:
Rafael DiMarco Barros:
Vasiliki Sofra:
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Location
Lectures will be held at Franklin Wilkins Building (FWB), which is part of
King’s College London. The Franklin Wilkins Building is named in
commemoration of Rosalind Franklin and Maurice Wilkins who, at King’s
College, jointly obtained the experimental data revealing the structure of the
genetic code. FWB is located in central London near Waterloo station, which
is served by the Northern, Jubilee, Bakerloo and Waterloo & City lines and
several bus lines. Alternative stations within walking distance (5-10min) are
Embankment and Southwark (check tube map on last page).
Please see attached maps for the travel connections, surrounding area, and
floorplan of the building.
Coffee breaks & lunch
Refreshments and lunch will be provided free of charge to participants
wearing name badges during the session breaks. They will be served in the
st
restaurant on the first floor of FWB. (see floor plan, 1 floor)
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Cloakroom
A cloakroom with luggage storage will be available on Thursday
(16.06.2015) and on Sunday (19.06.2015).
Conference Dinner
The conference dinner will be held at 8 Northumberland Avenue, which is a
short walk from FWB across the river Thames. A map with directions from
the conference venue to the dinner venue is included in this program.
Members of the Hayday laboratory will be happy to assist you. Please listen
out for further details on the day.
Dinner venue:
8 Northumberland Avenue, London, WC2N 5BY.
Currency
The UK currency is pound sterling (£) and cash can be withdrawn at cash
machines in Waterloo station and around the conference venue.
Electricity
UK voltage is 240V, 3pin plugs.
Emergencies
The UK emergency number is 999. The European emergency number 112 is
also accepted in the UK.
Travel
To obtain tickets there are different options. 1. You purchase an Oyster card
for which your deposit can be refunded at the end of your trip. 2. You use
ticket vending machines to obtain a paper ticket or a travel card. Travel
cards are more economical for those likely to make several journeys. Check
www.tfl.gov.uk for more details. Alternatively, you need not purchase tickets
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but can travel using contactless payment options on credit and debit cards.
However, be aware that additional foreign transaction fees may apply for
every time you touch in and touch out if your card is not a UK issue.
Card encryptions interfere with each other so we recommend that you do not
keep Oyster and contactless payment cards in the same wallet as you may
be overcharged.
The London underground, the tube, can get very busy, particularly at rush
hours so allow yourself plenty of time.
Mobile phones
Mobile phone charging boxes are available in the conference venue. Fees
may apply.
Internet access
At the venue, you can register for the cloud. Alternatively eduroam is
available. We suggest you register with eduroam with your home institution
prior to attending the conference.
How do I set up my phone or tablet to use The Cloud network of
hotspots?
There is a one-time authentication process to follow when you first visit a
Cloud hotspot. Simply follow the steps below to register or login...
1. Check your WiFi is on by selecting 'Settings' from the home screen and
selecting 'Wi-Fi'.
2. If you are in coverage of a Cloud hotspot, you will be offered '_The
Cloud', select this to connect.
3. Now return to the home screen and select web browser, you will see the
Cloud landing page. If you don't, click to refresh the page.
4. Now simply select 'Get Online' and follow the onscreen instructions.
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5. When the Wi-Fi session is established, you will see 'welcome to The
Cloud' and the session counter displays the session time. You can now
begin your Wi-Fi browsing session!
Insurance/Liabilities and Disclaimer
Franklin Wilkins Building including the lecture theatre, restaurant and poster
area are ALL spaces open to the general public. Please do not leave any
luggage and personal items unattended at any time.
The γδ Tcell conference organisers and conference venues (FWB, KCL and
8 Northumberland Avenue) will not be held liable for personal injuries or for
loss or damage to property incurred by participants at the 2016 γδ T cell
conference. Participants are encouraged to purchase insurance to cover loss
incurred in the event of cancellation, medical expenses or damage to or loss
of personal effects when travelling outside of their own countries.
Safety and Welfare
Basic precautionary measures should always be kept in mind in any city:
Do not carry all-important documents, money, credit cards and other
essential items in one bag and if possible leave important items in the hotel
safe during your stay. Take off your name badge when leaving the
conference venue. In heavily frequented tourist areas, be aware of attempts
of scam and pickpocketing.
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Maps
Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH
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Southbank, Food, Coffee, Relax!
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Social Event, 8 Nurthumberland Ave, WC2N 5BY
On A Differ
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On a different note…
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What to do in London
Museums:
http://www.soane.org
http://www.hunterianmuseum.org/
http://www.vam.ac.uk
http://www.tate.org.uk
http://www.nhm.ac.uk
http://www.britishmuseam.org
http://www.sciencemuseum.org.uk
http://www.somersethouse.org.uk
http://www.wellcomecollection.org
Parks:
https://www.royalparks.org.uk/parks/hyde-park Hyde Park
https://www.royalparks.org.uk/parks/the-regents-park
http://www.cityoflondon.gov.uk/things-to-do/green-spaces/hampsteadheath/Pages/default.aspx Favourite park in central London when the weather is
good. Also has a great view of London and a lido, which will be open in the summer
(and virtually empty on weekdays).
https://www.royalparks.org.uk/parks/greenwich-park World Heritage site, awesome
views, brilliant place to spend a sunny day and walking distance from black heath
which is a nice little centre of commerce full of shops/cafes/pubs and restaurants.
https://www.royalparks.org.uk/parks/richmond-park Enormous park – Richmond is
also a great place to spend a day.
Performance:
http://www.southbankcentre.co.uk/
http://wigmore-hall.org.uk/
http://www.wiltons.org.uk
http://www.nationaltheatre.org.uk
http://www.shakespearesglobe.com
https://www.ica.org.uk/
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Programme
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Abstracts
Session 1
What do γδ T cells recognize? CD1, haptens, novel
insights
Talks:
Thursday, June 16th
Poster presentation:
Thursday, June 16th, 6:30 – 8:30pm
Friday, June 17th, 1:00 – 2:30pm
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A-01
Examining the structure of terminal, antibody-like V domains of shark
NAR-TCR receptors
1
1
2
3
Caitlin D. Castro , Mary Coomes , Yuko Ohta , Michael F. Criscitiello , Martin F.
2
1
Flajnik , Erin J. Adams
1
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL;
Department of Microbiology and Immunology, University of Maryland, Baltimore, MD;
3
Department of Veterinary Pathobiology, Texas A&M University, College Station, TX
2
Cartilaginous fish (sharks, skates, rays) are the oldest extant animals with an
immunoglobulin (Ig) superfamily domain-based adaptive immune system that
includes B cell receptors (BCR) and T cell receptors (TCR). In addition to traditional
alpha beta (αβ) and gamma delta (γδ) TCRs, nurse sharks express another, unique
type of γδ TCR, called NAR-TCR. NAR-TCRδ chains are composed of three, rather
than two Ig domains: conventional Cδ and Vδ domains are positioned C terminally of
a third “NAR-TCR-V” domain, which is encoded by rearranging VDJ miniclusters
upstream of a conventional Vδ rearranging gene segment. The NAR-TCR-V domain
is evolutionarily related to the terminal, V domain of the single-chain Ig isotype,
IgNAR. The striking similarity of this TCR-V to an IgVH suggests that these TCRs may
bind to free antigen in a truly Ig-like fashion, rather than via presenting or adaptor
molecules. Although not described in placental mammals or bony fish, the presence
of several VH-like TCR domains in all other vertebrate taxa suggests they may be
playing important roles in adaptive immunity in many animals. Nothing is known,
however, about the structure of either the terminal NAR-TCR-V domain, or the
complete NAR-TCR protein. We have therefore begun structural studies to examine
these N-terminal single V domains, and have solved the structure of a nurse shark
type 1 NAR-TCR-V. By comparing this structure to previously published IgNAR-V
structures as well as models of other NAR-TCR-V types, including a type 3 NARTCR, we may begin to predict how these unusual TCRs bind antigen.
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A-02
Cholesteryl esters stabilise human CD1c conformations for recognition
by self-reactive T cells
a,b,1
a
c
b,d
Salah Mansour
, Anna S Tocheva , Chris Cave-Ayland , Moritz M Machelett ,
c
e
e
f
g
Barbara Sander , Nikolai M Lissin , Peter E Molloy , Mark S Baird , Gunthard Stübs ,
h
i
j
a
Nicolas WJ Schröder , Ralf R Schumann , Jörg Rademann , Anthony D Postle , Bent
e
a,b
c
a,b
b,k
K Jakobsen , Ben G Marshall , Rajendra Gosain , Paul Elkington , Tim Elliott ,
b,c
b,c
b,d
a,b,l,1
Chris-Kriton Skylaris , Jonathan W Essex , Ivo Tews , and Stephan D Gadola
a
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton,
Southampton, UK
b
Institute for Life Sciences, University of Southampton, Southampton, UK
c
School of Chemistry, University of Southampton, Southampton, UK
d
Centre for Biological Sciences, University of Southampton, Southampton, UK
e
Immunocore Ltd, Abingdon, UK
f
School of Chemistry, Bangor University, Bangor, Wales, UK
g
Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany
h
Institute for Pathology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
i
Institute for Microbiology and Hygiene, Charité University Medical Center, Berlin, Germany
j
Division of Medicinal Chemistry, Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany
k
Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK
l
Novartis Institutes of Biomedical Research, Basel, Switzerland
CD1c-dependent self-reactive T cells are highly abundant, comprise subsets of αβ
and γδ T cells, and have roles in human cancer and autoimmunity, but self-lipid
antigens presented by CD1c to the T cell receptors of these cells remain poorly
understood. We determined a new CD1c crystal structure at 2.4 Å revealing an
extended ligand binding potential of the antigen groove and a substantially different
conformation compared to known CD1c structures. Furthermore, our data reveal new
structural features for CD1c including the first description of a roof over the F’
channel. Computational simulations exploring different occupancy states of the
groove re-enacted these different CD1c conformations, and predicted the binding of
disease relevant lipid antigens such as cholesteryl esters (CE) and acylated steryl
glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that
binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T
cell receptors. Hence, human CD1c adopts different conformations dependent on
ligand occupancy of its groove, with CE and ASG stabilising CD1c conformations that
provide a footprint for recognition by self-reactive T cells.
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A-03
ABCA1 in cooperation with BTN3A1 and apo-AI regulates extracellular
IPP release and Vγ9Vδ2 T-cell activation by dendritic cells
1
1
1
2
Patrizia Sciancalepore , Barbara Castella , Myriam Foglietta , Joanna Kopecka ,
3
1
3
1
2
Nico Mitro , Giorgia Mandili , Donatella Caruso , Francesco Novelli , Chiara Riganti ,
1
and Massimo Massaia
1
Dipartimento di Biotecnologie Molecolari e Scienze della Salute, Divisione di Ematologia,
Università di Torino, Italy;
2
Dipartimento di Oncologia, Università di Torino, Italy;
3
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università di Milano, Italy
Phosphorylated metabolites of the mevalonate (Mev) pathway like isopentenylpyrophosphate (IPP) can induce Vγ9Vδ2 T-cell proliferation. Endogenous IPP levels
can be lowered or increased by statins or zoledronic acid (ZA) according to their
ability to target HMGR or FPPS in the Mev pathway. We have previously shown that
ZA-treated dendritic cells (DC) produce and release high amounts of soluble IPP and
are strong Vγ9Vδ2 T-cell activators. However, the mechanisms regulating IPP
release in DC and other cells are still unknown.
Here we demonstrate that the ATP binding cassette transporter A1 (ABCA1)
mediates extracellular IPP release from ZA-treated DC and leads to the activation of
Vγ9Vδ2 T-cells in cooperation with butyrophilin-3A1 (BTN3A1) and apoA-I. Vγ9Vδ2
T-cell activation is directly correlated with IPP levels released in the supernatants
(SN) and ABCA1 expression in DC.
Functional ABCA1 inhibition with probucol slightly increased intracellular IPP
accumulation, but abrogated extracellular IPP release and Vγ9Vδ2 T-cell activation.
Abca1 silencing experiments confirmed the key role played by ABCA1. ZA treatment
increased ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear
translocation and IPP-independent PI3K/Akt/mTOR pathway inhibition.
These results indicate that ABCA1 is involved in extracellular IPP release and
Vγ9Vδ2 T-cell activation induced by ZA-treated DC.
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A-04
Variegated expression of members of a pathogen recognition receptor
and co-receptor multi-gene family on γδ T cells known as WC1.
Damani-Yokota P, Telfer JC and Baldwin CL
Dept. of Veterinary & Animal Sciences, University of Massachusetts-Amherst
Cells of the immune system recognize disease-causing pathogens and respond in a
manner to stop the infection. While we know how conventional cells of the immune
system do this, for γδ T cell this process in less clear. We have shown that bovine γδ
T cells bear lineage-specific transmembrane glycoproteins known as WC1 coded by
a multigenic family and that these molecules function both as pattern recognition
receptors (PRR) and signaling co-receptors for cellular activation. For example, while
+
a subpopulation known as WC1.1 γδ T cells respond early and participate in a recall
response to the spirochetes Leptospira interrogans and Leptospira borgpetersenii,
other subpopulations that express a different set of the WC1 genes do not respond.
Other experiments showed that WC1 molecules on γδ T cells that respond to
Leptospira bind the bacteria. Thus, we hypothesize that WC1 is essential for the
recognition of bacterial pathogens by γδ T cells and that the WC1 family members
expressed by a particular cells will determine its ability to respond to an infection. We
are now evaluating both WC1 transcripts and proteins to further understand the roles
of WC1 molecules in directing γδ T cell responses during infection.
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A-05
HSP70 as a Candidate Ligand for Human Synovial Vδ1 T cells
1
1
Ralph C. Budd , Cheryl Collins , and Roy Mariuzza
1
2
2
University of Vermont
University of Maryland USA
A protective role of γδ T cells has been observed in various infectious models
including our own studies with murine and human infection with Borrelia burgdorferi.
γδ T cells also accumulate at sites of inflammation in autoimmune syndromes such
as the synovium in rheumatoid and Lyme arthritis. We have established several
human synovial γδ T cell clones of the Vδ1 subset from patients with Lyme arthritis.
We have noted that their response to Borrelia burgdorferii is indirect and that this is
augmented by dendritic cells (DC) undergoing necroptosis. This suggested that a
ligand(s) for the synovial γδ T cells might be exposed or upregulated in necroptotic
DC. We observed no detectable response of this γδ T cell clone to any CD1
molecules. We have now cloned one of the TCR-γδ from Vδ1 clone Bb15 and
expressed it as a soluble protein and used it as a bait to pull candidate ligands from
DC lysates. Screening the mass spec results particularly for cell stress/death- related
molecules, we identified several candidate ligands. Only one of these, Hsp70, was
able to activate several of our synovial γδ T cell clones. We are now performing
plasmon resonance for the interaction of the soluble TCR-γδ with Hsp70. We have
also transfected the same TCR-γδ into a Jurkat cell line for further high throughput
screening.
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A-06
Sensing of cell stress by human γδ TCR-dependent recognition of
Annexin A2
1
1
1,2
3
Romain Marlin , Angela Pappalardo , Hannah Kaminski , Carrie R. Willcox ,
1,4
1
1
5
Vincent Pitard , Sonia Netzer , Camille Khairallah , Anne-Marie Lomenech ,
1,6
6
1,7
6
Christelle Harly , Marc Bonneville , Jean-François Moreau , Emmanuel Scotet ,
3
1
1,4
Benjamin E. Willcox , Benjamin Faustin and Julie Déchanet-Merville .
1
CNRS, ImmunoConcept, Bordeaux University, Bordeaux, France ;
Renal Transplantation Department, CHU Bordeaux, Bordeaux, France;
3
Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United
Kingdom;
4
Flow cytometry facility, CNRS Bordeaux University, Bordeaux, France;
5
Proteomic facility, Functional Genomic Center, Bordeaux, France;
6
7
Center of Research in Cancerology, University of Nantes, Nantes, France; Immunology and
Immunogenetic laboratory, CHU Bordeaux, Bordeaux, France.
2
Human γδ T cells comprise a first line of defense through TCR recognition of
stressed cells. However, the molecular determinants and stress pathways involved in
this recognition are largely unknown. Here we show that exposure of tumor cells to
various situations of stress (heat shock, hypoxia or cytomegalovirus infection)
induced tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously
developed to identify antigenic ligands of γδ TCRs (Willcox et al, Nat Immunol, 2012,
13:872), Annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was
found to be expressed on tumor cells upon the stress situations tested in a reactiveoxygen species-dependent manner. Moreover, purified Annexin A2 was able to
neg
stimulate Vδ2
γδ T cell proliferation within PBMC incubated with IL-2. We thus
propose membrane exposure of Annexin A2 as an oxidative stress signal for a subset
of γδ T cells involved in lymphoid stress surveillance.
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A-07
EPCR expression
tumourigenesis
1
is
consistently
1
upregulated
2
in
colorectal
3
Neeraj Lal , Carrie R. Willcox , Andrew Beggs , Christopher J. Bagnall , Philippe
4
2
1
Taniere , Chris Tselepis and Benjamin E. Willcox
1
Institute of Immunology and Immunotherapy, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, UK
2
Institute of Cancer and Genomics, University of Birmingham, Edgbaston, Birmingham, B15
2TT, UK
3
Human Biomaterials Resource Centre, University of Birmingham, Edgbaston, Birmingham, B15
2TT, UK
4
Department of Pathology, Queen Elizabeth Hospital Birmingham, Edgbaston, Birmingham, UK
γδ Τ cells are able to recognise ‘altered self’, by detecting upregulation of host
components via germline-encoded receptors (eg NKG2D) or potentially via the γδ
TCR, in either microbial or non-microbial stress. We have previously identified EPCR
as a direct ligand for the Vγ4δ5 LES TCR, originally isolated from a patient with acute
CMV. Expressed on endothelial cells infected by CMV in vivo, EPCR was not
upregulated by CMV infection, but TCR recognition of EPCR in the context of a CMVinduced ‘multimolecular stress signature’ was sufficient to induce LES recognition of
target cells. In contrast, EPCR is abundantly expressed on various tumour lines
recognised by the LES clone, although the extent to which EPCR becomes
upregulated during in vivo tumourigenesis is unclear. Here, we show that EPCR is
commonly overexpressed on epithelial tumour cells in human colorectal cancer.
Bioinformatic methods revealed EPCR upregulation results from gene amplification
and DNA hypomethylation, effects likely relevant to multiple epithelial
malignancies. Moreover, EPCR is upregulated with a cohort of neighbouring genes
on chromosome 20q, a region previously implicated in chemoresistance. Also, in
vitro APC treatment had diverse effects on different EPCR+ CRC cell lines, including
ERK
phosphorylation,
protein
translation,
and
changes
in
chemoresistance/migration. Our results provide a compelling explanation for EPCR
upregulation in diverse epithelial malignancies, and may have relevance to in vivo
chemoresistance in cancer. They highlight that single γδ TCR ligands have the
potential to denote ‘altered self’ in different ways in distinct stress stimuli, including
infection and tumourigenesis.
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A-08
Human Vδ3 T cells induce maturation and IgM secretion by B cells in
vitro
Andreea Petrasca, Robyn Bruen, Derek G. Doherty
Discipline of Immunology, School of Medicine, Trinity College Dublin, Ireland
Human Vδ3 T cells constitute a minor subset of peripheral blood lymphocytes. They
have been reported to be CD1d restricted and are enriched in the liver and gut and
expanded in patients with HIV and B-cell chronic lymphocytic leukaemia. Like Vδ2 T
cells, they are capable of maturing DC into cytokine-producing antigen presenting
cells (APC), making them potential targets for DC-based immunotherapies. Since it is
unknown if Vδ3 T cells can also provide B cell help, we investigated if Vδ3 T cells
can promote B cell differentiation, antibody and cytokine production in vitro.
Vδ3 T cells were sorted from healthy human blood and expanded with
phytohemagglutinin in the presence of irradiated cells and IL-2 for 4 weeks.
Expanded, pure (>90%) lines of Vδ3 T cells were co-cultured with equal numbers of
freshly isolated human B cells for 24h or 7 days and the B cells and Vδ3 T cells were
then examined for expression of APC markers, cytokine secretion and
immunoglobulin production. Vδ3 T cells were also examined for their ability to
recognise glycolipids via CD1 molecules, but were found to be mostly CD1
unrestricted.
Vδ3 T cells and B cells reciprocally induced expression of maturation markers CD40,
CD86 and HLA-DR but not TH1, TH2 or TH17 cytokines after 24h. Furthermore, Vδ3 T
cells promoted the release of IgM, but not IgG or IgA by B cells.
These data demonstrate a reciprocal activating relationship between Vδ3 T cells and
B cells which could prove a useful target for cellular immunotherapy.
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Session 2
What do γδ T cells recognize? The roles of
Butyrophilin / Butyrophilin-like molecules in γδ
biology
Talks:
Friday, June 17th
Poster presentation:
Thursday, June 16th, 6:30 – 8:30pm
Friday, June 17th, 1:00 – 2:30pm
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A-09
BTN3 isoforms cooperate in PAg mediated activation of Vγ9Vδ2T cells
1,2
Mohindar Murugesh Karunakaran, Daniel Paletta, Lisa Starick, Marco J Herold ,
Ingolf Berberich, Thomas Herrmann
Institute for Virology and Immunobiology, University of Würzburg, Germany,
1
The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
2
Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
[email protected]
Vγ9Vδ2 T cells recognize pyrophosphate-containing metabolites of host and
microbial isoprenoid synthesis (phosphoantigens-PAg) such as IPP and HMBPP. The
PAg induced activation of Vγ9Vδ2 T cells requires its contact with BTN3A1
expressing cells. BTN3A1 belongs to BTN3 gene family which comprises also
BTN3A2 and BTN3A3. Though BTN3A1 is essential for PAg mediated activation of
Vγ9Vδ2 T cells, the contribution of other members of BTN3 still remains unknown. To
unravel the above in PAg mediated activation, we implemented CRISPR/Cas9
technique to knock out BTN3 genes. In our study, RAJI B-cell lymphoma cell line was
used as antigen presenting cell. With CRISPR/Cas9, we generated RAJI BTN3-/- and
RAJI BTN3A1-/-. RAJI, BTN3-/- and BTN3A1-/- cells were co-cultured with 53/4
hybridoma cells transduced with Vγ9Vδ2 TCR in the presence and absence of
zoledronate (ZOL) or HMBPP. The activation of TCR transductants was accessed by
measuring the IL-2 in co-cultures. Unlike RAJI, BTN3-/- and BTN3A1-/- cells were
unable to activate our TCR transductants in presence of HMBPP. It was reproduced
in the presence of ZOL except, at higher concentration of ZOL (100μM) BTN3A1-/resulted in weak activation of TCR transductants. Upon reconstitution of BTN3A1,
BTN3-/- cells activated TCR transductants but only marginal if compared with
reconstituted BTN3A1-/- cells or RAJI cells. Experiments investigating the exclusive
role of BTN3A2 and BTN3A3 in activating T cells in presence of HMBPP and ZOL
are underway. However, our data already suggest that BTN3A2 and/or BTN3A3 are
essential for the optimal activation of Vγ9Vδ2 T cells in presence of PAg.
Supported by Wilhelm Sanderstiftung
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A-10
Vγ9Vδ2 TCR mediated activation by agonistic butyrophilin 3 specific
mAb 20.1 is controlled by TCR-idiotypes and interferes with activation
by phosphoantigens
1
2
1
3
1
1
F. Riano*1, L. Starick,* , S. Gu. , M. M. Karunakaran , V. Kunzmann , J. Li , M. Kreiss , S.
4
5
6
7
2
1
Amslinger , E. Scotet D. Olive , G. De Libero , E. J Adams , T. Herrmann
1
Dept of Virology and Immunbiology, U. of Wuerzburg, GER,
Dept. of Biochemistry and Mol. Biol., U. of Chicago, IL, USA,
Med. Clinic and Policlinic II, U. of Wuerzburg, GER,
4
Institute of Organic Chemistry, U. of Regensburg, GER,
5
INSERM U892/CNRS UMR6299, U. Nantes, F,
6
CRCM Team Immunity and Cancer, INSERM U1068, Institut Paoli-Calmette, Marseille, F,
7
Experimental Immunology, U. of Basel, CH * equal contribution
correspondence: [email protected]
2
3
Phosphoantigens (PAg) such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate
and aminobisphosphonates activate Vg9Vd2 T cells in a TCR-mediated manner
which dependents on expression of butyrophilin 3A1 (BTN3A1) on a stimulator cell.
Another agonist is the BTN3 specific monoclonal antibody (mAb) 20.1 while the
BTN3 specific mAb 103.2 inhibits PAg and aminobisphosphonate induced activation.
Reports were contradictory on activation by mAb 20.1 of Vg9Vd2 TCR
transductants/transgenic and effects of mAb 20.1 on PAg induced stimulation. To
understand these different results, the TCRs originally used to generate the
respective TCR transductants/transgenic cells were expressed in the same reporter
cells and responses to native and single chain BTN3 specific antibodies in presence
or absence of PAg were directly compared. The major findings were: i) Culture of
murine TCR transductants with mAb 20. 1 and human stimulator cells revealed mAb
20.1-responder and non-responder Vg9Vd2 TCR idiotypes despite of that all
transductants responded readily to PAg and ABP. ii) The responder TCR
transductants were efficiently activated by mAb 20.1 and its single-chain (sc)
derivative and to some extent also by sc of antagonistic mAb 103.2. iii) Not only
103.2. mAb but also mAb 20.1 and sc 20.1 inhibited PAg induced activation of the
responder transductants. iv) The differences between responder and non-responder
TCR idiotypes were partially leveled in TCR transduced human Jurkat cells tested for
CD69 induction. These results allow to explain the apparent discrepancy in the
previous reports and to further elucidate BTN dependent Vg9Vd2-TCR mediated
activation. Supported by Wilhelm Sander Stiftung
Page 29 of 200
GDC 2016
A-11
Critical role of the intracellular coiled coil domain of butyrophilin 3A1
for prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells
Hong Wang
*, †
and Craig T. Morita
*, †, ‡
*
Division of Immunology, Department of Internal Medicine, University of Iowa Carver College of
Medicine, Iowa City, IA 52242, U.S.A.
†
Department of Veterans Affairs, Iowa City Health Care System, Iowa City, IA 52246, U.S.A.
‡
Interdisciplinary Graduate Program in Immunology, University of Iowa Carver College of
Medicine, Iowa City, IA 52242, U.S.A.
Vγ2Vδ2 T cells play important roles in human immunity to pathogens and in cancer
immunotherapy by responding to isoprenoid metabolites such as (E)-4-hydroxy-3methyl-but-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate. The
immunoglobulin superfamily protein, butyrophilin 3A1 (BTN3A1), is required for this
stimulation. Mutational studies by ourselves and others have shown that HMBPP is
not an antigen but instead is a sensed molecule that binds to the intracellular B30.2
domain of BTN3A1. How this binding is detected by the Vγ2Vδ2 TCR is unclear.
BTN3A1 is a dimer with extracellular IgV/IgC domains (ECD) linked to intracellular
B30.2 domains by an intracellular coiled coil domain. One possibility is that HMBPP
binding alters the conformation and/or distribution of the BTN3A1 dimer allowing
direct binding to the Vγ2Vδ2 TCR or to another molecule that binds the Vγ2Vδ2
TCR. Alternatively, the Vγ2Vδ2 TCR could bind to a protein(s) recruited to the
BTN3A1 intracellular tail upon B30.2 binding to HMBPP. To distinguish between
these mechanisms, we mutagenized 39 residues on the surface of the ECD and 8
anchor residues that would disrupt the coiled coil domain and tested the mutant
proteins for their function. Mutagenesis of ECD residues had no effect whereas
mutagenesis of coiled coil residues either abrogated (mid-region) or decreased (distal
region) HMBPP stimulation. Thus, these findings do not support direct binding by the
Vγ2Vδ2 TCR to the ECD of BTN3A1. Instead, Vγ2Vδ2 TCR recognition may be to
the ECD of a protein recruited to the coiled coil domain upon B30.2 domain binding to
HMBPP.
Page 30 of 200
GDC 2016
A-12
Phosphoantigen-Induced Conformational Change of the BTN3A1 and
its implication on Vγ9Vδ2 T cell Activation
1
1,2
1
1
Siyi Gu , Joseph R. Sachleben , Marta T. Borowska , Wioletta I. Nawrocka ,
3
1,4
Georgios Skiniotis , Erin J. Adams
1
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL
60637, USA
2
Biomolecular NMR Facility, University of Chicago, Chicago, IL 60637, USA
3
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109-2216, USA
4
Committee on Immunology, University of Chicago, Chicago, IL 60637, USA
Human Vγ9Vδ2 T cells circulate in peripheral blood and detect various microbial
infections and certain types of tumors. The antigen recognition process of Vγ9Vδ2 T
cells involves intracellular sensing of small pyrophosphate metabolites
(phosphoantigens, or pAgs) via the butyrophilin 3A(BTN3A) molecule. Here we
demonstrated that binding of pAg to BTN3A1 causes conformational change of its
intracellular domain by nuclear magnetic resonances (NMR) spectrometry. A tyrosine
residue near the pAg-binding pocket experiences largest chemical shift perturbation
(CSP) and is shown to be critical for differential binding of exogenous and
endogenous pAgs and T cell activation. We then showed by crystallography that like
its extracellular domains, BTN3A1 full-length intracellular domain with additional
membrane proximal region at the N-terminal of the B30.2 domain also adopts two
alternative conformations in the crystal lattice. The two dimer interfaces exhibit
intriguing features such as close proximity to the pAg-binding pocket and clustering of
residues that experience major CSP upon pAg binding. We also observed at least
one conformation of the extracellular domains of BTN3A1 that previously reported in
the crystal structure in a native lipid-like environment in vitro using negative staining
electron microscopy. Based on these evidences, we propose that pAg binding likely
facilitates interchange of two conformations of the intracellular domain of BTN3A1,
which then propagates to its extracellular domain. The conformational change of
BTN3A1 extracellular domains may present a critical signal to Vγ9Vδ2 T cells that
ultimately leads to activation.
Page 31 of 200
GDC 2016
A-13
Role of BTN3A molecules in activation of human gammadelta T cells
1
2
3
1
David A. Rhodes , Hung-Chan Chen , James C. Williamson , John Trowsdale ,
2
Matthias Eberl
1
Dept. of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB22 4PA
Division of Infection & Immunity, School of Medicine, and Systems Immunity Research Institute,
Cardiff University, Heath Park, Cardiff CF14 4XN;
3
Cambridge Institute for Medical Research, Hills Road, Cambridge, CB2 0XY, United Kingdom.
2
The phosphoantigen-dependent activation of human Vγ9Vδ2 T cells is dependent on
butyrophilin BTN3A molecules. We investigated the molecular basis of this complex
mechanism using structural studies, binding assays and by the use of BTN3A
knockdown in HeLa cells. Our results were consistent with: 1. direct binding of HMBPP and IPP to the cytosolic B30.2 domain of BTN3A1, 2. a role in transmitting
activation signals to T cells for the BTN3A isoforms BTN3A2 and BTN3A3, and 3.
involvement of the plakin protein periplakin, a BTN3A1 interacting molecule. These
results highlighted a number of additional complexities in this mechanism, including
1. how does externally delivered HMB-PP gain access to the cytosolic B30.2 domain,
2. Is there competition between endogenous IPP and HMB-PP for B30.2 binding, 3.
How does the agonist monoclonal antibody 20.1 mimic phosphoantigen dependent
activation, 4. how do the BTN3A isoforms interact to control T cell activation, and 5.
what is the precise role of periplakin and its interacting partners? We are addressing
these questions by investigating mutant Hela cell lines which show altered BTN3A
responses to phosphoantigen.
Rhodes, D. A., H. C. Chen, A. J. Price, A. H. Keeble, M. S. Davey, L. C. James, M.
Eberl, J. Trowsdale (2015). Activation of human gammadelta T cells by cytosolic
interactions of BTN3A1 with soluble phosphoantigens and the cytoskeletal adaptor
periplakin. J Immunol 194(5): 2390-2398.
Rhodes, D.A., W. Reith, and J. Trowsdale (2016) Regulation of Immunity by
Butyrophilins. Ann. Rev. Immunol. In press.
Page 32 of 200
GDC 2016
A-14
Evolution of γδ T cells – studying Vγ9Vδ2 T cells in Alpaca and
Armadillo
1
1
2
Alina S Fichtner , Mohindar Murugesh Karunakaran , Richard W. Truman , Thomas
1
Herrmann
1
2
Julius-Maximilians-Universität Würzburg, Würzburg, Germany and
National Hansen’s Disease Program, Louisiana State University, Baton Rouge, Louisiana, USA
1-5% of blood T cells are Vγ9Vδ2 T cells which express a TCR composed of a Vγ9JP (Vγ2-Jγ1.2) chain together with a δ-chain containing Vδ2 and a hydrophobic
amino acid at position 97. They respond to phosphoantigens like isopentenyl
pyrophosphate (IPP) or (E)-4-Hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP)
in a BTN3-dependent manner. These cells were thought to be restricted to primates,
but we demonstrated recently by analysis of genomic databases that Vγ9, Vδ2, and
BTN3 genes coevolved and emerged together with placental mammals. Furthermore,
we identified alpaca (Vicugna pacos) as species with typical Vγ9Vδ2 TCR
rearrangements and aim to directly identify Vγ9Vδ2 T cells. Another candidate to
study this coevolution is armadillo (Dasypus novemcinctus) which possesses inframe Vγ9 and Vδ2 genes and a BTN3 V-domain. It is of medical interest since it
serves as natural reservoir for Mycobacterium leprae. We analyzed the expression of
these genes by 3’RACE-PCR and performed further database analysis which
revealed neither a C-domain nor an intracellular B30.2 domain for BTN3 making it
likely to be non-functional in terms of phosphoantigen action. ORFs for BTN3ED-V
domain and Vγ9 were confirmed at DNA level but attempts to identify spliced
products by 3’RACE-PCR failed. In contrast, complete Vδ2 containing δ chains
rearranged with a Jδ4 homologue were cloned. So far, the lack of expression of Vγ9chains and BTN3 challenges armadillo as candidate species for phosphoantigenreactive Vγ9Vδ2 T cells but is in line with the postulated co-evolution of the three
genes, where occurrence of Vγ9Vδ2 TCR coincides with a functional BTN3.
Supported by DFG He 2346/7-1
Page 33 of 200
GDC 2016
A-15
The Role Of Chromosome 6 Located Genes In Vγ9Vδ2 T Cell Activation
Paletta D*, Fichtner AS*, Karunakaran MM*, Herrmann T*
*Institute for Virology and Immunobiology, University of Wuerzburg, Germany
The Vγ9Vδ2 T cell population is characterized by its phosphoantigen (PAg) reactivity.
Those phosphorylated antigens originate from microbes like HMBPP or are
expressed ubiquitously like IPP. IPP levels can be highly elevated in tumour cells or
can be increased artificially thereby marking those cells for lysis by Vγ9Vδ2 T cells.
While the anti-tumour potential of Vγ9Vδ2 T cells has been proven, the mechanism
of PAg-dependent Vγ9Vδ2 T cell activation has not been resolved yet: We could
show that butyrophilin 3A1 (BTN3A1) is substantial but not sufficient to mediate full
PAg-dependent activation (“phenotype x”). Rodent cells lack the ability to present
PAgs to Vγ9Vδ2 T cells and thus are a suitable tool to further analyse the
requirements for this process: By using hamster cells carrying human Chromosome 6
(huChr6), we were able to show that at least one additional gene present on huChr6
is needed to mediate this phenotype. In our current study we generate radiation
hybrids (RHs) as a strategy to identify further genes necessary to mediate PAg
presentation. huChr6 carrying, phenotype x positive rodent cells are irradiated and
fused with huBTN3A1 expressing, phenotype x negative rodent cells. The resulting
hybrid cells retain only parts of the previous huChr6 genetic content and can be
screened functionally for phenotype x, narrowing down potential regions. This allows
for selection of candidate genes from such regions which can be targeted for loss-offunction and reconstitution analysis. First results show the potential of this strategy
and confirm the importance of huChr6p.
Supported by Deutsche Krebshilfe
Page 34 of 200
GDC 2016
A-16
NKG2D contribution to effector functions of γ9δ2TCR expressing cells
is dictated by γ9δ2TCR-mediated avidity.
2
2
2
Anna Vyborova , Cordula Gründer , Trudy Straetemans , Zsolt Sebestyen
1,2
Jürgen Kuball
1
2
and
2
Department of Hematology , Laboratory of Translational Immunology , University Medical
Center Utrecht, Utrecht, The Netherlands
Gamma9 delta2 T cells are generally thought to share characteristics of both
adaptive and innate immunity, being characterized by expression of somatically
rearranged γ9δ2 TCR together with a set of inhibitory and activating NK-receptors.
As previously published by our group, diversity of CDR3 regions of the γ9δ2 TCR
clones may account for different affinities of the TCRs for the same putative ligand.
Next to diverse TCRs, NK-receptors may further shape the effector functions of γ9δ2
T cells, but the details of this interplay remain poorly understood.
In this study we addressed the role of the central activating NK-receptor NKG2D in
the context of γ9δ2 TCR’s which mediate different functional avidities. By coexpressing the two receptors in CD4+ αβT cells, which naturally lack NK-receptors,
we found that NKG2D acts in a co-stimulatory fashion, being unable to trigger T cell
response when co-expressed with a non-functional γ9δ2 TCR. Contribution of
NKG2D to functional avidity is substantial when NKG2D is co-expressed with a lowaffinity γ9δ2 TCR or when expression of putative γ9δ2 TCR ligands on a target cell is
low. In contrast, little or no contribution by NKG2D is observed when high amounts of
TCR ligands are present and the affinity of the γ9δ2TCR is high. In conclusion, γ9δ2
T cell selection and activation depends on different affinities of defined γ9δ2 TCR as
well as the presence or absence of co-stimulatory molecules. These data support a
concept of avidity thresholds for γ9δ2 T cells based on their individual composition of
activating receptors.
Page 35 of 200
GDC 2016
A-17
Structural studies on BTN3A1 support small molecule binding to the
B30.2 domain
1
2
1
1
Mahboob Salim , Timothy Knowles , Carrie R. Willcox , Martin S. Davey , Alfie
1
1
1
Baker , Fiyaz Mohammed and Benjamin E. Willcox
1
Institute of Immunology and Immunotherapy, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, UK
2
Institute of Cancer and Genomics, University of Birmingham, Edgbaston, Birmingham, B15
2TT, UK
The butyrophilin-like molecule BTN3A1 is thought to play a critical role in
phosphoantigen-mediated activation of Vγ9Vδ2 T cells, the predominant γδ T cell
subset in human peripheral blood. However the mechanisms involved are unclear,
with conflicting data suggesting presentation of phosphoantigen by the membranedistal immunoglobulin-like domain of BTN3A1, and alternatively suggesting
phosphoantigen sensing by the cytosolic SPRY/PRY B30.2 domain. To discriminate
between these two possibilities, we separately expressed the extracellular V-domain
and intracellular B30.2 domain, and carried out nuclear magnetic resonance studies
in the presence and absence of phosphoantigens. Whereas we were able to assign
1
15
all residues in the H- N HSQC spectrum of the V-domain, comparisons failed to
identify any chemical shifts in the presence of phosphoantigen, including in the
proposed phosphoantigen binding site. In contrast, comparable experiments
indicated substantial shifts in several residues in the B30.2 domain, indicative of
phosphoantigen binding and consistent with a subsequent conformational change. To
explore this further, we attempted but failed to co-crystallise the phosphonate
analogue HDMAPP with B30.2 domain. Instead, we serendipitously solved the
structure of the B30.2 domain with malonate, a negatively charged crystallisation
component of 160 Daltons. Malonate unequivocably bound at the proposed
phosphoantigen binding site, but did not alter protein conformation compared to the
unbound B30.2 domain. These data confirm binding of negatively charged small
molecules to the BTN3A1 B30.2 domain, and suggest the ability to induce a
conformational change in B30.2 may be a critical initiating step in phosphoantigenmediated sensing by Vγ9Vδ2 T cells.
Page 36 of 200
GDC 2016
A-18
Tissue-specific Butyrophilin-like molecules are master regulators of
intraepithelial γδ T cell composition
1-3
1
1,2,4
2
1
Rafael Di Marco Barros , Natalie Roberts , Robin Dart , Pierre Vantourout , Anett Jandke ,
2
1
1
1
2
Oliver Nussbaumer , Livija Deban , Sara Cipolat , Rosie Hart , Maria Luisa Iannitto , Adam
2
2
4
1
5
6
Laing , Deena Gibbons , Peter Irving , Bradley Spencer-Dene , Pablo Pereira , Ulrich Steinhoff ,
1-3
Adrian Hayday
1
Francis Crick Institute, London, WC2A 3LY, UK;
Peter Gorer Department of Immunobiology, King’s College London;
University College London
4
Department of Gastroenterology, Guy’s and St Thomas’ Foundation Trust, London SE1 7EH
5
Dept of Immunology, Pasteur Institute, Paris, France;
6
Institute for Medical Microbiology and Hospital Epidemiology, University of Marburg, Germany.
2
3
Many epithelial barriers are constitutively populated by large numbers of organspecific γδ T cells that collectively make up a substantial proportion of the body’s T
cells. In mice, such intraepithelial lymphocytes (IEL) display striking site-specific T
cell receptor (TCR) Variable (V)γ chain usage (Vγ5+- epidermis, Vγ6+-uterine
epithelium and Vγ7+-small intestinal epithelium). These signatory associations are
critically important, and multiple mouse models have linked deficiencies in canonical
Vγ5+ Dendritic Epidermal T Cells (DETC) to dysregulated cutaneous inflammation
and heightened susceptibility to cutaneous carcinoma.
Skint1 is a Butyrophilin-like (Btnl) molecule expressed exclusively by thymic
medullary epithelium and suprabasal keratinocytes, which shapes the DETC
compartment. However, as neither Skint1 nor DETC are conserved, a general
mechanism by which epithelia shape IEL composition remains unelucidated. Here we
show that enterocyte expression of Btnl1 shapes the small intestinal IEL
compartment by selectively driving extrathymic maturation and expansion of Vγ7+
IEL in trans upon their arrival in the gut. Uninfluenced by microbial or solid food
antigens, this mechanism evokes Major Histocompatibility Complex (MHC)-driven
selection of αβ T cell repertoires. Indeed, Btnl1 together with Btnl6 elicits specific
TCR-dependent responses of intestinal Vγ7+ cells in vitro.
From these data, a generalizable mechanism emerges whereby organ specific
expression of Btnl genes enables epithelia to actively shape the lifelong composition
of their signatory IEL. By identifying the selection elements that regulate signatory γδ
IEL compartments, our models provide novel avenues to study the unique
contributions of discreet IEL compartments to host immunity.
Page 37 of 200
GDC 2016
A-19
Butyrophilin-like 3 and 8 selectively regulate a subset of human tissue
resident colonic γδ T cells
1,2
1
1
Robin Dart , Pierre Vantourout , Oliver Nussbaumer & Adrian Hayday
1
2
1,2
Peter Gorer Department of Immunobiology, King’s College London;
Francis Crick Institute, London, WC2A 3LY, UK
-
Mechanisms governing the predilection of human Vδ2 γδ T cells to epithelia remain
unknown. Studying these subsets has been complicated by difficulties in isolating
them and a lack of clear parallels between mouse and human systems. The role of
Skint1 in DETC selection has provided insight in the shaping γδ repertoire by tissuespecific determinants. We recently demonstrated that a Butyrophilin-like (Btnl) 1/6
+
heterodimer regulates the maturation of murine intestinal Vγ7 cells. While there is no
direct orthologue of Btnl1/6 in humans, we identified BTNL3 and BTNL8 as potential
homologues as their expression is strongly enriched in human intestinal epithelium
and their surface expression is regulated by heterodimerisation. Using a novel culture
technique, we obtained tissue-resident T cells from human colonic biopsies,
facilitating their characterization. Co-culture with HEK293T cells co-transduced with
both BTNL3 and BTNL8 resulted in a marked downregulation of γδTCR expression
and CD25 upregulation, which was contact-dependent. A set of γ chain-specific
antibodies indicated that Vγ4+ cells, but not other γδ or αβ subsets, responded to the
coexpression of BTNL3 and BTNL8. Of note, this subset is particularly enriched in
human gut, and hence may be the functional equivalent to murine Vγ7+ IEL. Further
work will be focused on deciphering the mechanism by which BTNL3/8 regulate Vγ4+
cells, and the role of this axis in disease. In sum, our work further highlights the
critical role of Butyrophilin(-like) molecules in γδ T cell biology, and provides a unique
example of a direct parallel between mouse and human systems.
Page 38 of 200
GDC 2016
A-20
Skint1 mediates organ-specific epithelial normality-sensing
by resident epidermal T cells
1
1
2
1
2
Natalie Roberts , Rosie Hart , Pierre Vantourout , Anett Jandke , Martin Woodward ,
2
1, 2
Olga Sobolev and Adrian Hayday
1
2
The Francis Crick Institute, London, UK & Peter Gorer Department of Immunobiology, King’s
College London, London, UK
Murine intra-epidermal lymphocytes are prototypic innate-like T cells, responding to
tissue perturbations by engaging self-encoded stress-antigens and/or alarmins.
However, the basis of their selective, stable association with skin is unexplained.
Hypothesising that Skint1, a B7/PD1L-like and Butyrophilin-like molecule, expressed
at steady-state by keratinocytes, might communicate epithelial status; we found that
its expression was down-regulated by TPA and UVB induced stress. Interestingly,
quantification of DETC-keratinocyte interactions, by confocal microscopy, in mice
differentially expressing skint1 and/or the Vγ5Vδ1 TCR, revealed a reduced number
of interactions in the absence of Vγ5Vδ1 or WT skint1 and when the epidermis was
subject to stress. This reduction of Skint1 expression and DETC-keratinocytes
interactions concomitantly resulted in enhanced IL-2 production, suggesting
augmented DETC responsiveness. Thus, Skint1 expression by epithelial cells
communicates normality to neighbouring T cells through the formation of contact foci
that constrain T cell activation. Upon stress, skint1 is down-regulated, permitting
contact disruption and rapid T cell responsiveness.
Skint molecules on the surface may act as guardians of normality, this may work for
example, through either improved access to epithelium-derived cytokines or a contact
dependent mechanism that sustain the cells’ long-term viability. To further explore
this hypothesis we sought to establish an in vitro model whereby Skint molecules
transduced into host epithelial cell lines, and their capability to modulate DETC
responsiveness, can be tested. Readouts will include CD25 upregulation, CD3
downregulation and phosphoflow. Furthermore, coculture systems will allow
investigation and confirmation into whether DETC responses in this context require
direct interaction with Skint(s).
Page 39 of 200
GDC 2016
A-21
Triggering of BTN3A molecules considerably improve Vg9Vd2T cellsbased immunotherapy in Acute Myeloid Leukemia
1
1
2
1
Audrey Benyamine , Aude LeRoy , Emilie Mamessier , Julie Gertner-Dardenne , Céline
1
1
3
3
3
Castanier , Florence Orlanducci , Laurent Pouyet , Armelle Goubard , Yves Collette , Norbert
4,5
6
3
1, 3, 4
Vey , Emmanuel Scotet , Remy Castellano , Daniel Olive
1 Inserm, U1068, Centre de Recherche en Cancérologie de Marseille (CRCM), Immunity & Cancer, Institut
Paoli-Calmettes; Aix-Marseille Université UM 105; CNRS UMR 7258, Marseilles, F-13009, France;
2 Centre d'Immunologie de Marseille-Luminy (CIML), Laboratory Genomic Instability and Human
Hemopathies CNRS-INSERM-Université de la Méditerranée Parc Scientifique de Luminy, Case 906
Marseilles, F-13288, France
3 Inserm, U1068, CRCM, TrGET Plateforme d’Essais Précliniques; Institut Paoli-Calmettes; Aix-Marseille
Université UM 105; CNRS UMR 7258, Marseilles, F-13009 France
4 Aix-Marseille Univ, F-13284, Marseilles, France;
5 Institut Paoli Calmettes, 232 Bd Sainte Marguerite, F-13009, Marseilles, France
6 Inserm, U892, Nantes, F-44000, France
Given their recognized ability to kill Acute Myeloid Leukemia (AML) blasts both in
vitro and in vivo, Vγ9Vδ2 T cells are of growing interest in the design of new
strategies of immunotherapy. We report that Vγ9Vδ2 T cells can mediate potent
cytolytic activity against AML blasts with potency equal to that of NK cells. Although
differential killing of AML blasts by Vγ9Vδ2 T cells is observed, there is a positive
correlation between the expression of certain Natural Killer Receptors (NKR) ligands
on the AML blasts and their corresponding sensitivity to killing. We show that the
Butyrophilin3A (BTN3A, CD277) subfamily is a critical determinant of Vγ9Vδ2 TCRmediated recognition of human primary AML blasts ex vivo. Moreover, anti-BTN3A
20.1 agonist monoclonal antibodies (mAb) can trigger BTN3A on AML blasts leading
to further enhanced Vγ9Vδ2 T cell-mediated killing, but this mAb had no enhancing
effect upon NK cell-mediated killing. In addition, anti-BTN3A 20.1 mAbs could
specifically sensitize resistant blasts to Vγ9Vδ2 T cells lysis and overcome the poor
effect of AminoBisphosphonate (N-BP) treatment on those blasts. We confirmed the
enhancement of Vγ9Vδ2 T cells activity by anti-BTN3A 20.1 mAb using a human
AML xenotransplantation mouse model. We showed that anti-BTN3A 20.1 mAb
combined with Vγ9Vδ2 T cells immunotherapy could increase animal survival and
decrease the leukemic burden in blood and bone marrow. These findings could be of
great interest in the design of new immunotherapeutic strategies for treating AML.
Page 40 of 200
GDC 2016
A-22
RhoB is a novel mediator of phosphoantigen recognition by Vγ9Vδ2 T
cell receptor
1
1
1
7
1
Zsolt Sebestyen , Wouter Scheper , Anna Vyborova , Siyi Gu , Zuzana Rychnavska ,
1
1
1
1
Marleen Schiffler , Astrid Cleven , Coraline Chéneau , Martje van Noorden , Cassie2
3
4
5
5
Marie Peigné , Daniel Olive , Robert Jan Lebbink , Rimke Oostvogels , Tuna Mutis ,
6
7
2
1
Gerrit-Jan Schuurhuis , Erin J Adams , Emmanuel Scotet , Jürgen Kauball
1
Department of Hematology and Laboratory of Translational Immunology, University Medical
Center Utrecht, 3508 GA, The Netherlands;
2
INSERM, Unité Mixte de Recherche 892, Centre de Recherche en Cancérologie Nantes
Angers, 44000 Nantes, France;
3
INSERM, Centre de Recherche en Cancérologie Marseille, Institut Paoli-Calmettes, 13009
Marseille, France;
4
Department of Medical Microbiology, University Medical Center Utrecht, 3584CX, The
Netherlands;
5
Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, 3508
GA, The Netherlands;
6
Department of Hematology, VU University Medical Center, Amsterdam, 1081VH, The
Netherlands;
7
Department of Biochemistry and Molecular Biology, University of Chicago, 929 E. 57th St.
Chicago, IL 60615, USA
Human Vγ9Vδ2 T cells respond to tumour cells by sensing elevated levels of
phosphorylated intermediates of the dysregulated mevalonate pathway. This sensing
process is translated into activating signals by butyrophilin A1 (BTN3A1) through
unknown mechanisms. We developed an unbiased, genomewide screening method
and identified the GTPase RhoB in tumour cells as a critical mediator of Vγ9Vδ2TCR
activation, and we propose an activation mechanism comprised of two components.
We show that Vγ9Vδ2TCR activation is modulated by the GTPase activity of RhoB in
tumour cells, and by the relocalization of RhoB in proximity to BTN3A1. This
relocalization is associated with cytoskeletal changes that immobilize BTN3A1 in the
membrane, followed by the dissociation of RhoB from BTN3A1. Subsequently,
phosphoantigen accumulation induces a conformational change in BTN3A1,
rendering its extracellular domains recognizable by Vγ9Vδ2TCRs. These
complementary events provide further evidence for inside-out signaling, and suggest
potential tools to improve Vγ9Vδ2 T cell based therapies.
Page 41 of 200
GDC 2016
Session 3
What factors dictate γδ biology? Development,
regulation and signalling pathways
Talks:
Friday, June 17th
Poster presentation:
Thursday, June 16th, 6:30 – 8:30pm
Friday, June 17th, 1:00 – 2:30pm
Page 42 of 200
GDC 2016
A-23
TCR-ligand interactions are required for murine epidermal Vγ3Vδ1 T
cell development
Deborah A. Witherden, Olivia D. Garijo, Ryan Kelly, H. Kiyomi Komori and Wendy L.
Havran
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla,
California 92037
Dendritic epidermal T cells (DETC), which bear the Vγ3Vδ1 T cell receptor (TCR),
are the exclusive T cell population resident in the murine epidermis where they act as
sentinels for neighboring keratinocytes. γδ T cells differentiate from lymphoid
precursors largely within the thymic microenvironment in a highly organized manner.
Cellular selection processes are also believed to be involved and indeed several lines
+
of evidence suggest that proper development and homing of the Vγ3Vδ1 subset
does require positive selection events in the fetal thymus. To evaluate the role of TCR
interactions with cognate ligand in Vγ3Vδ1 T cell development and maturation, we
used a soluble DETC TCR tetramer to characterize ligand expression in the fetal
thymus and skin. We show that temporal expression of ligand in the thymus closely
mimics the window of DETC intrathymic development. Ligand expression is
independent of Skint1, a molecule shown to be important for thymic maturation of
DETC and their subsequent residence in the epidermis. Furthermore, using the
soluble DETC TCR tetramer as a ligand blocking reagent, we show that in fetal
thymic reaggregation cultures, TCR-ligand interactions are essential for DETC
development and maturation. Current experiments are investigating the nature of the
ligand-expressing thymic epithelial cell population. Understanding TCR-ligand
interactions involved in DETC development may provide important insight into
identifying the enigmatic DETC antigen.
Page 43 of 200
GDC 2016
A-24
MicroRNA-146a controls IFN-γ production and functional plasticity of
murine γδ T cells
1
1
Nina Schmolka , Tiago Amado , Anita Q. Gomes
1
2
1,2
and Bruno Silva-Santos
1
Instituto de Medicina Molecular de Lisboa, Faculdade de Medicina, Universidade de Lisboa.
Escola Superior de Tecnologia da Saúde de Lisboa.
γδ T cells have emerged as key providers of the proinflammatory cytokines
interleukin 17 (IL-17) and interferon-γ (IFN-γ) in various models of infection,
inflammation and autoimmunity. Our recent epigenetic and transcriptional analyses
+
have shown that whereas CD27 γδ T cells are committed to IFN-γ expression, the
IL-17 producing CD27 subset is functionally plastic and thus capable of coexpressing both cytokines under inflammatory conditions. To further understand the
molecular basis of this plasticity we addressed the role of miRNA-mediated posttranscriptional regulation. We identified miR-146a in a transcriptome-wide analysis of
+
miRNA expression in peripheral CD27 versus CD27 γδ T cell subsets as being
enriched in CD27 γδ T cells. Most importantly, through the analysis of miR-146adeficient mice we demonstrated that miR-146a prevents IFN-γ production by CD27
γδ T cells. In vitro polarisation of miR-146a-deficient CD27 γδ T cells under Th1driving conditions resulted in a marked production of IFN-γ, and their stimulation with
+
+
IL-1β plus IL-23 led to increased percentages of IFN-γ IL-17 γδ T cells in
comparison to miR-146a-sufficient counterparts. Consistently, in a Listeria infection
model, miR-146a-deficient mice showed an accumulation of this double producer
population compared to WT controls. Interestingly, forced expression of miR-146a in
+
γδ T cells reduced the frequency of IFN-γ γδ T cells. While we are identifying the
underlying mRNA targets, these results establish miR146a as a key regulator of IFNγ production and functional plasticity of γδ T cells. Importantly, this study is the first to
identify a non-redundant role for an individual miRNA in γδ T cell differentiation.
Page 44 of 200
GDC 2016
A-25
Casein Kinase 2 controls the survival of normal thymic and leukemic
γδ T-cells via modulation of AKT signaling
Sérgio T. Ribeiro, Julie C. Ribot, Francisco Caiado, João T. Barata, Bruno SilvaSantos
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa,
Portugal
The thymus is the major site for normal and leukemic T-cell development. In humans,
the molecular mechanisms that characterize T-cell subsets and their signatures of
transformation are largely unknown, especially for γδ T-cells. The serine-threonine
protein kinase CK2 (Casein Kinase 2) has been shown to support cell survival and
expansion, particularly of tumor cells. Here we disclose an unanticipated role of CK2
in human γδ T-cells, both normal thymocytes and transformed γδ T-cell acute
lymphoblastic leukemia (T-ALL) cells. By studying healthy thymic samples (obtained
from corrective cardiac surgery), we show that γδ T-cells display higher CK2 activity
than their αβ counterparts, and are strikingly susceptible to apoptosis upon treatment
with a specific and clinical-grade CK2 inhibitor, CX-4945. Similarly to several types of
tumors, γδ T-ALL cells acquired increased levels of CK2 activity compared to healthy
γδ thymocytes and presented increased sensitivity to G2/M cell cycle arrest and
apoptosis upon CK2 blockade. Moreover, γδ T-ALL cells were markedly more
sensitive than αβ T-ALL to CX-4945 treatment both in vitro and in vivo (xenograft
models). Mechanistically, we demonstrate that TCR/CD3 stimulation positively
regulates CK2 activity in normal γδ thymocytes, while γδ T-ALL cells further enhance
CK2 activity via CD27 costimulation. Importantly, we provide detailed biochemical
evidence that CK2 regulates the PTEN/ AKT signaling pathway towards promoting
the survival of normal and leukemic γδ T-cells. These data identify CK2 as a novel
determinant of normal and leukemic γδ T-cell survival, and may thus impact their
manipulation in both hematology and immunotherapy.
Page 45 of 200
GDC 2016
A-26
The critical roles of RhoH for development of IL-17-producing γδT cell
1,2
2
1
1
Muro Ryunosuke , Nitta Takeshi , Tamehiro Norimasa , Oda Hiroyo , Hiroshi
2
1
Takayanagi , Suzuki Harumi
1
Department of Immunology and Pathology, Research Institute, National Center for Global
Health and Medicine, Chiba Japan
2
Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The
University of Tokyo, Tokyo Japan
The γδ T cells expressing distinct TCR-Vγ develop in the thymus during ontogeny.
IL-17 producing γδ T (γδT17) cells, mainly Vγ4 and Vγ6 subsets, develop at
embryonic and neonatal stages and play a crucial role in pathogenic inflammatory
responses. RhoH is a non-classical small G protein and act as adaptor protein for
ZAP70 in T cells, therefore it is crucially required for the development of αβT cells. In
the current study, we present essential role of RhoH for γδT cell development. In the
adult periphery, the number of Vγ1 and Vγ5Vδ1 γδT cells in RhoH-deficient mice
was unchanged whereas the number of Vγ4 and Vγ6 γδT cells was reduced.
Consistently, development of thymic Vγ1 and Vγ5Vδ1 was normal, whereas
development of thymic Vγ4 and Vγ6 γδT cells was strongly inhibited both in embryos
and adults. Interestingly, none of the γδT cells in the RhoH deficient mice produced
IL-17, indicating the significance of RhoH in the development of γδT17 cells.
Impaired TCR-induced ERK-phosphorylation and reduced expression of CD5 suggest
that RhoH is required for γδTCR signal in vivo. Our current results inducate that
RhoH plays critical roles for γδTCR signaling to develop γδT17 cells in the thymus.
Page 46 of 200
GDC 2016
A-27
Examination of the γδ TCR ligand EPCR as a candidate molecular
exemplar of adaptive stress surveillance
1
1
1
1
Benjamin E. Willcox , Carrie R. Willcox , Martin S. Davey , Mahboob Salim and
1
Fiyaz Mohammed
1
Institute of Immunology and Immunotherapy, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, UK
Our recent studies of the Vδ2-negative γδ T cell compartment in human peripheral
blood highlight dramatic and relatively stable clonal expansions, accompanied by
cellular differentiation to an effector phenotype, consistent alterations in homing
markers, and upregulation of cytotoxic effector molecules. Collectively these findings
are strongly suggestive of an adaptive biology, which we term adaptive stress
surveillance. Here we propose four criteria by which to assess the potential in vivo
relevance of γδ TCR ligands to this process, and examine whether the LES TCR
ligand EPCR fulfils these criteria. In particular, in the context of the LES/TCR
interaction, we focus on (i) the relative frequency of the TCR clonotype, (ii) the
differentiation phenotype of the clonotype, (iii) reproducibility and involvement of
CDR3 regions in TCR binding, and (iv) potential biological relevance of candidate
ligand expression pattern. Previous analyses have shown the LES T cell clonotype
was expanded to high levels in vivo, and was characterised by a CD28-negative
CD45RO-negative phenotype consistent with effector memory status. Here we show
how the LES TCR CDR3γ and CDR3δ sequences relate to our recent TCR repertoire
data, highlighting a high level of N/P nucleotide addition characteristic of the largely
private Vδ2-negative repertoire; we use surface plasmon resonance with site-directed
mutagenesis to demonstrate involvement of both CDR3 regions in EPCR interaction;
we also note expression of EPCR on endothelium, a key site of CMV infection in vivo.
On this basis, we propose that the LES/EPCR interaction is a candidate molecular
exemplar of adaptive stress surveillance.
Page 47 of 200
GDC 2016
A-28
+
Studying the TCRγδ T-cell repertoire via Next-generation sequencing
(NGS)
1
1
2
1
2
Kallemeijn MJ , van der Klift MY , Kavelaars FG , Wolvers-Tettero ILM , Valk PJM ,
1
1
van Dongen JJM & Langerak AW .
1
2
Dept. of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands.
Dept. of Hematology, Erasmus Medical Center, Rotterdam, The Netherlands.
+
TCRγδ T-cells develop in the thymus where they undergo combinatorial and
junctional diversification via V(D)J-recombination and TdT-activity. During an early
+
stage of development TCRγδ T-cells split off. They generally localize to mucosal
epithelial tissues, and are less common in the peripheral blood (PB) (up to 5%).
+
During TCRγδ T-cell development both in the thymus and periphery selection takes
place, starting with γδ-expression in the thymus. Readily after birth and during aging
further selection takes place based on antigenic exposure, possibly contributing to
+
the circulating TCRγδ T-cell repertoire.
+
In order to obtain novel insights in the development and shaping of the TCRγδ T-cell
repertoire Next-generation Sequencing of TCRG and TCRD rearrangements was
applied. Following technical validation of multiplex-PCR-based NGS-methods using
+
artificial control samples, the optimized strategy was then applied to TCRγδ T-cells
isolated from different tissues, ranging from developing thymus and neonatal cord
blood (NCB), to young and elderly individuals.
+
Thymic and NCB TCRγδ T-cells show inter-sample homogeneity and intra-sample
high diversity with clear Vδ1–Jδ1 predominance, correlating with our previous Sanger
sequencing and flow cytometry data. The CDR3-lengths follow a normal distribution.
+
Conversely, adult PB circulating TCRγδ T-cells show higher inter-sample
heterogeneity generally with skewing towards Vγ9/Vδ2, again confirmed by Sanger
sequencing and flow cytometry data. Furthermore, selections for shorter CDR3regions of 13-15 amino acids are observed.
+
During TCRγδ T-cell development a broad immature repertoire is formed, with
tissue-specific predominant Vγ/Vδ usage. Upon aging, the circulating γδ-repertoire is
shaped, suggestive of an antigenic signature probably related to the individual’s
environment.
Page 48 of 200
GDC 2016
A-29
Adaptive immune-like γδT lymphocytes share many Common features
with their αβT cell counterparts
Amélie Lombes, Aurélie Durand, Céline Charvet, Matthieu Rivière, Nelly Bonilla,
Cédric Auffray, Bruno Lucas, and Bruno Martin
Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Paris 75014,
France; INSERM, U1016,
Cochin Institute, Paris 75014, France; and Cochin Institute, Paris Descartes University, Paris
75014, France
To better apprehend γδT cell biological functions in the periphery, it appears crucial
to identify markers highlighting the existence of distinct phenotypic and functional γδT
cell subsets. Interestingly, the expression of CD44 and Ly-6C subdivides murine
peripheral γδT cells into several subsets, with Ly-6C CD44hi γδT cells
corresponding to the IL-17-producing CD27 γδT cell subset exhibiting innate-like
+
features. By comparing the other subsets to naive and memory CD8 α/βT cells, in
+
lo
+
hi
this study, we show that Ly-6C or CD44 and Ly-6C CD44 γδT cells greatly
+
+
resemble, and behave like, their CD8 αβT cell counterparts. First, like memory CD8
+
hi
αβT cells, Ly-6C CD44 γδT cells are sparse in the thymus but largely increased in
lo
proportion in tissues. Second, similarly to naive CD8 αβT cells, CD44 γδT cells are
poorly cycling in vivo in the steady state, and their proportion declines with age in
lo
secondary lymphoid organs. Third, CD44 γδT cells undergo spontaneous
+
hi
proliferation and convert to a memory-like Ly-6C CD44 phenotype in response to
lo
lymphopenia. Finally, CD44 γδT cells have an intrinsic high plasticity as, upon
appropriate stimulation, they are capable of differentiating nonetheless into Th17-like
+
and Th1-like cells but also into fully functional Foxp3 induced regulatory T cell like
+
γδT cells. Thus, peripheral CD27 γδT cells, commonly considered as a functionally
related T cell compartment, actually share many common features with adaptive αβT
cells, as both lineages include naive-like and memory-like lymphocytes with distinct
phenotypic, functional, and homeostatic characteristics.
Page 49 of 200
GDC 2016
A-30
Metabolic requirements for γδ T cell development
1
1
Martin S , Sumaria N & Pennington DJ
1
1
Blizard Institute, Barts and The London School of Medicine, Queen Mary University of London,
4 Newark Street, London, E1 2AT, United Kingdom.
The generation of gammadelta (γδ) T cells in the thymus remains unclear,
particularly the mechanisms that underpin the differential development of γδ T cells
that secrete either IL-17A or IFN-γ. The rapidly developing field of
immunometabolism has provided significant evidence that metabolism underpins the
growth, proliferation and functionality of αβ T cells. With this newly evolving field in
mind, this project investigates the metabolic requirements for the development of γδ
T cells.
We used foetal thymic organ culture (FTOC) to analyse the development of IL17Asecreting and IFN-γ-secreting γδ T cells under different metabolic conditions; for
example, by using the metabolic inhibitor 2-deoxyglucose. γδ T cell subsets were
assessed using a new flow cytometry gating strategy that was recently established in
the lab.
IFN-γ-secreting γδ T cells appear to rely on glycolysis during development as culture
with 2-deoxyglucose impaired their generation. By contrast, the IL-17A-secreting γδ T
cell population significantly expanded in the presence of 2-deoxyglucose, with their
ability to produce IL-17A enhanced. Moreover, these cells had higher mitochondrial
mass than their IFN-γ-secreting counterparts. Further investigation showed
differential Vγ-usage by IL-17A-producing γδ T cells under glycolytically-impaired
conditions. Additionally and unexpectedly, γδ T cells seem better able to develop in
the presence of glycolytic inhibitors. These observations suggest that different
metabolic programs are required for thymic development of distinct cytokinesecreting γδ T subsets, and perhaps even influence αβ:γδ lineage choice in early DN
thymocytes.
Page 50 of 200
GDC 2016
A-31
ERK/MAPK and PI3K signalling dictate effector potential during γδ T
cell development
1
1
1
Sumaria N , Grandjean CLA , Pang DJ , Silva-Santos B
2,3
& Pennington DJ
1
1
Blizard Institute, Barts and The London School of Medicine, Queen Mary University of London,
4 Newark Street, London, E1 2AT, United Kingdom.
2
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal.
3
Instituto Gulbenkian de Ciência, Oeiras, Portugal.
Gamma delta (γδ) T cells play important roles in immune responses against
pathogens and tumours, which correlate with secretion of cytokines, such as IFNγ
and IL-17A. Recent evidence suggests that γδ cells are predominantly pre-committed
to certain effector fates during thymic development. T cell receptor (TCR) agonists
are thought to favour development of IFNγ-producing γδ cells, whereas absence of
ligand interactions is thought to generate IL-17A-producing γδ cells. The aim of this
study was to understand the underlying mechanisms that determine these effector
fates.
We used flow cytometry to establish an improved gating strategy to identify IFNγsecreting and IL-17A-secreting γδ cells in the lymph nodes and thymus of C57BL/6
mice. Subsequently, we used foetal thymic organ culture (FTOC) to analyse γδ cell
development and adoption of functional potential in conditions where TCR-induced
signal strength or various signalling pathways were manipulated.
We confirm that strong TCRγδ -mediated signalling favours the thymic development
of γδ cells that are associated with IFN-γ secretion in peripheral tissues. These
strong signals are mediated through the ERK/MAPK pathway which actively inhibits
the development of IL-17A-secreting γδ cells. Importantly, weak TCRγδ-mediated
signalling appears to promote the generation of IL-17A-secreting γδ cells through
Syk-family kinase-mediated activation of phosphoinositide 3-kinase (PI3K) in the
absence of ERK/MAPK signalling. Hence, these observations describe key signalling
events that dictate effector potential of developing γδ cells in the murine thymus.
Page 51 of 200
GDC 2016
A-32
Proximal Lck-Cre does not delete floxed genes in γδ T cells
1
1,2
1,3
1
Gina J. Fiala , Anna Morath , Miriam Hils , Susana Minguet , Wolfgang. W.
1,3
Schamel
1
Department of Molecular Immunology, BioIII, Faculty of Biology, University of Freiburg,
Germany
2
Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Germany
3
Centre of Chronic Immunodefciency (CCI), University Medical Center Freiburg, Germany
Conditional knock-out (ko) mice have substantially advanced analysis of gene
function in vivo. The gene of interest, flanked by loxp sites, is removed by expressing
Cre recombinase under the control of a cell-type and/or cell-stage specifc promoter,
enabling specific analysis of gene function while the remaining organism is
unaffected.
To create T cell-specific ko mice, a commonly used deleter strain is the proximal LckCre (pLck-Cre). Here, Cre is under the control of the proximal promoter of the
lymphocyte protein tyrosine kinase (Lck), which controls lck expression during
thymocyte development. Previous studies have demonstrated efficient deletion of
floxed genes in T cells starting at the CD4 CD8 stage and analyzed primarily αβ T
cells upon pLck-Cre-mediated deletion. Surprisingly, gene deletions causing
reductions in αβ T cells concurrently increased γδ T cells.
This discrepancy prompted us to analyze γδ T cells in the pLck-Cre ko system. When
αβ and γδ populations were sorted and pLck-Cre-mediated gene deletion
investigated by PCR, only αβ T cells deleted the gene of interest. Next, we crossed
flox-stop-floxdtomato
pLck-Cre mice to ROSA26
reporter mice, in which deletion of a floxed
+
+
stop cassette renders cells dtomato . While αβ T cells were dtomato , γδ T cells in
the thymus, lymph nodes, and spleen did not express dtomato.
These findings suggest that the proximal promoter of lck is not active during thymic
γδ T cell development and, therefore, pLck-Cre is not expressed in γδ T cells.
Hence, pLck-Cre mice are inappropriate for conditional gene deletion in γδ T cells.
Page 52 of 200
GDC 2016
A-33
T Cell Receptor expression levels and downstream signaling control
the functional differentiation of γδ T cells in the murine thymus
1,2
2
3
1
2
Muñoz-Ruiz M , Ribot JC , Pennington DJ , Regueiro JR , Silva-Santos B * and
1
Fernández -Malavé E *
1
Immunology Department, School of Medicine, Complutense University, Madrid, Spain
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa,
Portugal
3
Blizard Institute, Queen Mary University of London, London, UK
*Equal contributions
2
γδT cells are atypically programmed in the thymus to produce interferon-γ (IFN-γ) or
interleukin-17 (IL-17). However, the role of the signature T cell receptor (TCRγδ) in
+
+
controlling the relative pools of IFN-γ versus IL-17 γδT cells remains highly
controversial. Here we have generated CD3γ/ CD3d double haploinsufficient (CD3dh)
mice that displayed significantly reduced levels of TCRγδ expression on the cell
surface of γδ thymocytes. This resulted in reduced TCRγδ signal transduction, as
measured by ERK and AKT phosphorylation, calcium influx, and CD5 and CD73
expression in γδ T cells. Interestingly, the expression of TCRαβ, and the
+
development of αβ T cell subsets, including invariant NKT or Foxp3 regulatory T
cells, were unaffected in CD3dh mice. These therefore constitute a unique model to
+
+
investigate the TCR signaling requirements of IFN-γ versus IL-17 γδT cells. CD3DH
mice showed normal αβ thymocyte subsets but impaired differentiation of embryonic,
but not adult, IL-17-producing γδT cells and a marked depletion of IFN-γ-producing
+
+
CD122 NK1.1 γδT cells throughout ontogeny. Critically, adult CD3DH mice
displayed a reduced peripheral IFN-γ compartment and were resistant to
experimental cerebral malaria. Thus, TCR signal strength within specific thymic
developmental windows is a major determinant of the generation of proinflammatory
γδT cell subsets and their impact on pathophysiology.
Page 53 of 200
GDC 2016
A-34
Kruppel-like factor 10 regulates IL-17-committed CD27 γδ T cell
homeostasis
1
1
1
2
3
Girak Kim , MinJeong Gu , Soo Ju Kim , Seung Hyun Han , Jae-Ho Cho , Cheol1,
Heui Yun *
1
Immunology and Vaccine Development Laboratory, Department of Agricultural Biotechnology,
Seoul National University, Seoul, Republic of Korea;
2
Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of
Dentistry, Seoul National University, Seoul, Republic of Korea;
3
Academy of Immunology and Microbiology, Institute for Basic Science, Pohang, Republic of
Korea
γδ T cells are best known to be an important source of innate IL-17 that make critical
contributions to host immune responses. However, factors to control their
development and homeostasis are poorly elucidated. In the present study, we
examined the role of the zinc finger transcriptional factor, Kruppel-like factor 10
27—
(KLF10) in the development of IL-17-committed CD27 γδ T cells (γδ 17 cells). We
+
27found a selective increase of Vγ4 γδ cells with IL-17 production in KLF10-deficient
hi
+
27—
mice. Surprisingly, KLF10-deficient CD127 Vγ4 γδ 17 cells expressed CD5
higher than their wild-type counterparts did, with hyper-responsiveness to cytokines
+
27and T-cell receptor stimuli. The maturation of Vγ4 γδ
cells was enhanced in
newborn mice deficient in KLF10. Finally, mixed bone marrow chimera study
+
27—
indicated that intrinsic KLF10 signaling is mandatory to limit Vγ4 γδ 17 cells.
Collectively these findings demonstrated that KLF10 regulates thymic development of
+
27Vγ4 γδ cells and their peripheral homeostasis.
Page 54 of 200
GDC 2016
A-35
TCRαβ IELs but not TCRγδ IELs depend on mir181ab for their
selection, development and maturation.
1
1
1
Joana Barros-Martins , Inga Sandrock , Katrin Witzlau , Andreas Krueger
1
Immo Prinz
1
2
1,2
&
Immunologie Institute, Hannover Medical School, Hannover, Germany
Institute of Molecular Medicine, Goethe University Frankfurt am Main, Germany
The gut represents one of the major epithelial barriers to the external environment.
+
+
Intraepithelial lymphocytes (IELs), consisting of mainly TCRαβ and TCRγδ T
lymphocytes, are the major players in gut protection, preventing pathogen entry and
+
spreading. While the developmental stages of CD8αα TCRγδ expressing
+
lymphocytes remain unclear, CD8αα TCRαβ T cells are known to develop in the
thymus and migrate to the gut. In contrast to the development of conventional TCRαβ
lymphocytes, thymic TCRαβ IEL precursors (IELPs) probably undergo an alternative
+
agonist-selection. This means that these TCRαβ precursors strongly react to self+
+
antigens presented by cortical epithelial cells during their CD4 CD8 double-positive
stage leading to their differentiation into IELPs. The role of miRNAs in the
development and regulation of signalling pathways in lymphocytes is currently
emerging. In this work we show that along with the development of iNKT αβ cells, but
not γδ NKT lymphocytes, the unconventional TCRαβ IEL population depends on the
presence of mir181ab for their development and maturation. Mir181ab KO mice, as
compared to WT mice, show a large decrease in a specific population of
+
high
+
+
TCRβ TCRδ CD8β CD4 NK1.1 tet cells, which express CD5 CD127 CD44 in the
thymus. This leads to a reduced amount of TCRαβ IELs in the small intestine of the
mir181ab KO mice. These results stress the differential requirements for agonistselection of TCRγδ and TCRαβ T cells, in particular of TCRαβ IELs versus TCRγδ
IELs.
Page 55 of 200
GDC 2016
A-36
Negative and positive regulation of innate γδ T cells by the type I
interferon pathway
Rasmus Agerholm, John Rizk, Bengt-Johansson Lindbom, William W. Agace and
Vasileios Bekiaris
Section for Immunology & Vaccinology, Veterinary Institute, Danish Technical University,
Bülowsvej 27, 1870, Frederiksberg C, Denmark
The type I interferon (IFNs) pathways can regulate the immune system at steadystate and during infection its may lead to autoimmune diseases such as psoriasis.
Although many cell subsets could be directly regulated by type I IFNs, their impact on
γδ T cell populations is not well defined. We used mice deficient in the alpha subunit
of the type I IFN receptor (IFNAR1) to investigate its role in skin and lymph node (LN)
−
+
interleukin(IL)-17-producing CD27 CCR6 γδ T cells. We found that at homeostasis
+
−
−
+
IFNAR1 is a negative regulator of the turnover of Vγ4 and Vγ4 CD27 CCR6 γδ T
−
cells in LNs whereas in the skin this is restricted to the Vγ4 subset. Functionally,
−
+
IFNAR1 deficiency correlated with higher IL-17 in Vγ4 but not in Vγ4 cells
suggesting a differential regulation between the two subsets. Production of IFNγ was
also reduced when IFNAR1 was absent. Interestingly, expression of CCR6 was
significantly downregulated in IFNAR1 deficient cells. Despite the increased numbers
−
+
of CD27 CCR6 γδ T cells, IFNAR1 deficient mice were resistant to imiquimod (IMQ)
induced psoriasis. Notably, IFNAR1 mice presented with no or little skin reddening,
had significantly reduced epidermal thickening and expressed lower levels of γδ T
cell associated IL-17. In conclusion, our data show for the first time the critical role of
IFNAR1 signaling in the homeostasis of skin and LN resident innate γδ T cells and
link the effects of type I IFNs during psoriasis with their ability to sustain IL-17
expression by γδ T cells.
Page 56 of 200
GDC 2016
A-37
Controlled Initiation of T-Cell Development Reveals a Temporal
Regulation in the Generation of Specific γδ T-Cell Receptor
Repertoires and Functional Subsets
Edward L.Y. Chen, Patrycja K. Thompson, and Juan Carlos Zúñiga-Pflücker
Department of Immunology, University of Toronto, and Sunnybrook Research Institute, Toronto,
Canada
γδ T-cells are known mediators of both protective and destructive immunity. Thus,
elucidation of factors that dictate γδ T-cell differentiation remains an important
question. Here, we aim to better understand temporal regulation dictating γδ T-cell
differentiation. To study this, we used a novel mouse model whereby Notch signaling
can be temporally controlled in hematopoietic cells, allowing for the generation of Tcells to be restricted to specific developmental windows. This allows us to assess
whether fetal, neonatal, and adult time periods generate different γδ T-cell receptor
repertoires and functional subsets. In this model, induction of T-cell development can
occur in all three periods, leading to appearance of temporal-specific γδ T-cells in
+
+
peripheral tissues. We noted that the generation of Vγ5 and Vγ6 cells occurred
exclusively during the fetal period, suggesting that this model reflects physiological
+
+
outcomes. While all three periods generated Vγ1 and Vγ4 cells, there was a greater
+
+
propensity to produce Vγ4 cells during the fetal period, and Vγ1 cells postnatally.
+
Although fewer in numbers, fetal Vγ1 cells were enriched for PLZF-expression,
suggesting that the fetal period skews towards an NK-like functional phenotype. We
next assessed the generation of IL-17-producing γδ T-cells (γδT17). The fetal period
appeared to provide a favorable environment for the generation of this functional
subset, although its appearance was not restricted to this period as suggested by
previous reports. The neonatal and adult periods were still able to generate γδT17
cells, although at lower frequencies and with different tissue distributions. Our ability
to precisely regulate the appearance of γδ T-cells has provided us new insights as to
the unique temporal requirements for their differentiation.
Page 57 of 200
GDC 2016
A-38
Clonal selection and differentiation in the human Vδ2-negative γδ T
cell repertoire suggests TCR-dependent ‘adaptive stress surveillance’
1
1
2
1
Martin S. Davey , Carrie R. Willcox , Kristin Ladell , Stephen P. Joyce , Stuart J.
1
2
3
1
Hunter , David A. Price , Dmitriy M. Chudakov and Benjamin E. Willcox
1
Institute for Immunology and Immunotherapy, University of Birmingham, Edgbaston,
Birmingham B15 2TT, UK
2
Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff
CF14 4XN, Wales, UK
3
Shemyakin-Ovchinnikov Institute of bioorganic chemistry RAS, Moscow, Russia; Pirogov
Russian National Research Medical University, Moscow, Russia; Central European Institute of
Technology, Masaryk University, Brno, Czech Republic
γδ T-cells are typically characterised as pre-expanded “innate-like” lymphocytes
bearing semi-invariant TCRs, which mount rapid responses to microbial or nonmicrobial stress stimuli without a requirement for clonal selection and differentiation.
While human Vγ9Vδ2 T cells arguably conform to this paradigm, we applied RNAbased TCR repertoire analyses to 20 healthy adult donors (18-35yrs) to probe
whether it applied to Vδ2-negative T-cells, which are implicated in responses to viral
infection and cancer. Our results indicate the Vδ2-negative TCR repertoire is private
and initially unfocussed in neonates, typically becoming strongly focussed towards a
few high-frequency clonotypes by adulthood in both CMV-positive and CMV-negative
individuals. Dominant clonotypes featured highly complex CDR3 sequences resulting
from rare recombination events, which were relatively stable over ≥1.5-2 years. In
contrast, Vδ2-positive T-cells expressed semi-invariant TCRs already prevalent at
+
birth. Vδ1 clonal expansion was accompanied by differentiation from a CD27
+
CD45RA naïve phenotype to a predominant CD27-negative, CD45RA-positive
effector memory phenotype, concomitant with downregulation of CCR7 and CD62L,
suggestive of distinct homing capabilities. Compared to naive Vδ1 T-cells, expanded
effector memory populations displayed enhanced IL-15 vs IL-7 responsiveness,
preferentially became activated/proliferated in response to anti-CD3/anti-γδ-TCR
stimulation, and exhibited increased expression of the cytotoxic markers Granzyme
A/B and perforin. These data indicate the Vδ2-negative T-cell subset has evolved a
radically different biology relative to Vγ9Vδ2, MAIT and iNKT cells. They suggest a
central and personalised role for the γδ TCR in directing a highly adaptive form of
stress surveillance, in the face of microbial and non-microbial stress challenges.
Page 58 of 200
GDC 2016
A-39
Interleukin-9 production by human γδ T cells
1
2
1
Christian Peters , Robert Häsler , Daniela Wesch , Dieter Kabelitz
1
1
Institute of Immunology, University Hospital Schleswig-Holstein Campus Kiel, D-24105 Kiel;
and
2
Institute of Clinical Molecular Biology, University Hospital Schleswig-Holstein Campus Kiel, D24105 Kiel
Vδ2Vγ9 T cells are the dominant γδ T cell subset in human peripheral blood. Vδ2 T
cells recognize pyrophosphate molecules derived from microbes or tumor cells, and
hence Vδ2 T cells play a role in anti-microbial and anti-tumor immunity. TGF-β
together with IL-15 induces a regulatory phenotype in Vδ2 T cells, characterized by
FoxP3 expression and suppressive activity on CD4 T cell activation. We performed a
genome-wide transcriptome analysis and found that the same conditions (TGF-β plus
IL-15) strongly upregulate additional genes in Vδ2 T cells including IZKF4 (Eos),
ITGAE (αEβ7, CD103) and IL9. This is associated with potent IL-9 production as
revealed by flow cytometry and multiplex analysis of cell culture supernatants. Upon
antigen restimulation of Vδ2 T cells expanded in vitro in the presence of TGF-β and
IL-15, IL-9 was the most abundant among 16 analyzed cytokines and chemokines.
IL-9 is a pleiotropic cytokine which plays a role in diverse (patho)physiological
conditions including allergy and tumor defense where it can promote antitumor
immunity. Given the conspicuous sensitivity of many different tumors to Vδ2 T cellmediated killing, the conditions defined here for strong induction of IL-9 might be
relevant for the development of Vδ2 T cell-based immunotherapies.
Page 59 of 200
GDC 2016
A-40
Insights into γδ T cell biology by phenotyping a knockout mouse
library
1
1
1
1
1
Lucie Abeler-Dörner , Namita Saran , Keng Hng , Adam Laing , Anna Lorenc ,
1
1
1
Agnieszka Przemska-Kosicka , Dmitry Ushakov , Carmen Ballesteros Reviriego ,
1,2
2
2
2
Susana Caetano , Emma Cambridge , Heather Wilson , Jacqui White and Adrian
1,3
Hayday and the 3i consortium
1
King’s College London,
Wellcome Trust Sanger Institute,
3
The Francis Crick Institute
2
Immunology recently has seen enormous translational advances in the treatment of
infectious diseases and the harnessing of immunotherapy in the treatment of cancer.
And yet, we lack a full understanding of the shape of the immune system, and of
molecules and pathways underpinning the development and regulation of immune
cells, their response to challenge, and their interactions with tissues. Therefore, we
are conducting a large-scale, high throughput immunological screening of
approximately 800 knockout mouse lines generated by the Wellcome Trust Sanger.
The study is an open-access programme with data deposited into the public domain
for follow-up by those best-equipped to do so.
We analyse the immune cell compartments of spleen, mesenteric lymph nodes, bone
marrow, blood and serum in order to identify genes regulating the immune system in
the absence of challenge. Furthermore we investigate responses to chemical stress
(DSS colitis) and to viral, bacterial and nematode infections (Salmonella, influenza
and Trichuris muris). The screen is comprehensive as well as in-depth: In the steady
state we assess over 160 immune cell populations across five organs by 12-colour
flow cytometry and confocal microscopy.
The screen yielded several γδ T cell hits in skin, spleen and mesenteric lymph nodes.
Furthermore, analysing hundreds of wild type controls yielded new insights into γδ T
cells in the steady state. In this contribution we will present new aspects of γδ T cell
biology discovered by the programme and consider how the community can realize
the opportunity to benefit from these insights.
Page 60 of 200
GDC 2016
A-41
Generation of regulatory human γδ T cells in the presence of IL-35 and
TGF-β.
Leonce Kouakanou, Christian Peters and Dieter Kabelitz
Institute of Immunology, Christian-Albrechts-University of Kiel, Germany
Regulatory T cells (Treg) play a major role in controlling cellular immune responses
and in maintaining the peripheral immune tolerance.
We and others have previously demonstrated that human Vγ9Vδ2 γδ T cells, which
play an important role in infection and tumor defense, can be induced by TGF-β to
exhibit regulatory functions similar to conventional Treg. IL-35 is a cytokine which has
been implicated in immune regulation. The aim of this study was to characterize the
role of IL-35 for the induction of regulatory functions in human γδ T cells.
We first focused on the generation of regulatory gd T cells in vitro by stimulating
freshly isolated γδ T cells with the synthetic phosphoantigen BrHPP, in the presence
of IL-35, TGF-β and IL-2. Subsequently we evaluated the suppressive function of
+
these gd T cell lines on autologous CD4 αβ T cells in coculture experiments. IL-35
alone was not sufficient to support gd T cell proliferation after BrHPP stimulation, but
required the additional presence of IL-2. Furthermore the expansion of γδ T cells was
reduced in presence of TGF-β. Nonetheless gd T cells expanded in the presence of
+
IL-2, IL-35 and TGF-β exhibited a negative effect on the αβ CD4 T cell proliferation
(as determined after 7 days of coculture).
Our preliminary results suggest that the priming of gd T cells in presence of IL-35 in
combination with IL-2 and TGF-β, leads to a suppressive phenotype. The underlying
mechanisms need to be clarified in more detail in order to utilize regulatory γδ T cells
in therapeutic approaches.
Page 61 of 200
GDC 2016
A-42
Programming in utero and post-natal acquisition of effector functions
of Vγ9Vδ2 T cells
1,2
1,2
2
3
3
Maria Papadopoulou , Ling Ma , Tanya Dimova , Willem Hanekom , Elisa Nemes ,
1,2
David Vermijlen
1
Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Belgium
Institute for Medical Immunology (IMI), Université Libre de Bruxelles (ULB), Belgium
3
South African Tuberculosis Vaccine Initiative (SATVI), University of Cape Town, South Africa
2
The Vγ9Vδ2 T cell subset is the main γδ T cell population in adult peripheral blood.
Their main effector functions include IFN-γ production and killing of infected and
transformed cells. Their semi-invariant T-cell-receptor reacts towards microbe-and
host-derived phosphorylated prenyl metabolites (‘phosphoantigens’). Both
programming in utero (sterile environment) and postnatal acquisition early after birth
(exposure to phosphoantigen-producing microorganisms) of the effector functions
have been described.
The aim of this study is to understand at the molecular level the programming in utero
versus post-natal acquisition of a series of effector functions of Vγ9Vδ2 T cells. We
analysed Vγ9Vδ2 T cells derived from new-borns (cord blood), 10 week old infants
vaccinated or not at birth with the phosphoantigen-containing vaccine Bacillus
Calmette-Guérin (BCG), and from adult blood.
At birth, Vγ9Vδ2 T cells expressed no perforin or granzyme B (as determined by flow
cytometry) whereas at 10 weeks of age these killing effector molecules showed a
striking high level of expression. In contrast, IFN-γ production (after polyclonal
stimulation) within neonatal Vγ9Vδ2 T cells was already high at birth, which did not
change significantly at 10 weeks of age. Surprisingly, BCG vaccination at birth had no
eminent effect on the effector molecules investigated.
Overall, these data indicate that environmental exposure for 10 weeks has a potent
influence on the expression of particular effector molecules within Vγ9Vδ2 T cells.
Currently we are investigating in more detail the possible molecular mechanisms
involved in the programming in utero and post-natal acquisition of the effector
functions of Vγ9Vδ2 T cells.
Page 62 of 200
GDC 2016
A-43
Prolonged PD1 expression as a potential rheostat for neonatal Vδ2
lymphocyte Responses
Haoting Hsu*, Sarah Boudova†, Godfrey Mvula ‡, Titus H. Divala ‡, Randy G.
Mungwira ‡, Christopher Harman §, Miriam K. Laufer †, C. David Pauza *, Cristiana
Cairo*
* Institute of Human Virology, University of Maryland, Baltimore, MD, United States
† Division of Malaria Research at the Institute for Global Health, University of Maryland, School
of Medicine, Baltimore, MD, United States,
‡ Blantyre Malaria Project, University of Malawi College of Medicine, Blantyre, Malawi;
§ Obstetrics, Gynecology and Reproductive Health, University of Maryland, Baltimore, MD,
United States.
The fetal immune system, compared to its adult counterpart, follows an alternate
functional program favoring Th2 or regulatory responses over Th1 responses. This is
one of the mechanisms that foster tolerance and limit inflammation at the fetal
maternal interface, but its unintended consequence is high susceptibility to infections.
Unlike CD4 T cells, Vδ2 lymphocytes, which mount responses to a wide range of
microbial agents, are poised for Th1 responses before birth. Thus they are likely to
play a key role in protection against pathogens in infants, exerting their own Th1
effector functions and promoting Th1 polarization of adaptive responses. The
propensity of Vδ2 lymphocytes to release pro-inflammatory mediators may be tightly
controlled during fetal life to avoid triggering exaggerated inflammation that could lead
to adverse fetal outcomes. We investigated molecules that could act as rheostats for
fetal Vδ2 cells. PD1 is a negative regulator of T cell responses and a determinant of
tolerance, particularly at the fetal-maternal interface. Neonatal Vδ2 cells up-regulate
PD1 after activation and, unlike their adult counterparts, maintain its expression for at
least 28 days. Engagement of PD1 during TCR stimulation inhibits TNF-α production
and granule mobilization; the extent of the inhibition is directly proportional to the
+
fraction of PD1 Vδ2 lymphocytes. PD1 expression by neonatal Vδ2 cells is inversely
associated with promoter DNA methylation, and may be additionally regulated by the
transcriptional repressor Blimp1. We propose that prolonged PD1 expression is a
mechanism to control Vδ2 cell inflammatory responses during fetal life.
Page 63 of 200
GDC 2016
A-44
Human foetal haematopoietic stem and progenitor cells (HSPC)
generate invariant γδ T cells
1,2
1,2
3
4
Paolo Tieppo , Deborah Gatti , Françoise Gosselin , Glenn Goetgeluk , Naomi
5
5
2,
3
McGovern , Florent Ginhoux , Arnaud Marchant
Catherine Donner , Bart
4
1,2
Vandekerckhove , David Vermijlen
1
Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Bruxelles, Belgium
Institute for Medical Immunology, Université Libre de Bruxelles (ULB), Gosselies, Belgium
3
Department of Obstetrics and Gynecology, Hôpital Erasme, Bruxelles, Belgium
4
Department of Clinical chemistry, microbiology and immunology, Ghent University, Ghent,
Belgium
5
Singapore Immunology Network (SIgN), Singapore
2
Although there are common characteristics among γδ T cells, it is clear that γδ T
cells do not represent a homogenous population of cells with a single physiological
role. We have shown that γδ T cells expressing (semi)invariant germline-encoded T
cell receptors (TCR) dominate the γδ T cell repertoire in human foetal life. We
hypothesized that these ‘early’ γδ T cell subsets are made by specific haematopoietic
stem and precursor cells (HSPC) present at this period of life, and thus that human
γδ T cell development is layered according to age.
In an in vitro T cell development system, human foetal (blood and thymus, gestation
range 14-30 weeks) HSPC-derived γδ T cells and post-natal blood/thymus HSPCderived γδ T cells were enriched for different γ and δ chain combinations as
determined by flow cytometry. A more detailed analysis of the complementary
determining region-3 (CDR3)γ and CDR3δ repertoire by high-throughput sequencing
indicated that foetal HSPC-derived γδ T cells, in contrast to post-natal HSPC-derived
γδ T cells, were highly enriched for particular invariant germline-encoded CDR3
sequences. Importantly, some of these sequences were found in vivo.
These data indicate that HSPC in different periods during human development can
generate different types of γδ T cells. We are currently investigating the function of
these foetal HSPC-derived γδ T cells.
Page 64 of 200
GDC 2016
A-45
Necroptosis of Dendritic Cells Promotes Activation of γδ T cells
1
1
1
1
Cheryl C. Collins , Kathleen Bashant , Cuixia Erikson , Phyu Myat Thwe , Karen A.
1
2
2
1
Fortner , Hong Wang , Craig Morita , and Ralph C. Budd
1
Vermont Center for Immunology and Infectious Diseases Department of Medicine The
University of Vermont College of Medicine Burlington, VT USA 05405
2
Division of Rheumatology Department of Medicine University of Iowa Iowa City, IA 52246
γδ T cells function at the interface between the innate and adaptive immunity and
have well-demonstrated roles in response to infection, autoimmunity, and tumors. A
common characteristic of these seemingly disparate conditions may be cellular
stress. Yet, the conditions under which ligands for γδ T cells are induced or exposed
remain largely undefined. We have previously observed that Borrelia burgdorferi
stimulates both human and murine γδ T cells indirectly primarily via TLR2 signaling of
dendritic cells (DC). We now observe that induction of necroptosis of murine or
human dendritic cells by inhibition of caspase activity paradoxically augments their
ability to activate γδ T cells in response to Borrelia, or even in the absence of
Borrelia. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by
IL-4 not only greatly reduced the susceptibility of DC to necroptosis, but also
considerably decreased their ability to activate γδ T cells. Collectively these findings
suggest that induction of necroptosis in DC upregulates or exposes expression of γδ
T cell ligands, and supports the view that γδ T cells function in the immune
surveillance of cell stress.
Page 65 of 200
GDC 2016
A-46
Differential roles of mTOR signaling in IL-17 vs IFN-γ-producing γδ T
cell development and regulation
1
2
1
Yihua Cai , Feng Xue , Chris Fleming , Jun Yan
1
1
Department of Medicine and Department of Microbiology and Immunology, James Graham
Brown Cancer Center, University of Louisville, Louisville, KY, U.S.A.
2
Department of Dermatology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University,
Shanghai, P.R. China.
γδ T cells are defined as pre-programmed innate-like T lymphocytes and mainly
include two distinct subsets: IL-17-producing-γδ T cells (CD27 γδ T cells) and IFN-γ+
producing-γδ T cells (CD27 γδ T cells). However, little is known about the molecular
mechanisms underlying γδ T cell fate decision and activation. The
mechanistic/mammalian target of rapamycin (mTOR) is a key regulator controlling the
+
specification and differentiation of CD4 T effector cell subsets and NKT cell
development. In this study, we show that mTOR complex 1 (mTORC1) and mTORC2
+
have differential roles in the regulation of CD27 γδ T cells and CD27 γδ T cells.
Loss of Rictor, an obligatory component of mTOR2, selectively abolished the CD27
Vγ4 T cells in the neonatal thymus. In contrast, mTOR1 regulator Raptor is
+
indispensable for CD27 Vγ4 T cells development. Furthermore, the Vγ4 T cells in
the periphery significantly decreased in both
Raptor and Rictor conditional knockout (cKO) mice. However, Vγ4 T cells were
developmentally normal in Raptor cKO mice, suggesting that Raptor is required for
Vγ4 T cell egress from the thymus to the periphery. Interestingly, Rictor or Raptor
deficiency did not impair the development and function of Vγ6 T cells. Mice with
deficiency of Rictor or Raptor signaling in γδ T cells had significantly decreased skin
inflammation. These studies reveal the differential roles of mTOR1 and mTOR2
+
signaling in CD27 Vγ4 and CD27 Vγ4 T cells lineage determination and regulation,
implying a new molecular mechanism by which γδ T cells contribute to skin
inflammation.
Page 66 of 200
GDC 2016
A-47
TCR, Notch and Cytokine Signals are Integrated to Dictate the
Differentiation of Specific γδ T Cell Effector Programs
1
1
1
1
2
Payam Zarin , Edward Chen , Tracy S.H. In , Gladys W. Wong , David L. Wiest ,
1
Juan-Carlos Zúñiga-Pflücker
1
Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto,
ON, Canada
2
Blood Cell Development and Cancer Keystone, Immune Cell Development and Host Defense
Program, Fox Chase Cancer Center, Philadelphia, PA, USA
The thymic landscape provides an array of spatially and temporally controlled cues
that guide the development of functionally distinct effector immune cells. Despite
remaining an active area of research, the signals that give rise to different γδ T cell
functional subtypes remain undefined. Given the ability of these cells to perform a
wide range of tissue and disease specific functions, we set out to describe the signals
necessary for the development of functional γδ T cell subsets as defined by
-/differential cytokine production. To this end, Rag2 DN3 progenitors were transduced
to with a transgenic KN6 γδ T-cell receptor (TCR), and co-cultured on various stromal
cells engineered to express Notch, TCR ligands and supplemented with combinations
of cytokines, such as IL-1β, IL-7, and IL-23. Our results showed that specific
combinations of these signals are required to program IFN-γ, IL-4, IL-17, and IL-22
producing KN6 gd T cell subsets. More specifically, we establish that weak but not
absent TCR-ligand interactions are required for γδ17 development, and Notch
enables the survival of γδ T cells in an inflammatory milieu. This work provides a
framework for the integration of signals downstream of the Notch, TCR, and cytokine
receptors as they lead to the development of γδ T cell functional subsets that can
play a fundamental role in regulating health and disease.
Page 67 of 200
GDC 2016
A-48
Eomes expression identifies a γδ T cell population
1
Ciro Novaes Rosa Lino and Immo Prinz
1
1
Institute of Immunology, Hannover Medical School
+
Eomesodermin (Eomes) is a central T-box transcription factor for CD8 T and natural
Killer (NK) cells. Together with T-bet, it contributes to their cytotoxic properties
regulating the expression of IFN-γ, granzyme and perforin. Although some studies
have reported the presence of Eomes on γδ T cells, its role in those cells is still
largely unknown. Therefore, in this project we study the role of Eomes in γδ T cells.
+
Using Eomes-IRES-GFP mice, we show that Eomes γδ T cells are differently
+
distributed among organs. The highest proportion of Eomes γδ T cells is found in the
+
spleen with over 60% of total γδ, while no Eomes γδ T cells are present in the skin.
The distribution of Eomes expressing cells among γδ T cell subsets is also not
+
homogeneous. Eomes γδ T cells are mainly comprised within the Vγ1 subset,
+
followed by Vγ4, while no significant numbers of Eomes cells were found within Vγ5
–
+
or Vγ6 subsets. By comparing the gene expression between Eomes and Eomes
populations, we identified 388 genes presenting at least a 2 fold difference of their
expression levels and a p-value smaller than 0,01. Many of those genes are related
to chemokines, cytokines and cytotoxicity molecules. Besides, Eomes overlaps with
Th1-lineage related factors, but is not coexpressed with Th17-related genes in γδ T
cells, neither in steady state condition nor under inflammation induced by imiquimod.
+
Additionally, Eomes γδ T cells present a higher cytotoxicity towards YAC-1 cells
–
when compared to Eomes γδ T.
Page 68 of 200
GDC 2016
A-49
Mechanism of signal transduction by the γδ T cell receptor
Anna Morath
1,2,3
1,2
4
, Elaine P. Dopfer , Paul Fisch , Wolfgang W. Schamel
1,2
1
Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological
Signaling Studies and Centre of Chronic Immunodeficiency (CCI), University of Freiburg,
Schänzlestrasse 18, 79104 Freiburg, Germany.
2
Max Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, 79104 Freiburg,
Germany.
3
Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University
Freiburg, 79104 Freiburg, Germany.
4
Institute of Pathology, University Medical Center Freiburg, 79106 Freiburg, Germany.
The γδ T cell receptor (TCR) consists of the antigen-binding TCRγδ heterodimer and
the CD3 subunits that contain cytoplasmic signalling motifs, such as a proline-rich
sequence in CD3ε and ITAMs in all CD3 chains. Binding of the antigen to TCRγδ
leads to the activation of the T cell. It is still an open question how the TCRγδ
heterodimer transmits the signal of antigen binding to the CD3 subunits, which are
essential to initiate the intracellular signalling cascades. Signalling by αβ TCRs relies
on a conformational change in CD3 (CD3CC) that is induced by antigen-binding and
that leads to the exposure of the proline-rich sequence and the ITAMs. Without this
CD3CC the αβ TCR cannot be activated. In sharp contrast, the γδ TCRs fail to
induce the CD3CC upon antigen-binding. An artificial induction of the CD3CC at the
γδ TCR leads to an enhancement of signalling and increases tumor cell killing by γδ
T cells.
Our interest is to study the induction of signalling by αβ and γδ TCRs. Until now
comparative analyses of signalling were performed between αβ and γδ T cells.
However, differences in signalling from these studies cannot be ascribed to the
different TCRs but could also arise from differences in the cellular background. In our
study αβ and γδ TCRs were expressed in the same cellular background to focus on
differences in signalling only caused by the different TCRs. The first preliminary
results will be presented in the poster.
Page 69 of 200
GDC 2016
A-50
mTOR inhibition potentiates cytotoxicity of Vγ4 γδ T cells via upregulating NKG2D and TNF-α
Jianlei Hao
1, 2
3
, Guangchao Cao , and Zhinan Yin
1, 2, 3
1
Biomedical Translational Research Institute, Guangdong Province Key Laboratory of Molecular
Immunology and Antibody Engineering, Jinan University, Guangzhou 510632, China;
2
Department of Immunobiology
, Yale University School of Medicine, New Haven, CT 06520,
USA;
3
State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai
University, Tianjin 300071, China;
γδ T cells play a critical role in early anti-tumor immunity, and perform cytotoxicity via
NKG2D for recognition and multiple cytotoxic factors for tumor killing. Recent study
have demonstrated pivotal role of mTOR mediated metabolism in the maturation,
differentiation and effector function of diverse immune cells, including dendritic cells
(DC), natural killer cells (NK), CDT cells, but the role of mTOR signaling in γδ T cells
is barely known. Here, we showed that suppressing mTOR signaling via mechanistic
inhibitor rapamycin enhanced cytotoxicity of in vitro expanded Vγ4 γδ T cells against
multiple tumor cell lines, and performed better tumor suppressing effects upon
adoptive therapy. Further investigation revealed that elevated cytotoxicity was due to
up-regulation of NKG2D and TNF-α. Moreover, rapamycin treatment significantly
increased phosphorylated STAT5, and inhibiting the phosphorylation of STAT5
aborted the up-regulation of NKG2D and TNF-α in rapamycin treated Vγ4 γδ T cells.
These results uncovered an important role of mTOR signaling in cytotoxic γδ T cells’
effector function and provided a potential strategy to improve γδ T cells based cancer
immunotherapy.
Page 70 of 200
GDC 2016
Session 4
Where do γδ T cells function and how do they get
there? Tissues / tissue homeostasis / migration
Talks:
Saturday, June 18th
Poster presentation:
Friday, June 17th, 4:15 – 6:00pm
Saturday, June 18th, 1:15 – 2:30pm
Page 71 of 200
GDC 2016
B-01
The role of Nacc2 in epidermal γδT cell maintenance
Dmitry S. Ushakov, Markus Pasztorek, Natalie A. Roberts, Maria Luisa Iannitto, Lucie
Abeler-Dörner, Adrian C. Hayday
Dept Immunobiology, King's College London, London SE1 9RT
Francis Crick Institute, London WC2A 3LY
Skin is populated by a huge number of immune cells. However, little is known about
the mechanisms regulating their selective expansion and homeostasis within the skin.
Uniquely, mouse epidermis contains a clonotypic T cell repertoire, known as dendritic
epidermal T cells (DETC). DETC play key roles in epidermal homeostasis, wound
repair and tumor surveillance. Over 95% of DETC belong to Vγ5Vδ1 subset. This
property is exploited by the Infection, Immunity and Immunophenotyping (3i)
consortium in order to carry out immune screening of knockout mice generated by the
Wellcome Trust Sanger Institute. Nacc2 knock out mice provided by the 3i consortium
team for screening by confocal microscopy showed a phenotype in 16 week old
+
Nacc2 knockout (KO) mouse ear epidermis, where Vγ5 DETCs were replaced with
+
CD45 Vγ5 cells. Further analysis of Nacc2 KO epidermis by confocal microscopy
+
and FACS revealed that CD45 Vγ5 are composed of a mixture of γδT cells
+
+
belonging to Vγ1 and Vγ4 subsets. Although this phenotype resembled that of
+
Skint1 KO, a gene responsible for thymic selection and populating skin with Vγ5
DETC, RT-PCR analysis of Nacc2 KO did not show a significant change in Skint1
level despite complete loss of Vγ5 mRNA. Interestingly, 9 week old Nacc2 KO mice
+
had only partial reduction in the number of Vγ5 DETC and Vγ5 mRNA level. Nacc2
is a transcriptional repressor known to stabilise tumor suppressor p53 via MDM2
+
repression. Our data suggest that Nacc2 may be involved in the maintenance of Vγ5
DETC, which may have implications for tumor surveillance capabilities in skin.
Page 72 of 200
GDC 2016
B-02
New pathways regulating immunosurveillance by tissue-resident γδ T
cells
Maria Luisa Iannitto, Oliver Nussbaumer, Richard Woolf, Adrian Hayday
King’s College London & Francis Crick Institute, London, UK
γδ‐T cells are unique unconventional lymphocytes, mostly tissue‐resident, able to
mount rapid “lymphoid stress‐surveillance responses” to sentinels of tissue
dysregulation. We ignore the molecules regulating these potentially potent
responses. The importance of the PVR-TIGIT‐DNAM1 axis is emerging in
conventional T cell activation and tumour immunology, but its relevance to tissue‐
resident T cell populations, particularly γδ‐T cells, is unknown. PVR, expressed in
cell‐cell junctions and over‐expressed in tumours, can switch from a DNAM1‐
dependent activator to a TIGIT‐dependent suppressor. We found that human
+
+
peripheral blood Vδ2 cells and human skin‐resident Vδ1 cells both express
+
+
DNAM1, but Vδ1 cells constitutively express TIGIT whereas Vδ2 cells express
TIGIT only transiently post‐activation.
These data suggest that human tissue‐resident γδ‐T cells are pre‐activated cells
regulated by TIGIT that relays inhibitory signals in response to PVR engagement.
+
Indeed, rhPVR inhibited TCR‐induced cytokine production by Vδ1 cells, whereas
+
Vδ2 cells were unaffected. Constitutive TIGIT expression was likewise displayed by
+
+
murine, tissue‐resident, epidermal Vγ5 Vδ1 cells. TIGIT expression commenced
during their Skint1‐dependent developmental checkpoint, suggesting that
TIGIT is switched on to regulate the cells’ innate‐like immune‐ surveillance potentials.
Under physiological conditions, PVR is expressed primarily by Langerhans Cells, the
other large epidermal immune population, and only by a fraction of the CD45‐
compartment. This argues that the PVR‐TIGIT‐DNAM1 axis is utilized by local DCs to
suppress local T cell activation, until a threshold of immunogenicity is exceeded.
These findings have profound implications and clinical relevance to tissue‐mediated
regulation of anti‐tumour responses and of local T cell regulation in inflammatory
diseases.
Page 73 of 200
GDC 2016
B-03
Characterization of a novel NKp46pos/Vδ1-restricted
lymphocytes resident within the human intestine
1
1,2
1
γδ TCR
1
Ferdinando Oriolo , Joanna Mikulak , Kelly L. Hudspeth , Alessandra Roberto ,
1
1
3
1,2,4
Elena Bruni , Paolo Tentorio , Federico Colombo , Anna Villa
, Bruno Silva5
1,6
Santos , and Domenico Mavilio
1
Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center,
Rozzano, Milan,Italy;
2
Institute of Genetic and Biomedical Research (IRGB), CNR, Milan, Italy;
3
Flow Cytometry and Cell Sorting Unit, Humanitas Clinical and Research Center, Rozzano,
Milan, Italy;
4
Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene
Therapy, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific
Institute, Milan, Italy;
5
Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Portugal;
6
Department of Medical Biotechnologies and Translational Medicine (BioMeTra), University of
Milan, Milan, Italy.
NKp46 is a member of Natural Cytotoxic Receptors (NCRs) triggering Natural Killer
(NK) cell cytotoxicity and production of inflammatory cytokine against harmful cells.
Although NKp46 had been first identified as a specific NK cell receptors, recent
studies revealed that it is also expressed on other immune cells such as Innate
Lymphoid cells, αβ T and NKT lymphocytes. We previously reported that NKp46
expression is inducible upon in vitro activation on a subset of human peripheral blood
pos
Vδ1 T cells. Here, we identify a novel NKp46 /γδ T lymphocytes subset resident in
human intestine under homeostatic conditions within the intraepithelial (IEL)
compartment (average of 50% of all γδ T IELs) and, to a lower degree, in lamina
pos
propria (LP) (average of 10% of all γδ T LPLs). This NKp46 /γδ T IEL subset is
characterized by a cytotoxic phenotype expressing CD56, CD8, NKG2D and NKG2C.
Moreover, we found that the expression of NKp46 in intestinal gd IELs is restricted to
Vδ1 TCR chain repertoire that indeed distinguish their tissue-like phenotype from
peripheral blood gd T cells which mainly exhibited a Vδ2 TCR distribution. We also
observed that IL-2 and IL-15-stimulation induce a de-novo expression of NKp46 in
human thymic gd T cell precursors, but not in circulating gd T cells. Their co-culture
with intestinal epithelial cells, IL-10 and TGF-β can modulate the NKp46 expression
according to the activation steady state of these cells. These results suggest an early
pos
extra-thymic and gut-conditioned maturation of NKp46 /γδ T cells likely relevant
both in GALT homeostasis and physiopathology of the gut.
Page 74 of 200
GDC 2016
B-04
Dynamic chemokine receptor expression controls homeostatic and
inflammatory γδT17 cell trafficking
1
1
1
1
Duncan R. McKenzie , Ervin E. Kara , Timona S. Tyllis , Cameron R. Bastow ,
1
1
1
1,2
3,4
Carly E. Gregor , Rachelle Babb , Kevin A. Fenix , James C. Paton , Axel Kallies ,
3,4
1
1,5
Stephen L. Nutt , Iain Comerford , Shaun R. McColl .
1. Department of Molecular & Cellular Biology, University of Adelaide, Adelaide, South Australia,
Australia.
2. Research Centre for Infectious Diseases, University of Adelaide, Adelaide, South Australia,
Australia.
3. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
4. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia.
5. Centre for Molecular Pathology, University of Adelaide, Adelaide, South Australia, Australia.
Interleukin 17-producing γδ T cells (γδT17) exhibit unique trafficking characteristics.
They reside in homeostatic barrier tissues but also circulate and infiltrate inflamed
tissues. However, regulation of these processes remains poorly understood. We find
that chemokine receptor CCR6 regulates homeostatic distribution of γδT17 cells but
is irrelevant in their inflammatory trafficking. Resting γδT17 cells undergo constitutive
CCR6-dependent recruitment into uninflamed dermis, which is inhibited by γδT17 cell
activation. Correspondingly, IL-23 and IL-1β stimulation drives downregulation of
CCR6 in γδT17 cells by a RORγt/IRF4/BATF circuit. Using CCR6 expression as an
activation marker reveals novel homeostatic trafficking potential of microbiotareactive
γδT17 cells. In contrast, inflammatory trafficking is mediated by constitutive
expression of CCR2, which recruits γδT17 cells into the autoimmune central nervous
system and cancer lesions. Additionally, CCR2 is crucial for γδT17-dependent
protection against bacterial infection in mucosal tissue. We propose that programmed
expression of CCR6 and CCR2 in γδT17 cells allows surveillance in uninflamed skin
and rapid recruitment to inflammatory foci, respectively. Activation-induced
downregulation of CCR6 disrupts homeostatic circulation patterns and appears to
enhance efficiency of migration to inflamed tissue. These findings establish the
molecular regulation of γδT17 cell trafficking in health and disease, and reveal
substantial phenotypic distinction between resting and activated γδT17 cells.
Page 75 of 200
GDC 2016
B-05
γδ Τ cells are the major source of IL-17 in the meninges and control
brain cognitive functions.
Miguel Ribeiro, Joana E. Coelho, Mariana Temido-Ferreira, Diana G. Ferreira, Luisa
V. Lopes, Bruno Silva-Santos and Julie C. Ribot
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa
γδ T cells are known to populate multiple tissues, such as the skin, gut or lung, where
they make major contributions to local physiology. Here we investigated whether γδ T
cells could play a role in normal brain function, given that ab T cells were recently
shown to be involved in learning behavior of mice: whereas IFN-γ producing subsets
were detrimental, their IL-4-producing counterparts played a pro-cognitive role. We
found that γδ T cells infiltrate the meningeal spaces from the brain of naive C57/BL6
mice already at birth and persisted throughout life. Strikingly, at 1 week of age,
meningeal γδ T cells differentiated into IL-17 (but not IFN-γ) producers, which
seemingly depended on IL-1β/IL-23 and retinoic acid. In fact, γδ T cells were the
major source of IL-17, whereas ab T cells mostly provided IFN-γ in situ. To test
whether IL-17-producing γδ T cells influenced the cognitive performance of mice, we
scored the behavior of WT, TCRδ-/- and IL-17-/- mice in classical neuroscience
paradigms assessing learning capacities. We observed that, contrary to WT controls,
mice deficient for γδ T cells or IL-17 displayed impaired short-term/working memory
in the Y maze paradigm, but a normal long-term spatial memory in the Morris water
maze. We are currently identifying the underlying molecular mediators; interestingly,
we found reduced levels of Brain Derived Neurotropic Factor (BDNF), a major player
in memory and dementia, in the hippocampus of IL-17-/- mice compared to WT
controls. Altogether, our data suggest that γδ T cells regulate brain cognitive
functions through a novel IL-17-dependent mechanism.
Page 76 of 200
GDC 2016
B-06
Pathogen-specific amplification of local inflammation and tissue
remodeling by human γδ T cells and MAIT cells: implications for
diagnosis and therapy
1
1
1,2
1
1
Anna Rita Liuzzi , Ann Kift-Morgan , Melisa López Antón , Amy C. Brook , Ida M. Friberg ,
1
3
3
2
Jingjing Zhang , Gareth W. Roberts , Kieron L. Donovan , Chantal S. Colmont , Mark A.
1
1
4,5,6
7,8
1,8
Toleman , Timothy Bowen , David W. Johnson , Nicholas Topley , Bernhard Moser ,
1,2,8
1,8
Donald J. Fraser , and Matthias Eberl
1
Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK;
Wales Kidney Research Unit, Health and Care Research Wales, Cardiff, UK;
3
Directorate of Nephrology and Transplantation, Cardiff and Vale University Health Board,
4
University Hospital of Wales, Cardiff, UK; Department of Renal Medicine, University of
Queensland at Princess Alexandra Hospital, Brisbane, Australia;
5
Centre for Kidney Disease Research, Translational Research Institute, Brisbane, Australia;
6
Australia and New Zealand Dialysis and Transplant Registry, Adelaide, Australia;
7
Centre for Medical Education, School of Medicine, Cardiff University, Cardiff, UK;
8
Systems Immunity Research Institute, Cardiff University, Cardiff, UK
2
The characterization of unconventional human T-cell responses in vivo and their
relevance for homeostasis, immune surveillance and inflammation remains
challenging, with only limited access to relevant specimens from anatomical sites
other than blood, especially during acute disease. Here, we analyzed local and
systemic immune responses in individuals with end-stage kidney disease receiving
peritoneal dialysis (PD), before and during microbial infections. Our data confirm in
vitro and in vivo the ligand specificity of peritoneal Vγ9/Vδ2 T-cells and MAIT cells for
the
corresponding
microbial
ligands,
(E)-4-hydroxy-3-methyl-but-2-enyl
pyrophosphate (HMB-PP) and vitamin B2 derivatives, respectively, and demonstrate
that Vγ9/Vδ2 T-cells and MAIT cells accumulate locally at the site of infection with
organisms producing the corresponding ligands. We show that local unconventional
T-cells are major producers of IFN-γ and TNF-α in response to microbial pathogens,
and that these cytokines affect the cells lining the peritoneal cavity where they may
amplify local inflammation and trigger tissue remodeling, with potential consequences
for peritoneal membrane integrity. In fact, utilization of the HMB-PP and vitamin B2
pathways by the causative organism represents a useful predictive marker for
subsequent technique failure risk, implying that unconventional T-cell driven
responses may contribute to overall clinical outcomes. Our results identify key
pathways by which unconventional T-cells orchestrate early inflammatory responses,
and highlight potential diagnostic and therapeutic targets to improve early patient
management. In a wider context, we demonstrate the power of using PD as
experimental and clinical model for investigations into local immune responses and
monitoring individuals before, during and after defined microbial infections.
Page 77 of 200
GDC 2016
B-07
IL-15 Promotes Directed γδ T Cell Localization Within the Intestinal
Mucosa.
1
2
3,4
Alexander Ethridge , Bana Jabri , Jerrold R. Turner , Karen L. Edelblum
1
1
Center for Immunity and Inflammation, Department of Pathology and Laboratory Medicine,
Rutgers New Jersey Medical School, Newark, NJ
2
Department of Pediatrics, The University of Chicago, Chicago, IL
3
Departments of Pathology and
4
Department of Medicine, Brigham and Women’s Hospital, Harvard, Medical School, Boston,
MA
IL-15 is a critical factor in the proliferation and survival of intraepithelial lymphocytes
(IEL) expressing the γδ T cell receptor; however, little is known regarding the role of
IL-15 in γδ IEL migration. Since transpresentation of membrane bound IL-15 requires
cell cell contact between activating and responder cells, we hypothesized that IL-15
functions as a γδ T cell chemoattractant via direct interaction between γδ IELs and
the villous epithelium. We found that IL-15 enhanced γδ IEL migration into Caco-2
intestinal epithelial monolayers in a dose-dependent manner. To determine how
mucosal compartmentalization of IL-15 expression affects γδ T cell migration, GFP
γδ T cell reporter mice were crossed to transgenic (Tg) mice overexpressing murine
IL-15 either in the intestinal epithelium (vil-IL-15) or lamina propria via a MHC class-I
restricted promoter (Dd-IL-15). Consistent with the role of epithelial IL-15 in IEL
proliferation, vil-IL-15 Tg mice exhibited a 61% increase in γδ T cell number
compared to wildtype (WT).Time-lapse video microscopy revealed enhanced γδ IEL
migration into the epithelium in vil-IL-15 Tg mice relative to WT (44±8.0% vs
32±3.3%) leading to increased γδ IEL/epithelial interaction (4.5±0.2 vs 3.5±0.2
interactions/hr). In contrast, restriction of IL-15 to the lamina propria in Dd-IL-15 Tg
mice reduced the frequency of γδ IELs within the lateral intercellular space
(25±6.8%), resulting in increased γδ T cell localization within lamina propria. These
data indicate that IL-15 promotes γδ T cell chemotaxis, and thus, compartmentalized
IL-15 overexpression induces differential migration of γδ T cells within the intestinal
mucosa.
Page 78 of 200
GDC 2016
B-08
IL-23-dependent γδ T cells produce IL-17 and accumulate in tissues
exposed to mechanical stress including enthesis, aortic valve, and
ciliary body
1
2
1
3
1
Annika Reinhardt , Tetyana Yevsa , Tim Worbs , Stefan Lienenklaus , Inga Sandrock , Linda
1
4,5
1
1
1
Oberdörfer , Thomas Korn , Siegfried Weiss , Reinhold Förster , Immo Prinz
1
Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany,
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School,
30625 Hannover, Germany,
3
Institute for Laboratory Animal Science, REBIRTH Unit 8.4, Hannover Medical School, 30625
Hannover, Germany,
4
Department of Neurology, Klinikum rechts der Isar, Technische Universität München, 81675
Munich, Germany,
5
Munich Cluster of Systems Neurology (SyNergy).
2
Spondyloarthropathies (SpA) are a group of rheumatic diseases comprising
ossification and inflammation of entheseal tissue, the region where tendon attaches
onto bone. Interleukin (IL)-23 is involved in the pathogenesis of SpA by acting on IL23 receptor (IL-23R) expressed on enthesis-resident lymphocytes. Upon IL-23
binding, CD3+CD4–CD8– tissue-resident lymphocytes secrete IL-17A and IL-22,
leading to inflammation, bone loss, and ossification. Still, our knowledge about
enthesis-resident lymphocytes remains fragmentary, in particular the contribution of
entheseal gd _T cells is not clear.
We used two-photon microscopy and flow cytometric analysis to investigate
entheseal lymphocytes from C57BL/6, Tcrd-H2BeGFP, Rorc-GFP and IL-23R-GFP
mice. In non-inflamed entheseal tissues, fetal thymus-dependent Vγ6+CD27–γδ T
cells are abundant and constitute the large majority of RORγt+IL-23R+ enthesisresident lymphocytes. Upon in vitro stimulation wit IL-23 or PMA/ionomycin, γδ T
cells are the main source of IL-17A among entheseal lymphocytes.
To analyze entheseal γδ T cells in IL-23-induced inflammation, Tcrd-H2BeGFP mice
were crossed to the susceptible B10.RIII background. Hydrodynamic injection of IL23 minicircle DNA was performed for overexpression of IL-23 and induction of
inflammation. Light-sheet fluorescence microscopy was applied to visualize arthritic
inflammation. Under inflammatory conditions, γδ T cells increase in numbers at the
Achilles tendon enthesis, aortic root, and adjacent to the ciliary body.
In conclusion, we demonstrate that entheseal γδ T cells are derived from fetal
thymus and are maintained as self-renewing tissue-resident cells. As main IL-17A
producers within tissues exposed to mechanical stress including entheses, γδ T cells
are key players in the pathogenesis of IL-23-induced local inflammation.
Page 79 of 200
GDC 2016
B-09
IL-17-producing Vγ6+ γδ T cells enhance bone fracture healing
Takeshi Nitta, Takehito Ono, Kazuo Okamoto, Hiroshi Takayanagi
Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The
University of Tokyo
γδ T cells function not only in the protection against pathogens but also in the
maintenance of tissue homeostasis. Here we show that γδ T cells have promotive
effects on bone regeneration after injury. Using a bone fracture model, we found that
Vγ6+ γδ T cells increased in the injury site and produced IL-17A. Bone repair was
impaired in IL-17A-deficient mice due to a defect in osteoblastic bone formation. IL17A accelerated bone formation by stimulating the proliferation and osteoblastic
differentiation of mesenchymal progenitor cells. These results indicate that Vγ6+ γδ
T cells produce IL-17A, which promotes bone formation and facilitates bone fracture
healing, highlighting a novel role for γδ T cells in skeletal tissue regeneration.
Page 80 of 200
GDC 2016
B-10
It may seem inflammatory to some, but γδ T cells are innately healing
to the bone
Shirin Kalyan
Faculty of Medicine, University of British Columbia
We had first observed that bisphosphonate-associated osteonecrosis of the jaw
(ONJ), a rare serious adverse drug effect characterized by non-healing necrotic bone
tissue of the mandible or maxilla, was linked to a deficiency in the primary subset of
γδ T cells (Vγ9Vδ2 T cells) found in human peripheral blood. Patients who developed
ONJ while on bisphosphonate therapy for bone fragility not only lacked Vγ9Vδ2 T
cells, but they also all had underlying conditions that compromised their immune
integrity. We had followed up with our original observation by characterizing the
immune defects in the ONJ patients lacking γδ T cells and found that these patients
were deficient in a number of key homeostatic immune and wound healing factors
that are known to be regulated by these innate T cells. Intriguingly, a murine study
has just demonstrated that a subset of γδ T cells play an important role in bone
regeneration, particularly following trauma. This new work and other recent in vitro
studies using human cells have brought us back full circle to our initial observation
that a deficiency in γδ T cells was linked to a failure of bone regeneration and healing
in the context of osteonecrosis of the jaw. These findings seem to contradict the
prevailing view that “inflammatory” γδ T cells are bone degenerative rather than
restorative. This presentation will stitch together the emerging evidence of these socalled inflammatory γδ T cells in bone remodeling and healing, showing that they are
anything but all bad to the bone.
Page 81 of 200
GDC 2016
B-11
Analysis and characterization of γδ T cells in male reproductive organs
Wilharm A., Sandrock I., Reinhardt A., Oberdörfer L., Prinz I.
Hannover Medical School, Institute of Immunology, Carl-Neuberg-Str. 1, 30625 Hannover,
Germany
γδ T cells are frequent in mucosal tissues, where they contribute to antimicrobial
immunity and epithelial integrity. They are well-characterized in female, but not in
male reproductive organs. In this study, we investigated γδ T cells in the male
reproductive tract. Using TcrdH2BeGFP reporter mice and flow cytometry, we found
+
that in seminal vesicle as well as in testis and prostate nearly 20% of all CD3 cells
were γδ T cells. Immunofluorescence and two-photon laser-scanning microscopy
revealed that in all three organs γδ T cells were localized in the stromal connective
tissue near epithelial cells, which were highly important for the function of these
organs. While γδ T cells within the prostate and seminal vesicle showed a polyclonal
and diverse phenotype and were able to secrete IL-17 as well as IFN-γ, γδ T cells in
+
hi
the testis were more clonal (mainly Vγ6 ) and displayed a pre-activated CD44
+
phenotype. Investigating the ontogeny of γδ T cells, we found that Vγ6 γδ T cells
first infiltrate the testis during puberty, whereas seminal vesicles become populated
+
+
by Vγ1 CD27 γδ T cells at that time. In contrast, we could not detect any pubertydependent changes in γδ T cell populations in prostate. In all three male reproductive
tissues analyzed, γδ T cells were the main producers of all IL-17 after ex vivo
stimulation. Since approximately 15% of male infertility is due to infection and
inflammation of reproductive organs, we hypothesize that IL-17-producing tissueresident γδ T cells could contribute to regulation of male fertility.
Page 82 of 200
GDC 2016
B-12
Effects of intradermal BCG vaccination on circulating gamma delta Tcells in a non human primate (NHP) model
1
2
1
2
Joe R Fenn , Andrew D White , Angus G Dalgleish , Sally A Sharpe , Mark D
1
Bodman-Smith
1 Infection and Immunity Research Institute, St. George’s, University of London, Blackshaw Rd,
London.
2 Public Health England, Porton Down, Salisbury, Wiltshire.
Bacillus Calmette-Guérin (BCG) is the gold standard for vaccination against
tuberculosis but is also a well-established treatment for bladder cancer. The exact
mechanism of action of BCG as an intervention in cancer remains to be elucidated,
but it is clear that a strong immunological response is critical for clearance of the
tumour. It has been shown that Vγ9Vδ2 γδ T-cells have the ability to recognise
mycobacterial
pyrophosphates
including
(E)-4-Hydroxy-3-methyl-but-2-enyl
pyrophosphate (HMBPP) as well as tumour associated endogenous pyrophosphate
antigens like isopentenyl pyrophosphate (IPP). This duel recognition makes Vγ9Vδ2
T-cells an attractive candidate for investigation of mycobacteria-based cancer
intervention. A factor hampering current research in this area is that the Vγ9Vδ2 TCR
involved in pyrophosphate detection is unique to primates, necessitating the use of
non-human primates (NHPs) for infection and vaccination studies. In this series of
experiments we aimed to investigate the effect of intradermal (ID) BCG vaccination of
rhesus macaques on peripheral blood Vγ9Vδ2 T-cells with a view to exploit
responses in cancer immunotherapies. We see that macaque γδ T-cells are
phenotypically similar to their human counterparts and that ID BCG treatment induces
perturbations in circulating Vγ9Vδ2 cell frequency and expression of migrationassociated surface molecules including CXCR3. The results of these preliminary
investigations may be indicative of altered Vγ9Vδ2 migratory patterns which could be
favourable in cancer.
Page 83 of 200
GDC 2016
B-13
Human tissue-resident gd T cells: an innate-like surveillance system in
health and malignancy
1,4
1,2,3,4
1,2
Richard Woolf , Fernanda Kyle
, Robin Dart , Oliver Nussbaumer
1,3
3
1,2
Wu , Andrew Tutt , Adrian Hayday
1,2
, Yin
1
Peter Gorer Department Of Immunobiology, King’s College London
The Francis Crick Institute, Lincoln’s Inn Fields Laboratory
3
The Breakthough Breast Cancer Research Unit, King’s College London
4
These authors contributed equally to this work.
2
Mouse tissue-resident gd T cells contribute to cancer resistance and tissue
maintenance. Related to this is “lymphoid stress-surveillance” whereby the cells can
respond rapidly to tissue dysregulation, without requiring T cell receptor stimulation.
Conversely, tissue-resident gd T cells are poorly described in humans, and there is to
date no report of an innate-like stress-surveillance T cell compartment in human
tissues. This is despite the fact that a tumour-associated gd T cell signature was the
strongest gene expression correlate with overall survival across 18,000 cancer
patients. Addressing this, we have characterised tissue-resident T cell compartments
from skin, gut, and breast from over one hundred donors, reproducibly revealing a
substantial gd T cell compartment possessing overt innate-like responsiveness that
+
contrasted with the TCR-dependent responsiveness of co-localised CD8 T cells. The
cells’ abundance and cytolytic, Th1-like phenotype, coupled with a failure to express
IL-17, may explain their potential utility in tumour immunotherapy, consistent with
which such cells were readily isolated from primary human mammary carcinomas.
Page 84 of 200
GDC 2016
B-14
+
Human skin Vδ1 T cells: Mediators of stress-surveillance and a
potential tool for adaptive cell therapy.
1
1,
1
Oliver Nussbaumer , Richard Woolf Richard Beatson and Adrian Hayday
1
2
1,2
Peter Gorer Department of Immunobiology, King’s College London, London, UK
London Research Institute, The Francis Crick Institute, London, UK
Most studies looking at human γδ T cells study Vδ2 expressing T cells residing
almost exclusively in the blood. In mice, γδ T cells dominate tissues such as the skin
and respond rapidly to tissue stress. Since the biology of human tissue-resident γδ T
cells remains largely elusive, we have sought to characterise the human skin γδ T
cell compartment and their innate-like potential.
Human skin-resident lymphocytes contained a notable γδ T cell population
[8.3%±6.3], primarily expressing a Vδ1 TCR chain [74%±12.5] linked predominantly
with Vγ8 chains. Skin γδ T cells had a ‘memory’ phenotype, showed expression of
the activating receptor NKG2D, stained mostly CD25 negative but positive for the Th1
markers TIM3 and T-bet. Concordant with this, Vδ1 T cells produced IFN-γ, TNF-α,
CCL4 and GM-CSF but not IL-17 upon stimulation with PMA/Ionomycin or TCR cross
linking but also in response to NKG2D ligands alone. We have developed means to
expand these tissue-derived γδ T cells, making them for the first time accessible for
subsequent study. Their cytotoxic behaviour towards transformed cell lines, partially
mediated via the NKG2D receptor, implies a role of human non-Vδ2 γδ T cells in
tumour surveillance.
This places a tissue-resident T cell in the innate phase of the immune response,
implicating a human lymphoid stress-surveillance response in immune-protection.
Furthermore, availability of expansion methods for tissue resident γδ T cells from
human tissue such as skin, allows for the study of these cells and makes them
available for clinical adoptive cell therapy.
Page 85 of 200
GDC 2016
Session 5
γδ T cell function in health and medicine: 1.
infectious disease
Talks:
Saturday, June 18th
Poster presentation:
Friday, June 17th, 4:15 – 6:00pm
Saturday, June 18th, 1:15 – 2:30pm
Page 86 of 200
GDC 2016
B15
Selective destruction of IL-23 signal expansion of major Ag-specific γδ
T-cell subset in TB patients
‡
‡
¶
||
¶
Hongbo Shen *, Shanshan Liang , Dan Huang , Feifei Wang , Ling Shen , and
¶
Zheng W. Chen
‡
Unit of anti-tuberculosis immunity, Key Laboratory of Molecular Virology and Immunology,
Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China,
¶
Department of Microbiology & Immunology and Center for Primate Biomedical Research,
University of Illinois College of Medicine, Chicago, IL, USA,
||
Department of Medical Microbiology and Parasitology, Shanghai Medical College, Fudan
University, Shanghai 200032, China
*Correspondence address. Tel: +86-21-54923060; Fax: +86-21-54923061; E-mail:
[email protected]
Loses of Ag-specific T-cell responses due to cytokine signaling importency after
infections have not been demonstrated. We hypothesize that tuberculosis(TB) can
destruct signaling effects of selective cytokine(s), and blunt or exhaust Ag-specific T
cells in response to the respective cytokine. To test this, mechanistic studies were
conducted to examine whether and how TB destructed IL-23 and IL-2 signaling
effects on major human γδ T-cell population, phosphoantigen(HMBPP)-specific
Vγ2Vδ2 T cells. IL-23 and IL-2 distinctly expanded HMBPP-stimulated Vγ2Vδ2 T-cell
subpopulation from humans with latent TB infection(LTBI) and IL-2 synergized the IL23 effect. IL-23 expansion of the γδ T cells involved STAT3. Surprisingly, TB patients
exhibited selective destruction of IL-23 expansion of Vγ2Vδ2 T cells, as IL-23, but not
IL-2, failed to expand HMBPP-stimulated Vγ2Vδ2 T cells in TB patients. Selective
impairment of IL-23 signaling effects in TB could not be attributed to IL-23R nonexpression, APC dysfunction or PD-1 dysregulation. However, TB-driven impairing of
IL-23 signaling coincided with decreases in expression and phosphorylation of
STAT3, and reciprocal over-expression of antagonizing SOCS3 in Vγ2Vδ2 T-cell
subpopulation. Interestingly, impairing of STAT3 was linked to marked increases in
hsa-miR-337-3p and hsa-miR-125b-5p in Vγ2Vδ2 T cells from TB patients. Silence of
hsa-miR-337-3p and hsa-miR-125b-5p by miRNA sponge could improve IL-23mediated expansion of HMBPP-stimulated Vγ2Vδ2 T cells in TB patients and their
capability to express cytokines. These results support our hypothesis, and suggest
that TB can selectively impair a cytokine effect while sparing another, and induce
paralysis/exhaustion of T cells in response to the respective cytokine.
Page 87 of 200
GDC 2016
B-16
γδ T cells function in immunity against Bordetella pertussis as an early
source of innate IL-17 but also as adaptive tissue resident memory γδ
T cells.
Alicja Misiak, Mathilde Raverdeau, Mieszko M. Wilk & Kingston H.G. Mills
School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, Trinity College
Dublin, Dublin 2, Ireland
γδ T cells play a role in protective immunity to infection at mucosal surface, but also
mediate pathology in certain autoimmune diseases through innate IL-17 production.
Recent reports have suggested that γδ T cells can have memory analogous to
conventional αβ T cells. In this study we have examined the role γδ T cells in
immunity to the respiratory pathogen Bordetella pertussis. γδ T cells, predominantly
Vγ6, produced IL-17 in the lungs as early as 2 hours post infection. The bacterial
burden during primary infection was significantly enhanced in the absence of early IL17. γδ T cells, exclusively Vγ4, from the lungs of infected but not naïve mice
produced IL-17 in responses to heat killed B. pertussis in the presence of APC.
Furthermore, γδ T cells from the lungs of mice re-infected with B. pertussis produced
significantly more IL-17 than γδ T cells from infected un-primed mice. γδ T cells with
tissue resident memory T (TRM) cell phenotype were expanded in the lungs during
infection with B. pertussis and proliferated rapidly after re-challenge of convalescent
mice. Our findings demonstrate that lung γδ T cells, predominantly Vγ6 cells, provide
an early source of innate IL-17 to promote the production of antimicrobial peptides,
whereas antigen-specific Vγ4 cells function in adaptive immunological memory
against B. pertussis.
Page 88 of 200
GDC 2016
B-17
Hepatic γδ T cells dictate Plasmodium pathogenicity in experimental
cerebral malaria
Bruno Silva-Santos, Julie C. Ribot, Rita Neres, Liliana Mâncio-Silva, Vanessa
Zuzarte-Luís, Anita Q. Gomes, Maria M. Mota and Ana Pamplona
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon,
Portugal.
Symptomatic malaria infection has been associated with activation and expansion of
pro-inflammatory γδ T cells in the peripheral blood of Plasmodium falciparuminfected children. However, previous studies failed to identify a nonredundant role for
gd T cells in animal models of severe malaria. Here we show that mice lacking γδ T
cells (TCRδ-/-) are strikingly resistant to experimental cerebral malaria (ECM) when
infected with P. berghei ANKA sporozoites (or mosquito bite), while fully susceptible if
challenged with P. berghei ANKAparasitized red blood cells. This reveals an
unappreciated pathogenic role of murine γδ T cells that is strictly dependent on the
liver stage of infection. We further show that the presence of γδ T cells significantly
modulated the expression of ~20% of the parasite genome at the end of the liver
stage, with a focus on the induction of GPI-anchored proteins associated with
pathogenicity.
As consequence, TCRδ-/- mice displayed reduced immune activation and an intact
blood-brain barrier, in contrast with TCRδ+/+ controls. Of particular relevance was
the impairment in the pro-inflammatory cytokine interferon-γ (IFNγ), which was
reduced in the serum, in splenic CD4+ and CD8+ T cells and in the brain of TCRδ-/mice. Importantly, IFNγ-/- mice phenocopied the ECM resistance of TCRδ- /- mice.
We thus propose that hepatic γδ T cells modulate Plasmodium pathogenicity and the
inflammatory syndrome associated with CM. This unravels the importance of the liver
stage, which has been widely neglected in CM pathogenesis, while elucidating the
potential of targeting γδ T cells in future therapeutic approaches.
Page 89 of 200
GDC 2016
B-18
Monitoring of human γδ T cell receptor repertoires after allogeneic
stem cell transplantation to identify virus‐reactive γδ T cells
1
1,2
1
2
Sarina Ravens , Christian Schultze‐Florey , Solaiman Raha , Melanie Drenker ,
1
1
1,2
Linda Oberdörfer , Inga Ravens , Robert Geffers3, Christian Könecke* and Immo
1
Prinz*
1
Institute of Immunology, Hannover Medical School
Department of Hematology, Hemostasis, Oncology and Stem‐Cell Transplantation
3
Genome Analytics, Helmholtz Centre for Infection Research
*shared senior authorship
2
Cytomegalovirus (CMV) infection and graft‐versus‐host disease (GVHD) are major
complications after allogeneic hematopoietic stem cell transplantation (HSCT). γδ T
cells may be promising targets and cellular therapeutics since they show anti‐CMV
and anti‐tumor effects but don’t cause GVHD. Thus, it is fundamental to understand
how the human γδ T‐cell repertoire is regenerated in HSCT recipients and to identify
virus‐reactive expanded γδ T‐cell clones. We set up a longitudinal cohort study of
100 HSCT recipients either with or without CMV infection. We
monitored pre‐ and post‐transplantation γδ T‐cell kinetics of TCRγ or TCRδ V(D)J
regions. After FACSsorting, rearranged CDR3 regions were amplified from cDNA and
sequenced by Illumina Mi‐Seq analysis.
Result: TCR repertoires of healthy volunteers and of HSCT patients before
transplantation are private, extremely diverse, yet contain highly expanded clones.
Typically, the 20 most expanded clones make up 20‐40% of all γδ T cells. After post‐
natal expansion in response to microbial antigens, there is an overrepresentation of
Vγ9Vδ2 clones using CDR3 gamma sequences composed of Vγ9 and JγP gene
segments. After transplantation, we witnessed a quick reconstitution of γδ T cells.
Post‐transplantation repertoires were entirely changed as compared to pre‐
transplantation repertoires, but then remained surprisingly stable over 180 days.
Upon CMV reactivation, we monitored an additional expansion of Vγ9neg and Vδ2neg
γδ TCR sequences. Together, our high‐throughput TCR sequencing study of HSCT
patients establishes individualized clonal kinetics of a) γδ T cell reconstitution, and b)
γδ T cell‐mediated immune responses to viral infection, in particular CMV.
Page 90 of 200
GDC 2016
B-19
Vγ9Vδ2 T cells in Plasmodium falciparum infection: also an antigen
presenting cell ?
1,2
1,2
1,2
1,2
3
3
Howard, J. , Loizon, S. , Duluc, D. , Pitard, V. , Khan, M.W.A. , Moser, B. ,
4
4
4
5
1,2,4
Mechain, M. , Duvignaud, A. , Malvy. D. , Troye-Blomberg, M. , Moreau, J-F.
,
1,2
6
1,2
Déchanet- Merville, J. , Mercereau-Puijalon, O. , Mamani-Matsuda, M. and Behr,
1,2
C.
1
Centre National de la Recherche Scientifique UMR 5164 « Immuno ConcEpT »,
University of Bordeaux,
3
Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK;
4
Interdepartmental Section Tropical Medicine and Clinical International Health, University
5
Hospital Centre - CHU, Bordeaux, France;
6
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University,
Stockholm, Sweden;
7
Institut Pasteur, Immunologie Moléculaire des Parasites, Paris, France.
2
Clinical malaria, one of most deadly human diseases, is associated with the intraerythrocytic cycle of the Plasmodium parasites. A major limitation to achieve efficient
vaccine is the incomplete knowledge of induction and effector mechanisms of
immunity that control blood stage parasite biomass. It has been well established that
Plasmodium falciparum derived phosphoantigens induce potent activation and
expansion of Vγ9Vδ2 T cells, which contribute to the early immune response by
direct cytotoxic and inflammatory functions. Among the various mechanisms by which
the parasite evades the host immune response, the impairement of professional
antigen presenting cells (APC) by erythrocytic stages of P. falciparum has been
described. In vitro, phosphoantigen-activated Vγ9Vδ2 T lymphocytes have been
shown to act as APCs. However, whether this activity can be involved in a
pathophysiological context is not known. As P. falciparum infected red blood cells
activate Vγ9Vδ2 T cells via released phosphoantigens, we aimed to investigate the
potential of Vγ9Vδ2 T cells to participate to the activation of the adaptive immune
response in the context of malaria. Our data show that an APC-like phenotype on
Vγ9Vδ2 T cells can occur both in vivo in P. falciparum-infected patients and in vitro
during P. falciparum co-culture. Moreover, the ability of these stimulated cells to
induce alpha/beta T lymphocyte response is demonstrated. Vγ9Vδ2 T lymphocytes
could therefore represent an alternative route of adaptive immune response activation
in conventional APC-disabled P. falciparum infected hosts.
Page 91 of 200
GDC 2016
B-20
Toxoplasma gondii elicits fetal Vγ9Vδ2 T cell responses in utero
1,2
1,2
2
2
Ling Ma , Maria Papadopoulou , Martin Taton , Arnaud Marchant , Francesca
3
3,4
1,2
Genco , Valeria Meroni , David Vermijlen
1
Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Belgium
Institute for Medical Immunology, Université Libre de Bruxelles (ULB), Belgium
3
IRCCS San Matteo Polyclinic, Pavia, Italy
4
Internal medicine and medical therapy, University of Pavia, Italy
2
The Vγ9Vδ2 T cell subset is the main γδ T cell population in adult peripheral blood.
Their semi-invariant T cell receptor (TCR) reacts towards microbe-and host-derived
phosphorylated prenyl metabolites (‘phosphoantigens’), the most potent being the
microbe-derived HMB-PP. We have recently shown that this T cell subset is the main
γδ T cell subset in fetal blood and is already programmed at the level of their TCR
and their function, suggesting that they could play an important role upon infections in
utero. Here we explored this role by investigating the response of Vγ9Vδ2 T cells
towards congenital infection with the HMB-PP-producing parasite Toxoplasma gondii.
In congenitally-infected infants (peripheral blood sample within first week after birth),
Vγ9Vδ2 T cells, but not other γδ T cell subsets, where highly expanded compared to
age-matched controls. This was associated with differentiation of the Vγ9Vδ2 T cells
and increased expression of activation marker. Thus congenital infection with a
phosphoantigen-producing pathogen induces a clear and specific response in
Vγ9Vδ2 T cells in utero. Currently we are investigating this response in more detail at
the molecular level and we plan to follow these infants after birth in order assess the
longer-term effect of congenital infection on the γδ T cell repertoire.
Page 92 of 200
GDC 2016
B-21
Pre-treatment of staphylococcal cell wall component producing γδ T
cell-mediated IL-17A protected host from Staphylococcus aureus
infection
Min-Ja Lee, Min-Young Sung, Bo-Ram Lee, Hae-Su-Yun, Ji-Hye Park, Bok Luel Lee
Host Defense Laboratory of College of Pharmacy, Pusan national University, Busan, Korea
An effective vaccine against methicillin-resistant Staphylococcus aureus (MRSAs)
has not been developed yet. We supposed that γδ T-cells play an important role
against S. aureus infection. Mammalian γδ T cells are an important source of innate
IL-17A production, leading to the induction of neutrophil-mediated phagocytosis at the
early staged-infection. But, until this time, staphylococcal ligand molecule(s)
producing γδ T cell-mediated IL-17A is not identified yet. To identify ligand
molecule(s) from S. aureus cells, we generated 11 different S. aureus mutant strains,
which are mutated on genes involved in bio-syntheses of bacterial cell wall
components and then screened which strain can produce IL-17A at the early period.
When acetone-treated S. aureus mutant cells were injected into mouse peritoneal
cavity, one strain, which are unable to synthesize both acetylated peptidoglycan and
bacterial lipoproteins, highly induced IL-23 and IL-17A production. These two
cytokines were maximally induced after 6 h injection, but they were abrogated after
12 h injection. But, these cytokine productions were not observed on the γδ TCR KO
mice. To estimate in vivo effects of γδ T cell-mediated IL-17A production against S.
aureus infection, we pre-injected purified staphylococcal cell wall component onto
peritoneal cavity, and then cultured MRSA cells were infected after 5 h later. After 5
days later, when the survival rates, abscess and bacterial numbers in peritoneal
cavity were examined, mice pre-immunized with mutant-originated cell wall
component were completely protected from MRSA infection, suggesting that γδ T
cell-mediated host innate immunity protects host from S. aureus infection.
Page 93 of 200
GDC 2016
B-22
Bacterial exposure leads to drastic γδ T cell changes from a cytotoxic
Th1-type cytokine-producing to a pAPC phenotype accompanying
TCR-independent bacterial killing and extensive TCR-dependent
bacterial uptake.
Marta Barisa, Anne Kramer, Andrea Kakouri, Mona Bajaj-Elliott, John Anderson and
Kenth Gustafsson
UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
Following killing of cellular or microbial targets, γδ T cells are able to change
phenotype into professional antigen presenting cells (pAPC) and to take up target
material for antigen processing and MHC presentation. However, it has remained
unclear how this sequence of events proceed, what molecular interactions are
required and what the functional consequences are.
We have now employed a simplified dual bacterial model system to investigate
PBMC-derived γδ T cell responses upon primary encounter, and the evolution of their
phenotype prior to and after secondary exposure in in vitro co-cultures to the same
specific intact bacteria types. Our results indicate that human PBMC-derived γδ T
cells can efficiently kill co-cultured bacteria in a TCR-independent manner. The γδ T
cells strikingly switch from initial high TCR-dependent IFNγ and TNFa production,
and no or very low bacterial uptake, to TCR-dependent extensive bacterial uptake
after specific expansion. The extensive and specific bacterial uptake following
expansion was confirmed by using bacteria labelled with pH-sensitive dyes indicating
rapid acidification of the phago/lysosomal compartments. These functional changes
coincide with an evolution of effector phenotype from cytotoxic T cell to the
expression of myeloid-like molecules, such as MHCII and co-stimulatory molecules.
We conclude that following appropriate target stimulation, γδ T cells are indeed able
to drastically change from an ‘innate-like’ cytokine-producing cytotoxic phenotype to a
pAPC controlling uptake of microbes by its TCR. This γδ T cell behavior is likely to
constitute an important part of γδ T cell functions, which could be utilized in clinical
applications.
Page 94 of 200
GDC 2016
B-23
Identification of a locus on chromosome 1 regulating IL‐17‐producing
γδ T cells and mortality after influenza infection.
Oliver Dienz, Victoria L. DeVault, Camarie C. Spear, Laure K. Case, Cory Teuscher,
and Jonathan E. Boyson
γδ T cells can be divided into distinct functional groups based on their cytokine
expression profile. The genes regulating those different subsets remain incompletely
defined. We had previously identified a locus on chromosome 1 encompassing the
Slam gene family that regulates NKT and γδ T cells. To characterize this region
further, we generated a B6.129 congenic mouse strain in which a 1.1 Mbp region
(chr. 1: 171.05‐172.12) from the 129X1/SvJ strain was introgressed onto the
C57BL/6J background, and evaluated its effect on the host immune response in
influenza‐infected mice. Upon infection, we observed a significant and sustained
increase in the number of IL‐17A‐producing lung γδ T cells in B6.129c2 congenic
mice versus their B6 counterparts. The regulation of gd IL‐17 production by this locus
+
+
+
was subset‐specific, as it was observed in Vγ4 and Vγ6 , but not Vγ1 , γδ T cells.
+
+
IL‐17‐producing Vγ4 and Vγ6 subsets accounted for most of the increase in
B6.129c2 lung γδ T cell numbers, and characterization of these cells revealed
significant modifications in the cell surface expression of some Slam family receptors.
Since increased IL‐17 levels have been associated with immunopathology after
influenza infection, we assessed the effect of the congenic interval on survival. We
found that the survival of B6.129c2 congenic mice is significantly lower compared
with B6 mice after influenza infection. Taken together, our data suggest that we have
identified a genetic locus that regulates both IL‐17A producing γδ T cell subsets and
mortality after a primary influenza infection.
Page 95 of 200
GDC 2016
B-24
Novel vaccine approach to target protective Vγ2Vδ2 T cells and
pulmonary mucosal immunization.
Ling Shen, Frencher J, Huang D, Chen CY, Shen Y, Yan L and Zheng W. Chen
Dept of Microbiology and Immunology, Center for Primate Biomedical Research, University of
Illinois College of Medicine Chicago, Chicago, IL, USA
Major Vγ2Vδ2 T-cell subset recognizing phosphoantigen HMBPP can mount multifunctional/protective responses in infections. However, whether and how vaccine
design should target these antigen-specific γδ T cells need to be addressed.
Our previous studies in nonhuman primates(NHP) reproducibly demonstrated that
HMBPP-activated Vγ2Vδ2 T cells can readily traffic to and accumulate in the
pulmonary compartment in infections. In this study, we leveraged pulmonary
vaccination with vaccine/microbial vectors to elicit γδ T-cell immune responses. We
comparatively investigated HMBPP-producing BCG, B. anthracis and Listeria vectors.
Recombinant Listeria ΔactAprfA* expressing protein immunogens elicited remarkable
T-cell responses after vaccination via respiratory route. Interestingly, single
respiratory vaccination with non-replicating Listeria ΔactAprfA* also elicited
appreciable expansion and effector functions of Vγ2Vδ2 T cells, which were almost
comparable to those seen after systemic intramuscular vaccination with replicating
Listeria ΔactAprfA*. Consistently, single respiration vaccination with non-replicating
B. anthracis could significantly prime the ability of Vγ2Vδ2 T cells to mount recall-like
expansion in response to a subsequent BCG boosting. 4-fold greater recall-like
expansion of Vγ2Vδ2 T cells was detected in such prime-boost setting compared to
BCG only control. A mechanistic experiment showed that large numbers of Vγ2Vδ2 T
cells were detected in lymphoid tissues in respiratory lamina propria mucosa at the
peak expansion of Vγ2Vδ2 T cells after systemic HMBPP/IL-2 treatment.
Furthermore, respiratory vaccination with r-Listeria ΔactAprfA* conferred detectable
protection against TB infection. These findings support the view that rational vaccine
design may target potentially protective Vγ2Vδ2 T cells and pulmonary mucosal route
of immunization.
Page 96 of 200
GDC 2016
B-25
The protective role of γδ T cells in neonatal influenza
Xi-zhi J. Guo
a,b
a
, Pradyot Dash , Paul G. Thomas
a,b
a
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA, 38105;
Integrated Biomedical Sciences Program, University of Tennessee Health Science Center,
Memphis, TN, USA, 38163
b
Influenza virus infection is a continuing worldwide health threat, especially in infants
and the elderly. Neonates are highly susceptible to influenza infection, which is often
a cause of hospitalization. γδ T cells are the first T cells to develop during
embryogenesis and are a significant component of the neonatal immune system. In
the neonatal period, conventional αβT cell responses are functionally less prominent.
Hence, γδ T cells are poised to play a critical role in neonatal infections, however, the
specific role of γδ T cells in neonatal influenza infection remains to be elucidated.
Using a neonatal mouse model of intranasal influenza infection, we observed the
activation and proliferation of γδ T cells within two days after infection. By utilizing
Nur77-GFP reporter mice, we demonstrated that the activation of these γδ T cells
was via the T cell receptor and its downstream signaling. IL-17-producing γδ T cells,
rather than IFN-γ- producing cells, dominated in this response. A comparison
between wild-type and TCRδ- deficient neonates identified a protective function of γδ
T cells in neonates, as reduced weight loss and an increased survival rate were
observed in the wild-type compared to TCRδ-deficient animals. In infected lungs,
wild-type neonates also had more robust IFN-β production and increased
inflammatory cytokine production, especially among the Th2-relevant cytokines than
the TCRδ-deficient animals. Consistent with this observation, more eosinophils
infiltrated into the lungs of wild-type neonates. Our findings demonstrate a vital role
for γδ T cells in mediating protection during neonatal influenza virus infection via a
mechanism which appears to include promotion of a Type 2 immune environment
that may provide a new therapeutic target for pediatric influenza treatment.
Page 97 of 200
GDC 2016
B-26
NLRP3 inflammasome-dependent IL-1β secretion by neutrophils
regulates γδT17 cell response during respiratory bacterial infection
1,2
3
1
1
4
Maya Hassane , Dieter Demon , Daphnée Soulard , Josette Fontaine , Lance E. Keller ,
1
1
2
5
6
Emmanuel Patin , Rémi Porte , Fouad Dabboussi , Immo Prinz , Bernhard Ryffel , Aras
7
4
1
1
Kadioglu , Jan-Willem Veening , Jean-Claude Sirard , Christelle Faveeuw , Mohamed
3
1
1
Lamkanfi , François Trottein and Christophe Paget *
1
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection
and Immunity of Lille, F-59000 Lille, France.
2
Health and Environment Microbiology laboratory, Doctoral School of Sciences and Technology, Faculty of
Public Health, Lebanese University, Tripoli, Lebanon.
3
Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium.
4
Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for
Synthetic Biology, University of Groningen, Groningen, The Netherlands.
5
Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany.
6
CNRS, UMR7355, Orleans, France; Experimental and Molecular Immunology and Neurogenetics, University
of Orléans, Orléans, France.
7
Department of Clinical Infection, Institute of Infection and Global Health, University of Liverpool, Liverpool,
UK.
*Correspondence to Christophe Paget ([email protected]; Tel: +33 3 2087 7339).
Interleukin-17A (IL-17A) is a pro-inflammatory cytokine mainly produced by activated
T cells, which plays an important role in the development of a wide range of immune
responses including infection, allergy and auto-immunity. Using a model of invasive
pulmonary pneumococcal disease, we highlighted a protective role for IL-17A in
controlling neutrophil recruitment, bacterial loads and mice survival. As an early
source of this cytokine, we identified γδT cells, especially the pulmonary resident
CD3bright Vγ6Vδ1+ subset, as major producers during the early course of S.
pneumoniae infection. The rapid production of IL-17A by γδT cells depended on IL23 and IL-1β. Using gene-targeted mice, we demonstrated that lack of γδT cells
leads to decrease in neutrophil numbers and in the containment of the bacteria.
Moreover, the assessment of pro-IL-1β expression in several myeloid cells showed
that neutrophils were the major expressing cells of this pro-cytokine. Interestingly,
these latter were also able to produce bioactive IL-1β, an unexpected accessory role
for neutrophils which stimulate the activation of IL-17A-producing γδT (γδT17) cells.
IL-1β production by neutrophils was dependent on NLRP3 inflammasome activity
through a priming signal ensured by alveolar macrophage-derived TNF-α and an
activation signal mediated by bacterial pneumolysin. These results highlight
interesting cellular interactions leading to γδT17 cell activation and demonstrate the
existence of a biologically functional NLRP3 inflammasome in pulmonary neutrophils
that regulates a key immune axis in the development of protective innate immune
response to respiratory bacterial infection.
Page 98 of 200
GDC 2016
B-27
The influenza vaccine adjuvant AS03 promotes adaptive immunity via
early lymphoid activation and type I interferon
1-3
1-3
1-3
3,4
Sean O'Farrell , Olga Sobolev , Anna Lorenc , Andrew Cope , Mark
1,3
1-3
Peakman , Adrian Hayday
1
Peter Gorer Department of Immunobiology, King’s College London, UK
Immunosurveillance Laboratory, Francis Crick Institute, Lincolns‘s Inn Laboratories, London,
UK.
3
NIHR Biomedical Research Centre at Guy’s and St Thomas’ Hospital at King’s College
London, London, UK.
4
Academic Department of Rheumatology, Centre for Molecular and Cell Biology of
Inflammation, King’s College London, London, UK.
2
The innate mechanisms that drive effective adaptive immune responses to
adjuvanted vaccines are not fully understood. In particular, the contribution of
lymphocytes to the innate response has largely been overlooked, as these cells are
often viewed in the context of adaptive immunity. In a recent study, we observed a
rapid and transient myeloid and lymphoid cell response within 24 hours to an AS03adjuvanted swine flu (H1N1) vaccine (Pandemrix ® GlaxoSmithKline) in healthy
volunteers. To delineate the underlying mechanisms, we immunised adult mice with
AS03 plus H1N1 and tracked their responses.
AS03 elicited robust humoral and T cell immunity in wild-type mice. The innate
response was characterised by marked upregulation of IFNγ-inducible protein 10 (IP10) mRNA in skeletal muscle tissue as early as 4 hours post-vaccination. The
sustained local expression and serological protein production of IP-10 at 24 hours
were IFNα/β dependent, whereas acute IP-10 upregulation was IFNα/β independent.
Deletion of IP-10 resulted in an enhanced T cell response, but compromised humoral
immunity. Although AS03 did not induce an early IFNγ response in mice as it did in
humans, IL-13 mRNA was sharply increased locally at 4 hours. This was also a
feature of the recall response and was IFNα/β independent.
Our multidisciplinary, cross-species analysis of AS03 shows that its mechanism of
action includes lymphoid compartments and type I IFN. The rapid induction of
lymphoid effectors in humans and mice suggests an overt contribution of these cells
to the innate response to this adjuvant, leading to improved adaptive immunity.
Page 99 of 200
GDC 2016
B-28
Surveillance of γδ T Cells Predicts Cytomegalovirus Infection
Resolution in Kidney Transplants
Hannah Kaminski,* Isabelle Garrigue,†‡ Lionel Couzi,*§ Benjamin Taton,*
Thomas Bachelet,*§ Jean-François Moreau,§| Julie Déchanet-Merville,§ Rodolphe
Thiébaut,¶** and Pierre Merville*§
*Department of Nephrology, Transplantation, and Dialysis and †Virology and |Immunology laboratories,
Bordeaux University Hospital, Bordeaux, France;
‡National Center of Scientific Research(CNRS), Research Unit 5234, Bordeaux, France;
§National Center of Scientific Research, Mix Unit of Research 5164, Bordeaux, France;
¶French Institute of Health and Medical Research (INSERM), Institute of Public Health and Epidemiology and
Development (ISPED), Center U897–Epidemiology-Biostatistics, Bordeaux, France;
**National Institute for Research in Computer Science and Control (INRIA), Statistics In Systems biology and
Translational Medicine (SISTM) Team, Bordeaux, France
Cytomegalovirus (CMV) infection in solid-organ transplantation is associated with
increased morbidity and mortality, particularly if a CMVmutant strain with antiviral
resistance emerges. Monitoring CMV specific T cell response could provide relevant
information for patient care.We and others have shown the involvement of Vd2 neg
gd Tcells in controlling CMV infection. Here, we assessed if Vd2 neg γδ T cell
kinetics in peripheral blood predict CMV infection resolution and emergence of a
mutant strain in high–risk recipients of kidney transplants, including 168 seronegative
recipients receiving organs from seropositive donors (D+R-) and 104 seropositive
recipients receiving antithymocyte globulins (R+/ATG). Vd2 neg gd T cell
percentages were serially determined in patients grafted between 2003 and 2011.
The growing phase of Vd2neg γδ T cells was monitored in each infected patient, and
the expansion rate during this phase was estimated individually by a linear mixed
model. A Vd2neg γδ T cell expansion rate of ˃0.06% per day predicted the growing
phase. The time after infection at which an expansion rate of 0.06% per day occurred
was correlated with the resolution of CMV DNAemia (r=0.91; P,0.001). At 49 days of
antiviral treatment, Vd2 neg gd T cell expansion onset was associated with recovery,
whereas absence of expansion was associated with recurrent disease and DNAemia.
The appearance of antiviral–resistant mutant CMV strains was associated with a
delayed Vd2neg γδ T cell expansion (P,0.001). In conclusion, longitudinal
surveillance of Vd2 neg γδ T cells in recipients of kidney transplants may predict
CMV infection resolution and antiviral drug resistance.
Page 100 of 200
GDC 2016
B-29
Easier Control of Late-Onset Cytomegalovirus Disease Following
Universal Prophylaxis Through an Early Antiviral Immune Response in
Donor-Positive, Recipient-Negative Kidney Transplants
1
1,2
3,4
2,5
2
H. Kaminski , L. Couzi , I. Garrigue , J.-F. Moreau , J. Dechanet-Merville and P.
1, 2
Merville
1
Department of Nephrology, Transplantation and Dialysis, Bordeaux, France
National Center of Scientific Research (CNRS), Research Unit 5234, Bordeaux, France
3
Virology Laboratory, Bordeaux, France
4
National Center of Scientific Research, Mix Unit of Research 5164, Bordeaux, France
5
Immunology Laboratory, Pellegrin Hospital, Bordeaux, France
2
Universal prophylaxis for CMV prevention is viable but, compared to preemptive
strategy, leads to higher incidence of late-onset CMV disease (LOD) associated with
poor patient and graft survival. The purpose of this study was to compare LOD with
early-onset disease (EOD) focusing in the highest risk D+R- kidney transplant
recipients (KTR). CMV control depending on both antiviral treatment and specific
immune response, we also compared Vδ2 neg γδ T cells expansion involved in CMV
infection resolution. EOD was defined as occurring before 3 months and LOD after 3
months posttransplantation. Depending on the period, universal prophylaxis or
preemptive treatment was used. 168 D+R- KTR were included between 2003 and
2011. LOD was associated with a lower peak DNAemia (p=0.04), less recurrences
(OR=0.16; 95% CI=0.05-0.55; p=0.01), a shorter anti-CMV curative treatment (40
versus 60 days, p<0.0001). As a corollary, we found that Vδ2neg γδ T cells
expansion was faster in LOD than in EOD (31 versus 168 days after the beginning of
CMV disease, p<0.0001). In D+R- KTR, universal prophylaxis is associated with
more LOD which had a better infection management and a faster immune response.
These results support the use of universal prophylaxis over a preemptive strategy
and reappraise outcomes of LOD.
Page 101 of 200
GDC 2016
B-30
Concerted Control of the Cell Surface Proteome by a Human
Cytomegalovirus Gene Family Members Subverts Immune Recognition
1*
2, 6*
2
3
Ceri A. Fielding , Michael P. Weekes
, Luis V. Nobre , Eva Ruckova , Gavin
4
6
1,
2
2
Wilkie , Joao A. Paulo , James Davies Chiwen Chang , P. Robin Antrobus ,
1
1
1
3
Richard J. Stanton , Rebecca Aicheler , Hester Nichols , Borek Vojtesek , John
2
4
5
1**
Trowsdale , Andrew J. Davison , Steven P. Gygi , Peter Tomasec
Paul J.
2**
**1
Lehner , & Gavin W. G. Wilkinson
1
Institute of Infection and Immunity, School of Medicine, Cardiff University, Heath Park, Cardiff
CF14 4XN, United Kingdom
2
Cambridge Institute for Medical Research (CIMR), Wellcome Trust/MRC Building,
Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0XY, United Kingdom
3
Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno,
Czech Republic
4
MRC-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR,
United Kingdom
5
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA
02115, USA
Our interest in the US12 gene family stems from our previous observation that US18
and US20 co-operate to suppress cell surface expression of the NKG2D activating
receptor ligand MICA (Fielding et al., 2014). The 10 members of the family are
arranged sequentially within the Us region of the genome, and designated US12
through to US21. We sought to systematically characterize the entire US12 family. To
this end, a panel of HCMV deletion mutants was constructed for all members.
Functional assays identified 5 members of the US12 gene family as NK cell evasion
functions: US12, US14, US18, US20 and US21. Quantitative multiplexed proteomics
was deployed to compare expression of 1,300 cell surface and 7,200 whole-cell
proteins during infection for all US12 family mutants. The US12 family as a major new
hub of immune regulation, in which viral proteins act either individually or in concert to
regulate multiple cell adhesion molecules, cytokine receptors and NK ligands. The
NKp30 ligand B7-H6 was a novel target of the US12 family members, US18 and
US20, and a major regulator of the NK response to HCMV. These findings have
relevance for other immune subsets, including γδ T-cells.
Page 102 of 200
GDC 2016
B-31
Characterization of the γδ-T-cell-mediated immune control in mCMV
infections
1
1
2
1
2
Anne Maria Hahn , Sabrina Sell , Monika Dietz , Andrea Schneider , Michael Mach ,
1
Thomas Winkler
1
Chair of Genetics, Department for Biology, Friedrich-Alexander University of ErlangenNürnberg
2
Institute for Clinical and Molecular Virology, University Hospital Erlangen
The cytomegalovirus (CMV) is a widespread virus that can cause severe organ
disease or even death in immune-compromised patients. This includes transplant
recipients undergoing immunosuppressive treatment and new-borns, where the
immune system isn’t fully matured yet. The control of CMV infections relies on
multiple and redundant immune effector functions from both the innate and the
adaptive systems. Our previous work has shown the remarkable protective capacity
of γδ T cells against murine CMV (mCMV) infection, whereby full protection was
established even in the absence of other effector cells like αβ T cells, B cells or NK
cells. In adoptive transfer settings γδ T cells provide long-term protection in infected
-/RAG mice from the otherwise lethal course of disease. Data that will be presented
suggests an adaptive-like behaviour of γδ T cells during mCMV infection. Only γδ T
cells from mCMV-infected donors could provide protection, whereas equal numbers
of γδ T cells from uninfected animals were unable to provide significant protection.
Upon infection, γδ T lymphocytes undergo a significant expansion and a prominent
and long-lasting phenotypic change. We observed that the TCR repertoire and CDR3
length distributions focus upon infection, particularly in organs where the virus
persists. This implicates an antigen-driven response due to γδ T cell receptor (TCR)
-/involvement. Furthermore, secondary infections in TCRα are cleared faster and
more efficiently, underlining the adaptive feature of γδ T cell during mCMV infection.
The recognition mechanisms, the antigen specificity and the specific mode of antiviral capacity still remains enigmatic, however.
Page 103 of 200
GDC 2016
B-32
IL-18 Enhances Vγ9Vδ2 T Cell Responses
Murday AS, Pauza CD.
The Vg9Vd2 subset of human T cells is depleted during HIV disease and not
reconstituted after prolonged antiretroviral therapy (ART). Their loss is part of the
immunodeficiency syndrome likely linked to increased opportunistic infections or
cancer, and normal Vg9Vd2 function was observed in HIV natural virus suppressors
with little to no blood viremia in the absence of ART. Our goals are to understand the
mechanisms preventing full reconstitution of Vg9Vd2 T cells and discover conditions
in HIV+ patients that are limiting the functionality of existing cells. We know that
prolonged ART increases the T cell receptor complexity of Vg9 chains in blood,
showing that de novo cell synthesis is occurring in treated HIV patients.
Unfortunately, these cells remain incapable of responding to prototypical
phosphoantigens even while re-gaining responsiveness to aminobisphosphonate
drugs including zoledronic acid (Zol). Zol treatment increases stimulatory IPP and
promotes secretion of Caspase-1 processed cytokines IL-18 and IL-1β through its
effects on the NLRP3 inflammasome. The Vd2 T cell subset was particularly high for
IL-18 receptor expression compared to traditional IL-18 targets CD8+ T and natural
killer cells. To test the importance of IL-18, we showed that Vδ2 T cell activation was
blocked by IL-18 Binding Protein (IL-18bp) an inhibitor of IL-18, or the YVAD peptide
inhibitor of Caspase-1. IL-18 stimulation increased proliferation, enhanced the
accumulation of effector memory cells, and increased expression of TNFa and IFNg.
Our results focus on the direct effects of IL-18 on Vg9Vd2 T cells. We are currently
testing for links between IL-18 deficiency and the failed IPP response of Vg9Vd2 T
cells from HIV+ patients treated with ART.
Page 104 of 200
GDC 2016
B-33
Clostridium difficile induces expansion and cytokine production by
human Vγ9Vδ2 T cells
1
2
1
2
Andreea Petrasca , Micheál Mac Aogáin , Derek G. Doherty , Thomas R. Rogers .
1
2
Discipline of Immunology, School of Medicine, Trinity College Dublin, Ireland
Department of Microbiology, School of Medicine, Trinity College Dublin, Ireland.
Clostridium difficile is the main culprit for nosocomial antibiotic-associated diarrhoea
among hospital patients and in severe cases can result in pseudomembranous colitis
and can even be fatal. C.difficile mediates its pathogenesis via toxin production and
recently, hypervirulent strains have emerged, which exhibit increased toxin
production and increased resistance to treatment, resulting in increased fatality.
Isoprenoid biosynthesis is a pathway essential for cell survival, which occurs via
one of two pathways: the mevalonate pathway, which is used by eukaryotes and
some bacteria, and the MEP pathway, which is employed by most human pathogens.
An intermediate of the MEP pathway, hydroxyl-3-methyl-but-2-enyl pyrophosphate
(HMB-PP), is the most potent antigen for activating Vγ9Vδ2 T cells, and thus
expansion of Vγ9Vδ2 T cells is seen upon infection with a broad range of microbial
pathogens. Expression of MEP pathway genes by C.difficile suggests that C.difficile
may also activate Vγ9Vδ2 T cells. We compared the ability of C.difficile and HMB-PP
in inducing Vγ9Vδ2 T cell proliferation and cytokine production. Supernatants and
lysates of strain RT060 and hypervirulent strains RT001 and RT027 filtered with
0.22μm and 3kDa filters were used to stimulate peripheral blood Vγ9Vδ2 T cells.
C.difficile was found to induce 2-20 fold expansion and similar levels of IFN-γ and
TNF-α as HMB-PPstimulated Vγ9Vδ2 T cells. Furthermore, C.difficile, like HMB-PP,
did not stimulate IL-4, IL-10, IL-13 or IL-17 production by Vγ9Vδ2 T cells.
These results demonstrate a role for C.difficile in inducing an immune response
through stimulation of Vγ9Vδ2 T cells. Vγ9Vδ2 T cell stimulation was mediated by a
secreted factor of less than 3kDa, although the identity of this agent remains to be
elucidated. Therapeutic activation of Vγ9Vδ2 T cells may hold promise for treatment
of patients with C.difficile infection.
Page 105 of 200
GDC 2016
Session 6
γδ T cell function in health and medicine: 2.
autoimmune disease / inflammation/
transplantation
Talks:
Saturday, June 18th
Poster presentation:
Friday, June 17th, 4:15 – 6:00pm
Saturday, June 18th, 1:15 – 2:30pm
Page 106 of 200
GDC 2016
B-34
Hybrid αβ-γδ T cells, novel T cell populations with a critical pathogenic
role in CNS autoimmunity
1+
1+
2
2
Caroline E. Sutton , Sarah C. Edwards , James E. McLaren , Kristin Ladell , Julie
3
4
1
4
3
C. Ribot , Song Baik , Barry Moran , Graham Anderson , Bruno Silva-Santos , David
2
1*
A. Price and Kingston H.G. Mills
1
Immune Regulation Research Group, School of Biochemistry and Immunology, Trinity
Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland;
2
Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff,
3
UK; Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal;
4
MRC Centre for Immune Regulation, Institute for Biomedical Research, Medical School,
University of Birmingham, Edgbaston, Birmingham, UK.
+
These authors contributed equally to the project.
γδ T cells and CD4 T cells are the main sources of IL-17 during inflammation and
host immunity. Vγ4+ γδ T cells are found at high frequency in the central nervous
system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), an
animal model of multiple sclerosis. Here, we describe a novel population of T cells,
hybrid αβ-γδ T cells distinct from conventional αβ or γδ T cells, which play a critical
pathogenic role in CNS autoimmunity. Hybrid αβ-γδ T cells which express Vγ4+ cells
arise in the foetal thymus on day 16 of ontogeny, and constitute around 10% of Vγ4+
cells in the peripheral lymphoid organs. Hybrid αβ-γδ T cells are absent in TCRα-/and TCRβ-/- mice, however hybrid αβ-γ T cells were identified in TCRδ-/- mice. Coexpression of α, β, γ and δ chains was confirmed at the molecular level, revealing a
restricted Vγ repertoire with more heterogeneous Vα and Vβ usage. Hybrid αβ-Vγ4δ
T cells express the master transcription factor, RORγt, and IL-1RI, IL-23R, CD44,
CCR6 and α4β1 integrin. Furthermore, these cells secrete IL-17A, IFN-γ and GMCSF, following stimulation with IL-1β and IL-23 and respond to autoantigens when
purified from mice with EAE. Depletion of Vγ4+ cells from either WT or TCRδ-/- mice
dramatically attenuated EAE, and this effect was associated with a significant
reduction in CNS-infiltrating Th17 cells. These data identify novel populations of T
cells, hybrid αβ-γδ T cells which play a critical role in autoimmunity via the activation
of Th17 cells.
Page 107 of 200
GDC 2016
B-35
Retinoic acid suppresses IL-17 production by γδ T cells and attenuates
their
pathogenic
activity
in
experimental
autoimmune
encephalomyelitis
†
Mathilde Raverdeau*, Conor J. Breen , Alicja Misiak* and Kingston H.G. Mills*
*Immune Regulation Research Group, School of Biochemistry and Immunology, Trinity
Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland;
†
Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland.
Retinoic acid (RA) in the steady state enhances induction of Foxp3+ regulatory T
(Treg) cells and inhibits differentiation of Th1 and Th17 cells, thereby maintaining
tolerance, but can in inflammatory conditions promote effector Th1 and Th17 cells
that mediate inflammation. IL-17 producing γδ T cells have recently been shown to
play a major pathogenic role in autoimmune diseases. Here we examined the
immunomodulatory effects of RA on γδ T cells. We found that RA had a dramatic
suppressive effect on IL-17A and IL-17F production by γδ T cells stimulated with IL1β and IL-23. RA suppressed RORγt, IL-1R and IL-23R expression in γδ T cells.
Treatment of mice with RA suppressed IL-17 production by γδ T cells in vivo.
Furthermore, treatment of T cells with RA attenuated their ability to induce disease in
experimental autoimmune encephalomyelitis (EAE), a murine model for multiple
sclerosis. This was associated with a reduction in the number of CNS-infiltrating γδ T
cells, but also CD4+ T cells that produced IL-17A, IL-17F or GM-CSF. Interestingly,
treatment of γδ T cells with RA or removal of γδ T cells from a bulk population of T
cells significantly reduced their capacity to induce EAE, demonstrating a critical role
for γδ T cells in promoting pathogenic Th17 cells. Our findings demonstrate that the
anti-inflammatory properties of RA are mediated in part by suppressing STAT3mediated activation of cytokine production and cytokine receptor expression in γδ T
cells, which suppresses their ability to activate Th17 cells.
Page 108 of 200
GDC 2016
B-36
CD11d expression on blood-derived primary human gamma delta T
cells can be augmented in vitro upon administration of recombinant
human IL-2
1,3
3
Gabrielle M. Siegers , Lynne-Marie Postovit , Gregory A. Dekaban
1,2
1
Molecular Medicine, Robarts Research Institute, The University of Western Ontario, London,
Ontario N6A 5B7, Canada
2
Department of Microbiology and Immunology, The University of Western Ontario, London,
Ontario N6A 5B7, Canada
3
Department of Experimental Oncology, University of Alberta, Edmonton, Alberta, Canada
The CD11d integrin is an adhesion molecule expressed as a heterodimer with CD18,
classically found on neutrophils and monocytes/macrophages. Here, we report the
expression of CD11d on human peripheral blood γδ T cells (γδTc). CD11d
expression is consistently higher on freshly isolated γδTc compared to their
conventional αβ T cell (αβTc) counterparts (female n=7, male n=3 healthy donors),
yet varies within the same donor (n=5). We expanded primary human γδTc, initially
together with αβTc and then, after αβTc depletion, as a purified population. We also
cultured the depleted αβTc under the same conditions in parallel. Over time, we
consistently observed higher CD11d levels on γδTc compared to αβTc from the
same donor. Furthermore, CD11d expression on γδTc increased over time, and
correlated with levels of interleukin (IL-) 2 supplementation. Interestingly, Vδ1
express higher CD11d levels than Vδ2 γδTc, suggesting differential roles for this
integrin that may segregate with γδTc subsets. We are now exploring the functional
relevance of CD11d expression on γδTc. In cytotoxicity assays, blocking CD11d had
no effect; however, CD11d blockade prior to transendothelial migration assays
increased migration, suggesting a role for CD11d in γδTc retention. Finally, we
employed a multicolour flow cytometry panel to further identify CD11d expression on
B cells, CD4+ and CD8+ αβTc as well as NK cells in thawed peripheral blood
mononuclear cells (female n=5, male n=4 healthy donors). We found high CD11d
expression on NK and B cells, a phenomenon that deserves further study.
Page 109 of 200
GDC 2016
B-37
Cardiolipin dys-regulation of γδ T cells in systemic sclerosis (SSc)
through a CD1d dependent mechanism.
*#
Helena Migalovich Sheikhet and Ilan Bank
* #@
*Laboratory of Immune-regulation
#Department of Medicine
@Rheumatology Unit, Sheba Medical Center, Tel Hashomer
@Sackler School of Medicine, Tel Aviv University, Israel.
Vg9+ gd T cells (TC) decrease in SSc, a disease characterized by extensive tissue
fibrosis, whereas Vd1+ TC are activated and infiltrate afflicted organs. Antibodies to
cardiolipin, a auto-antigenic lipid which can bind to CD1d for presentation to Vd1+
TC receptors, are often detected in SSc. To further evaluate the role of cardiolipin in
SSc, expression of CD25 on cultured peripheral blood mononuclear cells of 12 SSc
patients and 8 healthy controls (HC) was evaluated after 4 days of culture in RPMI1640 with 10% fetal bovine serum containing 1000 IU/ml of IL-2 (FM), or FM with
zoledronate (zol, 2mM, FMzol) both with or without cardiolipin (2.5-10 mg/ml). In FM,
cardiolipin
decreased %CD25+Vd1+ TC in HC, whereas in FMzol,
cardiolipin
significantly increased HC %CD25+Vd1+ TC in a dose-dependent manner. Likewise,
in SSc, cardiolipin abrogated the significant decrease of %CD25+Vd1+ T cells that
was induced in FMzol. In FM + cardiolipin, %CD25+Vg9+ TC of SSc patients
decreased significantly, which was partially reversed by zol, whereas in FMzol HC,
cardiolipin significant increased %CD25+Vg9+ TC. As a result, Vd1/ Vg9+ TC ratio
increased in SSc but decreased in HC in FMzol with vs without cardiolipin.
Importantly, SSc and HC Vd1+TC were highly reactive with CD1d tetramers and
blocking anti-CD1d mAb abrogated cardiolipin-dependent enhancement of
expression of SSc %CD25+Vd1+ TC in FMzol and reversed the elevated Vd1/ Vg9+
TC ratio. CD1d dependent responses to cardiolipin may play a role in activation of
pro-fibrotic Vd1+ and suppression of potentially anti fibrotic Vg9+ TC in SSc.
Page 110 of 200
GDC 2016
B-38
γδ T cell homeostasis and function in childhood onset versus adult
onset obesity.
Christa Park, Kitty Cheung, Natalie Limon, Christine Yee, Anne E. Costanzo, Julie M.
Jameson
According to the Centers for Disease Control and Prevention, one in three adults and
one in six children and adolescents in the United States are obese. An excess of
chronic excess energy intake and low energy expenditure creates excess adipose
tissue that induces systemic inflammation in the body. Obesity is detrimental to
barrier tissues that protect the body from foreign pathogens and environmental
stressors. This metabolic disease disrupts normal homeostasis within the epithelium,
which then results in complications that include more severe inflammatory bowel
disease. We have identified that as obesity progresses, T cell numbers are reduced
within the epithelial layer of the intestine and proliferation of these immune cells in the
tissue becomes dysfunctional. Interestingly enough, we show that these same T cells
are partially restored in number with a four week low-fat diet. We examined the
relationship between obesity onset and the ability of intraepithelial lymphocytes to
seed and proliferate in the intestine to maintain homeostasis in the barrier tissue.
Here we demonstrate that obesity is particularly devastating in young mice that are
still establishing gd T lymphocyte populations in their epithelia. In addition, the longterm effects of chronic obesity from childhood to adulthood shows a dramatic
reduction in the number and function of gd T lymphocytes in the intestine. This study
helps shed light on how epithelial barrier maintenance differs in early versus late
onset of obesity and will direct research to explore alternative approaches to restore
intestinal intraepithelial T cells and promote optimal barrier integrity in obese patients.
Page 111 of 200
GDC 2016
B-39
Peripheral induction of IL-17-producing Vγ4+ γδ T cells in experimental
autoimmune encephalomyelitis
1
1
1
1
Pedro H. Papotto , Natacha Gonçalves-Sousa , Nina Schmolka , Sofia Mensurado ,
1
1
Julie C. Ribot , Bruno Silva-Santos
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa,
Portugal
The production of interleukin-17 (IL-17) is a major mechanism by which γδ T cells
contribute to (auto)immune responses. IL-17-producing γδ T cells (γδ17) have been
shown to be programmed in the thymus, and their generation is thought to be
constrained to a tight window during embryonic development. However, these innatelike “pre-programmed” features contradict the adaptive characteristics recently
attributed to peripheral γδ17 cells. Therefore, in the present study we hypothesized
that environmental triggers can promote de novo generation of γδ17 cells in the adult
periphery. In order to test this hypothesis, bone marrow chimeras (BMC; which lack
γδ17 cells) were challenged with pro-type-17 (infectious or autoimmune) stimuli and
in vivo generation of γδ17 cells was assessed. Both intraperitoneal
Lipopolysaccharide injection and Listeria monocytogenes infection, to which the
responders were mostly Vγ6+ γδ cells, failed to induce γδ17 cell differentiation.
Strikingly, using an experimental autoimmune encephalomyelitis (EAE) model, we
observed de novo generation of Vγ4+ γδ17 cells, which was coupled to cell
proliferation. γδ17 cells were found mainly in the brain and spleen on days 14 and 18
post-immunization (at the peak of inflammation). However, on day 7 postimmunization γδ17 cells were only found in the inguinal lymph nodes (LN; adjacent to
the immunization site), indicating that their generation does not occur in the target
tissue but rather in the draining LN. Our findings demonstrate that, beyond thymic
pre-programming, γδ17 (particularly adult Vγ4+) cells can be generated de novo in
peripheral lymphoid organs and migrate to tissues where they promote local
inflammation.
Page 112 of 200
GDC 2016
B-40
Temporarily depleted versus constitutively deficient - A new mouse
model to conditionally deplete γδ T cells
1
1
1
I. Sandrock , A. Reinhardt , L. Oberdörfer , I. Prinz
1
1
Hannover Medical School, Institute of Immunology, Hannover, Germany
Knowledge about specific functions of gd T cells is still limited as they share functions
with other immune cells such as NK cells, ILCs and αβ T cells. Investigations
addressing the distinct contributions of gd _T cells are currently limited due to the
lack of suitable experimental models. In the constitutive gd T cell-deficient mouse
(Tcrd–/–), gd _T cell niches are filled by other cells, indicative of a functional
compensation. Additionally, depletion of gd _T cells with specific antibodies is not
possible as the use of anti-gd _TCR antibodies leads to blocking and internalization
of the TCR but not to depletion of gd _T cells.
Here, we present a new knock-in mouse line, in which gd _T cells can be
conditionally depleted with diphtheria toxin. To this end, we inserted a cassette
encoding for eGFP, human diphtheria toxin receptor and luciferase into the 3´UTR of
the murine Tcrd constant gene (Tcrd-IRES-eGFP-2A-DTR-2A-luciferase, short TcrdGDL). Evaluation by flow cytometry, 2-photon microscopy and IVIS imaging revealed
efficient conditional depletion of gd _T cells and specific functional expression of
eGFP and luciferase.
Currently, we apply Tcrd-GDL mice to models relevant for human inflammatory
disease. In the Aldara-induced psoriasis model, gd _T cell-depleted Tcrd-GDL mice
developed a significantly milder disease phenotype compared to Tcrd–/– mice. This
finding supports our hypothesis that other cells may compensate for the lack of gd _T
cells in Tcrd–/– mice. Furthermore, Tcrd-GDL mice allow monitoring of recovery
kinetics of gd _T cell subpopulations after depletion.
Page 113 of 200
GDC 2016
B-41
Human gut-tropic and intestinal Vδ2+ T-cells mediate antigen
presentation and stimulate blood and colonic CD4+ T-cells to upregulate IL-22 via an ICOS-L/TNF-dependent mechanism
†
†
Neil E. McCarthy,* Christopher J. Tyler, Bernhard Moser, James O. Lindsay,*,
†
Andrew J. Stagg,* & Matthias Eberl.
‡
*Centre for Immunobiology, The Blizard Institute, Bart’s and The London School of Medicine
and Dentistry, Queen Mary University of London, UK;
†
Division of Infection & Immunity, School of Medicine, and Systems Immunity Research Institute,
Cardiff University, UK;
‡
Dept. Gastroenterology, Barts and The London Hospitals, Barts Health NHS Trust, London,
UK.
Vγ9/Vδ2+ T-cells populate the human intestine and modulate colonic αβT-cell
responses via an unknown mechanism that might constitute a novel therapeutic
target in inflammatory bowel disease (IBD). We investigated this mechanism using
flow-cytometry to assess Vγ9/Vδ2+ T-cell function in blood and colon from healthy
controls and IBD patients. Upon exposure to isoprenoid precursor antigen HMB-PP,
blood-derived Vγ9/Vδ2+ T-cells upregulated the gut-homing integrin β7 and
increased expression of HLA-DR and co-stimulatory molecules, which confered the
ability to stimulate allogeneic CD4+ T-cells to proliferate and produce cytokines
including IFN-γ and IL-22. When generated in the presence of growth factor IL-15,
antigen-presenting Vγ9/Vδ2 T-cells (γδT-APCs) promoted IL-22 expression in naïve
and memory CD4+T-cells more effectively than did monocytes or monocyte-derived
dendritic cells. These gut-tropic γδT-APCs polarized CD4+T-cells towards IL-22
expression without co-inducing IL-17A via a mechanism that was independent of IL-6
but involved TNF-α and ICOS-L. Consistent with these data, γδT-APCs isolated from
human gut tissue were potent stimulators of IL-22 responses in blood-derived naïve
CD4+T-cells. Similarly, gut-tropic γδT-APCs generated from peripheral blood
enhanced IL-22 expression by colonic memory CD4+T-cells, indicating potential to
influence ongoing immune responses in the gut. Moreover, patients with active IBD
displayed reduced potency of blood γδT-APC, perhaps due to sequestration of these
cells into the inflamed intestine, suggesting a possible role in disease pathology.
These data demonstrate a novel antigen-presenting function of human gut-tropic
Vγ9/Vδ2+ T-cells, which can polarize intestinal T-cell responses towards IL-22
expression and may promote mucosal protection in health, but potentially enhance
inflammation in IBD.
Page 114 of 200
GDC 2016
B-42
Distinct Subsets of γδ T Cells Play Opposing Roles in Regulating
Inflammation and Insulin Resistance in Adipose Tissue
1,2
3
4
1
Ayano C. Kohlgruber , Hui-Fern Koay , Derek Sant’Angelo , Michael B. Brenner ,
1,5
Lydia Lynch
1Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital;
2Division of Medical Sciences, Harvard Medical School, Boston, USA;
3University of Melbourne, Melbourne, Australia;
4Graduate School of Biomedical Sciences, Rutgers Robert Wood Johnson Medical School, New
Brunswick, NJ, USA;
5Division of Endocrinology, Department of Medicine, Brigham and Women’s Hospital, Harvard
Medical School, Boston, USA.
Adipose tissue harbors a substantial and unique immune system that is important for
coordinating homeostasis by maintaining an anti-inflammatory tenor. Obesity triggers
major changes in immune composition and is characterized by an influx of
neutrophils and accumulation of inflammatory macrophages, paralleled by a loss of
protective leukocytes including Tregs, iNKT cells, and eosinophils. However, the role
of γδ T cells in adipose tissue homeostasis and inflammation remain largely
unexplored. We found γδ T cells are significantly enriched, accounting for 10-15% of
the T cells in the adipose tissue. Moreover experiments using parabiotic mice
showed their unique residence and minimal recirculation, suggesting they play an
adipose specific role. Indeed, TCRδ KO mice have decreased numbers of
neutrophils and macrophages in the adipose tissue during obesity. Surprisingly, we
also found Treg numbers decreased in both the lean and obese states. Further
analysis revealed two γδ subsets based on promyelocytic leukaemia zinc finger
(PLZF) protein and CD3 expression. In order to better understand the role of the γδ
subsets, we performed RNAseq and single cell TCR-sequencing and defined these
two phenotypically distinct subsets as functionally discrete populations based on
gene expression, cytokine profiles, and TCR repertoires. Although efforts to fully
elucidate the unique enrichment, phenotype, and function of γδ T cells in the adipose
tissue are on going, our results point to two γδ T cell populations with opposing
inflammatory and anti-inflammatory functions that correspondingly help to maintain
healthy adipose homeostasis in the lean state, but can exacerbate adipose tissue
inflammation during obesity.
Page 115 of 200
GDC 2016
B-43
Dermal γδ T cells exhibit memory-like response after inflammation
1
1,2
1
1
Tom Hartwig , Stanislav Pantelyushin , Andrew L. Croxford , Paulina Kulig and
1
Burkhard Becher
1
Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva,
Switzerland
2
The concept of immunological memory is widely applied for adaptive immune cells
such as αβ T cells and B cells after encountering a foreign antigen. However, the
paradigm of non-adaptive immune branches to deliver a rapid and accelerated
response after recall or reinfection is just emerging. We identified a skin-resident
Vγ4+Vδ4+ γδ T cell subpopulation accumulating in the dermis after topical Aldara
(Imiquimod) treatment. Fate-mapping and reporter systems show that these Aldaraexperienced Vγ4+Vδ4+ T cells populate and persist in non-challenged skin for a long
period of time after initial stimulation and reveal enhanced IL-17 production after rechallenge resulting in an exacerbated skin inflammation. In addition to identifying a
unique feature of γδ T cells during inflammation, our results have direct relevance to
the human disease as this quasi-innate memory provides a mechanistic insight into
relapses and chronification of psoriasis.
This work is supported by: NIH Training Grant T32 AR07530
Page 116 of 200
GDC 2016
B-44
DETCs are pro-inflammatory mediators of contact hypersensitivity.
1,2,4
2
1,3
Morten M. Nielsen
, Paola Lovato , Beatrice Dyring-Andersen , Jonas D.
1
4
4
3
Schmidt , Amanda S. MacLeod , Deborah Witherden , Lone Skov , Sally
5
6
1
1
Dabelsteen , Steen S. Poulsen , Anders Woetmann , Niels Ødum , Wendy L.
4
1
1
Havran , Carsten Geisler and Charlotte M. Bonefeld .
1
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of
Copenhagen, Copenhagen, Denmark;
2
Department of Skin Inflammation Pharmacology, LEO Pharma A/S, Ballerup, Denmark;
3
Department of Dermato-Allergology, Gentofte Hospital, University of Copenhagen, Copenhagen, Denmark;
4
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA;
5
Department of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200
Copenhagen, Denmark
6
Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen,
Copenhagen, Denmark
The interaction between keratinocytes and skin-resident immune cells has an
important role in induction of contact hypersensitivity (CHS). Because the specific
subset of γδ T cells termed dendritic epidermal T cells (DETCs) are located in mouse
epidermis these are ideally poised to perform pro-inflammatory functions during the
initiation phase of CHS. Using a well-established model for CHS in which 2,4-dinitrofluorobenzene (DNFB) is used as allergen, we found that DETCs are important
players in CHS. Thus, IL-17A–producing DETCs appear in the skin following
exposure to DNFB in wild-type mice, and DNFB-induced ear swelling is reduced by
∼50% in both TCRδ-/- and IL-17A-/- mice. By in vitro assay we further find that
DETCs are activated and produce IL-17A in an IL-1β-dependent manner during CHS.
DETCs also express a variety of receptors important for the DETC response against
tumors and during wound healing, one of which is NKG2D. In mice, the ligands for
NKG2D are stress-induced proteins such as mouse UL16-binding protein-like
transcript 1 (Mult-1), histocompatibility 60 (H60), and retinoic acid early inducible-1
(Rae-1). Here we show that Mult-1 expression is up-regulated in epidermis 24 hours
after initial allergen exposure, and that DETC activation upon allergen exposure to
the skin can be partially inhibited by blocking NKG2D signaling. These findings
demonstrate that DETCs are pro-inflammatory mediators of CHS and that both IL-1β
and NKG2D signaling is involved in allergen-induced activation of DETCs and
indicate that these pathways might be potential targets for treatment of CHS.
Page 117 of 200
GDC 2016
B-45
γδT cells contribute to preterm brain injury via interleukin-17independent way
1
1
1
1
1
Anna-Maj Albertsson , Xiaoli Zhang , Dan Bi , Arshed Nazmi , Carina Mallard ,
1, 2, 3
4
5, 6
5, 6
Henrik Hagberg
Susanna Cardell , Jianmei W. Leavenworth , Harvey Cantor
1
and Xiaoyang Wang
1
Perinatal Center, Institute of Neuroscience and Physiology, Sahlgrenska Academy at University
of Gothenburg, Sweden
2
Perinatal Center, Department of Obstetrics and Gynecology, Sahlgrenska Academy at
University of Gothenburg, Sweden
3
Centre for the Developing Brain, Department of Perinatal Imaging and Health, King’s College
London, United Kingdom
4
Department of Microbiology and Immunology, Sahlgrenska Academy at University of
Gothenburg, Sweden
5
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute,
6
Department of Microbiology and Immunobiology, Division of Immunology, Harvard Medical
School, Boston, Massachusetts, USA.
Perinatal brain injury in preterm infants is a distinctive form of cerebral white-matter
injury accompanied by elements of gray-matter injury. Very early T-cell development
is disproportionately directed toward γδ T-cells in both mice and human. γδ T-cells
have been identified in demyelinating lesions in the CNS in human patients with
multiple sclerosis as well as in animal models of white-matter injury.
Using two mouse models: 1). hypoxia-ischemia (HI) induced -; 2) Inflammation (LPS)
induced- preterm brain injury, we found that γδ T cells infiltrating to the mouse brain
after HI; and the infiltrated γδ T-cells were mostly seen in the white matter of the
corpus callosum, the meninges of the ipsilateral hemisphere, choroid plexus in the
third ventricle and lateral ventricle areas around the blood vessels. Further, γδ TCR
deficient (γδ KO) mice show significantly decreased brain injury than the C57Bl/6
wild type (WT) mice after either HI induced- or LPS induced- preterm brain injury. At
7 days after HI there is a significant reduction in HI-induced brain tissue loss (%) in
the γδ T KO (9.3 ± 2.4 mm3, mean ± SEM, n = 20) compared to WT mice (18.1 ± 2.7
mm3, n = 24, p = 0.02). Moreover, blocking of IL-17/IL-22 signaling pathways using
either antibody depletion (for IL-17), or gene deficiency (for IL-22) did not show any
effect on brain injury in comparing to their WT control mice in the HI induced preterm
brain injury.
These results suggest that γδ T-cells contribute to the development of brain injury via
interleukin-17-independent way.
Page 118 of 200
GDC 2016
B-46
When human γδ T cells decide not to follow the rules: from
differentiation to senescence
1,2
1
3
Weili Xu , Tan Tze Ying Crystal , Tze Pin Ng , Anis Larbi
1,2
1 Singapore Immunology Network, 8a Biomedical Grove, 138648, Agency for Science,
Technology and Research (A*STAR)
2 Nanyang Technological University, School of Biological Sciences, 50 Nanyang Ave, 639798
3 National University of Singapore, Yong Loo Lin School of Medicine, Department of
Psychological Medicine National University Hospital 1E Kent Ridge Road, NUHS Tower Block,
Level 9 Singapore 119228
Aging has been associated with a decline of immune capacity. This decline might
suggest why there is a higher incidence of mortality due to infections or cancer in the
elderly. The classical αβ T cells (i.e. CD4 and CD8) have been extensively studied in
the context of human aging and/or persistent infections (eg. cytomegalovirus, CMV),
which are known to accelerate T cell differentiation ultimately leading to senescence.
However, the γδ T cells compartment are less studied in this context, despite their
essential role in immunosurveillance and a highly cytotoxic profile. It also remains a
question whether the markers that phenotypically and functionally define the αβ is
applicable to γδ. Our characterization shows that with CMV infection and aging, the
γδ T cells subsets are being impacted differently based on the classical markers
used for αβ T cells (including CD28, CD27, CD45RA, CD57). This shows the
importance of analyzing the γδ T cells subsets separately for future studies. Our data
also shows that the classical model of differentiation used in αβ T cells is not
applicable for certain γδ T cells subsets, whereas some markers remain relevant.
Finally, the Vδ2+ population is significantly less susceptible to senescence (as
defined in αβ T cells). Altogether our study suggests the differential homeostatic
signals between αβ and γδ T cells subsets. It also highlights the importance of
identify the markers specific to γδ T cell and their subsets to better study the biology
of γδ T cell in health and diseases.
Page 119 of 200
GDC 2016
B-47
Gammadelta T cell deficiency in the peripheral blood of patients with
Crohn’s disease: relationship with clinical and endoscopical activity.
1
2,5,6
3
Juan Carlos Andreu-Ballester , Ignacio Catalan-Serra
, García-Ballesteros C.
3
2
4
Victoria Amigo-Garcıa , Rafael Gil-Borras , Ferran Ballester , Amadeo Almela3
8
7
Quilis , , Monica Millan-Scheiding , Carlos Penarroja-Otero .
1
Head of Research Department. Arnau de Vilanova Hospital, Valencia (Spain).
Digestive Department. IBD Unit. Arnau de Vilanova Hospital, Valencia (Spain).
3
Hematology Department. Arnau de Vilanova Hospital, Valencia (Spain).
4
Center of Research in Public Health (CSISP), Valencia, (Spain)
5
Internal Medicine, Gastroenterology Department, Levanger Hospital, Levanger (Norway).
6
Centre for mollecular inflammation research (CEMIR), Norwegian Science and Technology
University (NTNU), Trondheim (Norway).
7
Catholic University of Valencia, Valencia, (Spain)
8
Department of Surgery, Bellvitge University Hospital, Barcelona, (Spain)
2
Email: [email protected]
Crohn’s disease (CD) is a chronic relapsing systemic disease affecting the
gastrointestinal tract. An altered immune reponse to commensal intestinal bacteria
takes place in genetically predisposed individuals, and it can be considered an
immune deficiency condition. γδ T lymphocytes (γδTL) are considered key cells in
the first line of defense against infections and can stimulate wound healing.
Aim: We aimed to determine the numbers of γδTL in the peripheral blood (PB) of a
large cohort of CD patients and matched controls and study its relation with the
clinical and endoscopical activity.
Methods: A prospective study of 102 patients with CD compared with 102 healthy
subjects (control group) matched by age and sex was undertaken. Lennard-Jones
criteria were used for the diagnosis of CD. Disease activity was measured with the
Crohn’s disease activity index (CDAI) and endoscopical activity by the SES-CD
index. New patients, patients in remission, and patients with active disease were
evaluated. Lymphocytic populations of CD3+, CD4+, CD8+, CD56+, and αβ and γδ
subsets were measured in the PB of all participants.
Results The number of total CD3+, CD4+, CD8+ and CD56+ lymphocytes was
decreased in CD patients compared with the control group (p<0.001, 0.003, <0.001,
and 0.001, respectively). Although both αβ and γδ T lymphocytes were lower in
patients, γδTL subsets showed the lowest levels in CD patients (mean 0.0332 x
109/l) vs controls (mean 0.0753 x109/l), p<0.001. This decrease was significant for
both CD4+γδ, CD8+γδ and CD56+γδ subsets (p<0.001), and for all the clinical
scenarios studied (new patients, remission or active disease) p<0.001. In addition,
Page 120 of 200
GDC 2016
we found and inverse correlation between the numbers of CD3+γδ and CD8+γδ
(and not the αβTL) with the clinical (-0.304 and -0.249; p=0.014 and p=0.017
respectively) and endoscopical activity (-0.275 and -0.249; p=0.017 p=0.017
respectively).
Conclusions: This study confirms a decrease in the global lymphocyte population in
the peripheral blood of patients with CD. This decrease is more evident in γδT
lymphocytes. Our results show for the first time an inverse correlation between γδTL
numbers in PB and disease activity, highlighting the importance of these cells in the
pathogenesis of the disease with potential therapeutical implications.
Page 121 of 200
GDC 2016
B-48
Zoledronic acid boosts γδ T-cell activity in children receiving αβ+ T
and CD19+ cell-depleted grafts from a haplo-identical donor
1†
2†
3†
2
3
A. Bertaina , A. Zorzoli , A. Petretto , G. Barbarito , E. Inglese , P. Merli1, C.
3
1
1
4
5
1,6‡
Lavarello , L. P. Brescia , B. De Angelis , G. Tripodi , L. Moretta , F. Locatelli
and
2*‡
I. Airoldi
1 Department of Pediatric Hematology and Oncology, IRCCS Ospedale Bambino Gesù, Rome,
Italy
2 Laboratorio di Oncologia, Istituto Giannina Gaslini, Genova, Italy
3 Core Facilities, Istituto Giannina Gaslini, Genova, Italy
4 Dipartimento Ricerca Traslazionale, Medicina di Laboratorio, Diagnostica e Servizi, Istituto
Giannina Gaslini, Genova, Italy
5 Area di Ricerca Immunologica, IRCCS Ospedale Bambino Gesù, Rome, Italy
6 Department of Pediatric Science, Università di Pavia, Italy.
A new method of graft manipulation based on physical removal of αβ+ T cells and
CD19+ B cells, leaving mature NK cells and γδ T cells in the graft, has been recently
developed for HLA-haploidentical HSCT. We demonstrated that γδ T cells collected
from transplanted patients are endowed with capacity of killing leukemia cells after ex
vivo treatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of
ZOL in patients receiving this type of graft, may boost γδ T cell cytotoxic activity
against leukemia cells.
Thirty-three patients were treated with ZOL every 28 days at least twice. γδ T cells
before and after ZOL treatments were studied till at least 7 months after HSCT by
high-resolution mass spectrometry, flow-cytometry, and degranulation assay.
Proteomic analysis of γδ T cells purified from patients showed that, starting from the
first infusion, ZOL caused up-regulation of proteins involved in activation processes
and immune response, paralleled by down-regulation of proteins involved in
proliferation. These findings are consistent with an induction of Vδ2 cell
differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against
primary leukemia blasts. Furthermore, a proteomic signature was identified for each
individual ZOL treatment. Patients given 3 or more ZOL infusions had a better
probability of survival in comparison to those given 1 or 2 treatments.
In conclusion, ZOL influences Vδ2 cell activity, determines a specific proteomic
signature and enhances anti-leukemia activity, this potentially resulting into an
increased anti-tumor effect.
Page 122 of 200
GDC 2016
B-49
Increased IFN-γ production by peripheral Vδ1 T cells in multiple
sclerosis correlates with neuronal damage and disease activity
1
2
2
2
Avadhesh Kumar Singh , Lenka Nováková , Markus Axelsson , Clas Malmeström ,
3
2, 4
1
Henrik Zetterberg , Jan Lycke and Susanna Cardell
1
Department of Microbiology and Immunology, Institute of Biomedicine, The Sahlgrenska
Academy, University of Gothenburg, Gothenburg, Sweden
2
The MS Center, Sahlgrenska University Hospital, Gothenburg, Sweden
3
Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology,
Sahlgrenska Academy, University of Gothenburg
4
Department of Neurology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg,
Sweden
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central
nervous system (CNS), clinically characterized by alternating periods of relapse and
remission. Whereas the cause of MS is multifactorial, a central role has been
attributed to autoimmune responses against components of the myelin sheath
coating nerve cells of the CNS. The myelin sheath is a rich source of sulfatides and
galactosylceramides. Recent studies have identified sulfatide-reactive TCR γδ T cells
among human peripheral blood lymphocytes (PBL). These γδ T cells were CD1d- or
CD1c-restricted, and preferentially used the TCR Vδ1-segment, a TCR previously
identified in MS lesions. We have investigated Vδ1 T cells in PBL and cerebrospinal
fluid (CSF) of newly-diagnosed MS (new-MS) patients using 14-color flow cytometry.
Vδ1 cells showed signs of recent activation in MS CSF and its frequency among total
γδ T cells was significantly increased in patients compared with healthy donors (HD).
Strikingly, new-MS patients had an increased frequency of IFN-γ-producing Vδ1 cells
in PBL compared with HD, while there was no change in other T cell subsets
analyzed. This contrasts to the anti-inflammatory profile normally associated with Vδ1
cells. The frequency of IFN-γ-producing Vδ1 cells positively correlated with markers
for neuronal damage and disease activity. MS patients treated with natalizumab
(blocking integrin a-4 and thereby leukocyte homing to the CNS) had normalized level
of IFN-g-producing Vδ1 cells. Taken together, this suggests that Vδ1 cells are
recently activated in new-MS patients and may contribute to the early MS
pathogenesis by producing proinflammatory cytokines.
Page 123 of 200
GDC 2016
B-50
Divergent roles of IL-12 and IL-23 in psoriasiform skin inflammation
1
2
3
1
Paulina Kulig , Stepahnie Musiol , Sandra Nicole Freiberger , Bettina Schreiner ,
4
5
1
6
Gabor Gyülveszi , Giancarlo Russo , Stanislav Pantelyushin , Kenji Kishihara ,
2
3
4
3
Francesca Alessandri , Thomas Kündig , Federica Sallusto , Günther FL Hofbauer ,
2
1
Stefan Haak and Burkhard Becher
1
Institute of Experimental Immunology, University of Zurich, 8057 Zurich, Switzerland
Experimental Immunology Unit, Centre of Allergy and Environment (ZAUM), Technical
University of Munich and Helmholtz Centre Munich, 80802 Munich, Germany
3
Department of Dermatology, University Hospital Zurich, 8091 Zurich, Switzerland
4
Institute for Research in Biomedicine, Cellular Immunology, 6500 Bellinzona, Switzerland
5
Functional Genomics Center Zurich, University of Zurich and ETH Zurich, 8057 Zurich,
Switzerland
6
Department of Immunology, Faculty of Pharmaceutical Sciences, Nagasaki International
University, 859-3298 Nagasaki, Japan
2
Neutralization of the common p40-subunit of IL-12/23 in psoriasis patients has led to
a breakthrough in the management of moderate to severe disease. Besides
neutralizing IL-23, which is thought to be responsible for the curative effect of antip40 therapy, it also interferes with IL-12 signalling and type-1 immunity. The goal of
our study was to dissect the individual contributions of these two cytokines to the
formation of psoriatic lesions and to understand the impact of therapeutic co-targeting
of IL-12 and IL-23 in psoriasis. Using a preclinical model for psoriatic plaque
formation we found that IL-12 in contrast to IL-23 exerted a regulatory function by
restraining the invasion of a distinct IL-17 committed γδT (γδT17) cell subset. We
discovered that IL-12 receptor signalling in keratinocytes initiates a protective
transcriptional program limiting skin inflammation, suggesting that collateral targeting
of IL-12 by anti-p40 monoclonal antibodies is counterproductive in the therapy of
psoriasis.
Page 124 of 200
GDC 2016
Session 7
γδ T cell function in health and medicine: 3.
cancer
Talks:
Sunday, June 19th
Poster presentation:
Saturday, June 18th, 4:30 – 6:30pm
Sunday, June 19th, 1:30 – 2:30pm
Page 125 of 200
GDC 2016
C-01
Interleukin-21 promotes regulatory functions of Vγ9Vδ2 T cells
Clément Barjon, Aurélie Gennetier, Nathalie Bonnefoy and Virginie Lafont
IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université
Montpellier , Montpellier, France
γδ T cells can contribute to the immune response against many tumor types directly
through their cytotoxic activity and indirectly by stimulating or regulating the biological
functions of other cell types required for the initiation and establishment of the antitumor immune response. However, the notion that tumor-infiltrating γδ T cells are a
favorable prognostic marker in cancer was recently challenged by studies showing
that the presence of γδ T cells in the tumor microenvironment was associated with
poor prognosis in both breast and colon cancer. Furthermore, recent studies
demonstrated that cytokines can confer some plasticity to γδ T cells and promote
their differentiation into cells with regulatory functions.
Here we show that activation of Vγ9Vδ2 T cells isolated from healthy donors in the
presence of IL-21 leads to a decreased expression of NKG2D by Vγ9Vδ2 T cells
whereas it increases their expression of NKG2A and their production of IL-8 and IL10. We also show that Vγ9Vδ2 T cells grown in the presence of IL-21 specifically
express CD39 and CD73 ectonucleotidases and generate adenosine, a potent
immunosuppressive molecule. Finally, we show that maturation of dendritic cells as
well as their capacity to produce IL-12 and to induce antigen specific T cell
proliferation are impaired when they are co-cultured with IL-21-amplified Vγ9Vδ2 T
cells.
Altogether these data indicates that IL-21 could favor regulatory functions of γδ T
cells and suggest that the presence of IL-21 in the tumor microenvironment might
negatively impact on their anti-tumor activity.
Page 126 of 200
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C-02
Genetic makeup of mammary tumors drives the balance between IL17- and IFN-producing γδ T cells
Seth B. Coffelt
CRUK Beatson Institute and Institute of Cancer Sciences, University of Glasgow
Metastasis remains the primary cause of death for breast cancer patients. This is due
in large part to the lack of knowledge about the mechanisms underlying cancer
F/F
F/F
spread. Recently, we showed that mammary tumors in K14-cre;Cdh1 ;p53 mice
elicit a systemic inflammatory cascade to dampen anti-tumor T cells and maximize
metastasis formation. This cascade is initiated by IL-1β that activates IL-17-producing
γδ T cells, leading to G-CSF-dependent neutrophil expansion and repolarization. In
+
turn, these neutrophils suppress CD8 T cells, allowing disseminated cancer cells to
+
go unnoticed. The IL-17-producing γδ T cells consist of Vγ4 and Vγ6, while CD27
IFNγ-producing γδ T cells mainly express Vγ1. However, it is unknown how different
breast cancer subtypes regulate these γδ T cell subsets. Macrophages are the main
producers of IL-1β in mammary tumors, so we phenotyped these cells in two different
models of triple-negative breast cancer. Interestingly, in a model of BRCA1-deficient
F/F
F/F
breast cancer (K14-cre;Brca1 ;p53
mice), we found that tumors contained
high
—
increased proportions of MHC-II TIE2 macrophages and lower expression of ILF/F
1β when compared with BRCA1-proficient tumors (K14-cre;p53 mice). Reduced IL1β expression in BRCA1-deficient mammary tumors correlated with decreased IL-17producing γδ T cells, lower circulating neutrophils and increased IFNγ-producing γδ T
cells. These data suggest that tumors regulate γδ T cell subsets through tumorassociated macrophages. Current efforts are underway to determine how the
mutational composition of cancer cells educates macrophages. Together, these data
provide further insight into how breast cancer metastasis occurs and uncover
potential new targets for γδ T cell-based immunotherapy.
Page 127 of 200
GDC 2016
C-03
Molecular and immunohistochemical analyses of γδ T cells infiltrating
human breast carcinomas
1
1
2
Jose Villacorta Hidalgo , Antonella Terrizzi , Matias Ruggieri, Kerstin Heise , Sylvia
1
3
2
1
Kock , Miroslav Malkovsky , Ralf Küppers , Paul Fisch
1
Department of Clinical Pathology, University of Freiburg Medical Center, Freiburg, Germany
Institute of Cell Biology (Tumor Research), University of Duisburg-Essen, Medical School,
Essen, Germany.
3
Department of Medical Microbiology and Immunology, UW School of Medicine and Public
Health, Madison, Wisconsin
2
Triple-negative breast carcinomas (TNBCs) lack expression of HER2, estrogen and
progesterone receptors and often contain lymphocytic infiltrates. The presence of
tumor-infiltrating γδ T cells (TIγδLs) was particularly prominent in medullary breast
carcinomas. In 11 out of the 26 TNBC patients, we isolated 20-50 TIγδLs from each
tumor using laser microdissection and examined them using a newly developed
single cell PCR method for the amplification of VγJγ and VδDδJδ rearrangements.
The CDR3 of the γ- and δ-chains were sequenced and aligned with the sequences in
the IMGT database. The TIγδLs displayed different Vγ and Vδ gene rearrangements
with marked junctional diversity. The V genes with in-frame rearrangements were (in
the order of their frequencies) Vγ8, Vγ2, Vγ3, Vγ9, Vγ5, Vγ4, and Vδ1, Vδ5, Vδ3, Vδ2,
Vδ4. The Vγ and Vδ pairing in the single cells was Vγ8Vδ1, Vγ9Vδ3, Vγ9Vδ1, Vγ3Vδ1,
Vγ8Vδ5, Vγ4Vδ1. Remarkably absent was the Vγ9Vδ2 pairing, which is the most
common combination in human peripheral blood γδ T cells. Most interestingly, TIγδLs
in several tumors had the same Vγ4, Vγ8 and Vδ5, Vδ1 rearrangements and
junctional sequences as found previously in many human CMV-reactive γδ T cells.
Our data provide a first molecular analysis of the γδ T-cell repertoire in TNBCs,
defining γ and δ TCR-chain combinations from single TIγδLs. Moreover, the results
strongly suggest that some TIγδLs in TNBCs could recognize CMV-induced antigens
and provide an explanation as to why some human breast cancers frequently show
infiltration by γδ T cells.
Page 128 of 200
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C-04
Blockade of PD-1/PD-L1 immune checkpoint enhances Vg9Vd2T cellsbased immunotherapy in prostate cancer
1,2
3
2
Christine Pasero , Julie Gertner-Dardenne , Gwenaëlle Gravis ,
1
2
2
1,2,4
Granjeaud , Mathilde Guerin , Jeanne Thomassin-Piana , Daniel Olive
Samuel
1
Centre de recherche en Cancérologie de Marseille, Inserm U1068 / CNRS U7258, Marseille,
F-13009 France ;
2
Institut Paoli-Calmettes, Marseille, F-13009 France ;
3
TxCell, Allée de la Nertière, Les Cardoulines, Valbonne Sophia Antipolis, F-06560 France ;
4
Aix Marseille Université, Marseille, F-13284 France
PD-L1 and PD-1 pathway blockade has elicited durable antitumor responses in
patients. Increased evidences that high frequency of Vγ9Vδ2 T cells infiltrate solid
tumors suggest their value in the design of new immunotherapeutic strategies.
Bisphosphonates, that treat prostate cancer patients with bone metastases, have
been shown to stimulate gd T cells in vitro and in vivo.
Here, we showed that high frequency of gdT cells infiltrate prostate tumors, with
activated but impaired phenotype (decreased NKG2D, DNAM, 2B4 activating
receptors and increased ILT2 inhibitory receptor). The inhibitory molecule PD-1 was
strongly expressed at the surface of infiltrating gd T cells, while prostate tumor cells
overexpressed its ligand PD-L1. The proliferation of gd T cells after exposition to PDL1+ prostate cancer cells was significantly reduced, thus PD-1/PD-L1 pathway
appears as a possible mechanism of immune escape by prostate cancer by
interfering with gd T cell antitumor activity.
Interestingly, we demonstrated the presence of an intracellular pool of PD-1 in resting
Vγ9Vδ2 T cells that is mobilized at the cell membrane after bisphosphonate
activation. Hence, prostate cancer cells subvert this homeostatic system through
upregulation of PD-L1, to inhibit the functions of PD-1+ gd T cells that infiltrate the
tumor microenvironment. Our data showed that PD-1/PD-L1 blockade by monoclonal
antibodies resulted in the enhancement of Vg9Vd2 T-cell proliferation. Potent
combination of bisphosphonates with anti-PD-1/PD-L1 antibodies to stimulate gd T
cells could be of great interest in the design of new immunotherapeutic strategies for
treating prostate cancer.
Page 129 of 200
GDC 2016
C-05
Human papillomavirus induces a reorganization
associated γδ T cells and promote tumour formation.
1
1
1
of
1
epidermal
Dorien Van Ηede , Virgine Renoux , Estelle Dortu , Inge Langers , David Vermijlen
1
and Nathalie Jacobs
2
1 Cellular and molecular immunology Lab, Giga-Cancer/Inflammation-infection and Immunity
(I3) University of Liège (ULG), Liège, Belgium.
2 Faculty of Pharmacy and Institute for Medical Immunology, Université Libre de Bruxelles
(ULB), Belgium.
γδ T cells have been shown to protect against cancer development in several
models. Yet, the role of γδ T cells in papillomavirus associated uterine cervical
cancer, the third cause of death by cancer in women worldwide, is unknown. Here,
we investigated the impact of γδ T cells using mice deficient for γδ T cells in a
transgenic model of carcinogenesis induced by HPV16 oncogenic proteins. In
contrast with their generally observed antitumoural role, γδ T cells promoted the
development of HPV oncogene-induced lesions. In fact, γδ T cells displayed a
significantly more rounded morphology and the number of epidermal resident Vγ5+
γδ T cells decreased dramatically in epidermis expressing HPV16 oncogenic
proteins. In addition, Vγ5+ γδ T were partially replaced by cells expressing
intermediate level of γδ TCR, some of which expressed Vγ4, but not Vγ1 or Vγ7. As
a potential mechanism explaining the protumoural role of γδ T cells in HPV
oncogene-driven tumour development, we observed a significantly higher density of
blood vessels in the dermis below HPV lesions of control mice compared to HPV
lesions of γδ T cells-deficient mice. We are currently investigating the hypothesis that
γδ T cells promote neoangiogenesis through the production of IL-17 in epidermis
expressing HPV16 oncogenic proteins. Supporting the clinical relevance of these
experimental observations, we observed that IL-17+ γδ T cells infiltrate HPV-induced
tumours in human patients, while only IL-17- γδ T cells are detected in normal
cervical biopsies. Our results suggest that γδ T cells promote cancer progression in
the context of HPV-induced lesions, which is linked with a reorganisation of the
epidermal-associated γδ T cells.
Page 130 of 200
GDC 2016
C-06
Lymphoid stress-surveillance by γδ T cells promotes protective IgE
responses in the skin
Greg Crawford, Rocio Castro Seoane and Jessica Strid
Division of Immunology and Inflammation, Department of Medicine, Imperial College London,
London, W12 0NN
Lymphoid stress-surveillance (LSS) refers to the capacity of resident intraepithelial
lymphocytes (IEL) to directly sense epithelial dysregulation and initiate immune
responses. γδTCR+ skin IEL have important host-protective functions in early
immune surveillance of cutaneous tumours and in maintaining skin homeostasis. We
have previously shown that the LSS response in the skin leads to potent induction of
IL-13 and IgE antibodies. Here we investigate the afferent pathways leading to IgE
following epithelial dysregulation and the role of IgE during DMBA-induced
carcinogenesis. We show that acute, topical skin exposure to DMBA carcinogen is a
potent inducer of IgE and IgG1 antibodies, promoting significant germinal centers
(GC) and plasma cell (PC) responses in draining LNs. Repeated DMBA exposure
during carcinogenesis leads to sustained IgE, whereas IgG1 and GC decline. The GC
and PC responses are dependent on the cytokines IL-13 and IL-4. αβ T cells also
play a non-redundant role, as TCRβ-/- animals cannot mount GC or PC responses.
However, the DMBA-induced IgE response is uniquely dependent on canonical IEL
as TCRδ-/- mice, as well as chimeric mice lacking only skin γδ T cells, have intact
IgG1 but significantly reduced IgE. This LSS-induced IgE accumulates inside skin
tumours and has physiological relevance in protecting against DMBA induced
carcinogenesis. IgE-/-, as well as TCRδ-/-, mice are more susceptible to DMBAcomplete carcinogenesis with earlier development, increased frequency and size of
tumours compared to controls. This work directly links IgE antibodies to tumour
control and demonstrates a novel facet of γδ T cell regulated immune surveillance.
Page 131 of 200
GDC 2016
C-07
Characterisation of human γδ T cell subsets in inflamed tissues of
oesophageal cancer
1*
1*
2
1
Dunne MR , Conroy MJ , Foley E, Clarke N, Reynolds JV , Lysaght J , O’Sullivan
1
JN
1
Department of Surgery, Trinity Translational Medicine Institute, St James’s Hospital, Dublin 8,
Ireland
2
Department of Clinical Medicine, James’s Hospital, Dublin 8, Ireland
*Joint first authors
γδ T cells are known to have tumouricidal properties and recent studies have linked
their presence in tumours with a strong favourable survival outcome. However, such
findings have yet to be successfully translated into improved clinical outcomes.
Immunotherapeutic strategies aiming to boost the frequency of activated γδ T cells at
tumour sites must consider potential exacerbation of tumourigenic inflammation.
The aim of this study was to characterise discrete γδ T cell subsets (Vδ1, Vδ2 and
Vδ3) present in oesophageal cancer (OC), an inflammation-associated cancer, at
different tissue sites and stages of cancer development. The relative frequency of γδ
T cell subsets in the blood and oesophageal tissues of patients with OC and the preneoplastic inflammatory disorder Barrett’s oesophagus (BO), were compared to ageand sex-matched healthy donors. γδ T cell subset frequencies were also assessed in
the liver and omentum of the OC patients, known sites for inflammatory cell
trafficking.
Tissues were collagenase digested and γδ subset frequencies were determined by
multi-colour flow cytometry, expressed as a percentage of total T cells.
Compared to healthy donor blood, all γδ T cell subtypes were reduced in OGA
patients but not BO patients (p<0.5). Subset frequencies in tissues were similar
however, comparing inflamed (BO), uninflamed and tumour tissues. In terms of sitespecific frequency, Vδ1 cells predominated in the liver (16.8%), and omentum
(16.6%) while Vδ2 cells predominated in blood (1.02%).
Future work will elucidate the contribution of γδ T cell subsets to pro-/antitumourigenic inflammation and mechanisms of tissue trafficking.
Page 132 of 200
GDC 2016
C-08
In vivo inhibition of tumour-associated IL-17-producing γδ T cells by
neutrophils
Sofia Mensurado, Margarida Rei, Telma Lança, Natacha-Gonçalves-Sousa, Hiroshi
Kubio, Karine Serre* and Bruno Silva-Santos*
(* equal contributions)
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa
γδ T cells are known to produce large amounts of anti-tumour mediators, such as
interferon-γ (IFN-γ) and cytolytic enzymes, but can also secrete interleukin-17 (IL-17)
that paradoxically promotes tumour growth. By contrast, we report here that, in a
peritoneal B16 melanoma tumour model, γδ T cells are suppressed and
unresponsive (and thus neutral) to tumour growth. Unlike in other models, γδ T cells
were unable to accumulate and to upregulate IL-17, IFN-γ, granzyme B or perforin
expression upon peritoneal B16 challenge (in comparison to steady-state).
Consistent with this, TCRδ-/- mice displayed tumour growth kinetics similar to wildtype (WT) controls. Interestingly, we observed a marked accumulation of neutrophils
in the peritoneal cavity, and thus hypothesized that this myeloid subset could inhibit
γδ T cells in this model. To modulate neutrophils, we employed an anti-Gr-1
monoclonal antibody (mAb), which led to their efficient depletion, and resulted in a
specific accumulation of γδ T cells and an increased proportion of IL-17-producing γδ
T cells. These were characterized by Vγ6+ TCR chain usage and lack of CD27
expression. Analysis of WT, TCRδ-/- and IL17-/- mice upon neutrophil depletion
revealed that both γδ T cells and IL-17 actively promoted tumour growth when not
suppressed by neutrophils. We are currently investigating the molecular
mechanism(s) underlying neutrophil-mediated suppression of γδ T cells. Collectively,
the work presented here suggests that pro-tumour IL-17-producing γδ T cells can be
regulated by neutrophils, which may have important implication for cancer
immunotherapy.
Page 133 of 200
GDC 2016
C-09
Novel Tumor-infiltrating CD39+ γδTregs Are the Predominant
Immunosuppressive T Cells in Human Colorectal Cancer that Facilitate
Tumor Progression
Guoming Hu,1,2,7 Pin Wu,1,3,7 Pu Cheng,1,2,7 Zhen Wang,1,2 Xiuyan Yu,2 Xuan
Shao,2 Dang Wu,4 Jun Ye,5 Tao Zhang,1 Xiaochen Wang,2 Fuming Qiu,1,2 Jun
Yan,6,* and Jian Huang1,2,*
1
Cancer Institute (Key Laboratory of Cancer Prevention & Intervention, National Ministry of
Education; Provincial Key Laboratory of Molecular Biology in Medical Sciences),
2
Department of Surgical Oncology,
3
Department of Thoracic Surgery,
4
Department of Radiation Oncology,
5
Department of Gastroenterology, Second Affiliated Hospital, Zhejiang University School of
Medicine, Hangzhou, 310009, China,
6
Department of Medicine and Department of Microbiology and Immunology, James Graham
Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA
7
Co-first authors
*Correspondence: [email protected] (J.H.); [email protected] (J.Y.)
Tumor microenvironment promotes immune suppression through recruiting and
expanding suppressive immune cells such as regulatory T cells (Tregs) to facilitate
cancer progression. In this study, we identify a novel CD39+ γδTreg in human
colorectal cancer (CRC). CD39+ γδTregs are abundant in CRC, express higher
levels of PD-1/CTLA-4, and have more potent immunosuppressive activity than CD4+
or CD8+ Tregs via the adenosine-mediated pathway but independent of TGF-β or IL10. They also secrete cytokines including IL-17A and GM-CSF which chemoattract
myeloid-derived suppressive cells (MDSCs) thus establishing an immunosuppressive
network. We further demonstrate that tumor-derived TGF-β1 induces CD39+ γδT
cells from paired normal colon tissues to produce more adenosine and become
potent immunosuppressive T cells. Moreover, CD39+ γδTreg infiltration is positively
correlated with TNM stage and other unfavorable clinic-pathological features,
implicating CD39+ γδTregs are the key player in establishment of
immunosuppressive tumor microenvironment in human CRC that may be critical for
tumor immunotherapy.
Page 134 of 200
GDC 2016
C-10
An attractive tool for γδ T cell-based immunotherapy
Daniela Wesch*, Daniel Gonnermann*, Christian Kellner§, Matthias Peipp§, Martin
Hermes*, Christian Peters*, Susanne Sebens#, Dieter Kabelitz*, Hans-Heinrich
Oberg*
*Institute of Immunology,
§Division of Stem Cell Transplantation and Immunotherapy,
#Institute for Experimental Medicine, Christian-Albrechts University of Kiel, Germany
γδ T cells (γδTc) are attractive effector cells based on our observation that γδTc
infiltrate in high numbers in the malignant ductal epithelium of pancreatic ductal
adenocarcinoma (PDAC) tissues indicating that γδTc infiltrate into the tumor.
Recently, we have shown the efficacy of a novel developed [(Her2)2xVg9] tribody to
selectively target gdTc to tumor-associated antigens such as human epidermal
growth factor 2 (HER2/neu) and to enhance γδTc-cytotoxicity in vitro and in vivo
upon transfer into immunocompromised mice.
In this study, we analyzed the impact of [(Her2)2xVg9] tribody on γδTc proliferation
and cell death as well as immunosuppressive mediators (e.g. Prostaglandin (PG)-E2,
TGF-b1) in the interaction with PDAC cells with the intention to better estimate a
possible clinical application in γδTc-based tumor immunotherapy. In contrast to
aminobisphosphonates (n-BP) or phosphorylated antigens (PAg) which are applied in
clinical trials to trigger γδTc proliferation, [(Her2)2xVg9] does not induce γδTc
proliferation. However, [(Her2)2xVg9] is better suited for triggering of gdTc-mediated
tumor cell lysis than PAg because in contrast to PAg it does not trigger activationinduced cell death in activated γδTc. In addition, [(Her2)2xVg9] overcomes the
suppression of γδTc-cytotoxicity against PDAC cells by PGE2 and TGF-b often
released in the tumor environment. [(Her2)2xVg9] mediates γδTc-cytotoxicity against
different HER2/neu expressing cancer cells (PDAC, breast-, gastric- and prostate
cancer cells).
Our results indicate that the initial administration of n-BP which induce γδTc
proliferation followed by infusion with [(Her2)2xVg9] tribody which enhance
cytotoxicity and avoid cell death of γδTc could be envisioned in the context of γδTcbased immunotherapeutic strategies.
Page 135 of 200
GDC 2016
C-11
Differential activation of γδ T cell cytotoxicity by bispecific antibodies
Hans-Heinrich Oberg*, Matthias Peipp§, Christian Kellner§, Martin Hermes*,
Christian Peters*, Daniel Gonnermann*, Susanne Sebens#, Dieter Kabelitz* and
Daniela Wesch*
*Institute of Immunology,
§Division of Stem Cell Transplantation and Immunotherapy,
#Institute for Experimental Medicine, Christian-Albrechts University of Kiel, Germany
The expression of human epidermal growth factor 2 (HER2/neu) on cancer cells is
often associated with poor clinical outcome. Clinical therapies with humanized HER2
monoclonal antibodies (mAbs) such as trastuzumab or pertuzumab have clearly
improved the outcome of patients with metastatic gastric or breast cancer. A novel
strategy to enhance cytotoxicity of immune cells is based on the usage of bispecific
antibody (bsAb) or tribody constructs, which selectively target T cells or NK cells to
tumor-associated antigens such as HER2/neu.
In our studies, the bsAb [HER2xCD3] with specificity for CD3 expressed on all T cells
enhanced the cytotoxic potential of αβ T cells (αβTc) against HER2/neu
overexpressing pancreatic ductal adenocarcinoma (PDAC) cells but had different
effects on the cytotoxic capacity of γδ T cells (γδTc). Although the stimulation via
CD3 induced in freshly isolated as well as in short-term activated γδTc enhanced
activation of PI3K/AKT, Ras/Erk and NFkB pathways, several PDAC cells were
susceptible while others were almost resistant to the lysis by freshly isolated γδTc in
the presence of bsAb [HER2xCD3]. Possible reasons for this different susceptibility
may be attributed to the different induction of conformational changes in αβTc vs
γδTc or the bsAb format.
Focusing on the bsAb format, we applied tribody [(HER2)2xVg9] comprising two
HER2-specific single chain fragment variable fused to a Fab directed to the Vγ9
subunit of the γδTc receptor. Our results revealed the superiority of [(HER2)2xVg9]
tribody in triggering γδTc-mediated lysis of HER2-expressing PDAC cells by the
enhanced release of perforin and granzyme B.
Page 136 of 200
GDC 2016
C-12
Analysis of anti-tumour and anti-viral reactivities of human gammadelta T cells
1,2
1
1,2
1,2
Knight Andrea , Piskacek Martin , Kralova Romana , Krchniakova Maria ,
1,2
1,2
1,2
3
3
Gallova Petra , Kubes Martin , Sustova Alena , Rihova Lucie , Vsianska Pavla ,
4
5
5
5
6
Pacasova Rita ,Penka Miroslav ,Adam Zdenek , Pour Ludek ,Hajek Roman ,Vasku
1
Anna
1 Faculty of Medicine, Department of Pathological Physiology, Masaryk University, Brno
2 Gamma-delta T cell Laboratory, Masaryk University, Brno
3 Department of Clinical Haematology, Faculty Hospital Brno
4 Transfusion and Tissue bank, Baculty Hospital Brno
5 Department of Internal Medicine, Haematology and Oncology, Faculty Hospital Brno
6 Clinic of Haematology, Faculty Hospital, Ostrava
Human gd T cells are currently the subject of intensive research and large
expansions of tumour-reactive gd T cells have been observed inpatients with
haematological malignancies including Multiple Myeloma (MM) and chronic
leukaemias (CML,CLL) in our laboratory. However, the role of innate effector gd T
cells subsets in patients progressing from monoclonal gammopathy of undetermined
significance (MGUS) to MM is currently unknown. Previously, we have also shown
expansion of gd T lymphocytes in cytomegalovirus (CMV) seropositive healthy
donors compared to CMV seronegative individuals (p=0.0002) suggesting their direct
involvement in anti-CMV immune response.
Here we performed detailed analyses of expansion, phenotype, clonality and function
of Vd1 and Vd2 gd T cells isolated from patients and age-matched healthy donors
(HD, n=53). We have analysed bone marrow (BM) and paired peripheral blood (PB)
samples from MGUS (n=30) and newly diagnosed myeloma (n=52) patients. Second,
we determined the whole genome profiles of tumour-reactive gd T cells and
compared these to healthy donors. Third, we analysed healthy donors with five most
commonly presented alleles for anti-CMV responses of gd T cells in parallel to CMVspecific ab T cells in the same HLA-A*02, HLA-A*01, HLA-A*24, HLA-A*07, and
HLA-A*35 donors. The microRNA (miRNA) expression profiles have been generated
from HLA-A*02, HLA-A801 CMV seropositive HD. Fourth, detailed analyses of the
TCR repertoire of the gamma (g1-8, g9, g10, g11) and delta (d1-d8) chains have
been determined in HD and patient cohorts. The summary of results will be presented
and discussed.
Page 137 of 200
GDC 2016
C-13
Analysis of the EphA2 Receptor Expression on Malignant Plasma Cells
and Plasmacytoid Dendritic Cells in MGUS and Multiple Myeloma
Patients
1
2
1
3
Krchniakova Maria , Rihova Lucie , Kralova Romana , Pacasova Rita , Penka
4
4
4
5
1
Miroslav , Adam Zdenek , Pour Ludek , Hajek Roman , Piskacek Martin , Knight
1
Andrea
1 Faculty of Medicine, Department of Pathological Physiology, Masaryk University, Brno
2 Department of Clinical Haematology, Faculty Hospital Brno
3 Transfusion and Tissue Bank, Faculty Hospital Brno
4 Department of Internal Medicine, Haematology and Oncology, Faculty Hospital Brno
5 Clinic of Haematology, Faculty Hospital Ostrava
Identification of ligands for human gamma-delta (γδ) T cells remains a major
challenge. The EphA2 (Ephrin receptor A2) has been identified as a ligand for the
Vδ1 γδ TCR transductants. The EphA2 is often over-expressed in aggressive
cancers mainly of epithelial origin; however, the expression in haematological
malignancies has not been determined. Multiple myeloma (MM) is a malignancy of
plasma cells located in bone marrow with a premalignant stage MGUS (monoclonal
gammopathy of undetermined significance). Plasmacytoid dendritic cells (pDC) are
known to promote the tumour plasma cell growth, survival and drug resistance in
myeloma.
The aim was to determine the expression of EphA2 on myeloma plasma cells (PC)
and pDCs (CD123+, CD303+, CD304+) using multicolour flow cytometry in MGUS
and newly diagnosed MM patients.
We have used bone marrow (BM) and paired peripheral blood (PB) samples from
MGUS (n=6) and MM patients (n=11). The age-matched healthy donors were used
as controls (BM n=9, PB n=16).
In MGUS and MM patients, we found prominent EphA2 expression on tumour plasma
cells in BM, while plasmablasts were mostly negative. PDC have been identified
uniformly EphA2+ in both PB and BM in healthy donors and in MGUS and MM
patients.
In summary, we showed the expression of EphA2 receptor on myeloma cells
correlating with malignant transformation. Since EphA2-positivity has been confirmed
on pDC in MGUS and MM patients, we hypothesize that large expansions of Vδ1 γδ
T cells are driven by the interactions with pDC and PC expressing the EphA2 in the
tumour microenvironment.
Page 138 of 200
GDC 2016
C-14
Analysis of gamma-delta T cells and plasmacytoid dendritic cells in
MGUS and Multiple Myeloma patients
1
2
2
1
Kralova Romana , Rihova Lucie , Vsianska Pavla , Krchniakova Maria , Piskacek
1
3
4
4
4
Martin , Pacasova Rita , Penka Miroslav , Adam Zdenek , Pour Ludek , Hajek
5
1
Roman , Knight Andrea
1 Faculty of Medicine, Department of Pathological Physiology, Masaryk University, Brno
2 Department of Clinical Haematology, Faculty Hospital Brno
3 Transfusion and Tissue Bank, Faculty Hospital Brno
4 Department of Internal Medicine, Haematology and Oncology, Faculty Hospital Brno
5 Clinic of Haematology, Faculty Hospital Ostrava
Multiple Myeloma (MM) remains incurable B-cell malignancy despite novel therapies.
Plasmacytoid dendritic cells (pDC) have been shown to mediate tumour plasma cell
growth, survival and drug resistance in MM patients. The role of effector γδ T cell
interactions with pDC in patients progressing from monoclonal gammopathy of
undetermined significance (MGUS) to MM is unknown.
Our aim was to determine frequencies and phenotypes of pDC together with Vδ1 and
Vδ2 γδ T cells. We used bone marrow (BM) and paired peripheral blood (PB)
samples from MGUS (n=8) and newly diagnosed myeloma (n=11) patients. Total of
n=24 age-matched healthy donors samples have been analysed. PDCs were
analysed by multicolour flow cytometry as CD123+, CD303+, and CD304+.
Phenotype of active pDC was identified as HLA-DR+ CD45RA+.
In MGUS and MM patients, the medians of Vδ1 T cells were comparable to normal
levels in contrast to Vδ2 T cells being significantly decreased in both patient cohorts.
In newly diagnosed MM patients, frequencies of pDCs were shown significantly
reduced compared to MGUS patients (p=0.012) or normal BM (p<0.0001). PBderived pDC from patient cohorts were numerically within the normal range. All
normal pDC samples presented an active phenotype in contrast to only 50% of
MGUS or MM patients.
In summary, we found dramatic differences in γδ T cells and pDC numbers and
phenotype in BM of myeloma patients. Further analyses of the key interactions
between the pDC, myeloma and γδ T cells through functional studies, gene
expression profiles in patient cohorts will be discussed.
Page 139 of 200
GDC 2016
C-15
Tumor microenvironment influences γδ T cells immune response in
Colorectal cancer
1
1
2
3
1
E. Lo Presti , V. Orlando , V. Catalano , M.Todaro , F.Dieli , S. Meraviglia
1
1 Dipartimento di Biopatologia e Metodologie Biomediche, Central Laboratory of Advanced
Diagnosis and Biomedical Research (CLADIBIOR), University of Palermo, Palermo, Italy
2 Dipartimento di Discipline Chirurgiche, Oncologiche e Stomatologiche (Di.Chir.On.S.),
Palermo, Italy
3 Dipartimento di Biomedico di Medicina interna e Specialistica, Palermo, Italy
Colorectal cancer (CRC) is the third most prevalent cancer in worldwide. Tumorinfiltrating lymphocytes are key mediators of tumor immune surveillance and are
important prognostic indicators in cancer progression. Previous studies have found
that γδ T cells are the major cellular source of IL-17 in human CRC and have
suggested they promote tumor progression. In this study, we have characterized
CRC-infiltrating γδ T cells in a cohort of 70 patients in terms of phenotype and
effector functions showing that Vδ1 T cells were the predominant population in the
vast majority of specimens while Vγ9Vδ2 T cells were found uniformly at lower
proportion in most of the patients, had a predominant terminally-differentiated
effector memory phenotype, expressed cytotoxic molecules and upon short term in
vitro stimulation produced pro-inflammatory cytokines as IL-17 and IFNγ. In
particular, we found that γδ T cells producing IFNγ predominated in patients at stage
I and II, while γδ T cells making IL-17 were the predominant population at advanced
tumor stages (III and IV). This suggests that signals from the microenvironment may
turn γδ T cells to protumoral activity. Accordingly, we found that supernatants from
cancer stem cells (CSC) but not cancer associated fibroblast (CAF) had profound
influence on γδ T cells causing: a) downregulation of surface CD3 expression; b)
inhibition of proliferation and IFNγ; c) differentiation to IL-17 production. In
conclusion, our study highlights a complex interplay between cancer stem cells,
tumor stroma and γδ T lymphocytes as a major determinant of the final outcome of
the γδ T lymphocyte response in CRC.
Page 140 of 200
GDC 2016
C-16
Negative correlation of circulating Vδ1+ γδ T-cells with overall survival
of stage IV melanoma patients
1,2
Kilian Wistuba-Hamprecht , Alexander Martens
2,3,4
1
Graham Pawelec
,Benjamin Weide
1,2
, Karin Hähnel
2
1
Claus Garbe ,
1 Department of Dermatology, University Medical Center, Tübingen, Germany
2 Department of Internal Medicine II, University Medical Center, Tübingen, Germany
3 School of Science and Technology, College of Arts and Science, Nottingham Trent University,
Nottingham, UK
4 Division of Cancer Studies, Faculty of Life Sciences and Medicine, King’s College London, UK
Interest has recently resurged in applying γδ T-cells for cancer immunotherapy,
because these cells can kill melanoma cells and possess regulatory and antigen
presenting capabilities, and could potentially influence the efficacy of
immunotherapies. The major subsets of γδ T-cells in the peripheral blood are Vδ2+
and Vδ1+ cells, about which little is known in melanoma. Thus, we investigated these
cells and their respective differentiation stages in stage IV melanoma patients using
cryopreserved PBMC from 6 clinical centers. Flow cytometry and a validated set of
monoclonal antibodies to assess γδ T-cell-phenotypes was used for data acquisition
and processing. In a discovery cohort of 27 patients treated in several different ways,
we found that a higher frequency of Vδ1+ cells was negatively associated with overall
survival (OS). This finding was validated in a second independent cohort of 109 stage
IV patients treated with ipilimumab. Consistent with this, total frequencies of Vδ1+
cells in patients were higher than in healthy controls. A detailed analysis of the
composition of the Vδ1+ subset in patients identified significantly higher proportions
of late-stage differentiated Vδ1+ cells in patients with the poorest outcome. We
therefore suggest that Vδ1+ T-cell frequencies may be a novel candidate biomarker
in melanoma, as we identified a significant association of these cells with patients’
OS in two different studies, independent of patient treatment regimens. Determining
the mechanism of the deleterious effect of Vδ1+ T-cells in late-.stage melanoma may
allow the development of treatment strategies targeting these cells.
Page 141 of 200
GDC 2016
C-17
Human γδΤ-APCs: Processing of Tumour Antigens and Induction of
Anti-Tumour Immunity
Kouzeli, Ariadni1., Rus, Teja1., Eberl, Matthias., Moser, Bernhard.
Division of Infection and Immunity, School of Medicine, Cardiff University
The recent discovery that activated human blood γδ T cells can behave like
professional antigen-presenting cells (γδT-APCs) has highlighted their potential use
as cellular vaccines for immunotherapy. γδΤ-APCs are ideal candidate for
immunotherapy as they are non-MHCrestricted, making them safe for use as they do
not mount unwanted/unpredicted crossreactivity in treated patients. They are also
capable of inducing potent antigen-specific and MHC-restricted CD8+ T cells that
may boost anti-tumour cytotoxic T cell responses. Lastly, they can be generated in
large numbers (>109) from small blood samples of cancer patients and
predominantly secrete proinflammatory cytokines (IFNγ, TNFα), which may help
overcome immune inhibitory conditions frequently associated with tumors.
It has been shown that γδT-APCs can excellently process microbial antigens
(influenza, CMV, Mtb), including small peptides, whole proteins and cell extracts, and
induce strong responses in allo-reactive γδT cells. This study focuses on the ability of
γδT-APCs to process and present tumour antigens, including the oncofoetal tumour
antigen 5T4 and NYESO-1, which is frequently expressed in various tumours but not
in healthy tissue. This is accomplished through the production and purification of
recombinant protein from E.coli and antigenspecific CD8+ T cells from peripheral
blood with the help of γδT-APCs, in an autologous system. These are then used to
examine the functionality of γδT-APCs in in vitro APC assays. Demonstration that
γδΤ-APCs are capable of inducing strong tumour-specific CD4+ and CD8+ γδT cell
responses is a prerequisite for carrying our first-in-man clinical trials in patients with
cancer. Further laboratory work will include localisation and generation of γδT-APCs
in colorectal cancer tissues, as well as the generation of a mouse immunotherapy
model to test the potential benefits of γδT-APCs as a cellular vaccine.
Page 142 of 200
GDC 2016
C-18
The innate immune system
glioblastoma multiforme
1
is compromised in patients with
2
1
2
Guranda Chitadze , Charlotte Flueh , Christian Peters , Michael Synowitz , Janka
2
1
Held-Feindt , Dieter Kabelitz
1
Institute of Immunology, Christian-Albrechts-University Kiel, Kiel, Germany
Department of Neurosurgery, University Medical Center Schleswig-Holstein UKSH, Campus
Kiel, Kiel, Germany
2
Vγ9Vδ2 T-cells elicit potent antitumor activity against various tumors including
glioblastoma multiforme (GBM), the most malignant brain tumor in adults. Vγ9Vδ2 Tcells recognize phosphoantigens over-produced in transformed cells and thus
distinguish tumors from healthy tissues. In addition Vγ9Vδ2 T-cells also recognize
tumor cells via Natural Killer Group 2, member D (NKG2D) receptor which binds to
stress-inducible ligands (NKG2DLs) expressed on GBM cells. These molecules like
MHC class I-related chain molecules A/B and UL-16 binding protein family members
when binding to corresponding NKG2D receptor trigger cytotoxic activity in Vγ9Vδ2
T-cells and hence, Vγ9Vδ2 T-cells play an important role in tumor
immunesurveillance. However, as an immune escape mechanism, tumor cells
including GBM release soluble NKG2DLs (sNKG2DLs).
Materials and methods: Here we analyzed expression of NKG2DLs on GBM primary
cells and defined the levels of sNKG2DLs in sera of GBM patients (n=37) and healthy
controls (HCs, n=20) via Luminex-based multiplex assay. In parallel we phenotyped
peripheral blood by 11-color based flow cytometry before and 3 months after
radio/chemotherapy.
Results: In comparison to HCs, GBM patients showed reduced numbers of Vγ9Vδ2
T-cells and NK cells. Serum levels of sNKG2DLs were elevated in GBM patients.
Radio/chemotherapy differentially affected the levels of serum NKG2DLs.
Conclusions: The innate arm of the immune system seems to be compromised in
GBM patients, which may be caused by high levels of immunosuppressive
sNKG2DLs. A better understanding of γδ T-cell functions and the role of sNKG2DLs
in clinical setup might help to develop new therapeutic strategies against GBM such
as γδ T-cell-based immunotherapies.
Page 143 of 200
GDC 2016
C-19
Procurement of broadly tumoricidal γδ T-cell clones from clinical grade
TIL products
1
1
1
1
2
Mateusz Legut , Garry Dolton , Meriem Attaf , Barbara Szomolay , Marco Donia ,
2
2
1
Per thor Straten , Inge Marie Svane , Andrew K. Sewell
1 Systems Immunity Research Institute, Division of Infection and Immunity, Cardiff University
School of Medicine, Henry Wellcome Building, University Hospital, Cardiff, CF14 4XN, Wales,
UK
2 Department of Haematology, Center for Cancer Immune Therapy, Copenhagen University
Hospital at Herlev, Herlev, Denmark
Adoptive cell transfer of tumour-infiltrating lymphocytes (TILs) is one of the most
promising treatment options for metastatic melanoma, resulting in objective response
rates of >40% and complete lasting remissions in >20% of patients. The anticancer
role of αβ T-cells in TIL products has been extensively studied while little is known
about the contribution of γδ T-cells to tumour clearance. Additionally, the TIL samples
can be substantially enriched in non-Vγ9Vδ2 T-cells, providing an excellent tool for
studying these T-cell subsets within clinically relevant tissue. Here we describe the
anticancer activity of γδ T-cells from five clinical grade TIL infusion products with γδ
T-cell fraction ranging from 10 to 90%. γδ T-cells derived from the TILs were capable
of producing cytokines in response to the autologous tumour, as well as exhibiting
cytotoxic activity similar to the parental TILs. Subsequently, we have derived a panel
of γδ T-cell clones that expressed a diverse TCR repertoire, were capable of
multifunctional response to the autologous tumour, and showed a robust cytotoxicity
against a wide range of HLA-mismatched cancer lines from different histologies.
Notably, few of the tumour-reactive clones expressed a Vγ9Vδ2 TCR but did not
mount a response to phosphoantigens, suggesting that other mechanisms may be
involved in tumour recognition. Finally, we have estimated the TCR diversity of
tumour-reactive γδ T-cells by deep sequencing of both TILs and peripheral blood
lymphocytes after treatment from a complete regression patient. In summary, these
studies pave the way for identification of novel cancer-associated γδ TCR ligands
and immunotherapies.
Page 144 of 200
GDC 2016
C-20
Chimeric antigen receptor transduced gamma delta T lymphocytes
combine effective tumour-cell killing and on-tumour specificity
1
1
1
Pierre Abramowski *, Jonathan Fisher *, Dilrukshi Wisidagamage Don , Barry
1
1
1
2
1
Flutter , Rebecca Wallace , Karin Straathof , Martin Pule , John Anderson
*equal contribution
1
Immunotherapy and Immune Regulation in Childhood Cancers Group, Developmental Biology
and Cancer Programme, Cancer Section, Institute of Child Health and Great Ormond Street
Hospital, University College London, London, United Kingdom,
2
Cancer Institute, Department of Haematology, Division of Medicine, University College London,
London, United Kingdom
Neuroblastoma affects 100 children/year in the UK. The 5-year average survival is
approximately 55% only. Neuroblastoma arises from cells of the sympathetic chain
and has cell surface expression of the disialoganglioside GD2. GD2 has been
successfully tested as a tumour antigen in antibody immunotherapy. A current clinical
trial programme tests a chimeric antigen receptor (CAR) against GD2 (Huk666-28zeta) expressed by alpha beta T cells after retroviral transduction.
In order to combine inherent T-cell functions, i.e. target-cell killing, with CARmediated specificity but also with additional safety, zoledronate-expanded gamma
delta T cells were transduced to express a GD2-specific, “co-stimulation-only” CAR
(GD2-DAP10). DAP10 is normally engaged by NKG2D and provides signal 2 for full
gamma delta T-cell activation upon phosphoantigen recognition by the TCR (signal
1). GD2-DAP10 CAR-mediated gamma delta T-cell activation involved defined
interferon gamma production only in presence of TCR and CAR engagement. Human
Lan-1 neuroblastoma target cells, which engage both the TCR (signal 1) and the
GD2-DAP10 CAR (signal 2), were effectively killed by GD2-DAP10 CAR-expressing
gamma delta T cells, although not by GD2-DAP10 CAR-expressing alpha beta T
cells. No increased killing was detectable in co-cultures of GD2-DAP10 CAR+
expressing gamma delta T cells with murine CT26-GD2 target cells, in which signal
1 was absent. GD2-28-zeta CAR expression on alpha beta and gamma delta T cells
+
led to efficient killing of CT26-GD2 target cells, respectively. In summary, our
strategy may open new avenues for anti-tumour CAR-cell therapies with diminished
side effects.
Page 145 of 200
GDC 2016
C-21
Liposomes encapsulating alendronic acid for cancer immunotherapy
and their effect on the in vivo biodistribution of Vg9/Vd2 T cells in
immunocompromised mice
1
1
2
3
2
N. Hodgins , J.Wang , A. Parente Pereira , W.T. Al-Jamal , J. Maher and K.T. Al1
Jamal
1
Institute of Pharmaceutical Science, King's College London, Franklin-Wilkins Building, 150
Stamford Street, London, SE1 9NH
2
CAR Mechanics Group, Research Oncology, 3rd Floor Bermondsey Wing, Guy's Hospital, St.
Thomas Street, London, SE1 9RT
3
School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ
Email: [email protected]
Nitrogen-containing bisphosphonates (N-BPs) including alendronic acid (ALD) inhibit
farnesyl diphosphate synthase, and sensitise tumour cells to destruction by Vγ9/Vδ2
T cells. N-BPs have limited in vivo activity due to rapid clearance from the circulation.
Liposomes lead to increased levels of N-BPs at tumour sites and can be used with
Vγ9/Vδ2 T cells for cancer immunotherapy. We hypothesise that uptake of ALD
liposomes (L-ALD) and Vγ9/Vδ2 T cells differs among tumour models. This work
aims at quantifying the amount of L-ALD and Vγ9/Vδ2 T cells in different tumour
models in order to assess their suitability for this immunotherapy.
L-ALD
were
formulated
with
DSPE-DTPA
(1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-diethylenetriaminepentaacetic acid), and radiolabelled with
111
Indium-111 ( In). Vγ9/Vδ2 T cells were isolated from whole blood and radiolabelled
111
with
In tropolone. Immunocompromised mice were inoculated with the melanoma
cell line A375Pβ6 to form subcutaneous, intraperitoneal or lung tumours. The
biodistribution of intravenously injected L-ALD or Vγ9/Vδ2 T cells in these different
tumour models was assessed by gamma counting.
111
In vivo,
In-labelled liposomes have shown accumulation of 1.9, 5.2 and 1.9 %
injection dose per gram (% ID/g) in subcutaneous tumours, intraperitoneal tumours
111
and in tumour bearing lungs, respectively. In the same three tumour models,
Inlabelled Vγ9/Vδ2 T cells had accumulation of 1.0, 1.4 and 22.5 % ID/g, respectively.
Pre-injection of mice with free ALD or L-ALD, did not lead to a significant change in
the tumour accumulation of Vγ9/Vδ2 T cells. Future work will focus on utilising this
quantitative biodistribution data for therapy studies.
Page 146 of 200
GDC 2016
C-22
Dichloroacetate inhibits aerobic glycolysis and proliferation of certain
leukemia cell lines and can enhance anti-tumor effects of gamma-delta
T-cells
1
1
1
1
Timm Hoeres , E. Holzmann. , M. Smetak , J. Birkmann and M. Wilhelm
1
1
Department of Hematology and Medical Oncology, Paracelsus Medical University, General
Hospital Nuremberg - [email protected]
Objective: Zoledronic acid (ZOL) activates human Gamma-delta T-cell (γδ T-cell) and
induces anti-tumor effects, but its therapeutic potential is limited. We aimed to
improve the efficiency of γδ T-cells in the treatment of leukemia by utilizing the effect
of the indirect pyruvate dehydrogenase (PDH) activator dichloroacetate (DCA).
Methods: We tested the impact of different concentrations and incubation periods of
sodium DCA on the proliferation, glucose consumption and lactate generation of
leukemia cell lines. Flow cytometric tests were used to measure the effect of DCA on
γδ T-cell degranulation and interferon-γ production alone and in response to coculturing with leukemia cell lines.
Results: DCA inhibits the proliferation of leukemia cell lines in a dose and time
dependent manner as well as glucose uptake and lactate production. A 3 weeks
treatment of KG-1 cells with 5mM or 10mM DCA significantly enhances IFN-γ
production and degranulation of co-incubated γδ T-cells. DCA neither influences γδ
T-cell degranulation or IFN-γ production in the absence of leukemia cells nor without
the addition of ZOL.
Conclusions: DCA inhibits growth and alters glucose metabolism of certain leukemia
cell lines in a concentration- and time-dependent manner. According to our
hypothesis DCA can additionally enhance the recognition of leukemia cells by γδ Tcells due to its impact on glycolysis and the downstream intermediary metabolism
when combined with ZOL. DCA or similar acting drugs could be used to preferentially
target cells that possess certain metabolic properties, thus simultaneously inhibiting
growth and enhancing γδ T-cell reactivity against these cells.
Page 147 of 200
GDC 2016
Session 8
γδ T cell function in health and medicine: 4. γδ T
cells in immunotherapy
Talks:
Sunday, June 19th
Poster presentation:
Saturday, June 18th, 4:30 – 6:30pm
Sunday, June 19th, 1:30 – 2:30pm
Page 148 of 200
GDC 2016
C-23
Epigenetic landscape of human γδ T-cells
1
2
3
2
Jaydeep Bhat , Swetlana Scheufele , Raheleh Sheibani , Anke Bergmann , Maren
3
3
2
1
Paulson , Philip Rosenstiel , Ole Ammerpohl , Dieter Kabelitz
1
Institute of Immunology, Christian-Albrechts University, Kiel, Germany
Institute of Human Genetics, Christian-Albrechts University, Kiel, Germany
3
Institute of Clinical Molecular Biology, Christian-Albrechts University, Kiel, Germany
2
Human γδ T-cells have emerged as key players in diverse immune responses both in
health and disease. Such a dynamic functional diversity of cells is controlled in an
orchestrated manner at various levels of (epi)genetic regulation. However, a
comprehensive overview of epigenetic mechanisms dictating human γδ T-cell
development, regulation and molecular function is largely missing. Our present study
aims at elucidating the epigenetic landscape of human γδ T-cells, comprising mainly two
distinct approaches. In a first approach, we decipher the “native state” of molecular
regulation of γδ T-cells in comparison to αβ T-cells using highly sensitive assays for
chromatin accessibility, DNA methylation and RNA expression. Next generation
sequencing based preliminary results clearly show differential regulation of genes
distinguishing γδ T-cell specificity as compared to other T-cell subsets. In a second
approach based on in vitro culture, we investigated the effect on functional responses of
γδ T-cells upon treatment with histone deacetylase inhibitor valproic acid, an epigenetic
drug widely used in the clinic. Valproic acid induces changes in molecular and cellular
responses of human γδ T-cells, specifically by enhanced expression of non-secretory
form of IL-4, regulation of cell death and induction of global histone acetylation (H3K9ac).
Furthermore, it modulates NKG2D receptor expression and function in co-culture with
PancTu-1 and PC-3 tumor cell lines. Our research based on (epi)genetic mechanisms
will broaden the characterization of human γδ T-cells and thereby contribute to a better
understanding of basic features of γδ T cells important for their future immunotherapeutic
application.
Page 149 of 200
GDC 2016
C-24
Aminobisphosphonates Reveal a Selective Anti-Viral Potential of Vg9Vd2
T Cells
Eric Champagne*, Hugo Barragué*, Charline Daguzan*, Florence Abravanel*, Floriant
Bellvert#, Suzanne Peyrottes$, Jacques Izopet*
* Centre de physiopathologie Toulouse Purpan, INSERM-U1043/CNRS-UMR5282/Université Paul
Sabatier, Toulouse, France
# LISBP, UMR INSA/CNRS 5504/INRA 792, Toulouse, France
$ IBMM, UMR 5247 CNRS-UM-ENSCM, Montpellier, France
Vg9Vd2 cells are activated by stressed cells producing endogenous phosphoantigens
following tumoral transformation or treatment with aminobisphosphonates. Alternatively
they respond to exogenous phosphoantigens produced by bacteria. Their potential
interest in viral infection is not well established. We analyzed in vitro their activation and
activity against HCMV-infected cells. Although they were not activated by HCMV-infected
fibroblasts, following treatment of targets with aminobisphosphonates, they were
preferentially and more potently activated by infected cells, resulting in a stronger IFNg
_production and induction of TNF. In conditions where uninfected cells were not affected,
they limited viral replication through an IFNg _and TNF-dependent mechanism, whereas
cytotoxicity did not play a significant role. This could be explained by an increased
uptake of a fluorescent zoledronate derivative by infected cells. In addition, HCMV
infection stimulated the transcription of mevalonate pathway enzymes HMG-CoA
reductase and synthase evaluated by RT PCR. As a result, the production of
endogenous phosphoantigens IPP and ApppI following low-dose treatment with
zoledronate was also boosted. Cytokines such as IL18, putatively produced by infected
cells did not play a significant role in the synergistic effect of HCMV and zoledronate. In
order to examine whether this was true for other viruses, we infected hepatocytoma cells
with the hepatitis E virus and treated them with zoledronate. In this case, viral infection
only moderately increased zoledronate-induced IFNg _production, but not TNF, by
Vg9Vd2 cells. Our results suggest that aminobisphosphonates could be used to target
the anti-viral potential of Vg9Vd2 T cells against selected viruses responsible for chronic
infections.
Page 150 of 200
GDC 2016
C-25
Activation of γδ T cells to improve healing in chronic wounds
1
2
2
2
Daisy Johnson , Cody Higginson , Srinivas Tekkam , Stephen Crooke , Deborah
1
2
1
Witherden , M.G.Finn , and Wendy Havran
1: The Scripps Research Institute, La Jolla, CA, USA.
2: Georgia Institute of Technology, Atlanta, GA, USA.
Chronic wounds, commonly caused by pressure sores and diabetic ulcers, are estimated
to affect 6.5 million patients in the US each year with the number of patients expected to
grow. Current treatment is typically limited to debridement to create fresh wound edges,
a process that also increases the size of the wound, making new approaches to
stimulating would healing a priority. γδ T cells contribute to the wound healing response
in humans and mice by producing cytokines and growth factors in and around the wound
that drive keratinocyte proliferation in response to damage. Although γδ T cells are
present in chronic wounds they remain in an unresponsive state, raising the possibility of
developing strategies to reactivate these in order to enhance healing in chronic wounds.
Activation of γδ T cells requires a costimulatory signal through the binding of the
junctional andhesion molecule-like (JAML) protein on their surface to the coxsackie and
adenovirus receptor (CAR) on keratinocytes, an interaction that has been shown to be
important in wound healing. Car is upregulated in and around acute wounds but its
expression is unchanged in chronic wounds, presenting a possible mechanism for the
unresponsive state of γδ
T cells in chronic wounds. In this study we are investigating the potential therapeutic
benefics of treating wounds with molecules that directly activate JAML using degradable
hydrogels as a delivery system. New strategies for costimulating of γδ
T cells in vivo may accelerate wound healing as well as impact treatment of additional
epithelial disorders.
Page 151 of 200
GDC 2016
C-26
Adoptive transfer of ex vivo expanded human Vγ9Vδ2 T cells in
combination with zoledronic acid inhibit cancer growth in bone
1
1
1
1
1
1
Aneta Zysk , M.O. DeNichilo , V. Panagopoulos , I. Zinonos , V. Liapis , S. Hay , V.
2
1
Ponomarev , A. Evdokiou
1
School of Medicine, Discipline of Surgery, Basil Hetzel Institute, University of Adelaide, Adelaide,
South Australia
2
Department of Radiology, Memorial Sloan-Kettering Cancer Centre, New York, USA
Bone metastases occur in more than 75% of patients with advanced breast cancer.
Cancer in bone is associated with bone destruction and is responsible for high levels of
morbidity and mortality but is notoriously difficult to treat. Bone destruction is also the
primary cause of morbidity in patients with primary bone cancer, such as osteosarcoma,
with metastatic spread to the lungs correlating with poor survival. Therefore, it is clear
that new therapies are desperately needed to target cancers in the bone. In this study we
explored the therapeutic potential of gamma delta (Vγ9Vδ2) T cell based adoptive
transfer using animal models of osteolytic breast cancer and osteosarcoma. Cytotoxic
Vγ9Vδ2 T cells were expanded ex vivo from peripheral blood using IL-2 and zoledronic
acid (ZOL). In vitro, expanded Vγ9Vδ2 T cells were cytotoxic against a panel of breast
cancer and osteosarcoma cell lines and pre-treatment with ZOL sensitized all cancer
cells to rapid killing by Vγ9Vδ2 T cells. Adoptive transfer of fluorescently labeled ex vivo
expanded Vγ9Vδ2 T cells into NOD/SCID mice localized to cancer lesions in bone.
Multiple infusions of Vγ9Vδ2 T cells reduced breast cancer growth, but had no effect on
osteosarcoma growth in the bone marrow. However, in both cases, ZOL pre-treatment
potentiated the anti-cancer efficacy of Vγ9Vδ2 T cells in bone, protected the bone from
cancer-induced osteolysis and decreased the incidence of pulmonary metastases.
Collectively our data suggests this treatment regimen to be an effective
immunotherapeutic approach for the treatment of primary and metastatic bone cancers.
Page 152 of 200
GDC 2016
C-27
CCL4 Secretion By Ineterleukin-15 Dendritic Cells Directs Superior
+
Recruitment Of CD56 Cytolytic Lymphocytes
1
2
1,3
1,4
Heleen H. Van Acker, Ottavio Beretta, Sébastien Anguille, Lien De Caluwé, Angela
2
1
1
1
Papagna, Johan M. Van den Bergh, Yannick Willemen, Herman Goossens, Zwi N.
1,3
1
1,3,5
2,#
1,3,#
Berneman, Viggo F. Van Tendeloo, Evelien L. Smits,
Maria Foti, Eva Lion
1
Laboratory of Experimental Hematology, Tumor Immunology Group (TIGR), Vaccine & Infectious
Disease Institute (VAXINFECTIO), University of Antwerp, Faculty of Medicine and Health Sciences,
Antwerp, Belgium
2
Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy
3
Center for Cell Therapy & Regenerative Medicine, Antwerp University Hospital, Edegem, Belgium
4
Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
5
Center for Oncological Research (CORE), University of Antwerp, Faculty of Medicine and Health
Sciences, Antwerp, Belgium
#
Share senior authorship
The past decade witnessed the advent of a new generation of dendritic cell (DC)-based
vaccines with improved potency, epitomized by the promising interleukin (IL)-15 DC
cancer vaccine (Anguille J Transl Med 2009). Although critical for therapeutic efficacy,
immune cell recruitment is a largely overlooked aspect concerning optimization of DC
therapy. To this extent, we have made a head-to-head comparison of IL-15-cultured DCs
and conventional IL-4-cultured DCs regarding their proficiency in the recruitment of
(innate) immune cells. We demonstrated that conventional IL-4 DCs are suboptimal in
attracting effector lymphocytes, while IL-15 DCs provide a favorable chemokine milieu for
+
recruiting CD8 T cells, natural killer (NK) cells and gamma delta (γδ) T cells. Gene
expression analysis revealed that IL-15 DCs exhibit a high expression of chemokines
involved in antitumor immune effector cell attraction, while IL-4 DCs display a more
immunoregulatory profile characterized by the expression of Th2 and regulatory T cellattracting chemokines. A possible explanation for the superior recruitment of effector
lymphocytes by IL-15 DCs could be ascribed to the CCL4-CCR5 signaling pathway
because of higher CCL4 chemokine gene expression in IL-15 DCs and lowered CCR5
expression on both migrated γδ T cells and NK cells. Following validation of significant
higher CCL4 secretion by IL-15 DCs then by IL-4 DCs, we demonstrated that
neutralization of CCR5 on PBMC prior to migration resulted in a significant inhibition of
γδ T cell and NK cell recruitment by only IL-15 DCs. These findings further underscore
the strong immunotherapeutic potential of IL-15 DCs.
Page 153 of 200
GDC 2016
C-28
Characterization of CD4+ Vγ9Vδ2 T cells as efficient cancer cell killers
1
1
1
1
Gitte Holmen Olofsson , Sara Ram Pedersen , Maria Olsen , Manja Idorn , Inge Marie
1,2
1,3
Svane and Per thor Straten .
1 Center for Cancer Immunterapi, CCIT, Copenhagen University Hospital Herlev, Herlev, Denmark,
2 Department of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark,
3 Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University
of Copenhagen,Copenhagen, Denmark.
Human Vγ9Vδ2 T cells holds great potential in immunotherapy, hence they are both
antigen presenting cells (APC) and cytotoxic towards cancer cells. The dogma states
that Vγ9Vδ2 T cells express CD8+ (20-30%) or are double negative (DN) for the costimulatory molecules CD4 and CD8. We have discovered a subtype of Vγ9Vδ2 T cells
that express the co-stimulatory molecule CD4. These cells comprise 0.1-7% of the
peripheral blood Vγ9Vδ2 T cells, and can be expanded in vitro using zoledronic acid,
pamidronic acid or CD3 antibodies combined with IL2 and feeder cells. Unlike
conventional CD4+ αβ-T cells, the CD4+ Vγ9Vδ2 T cells are potently cytotoxic and kill
cancer cell lines of different histological origin. Thus, Vγ9Vδ2 CD4+ T cells can kill breast
cancer, leukemia, and melanoma cell lines, upon treatment of the with Zoledronic acid.
Capacity to kill tumor cell targets correlated with co-expression of CD56. Combined with
the ease of expanding Vγ9Vδ2 T cells in vitro to billions of cells, makes Vγ9Vδ2 CD4+ T
subtype, a potential candidate for future clinical application.
Page 154 of 200
GDC 2016
C-29
Combined killing of cancer cells and cross presentation of tumor antigen
by Vγ9Vδ2 T cells
1
1
2
2
Gitte Holmen Olofsson , Manja Idorn , Ramona Schenker , Elfriede Nössner , Reno
3
4
1,5
1,6
Debets , Bernhard Moser , Özcan Met , and Per thor Straten .
1 Center for Cancer Immunterapi, CCIT, Copenhagen University Hospital Herlev, Herlev, Denmark,
2 Helmholtz Zentrum München, Germany research Center for Environmental Health, Institute for
Molecular Immunology, München, Germany,
3 Laboratory of Experimental Tumor Immunology, Erasmus MC Cancer Institute, Rotterdam,
Netherlands, 4Department of Infection, Immunity & Biochemistry, School of Medicine, Cardiff, UK, 5
Department of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark,
6 Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University
of Copenhagen, Copenhagen, Denmark.
The human Vγ9Vδ2 T cells are a unique T cell type, and recent studies of the biology of
Vγ9Vδ2 T cells emphasize the potential exploitation of these cells in immunotherapy of
cancer. Vγ9Vδ2 T cells exhibit dual functionality in that they are both antigen presenting
cells (APC) and cytotoxic towards cancer cells. We show that Vγ9Vδ2 T cells can kill
cancer cell lines from various cancer types such as leukemia, melanoma, prostate-, and
breast cancer, with a significantly increased killing upon treatment of the cancer cells
with Zoledronic acid. In addition, we show that Vγ9Vδ2 T cells take up tumor antigens
gp100 and MART-1 (long peptide and recombinant protein, respectively), and process
these antigens for presentation of class I restricted peptides in the context of the HLAA02.01 molecule, to be recognized by peptide specific cytotoxic CD8 T cells. Moreover,
we show that specific inhibition of the proteasome by lactacystin impair recognition by
peptide specific CD8 T cells, strongly suggesting proteasome involvement in
presentation of the relevant class I restricted peptides. The dual functions; killing and
antigen presentation combined with the ease of expanding Vγ9Vδ2 T cells in vitro from
peripheral blood lymphocytes to billions of cells, makes Vγ9Vδ2 T cells attractive
vehicles for adoptive cell therapy (ACT) in cancer therapy. Thus, Vγ9Vδ2 T cells are
broadly tumor specific killers that concurrently could induce or support tumor specific αβT cell responses.
Page 155 of 200
GDC 2016
C-30
Human Vγ9Vδ2 T Cells Spontaneously Eliminate Particular Subsets Of
Primary Glioblastoma Tumor Cells
Cynthia Chauvin1.2,
Emmanuel Scotet1.2
Ulrich
Jarry1.2,
Noémie
Joalland1.2,
Claire
Pecqueur1.2,
1 Inserm, U892, Nantes, F-44000, France; Univ Nantes, Nantes, F-44000, France; CNRS, UMR
6299, Nantes, F-44000, France
2 LabEx IGO “Immunotherapy, Graft, Oncology”, Nantes, F-44000, France
Glioblastoma multiform (GBM) is the most frequent and aggressive primary brain tumor
in adults,with a dismal prognosis and few therapeutic advances made over the last
decade. Cellular immunotherapies are currently being explored to eliminate highly
invasive GBM cells likely involvedin rapid disease relapse. Non-alloreactive human
Vγ9Vδ2 T cells are able to kill a wide range of human tumor cells and display effector
functions, setting them up as promising cell candidates forefficient immunotherapies.We
recently showed that immunodeficient NSG mice carrying orthotopic primary human
GBM tumor xenografts recapitulate GBM tumor development in patients. Furthermore,
we demonstrated that allogeneic human Vγ9Vδ2 T cells are able to survive and patrol for
days within the brain parenchyma following adoptive transfer, successfully eliminate
infiltrative GBM primary cells upon orthotopic aminobisphosphonate treatment. However,
in order to bypass this sensitization, various allogeneic human Vγ9Vδ2 T cells have
been prepared from PBMCs of healthy donors and screened for their ability to naturally
and specifically react against primary human GBM tumor cells. Our results showed that
some Vγ9Vδ2 T cell lines efficiently and preferentially eliminate a particular subtype of
primary GBM tumor cells through cellular stress-associated molecular pathways that are
currently under final characterization.
Taken together, our results provide an important preclinical proof of concept for
optimized immunotherapies of GBM and also indicate that optimal conditions need to be
defined before considering them in clinical approaches.
Page 156 of 200
GDC 2016
C-31
TCRVγ9 γδ T cell response to IL-33: a CD4 T cell-dependent mechanism
1
1
1
2
Caroline Duault , Don Marc Franchini , Julien Familliades , Corinne Cayrol , Stéphane
2
2
1
1
Roga , Jean-Philippe Girard , Jean-Jacques Fournié and Mary Poupot
1 INSERM UMR1037, Cancer Research Center of Toulouse, Université Toulouse III, CNRS ERL
5294, Toulouse, France
2 CNRS UMR 5089, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France
email : [email protected]
Due to their anticancer cytotoxicity, TCRVγ9-expressing T lymphocytes have allowed the
development of γδ T cell-based cancer immunotherapies. However, such strategies are
dependent on the use of exogenous IL-2 which is too much toxic for patients. In our
quest to find an alternative method for inducing γδ T cell stimulation, we found IL-33, a γ
chain receptor-independent cytokine of the IL-1 superfamily, able to sustain in vitro
activation of Vγ9 T lymphocytes. This cytokine is expressed by endothelial cells from a
tumor microenvironment and was shown as can sustain Th1 and Th2 immune
responses.
Thus, we showed that IL-33 efficiently sustained the in vitro activation of Vγ9 T
lymphocytes by synthetic phosphoantigens, zoledronate and a BTN3A1 antibody in the
absence of an exogenous supply of IL-2. IL-33 was as potent as IL-2 in allowing the
proliferative amplification of Vγ9 T cells isolated from PBMC following activation by the
synthetic phosphoantigen BrHPP. IL-33 also induced an identical maturation into TNF-αand IFN-g-producing Th1 effector memory cells, and IL-33-stimulated cells showed an
equivalent cytotoxicity for various tumor cells in vitro. Finally, we found that the bioactivity
of IL-33 on the Vγ9 T cell was indirectly mediated through contact with CD4 T cells and
endougenous IL-2 production by CD4 T cells and Vγ9 T cells themselves. These data
posit IL-33 as an alternative to IL-2 for Vγ9 T cell-based cancer immunotherapies.
Page 157 of 200
GDC 2016
C-32
+
Vδ1 T cells expressing natural cytotoxicity receptors for immunotherapy
of leukemia: clinical-grade expansion/ differentiation and preclinical
proof-of-concept
1,2
2
3
2
Daniel V. Correia *, Afonso Almeida *, Maria G. Silva , Diogo Anjos and Bruno Silva1,2
Santos
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
Lymphact – Lymphocyte activation Technologies S.A., Coimbra, Portugal.
3
Instituto Português de Oncologia de Lisboa, Lisbon, Portugal.
* Equal contributions.
2
+
The Vδ1 subset of human γδ T lymphocytes is an attractive candidate for cancer
immunotherapy given its abundance in tissues, substantial infiltration into tumors, and
+
broad recognition of transformed cells. However, Vδ1 T cells have never been tested in
clinical trials due to major difficulties in expanding these cells ex vivo (for adoptive
transfer) using clinical-grade reagents. We have devised a very robust protocol for the
+
selective expansion and differentiation of type 1 cytotoxic Vδ1 T cells for clinical use.
+
Vδ1 T cells generated from blood of healthy donors or chronic lymphocytic leukemia
(CLL) patients selectively recognized malignant (but not normal) leukocytes in vitro and
in vivo. The differentiated lymphocytes express a broad repertoire of natural killer cell
receptors, including NKG2D and the natural cytotoxicity receptors (NCRs), NKp30,
NKp44 and NKp46. Critically, NCR expression synergizes with the TCR to endow these
®
lymphocytes, termed Delta One T (DOT-) cells , with enhanced cytotoxicity against
lymphoid and myeloid leukemia cells in vitro. When transferred in vivo (NSG mice), DOT
cells infiltrated tumors and other peripheral tissues, and persisted until the end of the
analysis (55 days post-transfer) without showing any signs of exhaustion; indeed, DOT
cells produced abundant IFN-γ and TNF-α, but importantly no IL-17, in vivo. Critically,
DOT cells were capable of inhibiting tumor growth and dissemination in two xenograft
models of CLL, without any signs of treatment-associated toxicity (biochemistry and
histology). Collectively, our results provide new means and the proof-of-principle for
clinical application of DOT cells in adoptive immunotherapy of leukemia.
Page 158 of 200
GDC 2016
C-33
Macrophages become susceptible to γδ T cell cytotoxicity following
zoledronic acid treatment
Fowler DW, Copier J, Dalgleish AG and Bodman-Smith MD
Institute for Infection and Immunity, St. George’s University of London, UK
In humans the nitrogen-containing bisphosphonate drug zoledronic acid (ZA) can render
certain cell types susceptible to attack by a subset of cytotoxic γδ T cells. In vitro studies
have demonstrated this effect in cell lines from a broad range of solid and
haematological malignancies; however, little is known about how ZA affects γδ T cell
targeting of non-malignant cell types. In this study we have focussed on targeting of
macrophages (Mϕs), as these cells have recently been shown to take up
bisphosphonates. We isolated monocytes from the peripheral blood of healthy donors,
and differentiated these cells in vitro into prototypic Mϕs. We generated pro- and antiinflammatory Mϕs by treating cells with either IFN-γ or IL-4, respectively. IFN-γ-treated
Mϕs displayed elevated expression of CD64 and increased production of IL-12p70;
whereas, IL-4-treated Mϕs displayed elevated expression of CD206 and increased
production of CCL18. The susceptibility of these Mϕs to γδ T cell cytotoxicity was
assessed using flow cytometry-based assays, and found to be markedly increased by ZA
treatment. Investigations into the mechanism by which γδ T cells kill these ZA-treated
Mϕs are currently underway. The findings presented here suggest that ZA can increase
the susceptibility of Mϕs to γδ T cell killing, which has important implications regarding
the use of ZA in macrophage-driven disease.
Page 159 of 200
GDC 2016
C-34
Ex vivo expanded γδ T cells can kill patient-derived breast cancer stem
cells (BCSC) in vitro and lead to tumor regression in BCSC-derived
xenografts
1,3
2
1
2
Katrin Raute , Juliane Strietz , Gina Fiala , Jochen Maurer and Susana Minguet
1,3
1 Department of Molecular Immunology, Faculty of Biology, and BIOSS Centre for Biological
Signaling Studies,
University of Freiburg, Germany
2 University Hospital Freiburg, Germany
3 Spemann Graduate School of Biology and Medicine, University of Freiburg, Germany
T cell-based cancer immunotherapies gained a lot of attention in the last few years.
Clinical trials have revealed that T-cell immunotherapies are mainly successful against
non-solid cancers, such as hematopoietic malignancies1. This effect is among others
mainly attributable to the need for specific T-cell receptors against tumor antigens and for
T cells being capable of infiltrating the sites of disease. A further emerging challenge in
the field of treating solid tumors has been the discovery of cancer stem cells. These cells
are more resistant to conventional chemotherapeutical approaches and eventually lead
to higher relapse cases. Two T-cell subsets are currently being investigated in clinical
trials: alpha/beta T cells (αβTc) and gamma/delta T cells (γδTc). In contrast to αβTc,
γδTc display the advantage of being major histocompatibility complex-unrestricted and
recognizing stress-induced molecules as well as phosphoantigens accumulating during
cancer cell metabolism2.
Based on previously reported protocols3, we expanded ex vivo human highly cytotoxic
effector memory γδTc. In vitro, these cells efficiently kill breast cancer stem cells (BCSC)
derived from patient tumor samples. Furthermore, the treatment of BCSC-derived
xenograft tumors in NOD/SCID mice with our ex vivo expanded γδTc showed a
statistically significant amelioration of the tumor burden. Based on these results, our
xenograft model using primary human BCSCs can be further explored to ameliorate anticancer responses by ex vivo expanded cytotoxic γδTc.
References:
1. Kalos et al., Science Translational Medicine, 2011
2. Uchida et al., Biochemical and Biophysical Research Communications, 2007
3. Siegers et al., Cancer Immunology and Immunotherapy, 2013
Page 160 of 200
GDC 2016
C-35
Immune checkpoint blockade combinations as promising strategy for
cancer immunotherapy in multiple myeloma patients
1,2
1,2
1,2
Barbara Castella , Myriam Foglietta , Patrizia Sciancalepore , and Massimo
1,2
Massaia
1Department of Molecular Biotecnology and Health Science, Hematology Division, University of
Turin, Italy;
2 Center for Experimental Research and Medical Studies (CeRMS)
Vg9Vd2 T cells (γδ) are very attractive candidates for adoptive immunotherapy in
multiple myeloma (MM), but clinical trials with autologous γδ have not fulfilled
expectations. The immune microenvironment is highly dysfunctional in the bone marrow
(BM) of MM patients. We have recently shown that γδ are anergic in the BM of MM
patients and we have identified the PD-1/PD-L1 inhibitory pathway as responsible for the
BM γδ immune paresis. There is emerging evidence that hematological tumors exploit
immune checkpoints as a major immune resistance mechanism.
Some checkpoint blockade combinations are in the early clinical development stage for
solid tumors, combining anti-PD1 with antibodies blocking BTLA, LAG-3 or TIM-3. Our
preliminary data show that BM γδ express other inhibitory receptors beyond PD-1. We
analyzed TIM-3 expression in peripheral blood (PB) and BM γδ from healthy donors
(CTRL) and MM patients and observed a significantly up-regulation of this inhibitory
receptor selectively in BM γδ T cells. Moreover, TIM-3 expression in γδ after 7-day
stimulation of PB and BM mononuclear cells from CTRL and MM with IL-2 and ZA+IL2
increased only in γδ anergic to pAg stimulation, suggesting a further up-regulation of
immune checkpoint receptors upon ineffective TCR engagement by pAgs. Later, ZA
stimulation was carried out in the presence of neutralizing anti-PD-1 and anti-TIM-3
antibody and we observed a significantly recovery of BM γδ proliferation after the
combined treatment. In conclusion, our preliminary data suggest that immune checkpoint
blockade combinations could be a potentially promising approach for cancer
immunotherapy in MM patients.
Page 161 of 200
GDC 2016
C-36
The effect of activation on the intracellular production, and subsequent
+
release, of the cytotoxic effector molecule granulysin by Vδ2 γδ T cells.
Miss Emma Sparrow, Dr Daniel Fowler, Professor Angus Dalgleish & Dr Mark BodmanSmith,
γδ T cells are capable of killing tumour directly using cytotoxic molecules, or indirectly
through activation of other immune effectors. The link between innate and adaptive
+
immune responses to tumour can be partly attributed to granulysin secretion by Vδ2 γδ
T cells. A cytolytic 9kDa isoform of granulysin kills tumour directly, while a 15kDa
precursor is thought to be a chemoattractant. Attraction of dendritic cells to tumour would
allow initiation of adaptive immune responses, through internalization of tumour material
+
+
and subsequent migration to lymph nodes, activating CD4 and CD8 T cells.
Peripheral blood mononuclear cells were treated with stimulants known to activate γδ T
cells. Flow cytometry allowed identification of γδ T cell populations and changes in
intracellular granulysin, while ELISA determined secreted granulysin levels. 75% of γδ T
cells expressed 15kDa granulysin. 9kDa granulysin was low in resting γδ T cells, but
increased by 40% on BCG treatment. Granulysin secretion was observed on treatment
+
with ZA or BCG, and was associated with an increase in CD107a expression on Vδ2 γδ
T cells. Identical stimulation of isolated γδ T cells yielded no activation, as shown
through an absence of IFNγ expression, suggesting additional immune cells are required
+
for γδ T cell activation. However, activation of Vδ2 γδ T cells and subsequent secretion
of granulysin was observed when isolated cells were exposed to a tumour target in vitro.
These data support the hypothesis that γδ T cells, activated by tumour, release
granulysin, which may recruit DCs and initiate adaptive immune responses.
Page 162 of 200
GDC 2016
C-37
Galectin-3 is released in the interaction of PDAC tumor cells and T cells
Daniel Gonnermann, Hans-Heinrich Oberg, Dietrich Kabelitz, Daniela Wesch
Institute of Immunology, Christian-Albrechts-University Kiel, Germany
Pancreatic ductal adenocarcinoma (PDAC) is a malignant disease with poor prognosis
and fewtreatment options. To establish an immunotherapy for PDAC with gd T
lymphocytes the dissection of the gd T cell and PDAC cell interaction is of great
importance. In this study, we focused on galectin-3, a multifunctional b-galactosidebinding protein, which is described to be highly expressed in PDAC cells. Extracellular
galectin-3 binding to cell surface Nglycans
of T cells has been shown to suppress T cell activation and induce apoptosis. To test
whether Galectin-3 is involved in PDAC cell and gd T cell interaction, we measured
galectin-3 expression and galectin-3 secretion of PDAC cell lines and ab as well as gd T
cells alone or in coculture with PDAC cells. Additionally, we studied effects of exogenous
galectin-3 on T cells. Intracellular galectin-3 is expressed in all applied PDAC cells and,
interestingly, also in gd and ab T cells, respectively. gd T cells but not PDAC cells alone
slightly released galectin-3 after their activation. In co-culture of PDAC cells with either
ab or gd T cells, however, large amounts of galectin-3 were secreted. Ongoing
experiments with siRNA and Imagestream technology will clarify whether galectin-3 in
co-culture is produced by PDAC cells as a kind of tumor-escape mechanism or by ab or
gd T cells as a cytotoxic mediator.
Overall our preliminary results suggest that galectin-3 is involved in the interaction of gd
T lymphocytes and PDAC cells, which has to be further dissected before establishing an
immunotherapy with gd T cells.
Page 163 of 200
GDC 2016
C-38
γδ T cell Apoptosis Induced via “Blocking” Antibodies: A Cautionary Tale
1
2
1
Indrani Dutta , Dalam Ly , Lynne-Marie Postovit and Gabrielle M. Siegers
1
1 Department of Experimental Oncology, University of Alberta, Edmonton, AB, Canada
2 University Health Network, University of Toronto, Department of Immunology, Toronto, ON,
Canada
Mechanistic studies contribute greatly to our understanding of γδ T cell (γδTc) biology,
aiding development of these cells as immunotherapeutic agents. The antibody blocking
assay is an accepted method to determine the receptors involved in γδTc killing of
tumour targets. Effectors and/or targets are pre-ˇ‐incubated with microgram quantities of
so-ˇ‐called “blocking” antibodies, advertised as such by commercial sources. We and
others have used such assays extensively in the past, correlating decreases in
cytotoxicity against specific targets with involvement of the blocked receptor(s). However,
we wondered whether other mechanisms might be at play beyond cytotoxicity inhibition.
Indeed, administration of certain blocking antibodies induced γδTc death, proportional to
the observed decrease in cytotoxicity. Upon further investigation, we discovered that
γδTc undergo apoptosis triggered by incubation with certain “blocking” antibodies. This
induction of activation-ˇ‐induced cell death (AICD) also explained flow cytometry results
in which we were unable to consistently detect blocking antibody binding to γδTc using
fluorophore-ˇ‐conjugated secondary antibodies. The γδ TCR undergoes rapid
internalization upon stimulation with an activating antibody, thus it seems that some of
these “blocking” antibodies may actually be “activating”. We are now testing a panel of
purified anti-ˇ‐γδ TCR antibodies to determine which clone(s) may prove better blocking
antibodies than those currently in use.
Page 164 of 200
GDC 2016
C-39
Interleukin-17 Producing Vγ9Vδ2 T cells; Frequency, Phenotype and
Differentiation
1
1
1
Simone Kloch Bendtsen , Manja Idorn , Gitte Holmen Olofsson , Per thor Straten
1,2
1 Center for Cancer Immune Therapy, Department of Hematology, University Hospital Herlev,
Denmark
2 Department of Immunology and Microbiology, University of Copenhagen, Denmark
Promising results of adoptive cell transfer (ACT) with tumor reactive T cells expanded
from tumor infiltrating lymphocytes (TILs) are limited to cancer patients where the tumor
can be surgically removed and/or tumor reactive T cells expanded. Therefore research
into other cell types like the major γδ T cell subtype in the circulation, Vγ9Vδ2, are
ongoing. These cells can be isolated from a simple blood sample and expanded in vitro
using drugs already approved for therapy. Furthermore they respond to
phosphoantigens, which are upregulated on cancer cells. For many different cancer cell
lines a direct correlation between the recognition of the cells by Vγ9Vδ2 T cells and
accumulation of phosphoantigens have been found.
However, Vγ9Vδ2 T cells are capable of producing IL-17. This cytokine has been linked
to several different pro-tumor activities like promotion of proliferation and metastatic
potential of the cancer cells. Vγ9Vδ2 T cells therefore need careful scrutiny before being
used in adoptive therapy, as the tumor microenvironment contains many different stimuli
and thus could support the differentiation of IL-17- producing γδ T cells.
We wish to investigate the parameters that drive in vitro expanded γδ T cells in an IL-17producing direction using cytokine cocktails of IL-1β, IL-6, IL-23 and TGF-β. Importantly
we aim to study if co-culture with different cancer cell lines or supernatants from these
can push γδ T cells into secreting IL-17. IL-17-producing γδ T cells will subsequently be
investigated for capacity to kill cancer cells and their impact on cancer cell growth in vitro
and in vivo.
Page 165 of 200
GDC 2016
C-40
Generating highly proinflammatory cytolytic gamma-delta T cells for
targeting cancers
Cheryl Lai-Lai Chiang, Peter Wong and Han Chong Toh.
National Cancer Centre Singapore, 11 Hosptial Drive, Singapore 169610.
Gamma-delta T cells are important innate immune cells for first-line defense against
transformed and pathogen-infected epithelial cells. In particular, Vgamma9delta2 T cells
can be expanded up to 30–50% upon bacterial or protozoan infection. Gamma-delta T
cells have been used for treating cancers. Two main methods are the ex vivo expansion
of gamma-delta T cells from peripheral blood and the in vivo expansion of these cells
with zoledronic acid administration. Ex vivo expansion of gamma-delta T cells is a
versatile approach as we can manipulate the cells before administrating back to patients.
We have optimised a simple yet robust protocol to expand gamma-delta T cells ex vivo
for clinical use. We evaluated the use of pooled human AB serum versus fetal bovine
serum (FBS) for culturing gamma-delta T cells and different cytokine combinations (i.e.
IL-2 alone, IL-2 + IL-7 and IL-2 + IL-15). Highest number and purity of gamma-delta T
cells was seen with Click’s media containing 10% FBS, zoledronic acid and IL-2. We
found that gamma-delta T cells expanded with different cytokines exhibited different
levels of cytotoxic and antigen-presentation phenotypes. Upon stimulation with PMA and
ionomycin, these gamma-delta T cells rapidly produced proinflammatory IFN-gamma,
TNF-alpha and no IL-10. Finally, we demonstrated that these gamma-delta T cells
directly kill many tumour types through the release of granzymes and perforin. Thus, we
have optimized a novel and robust for ex vivo generation of highly proinflammatory and
highly cytolytic gamma-delta T cells for targeting many types of tumours.
Page 166 of 200
GDC 2016
C-41
Redirecting vδ1 and vδ2 γδ T cell specificity by genetic modification with
a GD2 specific chimeric antigen receptor
1
1
1
1
1
Anna Capsomidis , Gabriel Benthall , Talia Gileadi , Rebecca Wallace , Karin Straathof ,
2
1
1
1
Martin Pule , Kenth Gustafsson Barry Flutter and John Anderson
1
Immunotherapy and Immune Regulation in Childhood Cancers Group, Developmental Biology and
Cancer Programme, Cancer Section, Institute of Child Health and Great Ormond Street Hospital,
University College London, London, United Kingdom
2
Cancer Institute, Department of Haematology, Division of Medicine, University College London,
London, United Kingdom
Chimeric antigen receptor-engineered T-cell (CAR-T-cell) therapy has shown great
promise, particularly against CD19-expressing haematological malignancies. Translation
to solid tumours is hindered partly due to the immunosuppressive tumour
microenvironment impairing T-cell homing, cytotoxicity and survival. Unconventional Tcells, including vδ1 and vδ2 γδ T-cells, have additional combined functional properties
potentially advantageous for cancer immunotherapy. Here we describe an approach for
the propagation and transduction of GD2-specific vδ1 and vδ2 CAR-T-cells, their
cytotoxicity profile, and desirable phenotypic characteristics for immunotherapy.
Concanavilin A (ConA) or zoledronate (ZOL) activated peripheral blood mononuclear
cells were efficiently transduced with a second-generation GD2-specific CAR, consisting
of CD3ζ and CD28 signalling domains. Using ConA or ZOL, vδ1 and vδ2 CAR-T-cells,
respectively, were propagated to clinically relevant number for adoptive transfer following
patient leukapheresis. Efficacy was measured by chromium-release assay, phenotypic
analysis, cytokine quantification, and CFSE cell proliferation.
+
αβ, vδ1 and vδ2 CAR-T-cells showed equivalent cytotoxicity towards GD2 cell lines.
Antibody dependent cellular cytotoxicity by non-transduced vδ2 was equivalent to vδ2
CAR-T-cells following target opsonisation with anti-GD2 antibody. Activated vδ2 CAR-Tcells were predominantly ‘effector memory’ phenotype (CD45RA /CD27 ), with high
expression of CD86 and HLA-DR. Conversely, vδ1 CAR-T-cells had the highest
proportion of ‘naïve’ cells with lowest expression of exhaustion markers, PD-1 and Tim3.
In summary, γδ CAR-T-cells can be generated in sufficient number for adoptive transfer.
Further study is required including whether ‘naïve’ vδ1 cells have greater proliferative
capacity, homing ability, and survival in vivo, and whether expanded vδ2 CAR-T-cells
with a ‘professional antigen presenting cell’ phenotype can cross-present antigen.
Page 167 of 200
GDC 2016
C-42
(+)
Identification of variation in cytotoxic potential of tumour-reactive Vδ2 Tcells among a healthy cohort
1
1
1
Christopher J. Holland , Paul Ryan , Nital Sumaria , and Daniel Pennington
1
1
Blizard Institute, Barts and the London Medical School, Queen Mary University
γδ T-cells have demonstrated important functional roles in a range of disease settings
+
including cancer. γδ T-cells expressing the TCR Vγ9Vδ2 (Vδ2 ), which can be activated
by phosphoantigens, such as IPP, which accumulates in transformed eukaryotic cells,
have shown potential in phase I/II clinical trials of haematological malignancies.
However, objective responses have varied within trial cohorts and among trials.
+
Phenotyping of the δ2 T-cell subset, within a health human donor cohort (unpublished
data), has identified an inherent disparity in CD27, CD28, and CD16 expression across
+
the cohort. These markers allow an individuals’ δ2 T-cell subset to be categorised into
six differing expression profiles. Moreover, gene micro array data comparing the two
+
+
+
extreme profiles (Profile 1: CD28 , CD27 , CD16; Profile 6: CD28 , CD27 , CD16 ) signify
functional differences such as cytotoxicity and chemotaxis.
+
Our data indicates that δ2 T-cell profiles possess differing cytotoxic potentials that
appear dependent upon which cytolytic granules they express. Specifically, profile 1
individuals express granzyme K whereas profile 6 individuals co-express granzyme B
+
and granulysin. We hypothesise that clinical outcome of δ2 T-cell adoptive cell therapies
+
will be improved by matching the most suitable δ2 T-cell profile to tackle a
haematological malignancy. Specifically, matching the cytotoxic mechanism possessed
+
by a δ2 T-cell profile to potential tumour escape mechanisms employed, such as
expression of PI-9, a granzyme B inhibitor, which has been associated, for example, with
high grade malignancy in B-cell Non-Hodgkin lymphoma patients.
Page 168 of 200
GDC 2016
C-43
(+)
Phenotypic and functional re-evaluation of human Vδ2 T cells across
healthy individuals
12
1
1
1
1
3
P.L. Ryan , N. Sumaria , C.J. Holland , C.M. Bradford , N. Izotova , A.S. Jawad , L.A.
4
1
Bergmeier , D.J. Pennington
1
Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine and
Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, United Kingdom;
2
Centre for Adult Oral Health, Institute of Dentistry, Barts and The London School of Medicine and
Dentistry, Queen Mary University of London, Turner Street, London, E1 2AD, United Kingdom;
3
Rheumatology Department, Royal London Hospital, London, United Kingdom;
4
Centre for Clinical and Diagnostic Oral Sciences, Institute of Dentistry, Barts and The London
School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1
2AT, United Kingdom.
Human γδ T cells display potent responses to a spectrum of pathogens and
malignancies. Of particular interest are those expressing a γδ T cell receptor
(+)
incorporating TCRδ-chain variable-region-2 (Vδ2 T cells). These cells are uniquely
activated by phosphoantigens (pAgs) such as HMB-PP and IPP that accumulates in
transformed eukaryotic cells, or in healthy cells exposed to aminobisphosphonates.
(+)
Indeed, methods used to boost Vδ2 T cell cytotoxic activity against solid tumour targets
have shown mixed patient responses in clinical trials to date. Therefore, there is a
(+)
pressing need to re-evaluate Vδ2 T cell functional variation at the individual level to
help improve immunotherapeutic outcomes.
(+)
Re-evaluation of conventional Vδ2 T cell surface markers in 63 healthy individuals
(+)
using CD28, CD27, and CD16 unambiguously identifies four Vδ2 T cell subsets
subdividing human individuals into six “Vδ2-profiles”.
Analysis of these profiles
(+)
demonstrate the spectrum of Vδ2 T cell phenotypes, ranging from individuals with a
(28+)
+
+
high proportion of the highly proliferative γδ
[CD28 CD27 CD16 ] subset to those with
(16+)
-
-
+
a γδ
[CD28 CD27 CD16 ] phenotype exhibiting reduced proliferative ability but
increased cytotoxic marker expression and function. These profiles appear to be stable
over time and do not correlate with age, gender, ethnicity, or history of phosphoantigen
activation.
(+)
In conclusion, the use of CD28, CD27 and CD16 to phenotype Vδ2 T cells highlights
the significant heterogeneity in the human population, dividing individuals into distinct
“Vδ2-profiles”. Stratification of individuals by “Vδ2-profile” may be useful in predicting
(+)
Vδ2 T cell responses in infectious scenarios and in development of more predictable
cancer immunotherapies.
Page 169 of 200
GDC 2016
C-44
Optimizing adoptive cancer immunotherapy with Vγ2Vδ2 T cells through
zoledronate pulse stimulation
*, †, ‡, §
Mohanad H. Nada,
Hong Wang,
¶
*, †, ‡
Tanaka, and Craig T. Morita
*, †
Grefachew Workalemahu,
*, †
Yoshimasa
*
Division of Immunology, Department of Internal Medicine, University of Iowa Carver College of
Medicine, Iowa City, IA 52242, U.S.A.
†
Department of Veterans Affairs, Iowa City Health Care System, Iowa City, IA 52246, U.S.A.
‡
Interdisciplinary Graduate Program in Immunology, University of Iowa Carver College of Medicine,
Iowa City, IA 52242, U.S.A.
§
Department of Pathology, College of Medicine, Tikrit University, Iraq
¶
Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences,
Nagasaki University, Nagasaki 852-8521, Japan
Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl
pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes
and tumors. Vγ2Vδ2 cells have been used for adoptive cancer immunotherapy with
some partial and complete remissions. Most trials have used continuous zoledronate
exposure to expand Vγ2Vδ2 cells. Zoledronate inhibits farnesyl pyrophosphate synthase
causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells.
Because zoledronate exposure is toxic, we hypothesized that a short period of exposure
would reduce T cell toxicity but still be sufficient for monocytes uptake. Supporting this
hypothesis, pulse zoledronate exposure with IL-2 resulted in more uniform expansion of
Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous
exposure. These Vγ2Vδ2 cells had higher levels of CD107a and perforin and slightly
increased tumor cytotoxicity. Importantly, adoptive immunotherapy with Vγ2Vδ2 cells
derived by pulse stimulation controlled human PC-3 prostate cancer tumors in
immunodeficient NSG mice significantly better (halting tumor growth) than those derived
by continuous stimulation. Pulse zoledronate stimulation of Vγ2Vδ2 cells with IL-15 also
resulted in higher purity and cell numbers. Like with CD8 αβ T cells, IL-15 preserved
early memory Vγ2Vδ2 T cell subsets better than IL-2. However, adoptive immunotherapy
with Vγ2Vδ2 cells derived with IL-15 showed similar inhibition of PC-3 tumor growth as
those derived with IL-2. Thus, pulse zoledronate stimulation maximizes the purity,
quantity, and quality of expanded Vγ2Vδ2 cells. This simple modification to existing
protocols would likely enhance the effectiveness of adoptively transferred Vγ2Vδ2 T
cells.
Page 170 of 200
GDC 2016
C-45
Heteromeric interactions regulate Butyrophilin trafficking, expression and
function
P. Vantourout, A. Laing, M.J. Woodward, R. Dart, R. DiMarco Barros, A. Hayday
Peter Gorer Department of Immunobiology, King’s College London;
Francis Crick Institute, London, WC2A 3LY, UK
An emerging commonality of gd T cell biology is their critical regulation by Butyrophilin(like) genes (BTN and BTNL). Thus, BTN3A1 is essential for the activation of human
Vg9Vd2 T cells, while Btnl1 and the related gene, Skint1, are respectively implicated in
murine intestinal and epidermal gd T cell selection. However, the precise mechanism by
which these molecules perform their function is largely unknown and debated. Most
BTN(-L) are grouped in sub-families. For instance, BTN3A2 and BTN3A3 are closely
related to BTN3A1, but their function has not been explored.
Disrupting BTN3A genes using CRISPR/Cas9, we established that all BTN3A genes
contribute
to
Vg9Vd2 T
cell
activation.
Moreover,
their
expression
displayed
characteristics of intracellular retention by an active mechanism affecting their trafficking,
which was regulated by their co-expression and dependent on their interaction via the
IgC domain. Using several approaches we could determine that BTN3A gene products,
particularly BTN3A1 and 3A2, were retained in the Endoplasmic Reticulum (ER). This
was attributed to the presence of canonical ER retention/retrieval signals in their
intracellular domains. Importantly, we show that ER retention is a critical factor
contribution to their activity. Finally, regulation of expression by heteromerization was a
common feature of several human and murine butyrophilin(-like) proteins.
In sum, we identified a functional role for human BTN3A2 and BTN3A3 and
characterized key aspects of the regulation of BTN3A, which extend to several members
of the butyrophilin family. This new insight may help us decipher the mechanisms by
which they uniquely regulate gd T cell selection and activation.
Page 171 of 200
GDC 2016
Poster Session A
combining Sessions 1, 2 and 3
Poster presentation: June 16th, 6:30 – 8:30pm & June 17th, 1:00 – 2:30pm
Author
PosterID
Author
PosterID
CaitlinCastro
1
MuroRyunosuke
26
SalahMansour
2
BenjaminWillcox
27
SciancaleporePatrizia
3
MartineKallemeijn
28
PayalDamani-Yokota
4
BrunoMartin
29
5
StefaniaMartin
30
31
RalphBudd
AngelaPappalardo
6
NitalSumaria
CarrieWillcox
7
GinaFiala
32
AndreeaPetrasca..
8
MiguelMuñozRuiz
33
MohindarMurugeshKarunakaran
9
Cheol-HeuiYun
34
T.Hermann
10
JoanaBarrosMartins
35
HongWang...CraigT.Morita
11
RasmusAgerholm
36
12
EdwardL.Y.Chen
37
13
MartinDavey
38
39
SiyiGu
Dr.DavidRhodes
AlinaFichtner
14
ChristianPeters
DanielPaletta
15
LucieAbeler-Dörner
40
MsAnnaVyborova
16
LeonceKouakanou
41
MahboobSalim
17
MariaPapadopoulou
42
RafaelDiMarcoBarros
18
CristianaCairo
43
RobinDart
19
PaolaTieppo
44
NatalieA.Roberts
20
CherylCollins
45
21
YihuaCai
46
47
AudreyBenyamine
ZsoltSebestyen
22
PayamZarin
DeborahWitherden
23
CiroNovaesRosaLino
48
NinaSchmolka
24
AnnaMorath
49
SérgioT.Ribeiro
25
JianleiHao
50
Page 172 of 200
GDC 2016
Poster Session B
combining Sessions 4, 5 and 6
Poster presentation: June 17th, 4:15 – 6:00pm & June 18th, 1:15 – 2:30pm
Author
PosterID
Author
PosterID
1
Maya Hassane 26
2
Sean O'Farrell
27
Ferdinando Oriolo
3
Hannah Kaminski, 28
Duncan McKenzie
4
Hannah Kaminski, L. Couzi
29
Julie C. Ribot
5
Dr Ceri Fielding
30
6
Thomas Winkler 31
7
Alanna Murday
32
33
Dmitry Ushakov
Maria Luisa Iannitto
Anna Rita Liuzzi
Karen Edelblum
Annika Reinhardt
8
Andreea Petrasca
Takeshi Nitta
9
Dr. Caroline Sutton
34
Shirin Kalyan
10
Mathilde Raverdeau
35
Anneke Wilharm
11
Gabrielle M. Siegers
36
Joe Fenn
12
Migalovich Sheikhet Helena
37
Fernanda Kyle
13
Julie Jameson
38
14
Pedro Papotto
39
15
Inga Sandrock 40
41
Oliver Nussbaumer Hongbo Shen
Alicja Misiak
16
Neil McCarthy
Bruno Silva-Santos
17
Ayano Kohlgruber
42
Immo Prinz
18
Tom Hartwig 43
Maria Mamani Matsuda
19
Morten M. Nielsen
44
Ling MA 20
Xiaoyang Wang
45
Bok Luel lee
21
Xu Weili
46
22
Ignacio Catalan-Serra
47
23
Irma Airoldi
48
24
Avadhesh Kumar Singh
49
25
Paulina Kulig
50
Kenth Gustafsson
Jonathan Boyson
Dr. Ling Shen
Xizhi Guo
Page 173 of 200
GDC 2016
Poster Session C
combining Sessions 7 and 8
Poster presentation: June 18th, 4:30 – 6:30pm & June 19th, 1:30 – 2:30pm
Author
PosterID
Author
PosterID
ClementBarjon
1
EricChampagne
24
SethCoffelt
2
DaisyJohnson
25
JoseVillacortaHidalgo
3
AnetaZysk
26
ChristinePasero
4
HeleenVanAcker
27
NathalieJacobs
5
GitteHolmenOlofsson
28
6
GitteHolmenOlofsson.
29
30
GregCrawford
MargaretDunne
7
CynthiaChauvin
SofiaMensurado
8
MaryPoupot
31
JianHuang
9
DanielVargasCorreia
32
DanielaWesch
10
DanielFowler
33
Hans-HeinrichOberg
11
KatrinRaute
34
AndreaKnight
12
BarbaraCastella
35
13
MissEmmaSparrow
36
14
DanielGonnermann
37
38
MariaKrchniakova
RomanaKralova
ElenaLoPresti
15
IndraniDutta...GabrielleM.Siegers
KilianWistuba-Hamprecht
16
SimoneKlochBendtsen
39
AriadniKouzeli
17
CherylChiang
40
GurandaChitadze
18
DrAnnaCapsomidis
41
MateuszLegut
19
ChrisHolland
42
PierreAbramowski
20
DrPaulLeoRyan
43
NaomiHodgins
21
MohanadNada...CraigT.Morita
44
22
PierreVantourout
45
23
TimmHöres
JaydeepBhat
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Notes
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Thank you for coming, have a
great time. Don’t forget to ask
us if there is anything you
think we can help with.
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