Download Chapter 3. Characterising αCXCR3-9C5

CHARACTERISATION OF THE
IMMUNOMODULATORY ACTION OF A UNIQUE
ANTI-CXCR3 MONOCLONAL ANTIBODY
EXPLOITING A CONSERVED HUMAN AND
MURINE EPITOPE FOR TRANSPLANTATION
Suzanna M. Pilgrim
A thesis in fulfilment of the requirements for the degree of
Masters of Philosophy
Department of Physiology, The University of Sydney,
January 2016
Department of Immunology, Garvan
Institute of Medical Research
Table of Contents
Acknowledgements ....................................................................................................... vi
Tables .................................................................................................................................. vii
Figures ............................................................................................................................... viii
Abbreviations ................................................................................................................... xi
Awards, Publications and Presentations arising from this Thesis ...... xv
Abstract............................................................................................................................. xvi
Experiments that were not the sole work of the author ......................... xvii
Chapter 1. Introduction
The role of CXCR3 in type 1 diabetes and islet transplantation ...........1
Overview .........................................................................................................................1
Type 1 Diabetes Mellitus .........................................................................................2
Classification and Epidemiology .............................................................................2
Symptoms .......................................................................................................................2
Genetic and Environmental Susceptibility .............................................................3
Human Pathogenesis and Natural History.............................................................4
Animal Models ..............................................................................................................5
Therapies ........................................................................................................................8
Islet Transplantation
............................................................................................ 10
Transplantation ......................................................................................................... 10
Allograft Immune Response – A role for T cells ................................................ 10
Antigen Presentation (Signal 1) ............................................................................. 11
T cell Co-stimulation (Signal 2) .............................................................................. 11
i
Cytokine and Chemokine Milieu (Signal 3) ......................................................... 11
T cell Effectors and Immune Regulation ............................................................... 12
Islet Allograft Success and Immunosuppressive Therapy ............................... 13
Current Immunosuppressants.................................................................................. 15
Chemokines
CXCR3
............................................................................................................... 17
......................................................................................................................... 19
CXCR3 Expression ................................................................................................... 19
CXCR3 Chemokines................................................................................................. 20
Receptor Structure .................................................................................................. 21
Signaling Pathways ................................................................................................... 23
Experimental Models .............................................................................................. 25
CXCR3 in Inflammation .......................................................................................... 25
CXCR3 in Diabetes .................................................................................................... 28
CXCR3 in Allograft.................................................................................................... 30
Thesis Aims ................................................................................................................. 34
Chapter 2. Materials and Methods .......................................................... 36
Materials ....................................................................................................................... 37
Methods......................................................................................................................... 39
Mice .............................................................................................................................. 39
Islet Isolation and Transplantation ....................................................................... 40
Islet Isolation............................................................................................................... 40
Islet Transplantation.................................................................................................. 41
Antibody ...................................................................................................................... 41
Antibody Treatments ............................................................................................... 42
Allograft ....................................................................................................................... 42
Type 1 Diabetes ........................................................................................................... 42
ii
CD25 Depletion – PC61 ............................................................................................ 42
Cell Population Analysis .......................................................................................... 43
Lymphocyte Isolation................................................................................................. 43
Flow Cytometry ........................................................................................................... 43
CFSE Proliferation Assay/Mixed Lymphocyte Reaction ................................ 44
Antibody Characterisation ..................................................................................... 45
Chemotaxis Assay....................................................................................................... 45
Receptor Internalisation Assay ................................................................................ 45
Competitive Binding Assay ....................................................................................... 46
Antibody Titrations .................................................................................................... 46
Adoptive Transfer ..................................................................................................... 46
Histology ...................................................................................................................... 47
H&E .............................................................................................................................. 47
Insulin Staining .......................................................................................................... 48
Foxp3+ Staining ......................................................................................................... 48
Statistics ....................................................................................................................... 49
Chapter 3. Characterising αCXCR3-9C5....................................... 51
αCXCR3-9C5 Impairs Chemotaxis of CXCR3 transfected cells ....................... 52
Effects of αCXCR3-9C5 on CXCR3 expression and cell stability ................... 53
Receptor internalisation and competitive binding assays .................................... 54
Summary & Conclusions ............................................................................................. 58
Chapter 4. αCXCR3-9C5 in the Islet Allograft Model ............ 61
Peripheral Immune cell characterisation of CXCR3 Expression ..................... 61
αCXCR3-9C5 in C57BL/6 Islet Allografts .............................................................. 65
Basal and Islet Allograft CXCR3 Expression ......................................................... 66
iii
αCXCR3-9C5 Binds CXCR3 and Reduces Memory and Effector CD8+ T cells
............................................................................................................................................ 68
αCXCR3-9C5 Prolongs C57BL/6 Islet Allograft Survival ................................. 77
αCXCR3-9C5 and Rapamycin Therapy Preserve Islet Allograft Architecture
............................................................................................................................................ 79
A role for Regulatory T cells in αCXCR3-9C5 and Rapamycin treated Islet
Allograft survival........................................................................................................... 80
Summary & Conclusions ............................................................................................. 82
Chapter 5. αCXCR3-9C5 in the NOD Autoimmune
Diabetic Model.......................................................................................................... 86
Peripheral Immune cell characterisation of CXCR3 Expression ..................... 87
Basal CXCR3 Expression ............................................................................................ 89
NOD Insulitis Scoring .................................................................................................. 90
NOD Diabetes Incidence .............................................................................................. 91
αCXCR3-9C5 does not alter Diabetes Incidence in the NOD mouse ................ 92
αCXCR3-9C5 does not alter Insulitis or Regulatory T cell Migration in the
NOD mouse ..................................................................................................................... 95
αCXCR3-9C5 reduces CXCR3 but not CD44 expression in the NOD mouse 97
Summary & Conclusions .......................................................................................... 102
Chapter 6. αCXCR3-9C5 in the NOD Islet Allograft
Model................................................................................................................ 105
αCXCR3-9C5 Prolongs C57BL/6 Islet Allograft Survival ................................ 106
Summary & Conclusions .......................................................................................... 111
Chapter 7. Clarifying the Role of αCXCR3-9C5 ..................... 114
Summary & Conclusions .............................................................................................. 118
Chapter 8. Discussion ....................................................................................... 121
iv
Overview
................................................................................................................... 121
Allograft Transplantation ................................................................................. 121
T cell Activation & Regulatory T cells ............................................................... 122
Allograft Transplantation in the NOD mouse
....................................... 124
The NOD Mouse ..................................................................................................... 124
Conclusion ................................................................................................................. 126
Chapter 9. References....................................................................................... 127
v
Acknowledgements
I would like to thank my supervisor, Shane Grey, for all the support and advice you
have given me over the past two years. Without you I would not have been able to
complete this thesis.
I would like to thank all of the members of Lab 54 for your help, trouble shooting
and moral support, which have been invaluable. In particular; thank you to the ever
patient Stacey Walters without whom I could not have succeeded in my Masters and
to Nathan Zammit who laid the foundation for this work. It has been a pleasure to get
to know and work with you all.
Thank you to my amazing partner Luke, who has remained my rock over the past
two years, through all of my academic and personal struggles. I would not have made
it this far without your love, support and patience.
I would also like to thank my family for all their support and encouragement without
whom, I would not have my love of science or my drive to succeed. Thank you for
the experiences I was given growing up, which taught me that hard work and
perseverance pay off, I wouldn’t be here without those lessons, so thank you.
Lastly but certainly not least, I dedicate this thesis to my amazing dad who sparked
my interest and nurtured my love for science.
vi
Tables
Table 1.1: Immunosuppressant Side-effects .................................................................. 16
Table 2.1: Buffers/Reagents ............................................................................................. 37
Table 2.2: Flow Cytometry Staining Conditions........................................................... 49
Table 2.3: Antibodies for Flow Cytometry ..................................................................... 50
Table 4.1: C57BL/6 CXCR3 Expression Profiles .................................................... 68
Table 5.1: NOD CXCR3 Expression Profiles .......................................................... 90
vii
Figures
Figure 1.1: Comparison of Human T1D and NOD diabetes. .................................. 7
Figure 1.2: Islet Transplantation ............................................................................. 14
Figure 1.3: Leukocyte Chemotaxis ........................................................................... 18
Figure 1.4: CXCR3 in immune cell migration ........................................................ 21
Figure 1.5: Snakeplot structure of CXCR3 amino acid sequence .......................... 22
Figure 3.1: αCXCR3-9C5 antibody binds a conserved epitope and impairs
Lymphocyte Chemotaxis ........................................................................................ 53
Figure 3.2: Equivalent Binding of CXCR3+ T cells by αCXCR3-9C5 and
commercial51 available CXCR3-173 antibody. ................................................. 54
Figure 3.3: Internalisation and competitive binding assay. ........................... 55
Figure 3.4: αCXCR3-9C5 competitively binds CXCR3 receptor but does not
cause internalisation or lymphocyte depletion. ................................................ 56
Figure 4.1: αCXCR3-9C5 Antibody Titrations – C57BL/6 .................................. 63
Figure 4.2: αCXCR3-9C5 Antibody Titrations – C57BL/6 .................................. 64
Figure 4.3: Allogeneic Experimental Model ........................................................ 66
Figure 4.4: C57BL/6 CXCR3 Expression Profiles ................................................ 67
Figure 4.5: Untouched and allograft recipient mice total splenocyte counts 69
Figure 4.6: αCXCR3-9C5 treatment in untouched C57BL/6 mice – CD4+ T
cells ............................................................................................................................ 69
Figure 4.7: αCXCR3-9C5 treatment in untouched C57BL/6 mice – CD4+
Foxp3+ T cells .......................................................................................................... 70
Figure 4.8: αCXCR3-9C5 treatment in untouched C57BL/6 mice – CD8+ T
cells ............................................................................................................................ 70
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Figure 4.9: αCXCR3-9C5 treatment in untouched C57BL/6 mice – T cell
activation.................................................................................................................. 71
Figure 4.10: αCXCR3-9C5 treatment in transplanted C57BL/6 mice – CD4+ T
cells ............................................................................................................................ 73
Figure 4.11: αCXCR3-9C5 treatment in transplanted C57BL/6 mice –
CD4+Foxp3+ T cells ................................................................................................. 74
Figure 4.12: αCXCR3-9C5 treatment in transplanted C57BL/6 mice – CD8+ T
cells ............................................................................................................................ 74
Figure 4.13: αCXCR3-9C5 treatment in transplanted C57BL/6 mice – T cell
activation.................................................................................................................. 75
Figure 4.14: Altered activation of naïve and allogeneic C57BL/6 splenocytes
.................................................................................................................................... 76
Figure 4.15: Altered CD44 expression of un-transplanted and allogeneic
C57BL/6 splenocytes .............................................................................................. 77
Figure 4.16: Allogeneic C57BL/6 Islet Transplant ............................................ 78
Figure 4.17: Long-term surviving allograft pathology ..................................... 79
Figure 4.18: A role for Regulatory T cells in αCXCR3-9C5 allograft survival 81
Figure 5.1: αCXCR3-9C5 Antibody Titrations – NOD ......................................... 88
Figure 5.2: NOD CXCR3 Expression Profiles ........................................................ 89
Figure 5.3: NOD Insulitis Grading ........................................................................ 91
Figure 5.4: NOD Diabetes incidence .................................................................... 92
Figure 5.5: Autoimmune NOD Experimental Model........................................... 93
Figure 5.6: αCXCR3-9C5 does not alter NOD Diabetes incidence .................... 94
ix
Figure 5.7: Equivalent diabetes incidence for 1mg/kg or 2mg/kg αCXCR39C5 ............................................................................................................................ 94
Figure 5.8: αCXCR3-9C5 does not affect NOD pancreatic insulitis ................. 95
Figure 5.9: αCXCR3-9C5 does not affect NOD pancreatic regulatory T cell
Infiltrate ................................................................................................................... 97
Figure 5.10: Pre-diabetic NOD αCXCR3 Treatment – CD4+ T cell &
CD4+Foxp3+ T cell analysis ................................................................................... 99
Figure 5.11: Pre-diabetic NOD αCXCR3 Treatment – CD8+ T cell & NK cell
analysis ................................................................................................................... 100
Figure 5.12: Unaltered activation of NOD splenocytes ..................................... 101
Figure 6.1: NOD Allogeneic Experimental Model ............................................. 105
Figure 6.2: αCXCR3-9C5 Prolongs NOD Islet Allograft Survival .................... 116
Figure 6.3: αCXCR3-9C5 NOD Islet Allograft .................................................... 108
Figure 6.4: αCXCR3-9C5 NOD Islet Allograft Long-term survival ................. 109
Figure 6.5: αCXCR3-9C5 NOD Islet Allograft Long-term survival shows
minimal islet insulin staining ............................................................................. 109
Figure 7.1: αCXCR3-9C5 treatment does not alter CD4+ T cell proliferation or
activation ............................................................................................................... 115
Figure 7.2: αCXCR3-9C5 treatment alters expression of CD8+ CD44high T cells
.................................................................................................................................. 117
x
Abbreviations
αCXCR3-9C5 – anti-CXCR3 monoclonal antibody, clone 9C5
αCXCR3-173 – anti-CXCR3 monoclonal antibody, clone 173 (PerCpCy5.5 labelled)
γIRE-1 - IFN-response element
ADCC - antibody-dependent cellular cytotoxicity
AEC - Animal Ethics Committee
Akt - Protein kinase B
APC - antigen presenting cells
APC – Allophycocyanin
ARC - Animal Resource Centre
ASM – airway smooth muscle
ATG - anti-thymocyte globulin
BAL - bronchiolar lavage fluid
BGL - blood glucose levels
CDC - complement-dependent cytotoxicity
CFSE - Carboxyfluorescein succinimidyl ester
CITR - Collaborative Islet Transplant Registry
CNI - calcineurin inhibitor
CTLA-4 - cytotoxic T-lymphocyte-associated protein 4
CTL - cytotoxic T lymphocyte
CXCR3 – chemokine receptor 3
CXCL9/10/11 – chemokines of CXCR3
CXCL9 - monokine induced by gamma interferon (MIG or CXCL9)
CXCL10 - interferon-induced protein of 10kDa, (IP-10 or CXCL10)
CXCL11 - interferon-inducible T cell alpha chemoattractant (I-TAC or CXCL11)
DC - dendritic cell
DT - diphtheria toxin
dLN – distal lymph node
DRY - aspartate-arginine-tyrosine motif
xi
DM - Diabetes mellitus
EAE - experimental allergic encephalomyelitis
Eff – effector
FACS - Fluorescence-activated cell sorting
Foxp3 - forkhead box P3 transcription factor
Gαi – G protein subunit
GADA - glutamate decarboxylase
GITR - glucocorticoid-induced TNFR-related protein
GP – glycoprotein
GPCR - G-protein coupled receptor
GVHD - graft versus host disease
H&E – haematoxylin and eosin
HLA - human leukocyte antigen
HO-1 - heme oxygenase 1
IAA - insulin islet antigens/insulin autoantibodies
IA-2A - protein tyrosine phosphatase-like protein IA-2
IBD - inflammatory bowel disease
ICAM-1 - Intercellular Adhesion Molecule 1
ICI - insulin containing islets
IDI - insulin deficient islets
IDDM1 – insulin dependent diabetes mellitus 1
IDDM2 – insulin dependent diabetes mellitus 2
IFN – interferon
IgG1 – immunoglobulin G1
IGRP - Islet-specific glucose-6-phosphatase catalytic subunit-related protein
IL – interleukin
IL2R – interleukin 2 receptor
IMPDH - Inosine Monophosphate Dehydrogenase
i.p. – Intraperitoneal
xii
IRSE - IFN-stimulated response elements
i.v. – intravenous
JAK - c-Jun N-terminal kinase/ Janus kinase
kDa – kilodalton
LAT - Linker of Activated T cells
LCMV - Lymphocytic choriomeningitis
MØ – macrophages
M199 - Islet Isolation Media
mAb – monoclonal antibody
MACS – magnetic-activated cell sorting buffer
MAPK - Mitogen-activated protein kinase
Mem – memory
MFI - Mean fluorescence intensity
MHC - major histocompatibility complex
MLR – mixed lymphocyte reaction
mRNA – messenger RNA
MST - median survival time
mTOR - mammalian target of rapamycin
NFAT - Nuclear factor of activated T-cells
NFκB - p65 nuclear factor kappa-light-chain-enhancer of activated B cells
NK - Natural Killer
NKT - Natural killer T cells
NLRP3 - nucleotide-binding oligomerisation domain leucine rich repeat and pyrin
domain-containing protein
NOD – non-obese diabetic mouse
PBS - Phosphate-buffered saline
PC61 – anti-CD25 antibody, clone PC61
PI3K - phosphatidylinositide 3-kinases
PLC - Phospholipase C
pLN – pancreatic lymph node
xiii
POD – post-operative days
PTX - pertussis toxin
Rapa – rapamycin
RBCL - red blood cell lysis buffer
RIP - rat insulin promoter
SEM – standard error of the mean
STAT - Signal Transducer and Activator of Transcription
STZ – streptozotocin
T1D – Type 1 diabetes
T2D – Type 2 diabetes
T-bet - T-box expressed in T cells
TCR - T cell receptor
TGF - Transforming growth factor
TKCC – the Kinghorn Cancer Centre
Th – CD4+ T helper cell
TLR - toll-like receptor
TNF - tumour necrosis factor
Treg – regulatory T cells (CD4+ Foxp3+)
VCAM-1 - vascular cell adhesion molecule 1
ZAP-70 - Zeta-chain-associated protein kinase 70
xiv
Awards and Presentations arising from this Thesis
Awards
 Mentee-Mentor Award, TSS, Melbourne, (2015)
 Young Investigator Award, Annual Scientific Meeting, TSANZ, Canberra, (2015)
 JDRF Top-up Scholarship (2014)
Presentations
 Characterisation of the immunomodulatory action of a novel anti-CXCR3
monoclonal antibody exploiting a conserved human and murine epitope for
transplantation. Mini Oral, Annual Scientific Meeting, IPITA, Melbourne,
(2015)
 Characterisation of the immunomodulatory action of a unique anti-CXCR3
monoclonal antibody exploiting a conserved human and murine epitope for
transplantation. Mini Oral, Annual Scientific Meeting, TSS, Melbourne,
(2015)
 Characterisation of the immunomodulatory action of a novel anti-CXCR3
monoclonal antibody exploiting a conserved human and murine epitope for
transplantation. Mini Oral, Annual Scientific Meeting, TSANZ, Canberra,
(2015)
xv
Abstract
Transplantation of foreign tissues into genetically dissimilar recipients, called an
allograft, results in an inflammatory immune response against the allograft,
necessitating
suppression
of
the
rejection
response.
However,
current
immunosuppressive drugs broadly supress the immune system and have number of
associated side-effects. More selective, alternative therapies such as inhibition of T
cell migration to sites of allograft are for this reason of great interest. CXCR3 is a
chemokine receptor highly expressed on activated Th1 and CD8+ T cells, with
elevated expression in patients undergoing allograft rejection and animal models of
allograft rejection and autoimmune disease.
Due to the clinical application of islet transplantation for use in patients with diabetes
we investigated the efficacy of a novel anti-CXCR3 mAb in models of islet
transplant, autoimmune diabetes and islet transplant into autoimmune diabetic
recipient mice. We hypothesised that transient blockade of CXCR3 signalling would
delay islet allograft rejection and alter diabetes incidence.
As anticipated inhibition of CXCR3 signalling impaired chemotaxis of CXCR3
expressing cells and prolonged islet allograft survival in models of islet transplant.
This was associated with reduced CD8+CD44 expression and reduced CD8+ effector
and memory T cells. An increased frequency of Treg was also seen in islet allograft
recipients. Further, Islet allograft survival was prolonged in autoimmune diabetic
allograft recipients, a finding yet to be shown within the literature.
The role of CXCR3 in diabetes is ambiguous; with some prior studies indicating
accelerated diabetes onset, while others show a delay. Of note treatment with our
anti-CXCR3 antibody showed no change in diabetes incidence or pathology. In
particular recruitment of Tregs from the pancreas to the pancreatic lymph node was
unaffected, in contrast to previous literature showing impaired Treg migration and
resultant accelerated diabetes onset.
Therefore transient antibody targeting of CXCR3 signalling with our antibody shows
promise for use in allograft, in particular in islet allograft with safety in models of
diabetes.
xvi
Experiments that were not the sole work of the author
All experiments were performed solely by the author at the Garvan Institute of
Medical Research, with the exception of:
Figure 3.1c – experiment performed by Remy Robert
Figure 4.16 – experiment performed with Nathan Zammit
Figure 4.17a – experiment performed with Nathan Zammit
Figure 4.18 - experiment performed with Nathan Zammit
Figure 5.4 - experiment performed by Eliana Marino
xvii
Chapter 1. General Introduction
The role of CXCR3 in type 1 diabetes and islet
transplantation
General Introduction
Overview
The immune system is highly mobile, this key element of its function is determined
by the secretion and response to a large family of small proteins called
“chemokines”, taken from the Greek –kinos or movement. The involvement of
chemokines and their receptors in recruitment of immune cells to sites of
inflammation is well established8. In particular recruitment of T cells to sites of
inflammation under conditions of damage or infection is crucial for clearance of
infection and resolution of inflammatory processes9,10. Under conditions of
inflammation in the autoimmune as well as the allogeneic rejection response,
recruitment of immune cells is unwanted and results in damage to either self-tissue or
to allogeneic replacement tissues, respectively11,12. Additionally, chemokines play an
important role in immune cell development and homeostasis, dictating the flow of
immune cells from the bone marrow to peripheral sites for further development13,14.
The immune system plays a key role in many known pathologies, making targeting
the immune system an appealing therapeutic strategy for intervention15. However
cellular based therapies suffer numerous caveats. For instance current therapeutic
approaches that target T cells by depletion or by impairing their activation result in
systemic suppression of the immune system and are often not T cell specific resulting
in toxicities in other tissues16-20. For these reasons other approaches need to be
explored. Targeting chemokine signalling pathways, which are primarily involved in
migration of activated T cells towards inflammation, is one appealing therapeutic
strategy to intervene in these inflammatory responses3,21-26. In this thesis we will
develop this concept further and explore in a series of in vitro and in vivo models, the
potential for targeting one such chemokine ligand-receptor system, namely the
CXCR3–CXCL9/10/11 family.
1
General Introduction
Type 1 Diabetes Mellitus
Classification and Epidemiology
Diabetes mellitus (DM) is a heterogeneous metabolic disorder characterised by
hyperglycaemia, which can be subdivided into several distinct subtypes based on the
pathogenic processes leading to this hyperglycaemia. Of these the two major
subtypes include type 1 and type 2 diabetes mellitus. Type 2 diabetes mellitus (T2D)
is a heterogeneous group of disorders characterised by varying degrees of insulin
resistance, impaired insulin secretion and increased glucose production and accounts
for approximately 85% of individuals with diabetes. Type 1 diabetes mellitus (T1D)
is characterised by insulin deficiency, typically due to autoimmune beta cell
destruction (type 1A) although a small proportion of individuals lack markers
indicative of autoimmune disease (type 1B).
Of all individuals with diabetes,
individuals with T1D account approximately 10%27-30.
Diabetes mellitus currently affects an estimated 382 million people, 8.3% of adults,
with estimates for 2035 predicting a rise to 592 million individuals affected by the
disease globally29. Australia in particular, has an estimated diabetes prevalence of
7.4%, with approximately 7.7% of these individuals having T1D and one of the
highest incidence rates of T1D in children globally (22.3 cases per 100,000
children)29,31,32.
Symptoms
In individuals with T1D the insulin-producing pancreatic beta cells, within the islets
of Langerhans, are destroyed through an autoimmune process. Due to the importance
of insulin secretion in glucose metabolism the loss of insulin results in a number of
symptoms. Under physiological conditions the pancreatic islets produce both insulin
and glucacon, which act in concert to regulate blood glucose levels. Insulin secretion
triggers glucose uptake and storage as glycogen by insulin sensitive tissue such as the
muscle and liver. In contrast glucagon mediates glycogenolysis, gluconeogenesis and
ketone body formation by liver and lipolysis by adipocytes in order to breakdown
glucose stores27,28,33. The lack of insulin produced in individuals with T1D results in
impaired glucose uptake by liver and muscle and dysregulated, elevated glucagon
levels. If left untreated this results in elevated blood glucose (hyperglycaemia) and
2
General Introduction
symptoms of polyuria, polydipsia and polyphagia, volume depletion, tachycardia,
hypotension and metabolic acidosis. In extreme cases, if unchecked, hyperglycaemia
can result in coma and be fatal27. In addition to these acute symptoms, chronic
hyperglycaemia results in an increased risk of severe micro and macro-vascular
complications including; retinopathy, nephropathy, neuropathy, ischaemic heart
disease, stroke and peripheral vascular disease28,30.
Genetic and Environmental Susceptibility
Type 1 diabetes (T1D) results from the interaction of environmental factors within
genetically susceptible individuals. A role for genetics in T1D is demonstrated by the
high concordance rate seen within monozygotic twins, which are genetically
identical, showing between 22.7 to 70% concordance, particularly where T1D
develops early in life i.e. within the first 10 years. This rate is much higher than that
seen for dizygotic twins (0-13%) indicating an important genetic component for early
diabetes, but also highlighting an environmental component, as genes do not account
for 100% of disease susceptibility34-37. The increased risk of T1D in individuals with
first degree relatives who have T1D provides further evidence for the role of
genetics36. Moreover individuals without diabetes who have first degree relatives
affected by TID, have significantly greater levels of autoantibodies against islet
antigens (insulin (IAA), glutamate decarboxylase (GADA), and the protein tyrosine
phosphatase-like protein IA-2 (IA-2A)), with 31% found to have at least one positive
autoantibody compared to none in controls who had no first degree relatives with
T1D38. Islet autoantibodies are strong predictors of T1D risk with higher
autoantibody titer, presence of particular epitopes and IgG subclasses in patient
serum correlating to increased risk of diabetes development39. In addition these
individuals have worse beta cell function and reduced insulin sensitivity showing
heritability of the disease38.
T1D is a polygenic disease with at least 50 loci associated with susceptibility to the
disease40, of these the largest contribution comes from the so called InsulinDependent Diabetes Mellitus (IDDM) 1 locus. Linkage of this region to the human
leukocyte antigen (HLA) class II major histocompatibility complex (MHC) allele
accounts for over 50% of the genetic risk of developing T1D40. Many other nonHLA genes show strong genetic linkage associated with TID susceptibility, such as
3
General Introduction
IDDM2 a polymorphic locus near the human insulin gene and IDDM12 a
polymorphism of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) gene4143
.
In addition to the genetic component of T1D susceptibility a number of
environmental factors have been suggested to contribute to disease development,
though to date this aspect of T1D is not well understood. Of the suggested
environmental factors birth route via caesarean birth has been shown to increase the
incidence of T1D, possibly to due lack of exposure to the vaginal microbiome of the
mother, leading to an altered gut microbiota44. Diet has also been suggested to
contribute to T1D development with early exposure to cows milk leading to an
increased incidence of T1D. Shorter duration of breastfeeding and earlier exposure to
cow milk proteins are both thought to alter the gut microbiome which plays an
important role in development of oral and peripheral tolerance45,46. Further, exposure
to some infectious agents, such as enteral viruses, has been linked to an increased
diabetes incidence47.
However controversy surrounds many of these proposed
environmental triggers due to a number of conflicting studies44-46,48-51. As such more
research is needed to determine with more certainty the roles of these environmental
triggers in T1D development.
Human Pathogenesis and Natural History
T1D results from an autoimmune destruction of the insulin-producing pancreatic beta
cells, found within the islets of Langerhans. In 1965 a pivotal article by Gepts et. al.,
noted the presence of a lymphocytic infiltrate, now termed insulitis, and significantly
reduced number of insulin-secreting beta cells in the pancreatic islets upon autopsy
of patients with T1D. However glucagon (alpha), somatostatin (delta) and pancreatic
polypeptide secreting cells were unaffected in these individuals52,53. This led Gepts
and colleagues to propose that; “the islet inflammation may be secondary to a viral
infection or represent an immunological autoimmune process”52.
Much research has been since conducted to test the hypothesis that T1D is an
autoimmune condition. Key studies that support this notion are those which have
identified islet cell autoantibodies against insulin (IAA), glutamate decarboxylase
(GADA) and the protein tyrosine phosphatase-like protein IA-2 (IA-2A) in people
with T1D as well as murine models54-56. In addition the identification of islet specific
4
General Introduction
T cell clones, including those against insulin and Islet-specific glucose-6phosphatase catalytic subunit-related protein (IGRP) further support the idea that
T1D exhibits an autoimmune atiology57,58. In line with these findings T cell
suppression using prednisone and anti-thymocyte globulin (ATG) or cyclosporine
were found to delay onset of T1D59,60.
The major leukocytes found within the islet infiltrate are CD8 cytotoxic T cells
(CTL) and B cells, with a small proportion of CD4 T cells and macrophages
occasionally present11,61-63. Within the affected pancreas all cells of the islet hyperexpress MHC I and secrete interferon (IFN)-α and this appears to precede insulitis,
suggesting a role for the islets themselves in the initial activation of an immune
response against the beta cells61-65. Beta cells are also able to secrete chemokines
(e.g. CXCL10) involved in recruitment of both CD8+ and CD4+ T to the islet,
providing further evidence for a beta cell specific role in the immune response66.
Collectively these data indicate a role for T cells in the pathogenesis of the disease,
but most significantly support the concept that T1D has an autoimmune aetiology5460
.
Study of the natural history of the disease in humans has been limited by its reliance
on specimens obtained from autopsy of deceased patients with T1D. However our
current understanding of the pathogenesis of TID has evolved over time with initial
models suggesting a linear decline in beta cell mass and onset of overt diabetes with
only 10% residual beta cell mass. These models have now been expanded to
encompass a relapsing-remitting decline in beta cell mass and onset of overt diabetes
occurring with variable beta cell mass remaining (40-50% in some individuals)40,52,6769
. The pancreatic pathology also suggests a heterogeneous and lobular distribution
of affected islets with some islets heavily infiltrated while other neighbouring islets
are unaffected. Beta cells may also be classified based on insulin content as insulin
deficient islets (IDI) or insulin containing islets (ICI)53,65. The medical significance
of these patterns is unknown.
5
General Introduction
Animal Models
Due to the limited availability of human pancreas specimens our current
understanding of T1D has been heavily reliant on experimental animal models, of
which the non-obese diabetic (NOD) mouse has been extensively studied. The NOD
mouse shares a number of important features with the human disease although a
number of differences should also be noted (reviewed by70-72). As for patients with
T1D, NOD mice show destruction of the pancreatic beta cell with loss of insulin
production and hyperglycaemia70 (Figure 1.1). There is a strong genetic component
to diabetes in the NOD mouse, with a significant contribution by the MHC and other
loci that overlap with human loci involved in T1D73,74. Further to this, there is also a
strong environmental influence on disease susceptibility, which appears to relate to
infection and diet. As an example of this environmental susceptibility, the
‘cleanliness’ with respect to viral load of the NOD colony is known to significantly
affect disease penetrance in this model70,75. Increased viral load results in a decreased
diabetes incidence in the NOD colony while specific-pathogen-free mice show a
greater diabetes incidence75.
As understood for T1D, NOD mice also show islet
specific autoantibodies and T cell autoreactivity against beta cell specific antigens
(insulin and IGRP) with disease driven by CD4+ and CD8+ T cell cytotoxicity,
which in the later stages requires the presence of B cells76-80. NOD mice also show
beta cell replication and a type 1 IFN signature early in disease progression, as in the
human disease63,77,81-83.
The age of onset and staging of disease in the NOD mouse differs from that seen in
individuals with T1D, with insulitis beginning at 4 weeks of age and overt diabetes
developing from 12 to 30 weeks of age in 80% of female mice. In comparison, onset
of overt TID in humans is variable and can present at any age starting from infancy
into old age68,71. The NOD mouse also shows a clear female gender bias, with 80%
of female but only 10-40% of males developing diabetes, another aspect of the NOD
disease not seen in humans72,75. The inbred nature of the NOD strain also means
study of this model is representative of a single case study of diabetes, rather than the
heterogeneous human population68,71. Finally NOD mice are able to survive 1-2
months with hyperglycaemia without requirement of insulin for survival, which is
not seen in humans and results from their resistance to ketoacidosis75.
6
General Introduction
Figure 1.1: Comparison of Human T1D and NOD diabetes.
Left; H&E stained islets infiltrated with immune cells (blue) from a pre-diabetic NOD
mouse and a person with T1D and pancreas transplant showing recurrent autoimmunity,
with two neighbouring islets, one heavily infiltrated and one without infiltrate, courtesy of
S. Pilgrim, Sydney (top) and G. Burke, Miami, (bottom)
Middle; Insulin stained islets from a diabetic NOD mouse showing heavily infiltrated islets
(blue) and residual insulin positive cells (brown) and from a healthy person showing an
islet without infiltrate and insulin positive staining (brown) courtesy of S. Pilgrim, Sydney
(top) and S. Bonner Weir, Cambridge, Massachusetts (bottom)
Right; T cell infiltration (green) of an islet from a NOD mouse showing residual insulin
staining (red) and T cell infiltration (αCD3, brown) of an islet from a person with T1Ds,
courtesy of S. Grey, Sydney (top) and T. Kay, Melbourne (bottom)
Within the NOD model disease development can be divided into 4 major stages
based on age and immune infiltrate. Initial infiltrate from 4-6 weeks of age consists
primarily of antigen presenting cells (APC), as well as CD4+ T cells, found in 1030% of islets and accompanied by a type 1 IFN signature. From around 8 weeks of
age up-regulation of chemokines (e.g. CXCL9, CXCL10, CXCL11, CCL4, CCL5,
CCL2, CCL22) and adhesion molecules (ICAM-1, VCAM-1), as well as a 7 fold
increase in T cells, and CD11c+ cell infiltration occurs, with 50-60% of islets
showing predominantly peri-insulitis77. At 11-12 weeks, the point at which
development of overt diabetes is first seen, a 3 fold increase in marginal zone B cells
is evident1,77,81. An important role for B cell presentation of beta cell antigen, in a
MHC class 1-restricted manner, to cytotoxic CD8+ T cells for their activation and
proliferation has been identified1. Together this results in progressive beta cell loss
7
General Introduction
and onset of overt diabetes. This time point is associated with a cytotoxic related
gene profile77. Development of diabetes in the NOD mouse is dependent on both
CD4+ and CD8+ T cells and a breakdown of regulatory and tolerance mechanisms is
thought to contribute to disease development in the NOD mouse72,84-88.
A number of other models of diabetes exist, briefly these include; the BB rat, which
develops insulitis and spontaneous diabetes, characterised by hypoinsulinaemia,
hyperglycaemia, glycosuria, ketonaemia and ketosis. However unlike human or
NOD diabetes the BB rat displays lymphopenia89,90. Models of experimentally
induced diabetes are also used, one such model is the RIP-LCMV model. In this
model mice transgenically express the Lymphocytic choriomeningitis (LCMV) viral
glycoprotein (GP) under the control of the rat insulin promoter (RIP). This does not
itself result in diabetes but leads to beta cell specific expression of the LCMV GP
and upon infection of mice with LCMV leads to generation of GP specific CD8+ T
cell clones, which recognise and destroy GP expressing beta cells leading to
hyperglycemia90-92.
Therapies
Currently no cure for diabetes exists and the primary treatment involves
administration of exogenous insulin typically via multiple daily injections or a pump
system, which aims to maintain euglycaemia artificially93. While most individuals
with T1D are able to maintain blood glucose levels (BGL) with regular
administration of insulin, it is an imperfect solution and requires constant monitoring
to maintain euglycaemia, preventing hyper- or hypoglycaemia27,93. The risk of
complications in individuals with diabetes is significantly reduced through tight
management of blood glucose, although fear of hypoglycaemia prevents many
individuals from maintaining optimal glycaemic control27,93-96.
Hypoglycaemia is a severe complication of insulin therapy, characterised by
palpitations, trembling, sweating, hunger and confusion due to low blood glucose
levels97. Many individuals are able to recognise and respond appropriately to these
symptoms. However individuals with hypoglycaemic unawareness lack the
physiological response to hypoglycaemia and have an impaired ability to
appropriately manage their blood glucose levels. In addition to those with
8
General Introduction
hypoglycaemic unawareness a small group of individuals with diabetes show
significant variability in their blood glucose level, which is poorly managed by
exogenous insulin, known as ‘brittle diabetes’. Individuals suffering these more
extreme episodes make up approximately one third of individuals with T1D and
suffer recurrent episodes of severe, disabling hypoglycaemia, increasing the risk of
complications
and
mortality27,97-99.
These
individuals
with
hypoglycaemic
unawareness and brittle diabetes have a significantly reduced quality of life and
diminished ability to carry out everyday tasks and it is in these individuals that islet
transplant is advised27,98,100-102.
9
General Introduction
Islet Transplantation
Transplantation
Transplantation is a medical procedure involving the transfer of organs from a donor
into a recipient, primarily to replace damaged or absent organs. The evolution of
modern transplantation began as early as the 2nd century B.C. with incomplete
accounts of skin autograft performed by Sushruta followed in the 16th century A.D by
the better documented works of Tagliacozzi103,104. In the 1940s the role of the
immune system in failure of transplants was recognized when skin grafts received by
burns patients from unrelated donors underwent a process of inflammatory necrosis,
while skin grafts from the patient themselves successfully engrafted105,106. This antigraft immune response was termed ‘rejection’ and was followed in the mid-1970s
with drugs able to suppress the rejection response. These immunosuppressive drugs
revolutionised the practice of transplantation, impairing the host immune response
against the donor tissue and allowing successful engraftment of allogeneic tissue107.
Today the use of transplantation is much more widespread, with significant
improvements in immunosuppressants and surgical techniques leading to higher rates
of graft and patient survival. A number of organs and tissues are transplantable today
including; heart, liver, kidney, pancreas, lung, skin and pancreatic islets among
others. There are however two major caveats to transplantation; firstly the
availability of donor organs and secondly the immune response to foreign
tissues106,107.
Allograft Immune Response – A role for T cells
Transplant of tissues between genetically dissimilar individuals of the same species
is termed an allograft, with foreign tissues recognised by the recipient immune
system and rapidly destroyed, leading to organ/tissue rejection. This occurs due to
the introduction of MHC antigens on the surface of donor cells, designated HLA in
humans, which differ to those of the recipient108,109. These are expressed on all cells
of the body, with MHC class II molecules expressed by professional APC, primarily
dendritic cells (DC), macrophages (MØ), monocytes and B cells and MHC I
expressed ubiquitously on all nucleated cells109. The recognition of these genetic
10
General Introduction
MHC variants, termed ‘mismatches’, between the donor and recipient result in a T
cell mediated immune response against the donor tissue leading to acute allograft
rejection responses110.
Antigen Presentation (Signal 1)
Cell mediated immune reactions involve T cells, which can be subdivided into CD4+
and CD8+ T cells based on their expression of cluster of differentiation (CD)4 and
CD8 surface glycoproteins. CD4 and CD8 T cells recognise antigen presented by
either MHC II or MHC I, respectively, via their T cell receptor (TCR)(Signal
1)108,109. In the transplant setting this interaction between the MHC molecules and
TCR can occur through direct, indirect or semi-direct antigen presentation and
represents the first signal of T cell activation. Indirect presentation requires uptake
and processing of donor expressed MHC into peptides and presentation by recipient
APCs. Direct presentation involves transfer of donor derived APCs within graft
tissue, which migrate to the recipients draining lymph nodes and are recognised, via
their MHC expression, as foreign by recipient T cells109,111,112. A more recently
proposed pathway, the semi-direct pathway of allorecognition, involves uptake of
intact donor MHC by recipient APC and presentation of the intact allogeneic MHC
on the surface of recipient APCs112,113. The direct and semi-direct pathways of
recognition are unique to the allograft setting, where foreign MHC II and MHC I are
introduced with the graft tissue112-114.
T cell Co-stimulation (Signal 2)
Cell mediated immune reactions also require a second costimulatory signal for
activation (Signal 2). This involves binding of T cell expressed CD28 to CD80/CD86
(B7.1/B7.2) expressed by APCs, in addition to a number of other costimulatory
signals such as CD40-CD40L (CD154). Together with MHC interactions these
signals activate T cells resulting in expansion and differentiation of antigen activated
T cells115-118
Cytokine and Chemokine Milieu (Signal 3)
In addition to TCR and co-stimulation, CD4+ T cells are driven to differentiate into
effector lineages depending on the cytokine milieu and the cytokine receptors
11
General Introduction
induced during activation119. Within the allograft immune response damage at the
site of the allograft releases pro-inflammatory cytokines and chemokines that
contribute to T cell activation, differentiation and migration of T cells (signal
3)8,16,120-124. Some of the triggers leading to this damage include mechanical injury
and infection. Ischemia reperfusion injury, which results from storage, transport,
transplantation and reperfusion of the organ also results in release of danger
associated molecules (e.g. DAMPs), damaging the organ125.
The CD4+ T helper (Th) cell subsets involved in allograft rejection include; Th1,
Th17 and CD4+ regulatory T cells, Foxp3+Treg126-128.
These 3 signals result in activation of antigen specific T cell subsets, which are able
to both directly damage or provide help to other immune cell subsets, mediating
allograft rejection and offer attractive therapeutic targets for the prevention of
allograft rejection.
T cell Effectors and Immune Regulation
T cell subsets differentiate based on different environmental cues, signal 1-3 above
and are characterised by their expression of different transcription factors, cytokines
and chemokines. Differentiation of the Th1 subset is driven by interleukin (IL)-12,
IFN-γ, activation of Signal Transducer and Activator of Transcription (STAT) 4 and
the transcription factor T-bet123,129,130. Activated Th1 highly express CXCR3 and
IFN-γ and are associated with allograft rejection123,131. Alternatively Th cells may
become Th17 cells, which differentiate in response to Transforming growth factor
(TGF)-β and IL-6 and release IL-17 and IL-6. Their exact role in allograft rejection
remains uncertain, although the ratio of Th17 to Tregs appears to predict allograft
dysfunction and production of IL-17 enhances local inflammation132-137. Regulatory
CD4+ T cells (Tregs) are characterised by expression of the transcription factor
forkhead box P3 (Foxp3). These may be thymically derived or induced from naïve
CD4+ T cells in the periphery upon exposure to TGF-β138,139. Tregs are essential to
peripheral tolerance and can control allograft responses. Indeed, in experimental
models that allow allograft survival many have found expanded Tregs to be essential
for this phenomenon. For instance, studies indicate an increased ratio of Tregs to
CD3+ T cells positively correlates with allograft function. Treg transfer was also able
12
General Introduction
to induce antigen-specific tolerance in allograft models119,140-144. The suppressive
activity of Tregs is attributed to multiple mechanisms but close proximity to the cell
targeted for regulation appears to be key. Mechanisms of regulation include;
impaired T cell activation and maturation via IL-10 and TGF-β secretion, inhibition
of pro-inflammatory transcription factors, cytolysis and cell contact mediated
inhibition of T cell co-stimulation via CTLA-4139,145-148.
Islet Allograft Success and Immunosuppressive Therapy
The allograft immune response is a major factor hindering the application of islet
transplantation. However recent improvements in islet isolation techniques as well as
the introduction of the Edmonton Protocol of immunosuppressive therapy have
significantly improved islet allograft success rates101,149,150 and from 1999 to 2012 a
total of 864 allogeneic islet transplants were performed (figure 1.2)151.
Although islet allograft success rates have improved from initial transplantation
outcomes to those seen in 2010, discussed below, a number of issues including; the
availability of HLA matched donor organs, the risk of surgical complications,
particularly for pancreas transplant, and most importantly the need for life-long
treatment with immunosuppressant drugs to prevent graft rejection exist16,100. The
current immunosuppressant regimens required to maintain allograft function broadly
supress immune function, resulting in a number of side-effects16,17. These include; an
increased susceptibility to infections, development of cancer18 and toxic effects of
the drugs such as nephrotoxicity19 and impaired beta cell function (diabetogenic
drugs)20. These toxic side effects may also damage the replacement organ resulting in
impaired function152. In addition current immunosuppressive therapy appears to
impede development of long-term immunological tolerance153.
These issues limit pancreas and islet cell allograft to the most serious cases100,154.
Islet transplant is also not performed in children or those individuals with diabetes,
where the benefits of transplant for the individual are outweighed by the requirement
for life long immune suppression16,154. However earlier transplantation in these
individuals has the potential to improve glycaemic control and may minimise longterm complications.
13
General Introduction
The advent of the Edmonton protocol, by Shapiro et al., in 2000 revolutionised the
course of islet transplant with 7 out of 7 patients achieving insulin-independence149.
This drug regimen was corticosteroid free, using an IL-2 receptor antagonist for
induction together with the mammalian target of rapamycin (mTOR) inhibitor
(sirolimus/rapamycin)
149
maintenance
and
a
calcineurin
inhibitor
(CNI)
(tacrolimus)
for
. The Collaborative Islet Transplant Registry (CITR) indicates rates of
51% insulin-independence at 1 year, 36% at 2 years and 27% at 3 years for
transplants performed from 1999-2002. Transplants performed from 2007-2010
showed significantly improved insulin-independence rates of 66%, 55% and 44% at
1, 2 and 3 years post-transplant, respectively. The period from 2007-2010 has also
seen the use of a T-cell depleting antibody in addition to an antibody that blocks
activity of the inflammatory cytokine tumour necrosis factor (TNF)-α (etanercept)
and involves maintenance therapy with an mTOR inhibitor or an inosine
monophosphate dehydrogenase inhibitor (mycophenolic acid) combined with a
CNI149,150.
Figure 1.2: Islet Transplantation
Collection, isolation and infusion of pancreatic islets from a donor into the portal vein of
an islet transplant recipient with T1D. Bright field image of human islets curtesy of S.
Grey and islet transplant image adapted from7.
Although a number of individuals are no longer insulin-independent by the 3rd year
post transplant, islet graft survival (C-peptide ≥0.3 ng/mL) remains in 83% of
individuals and over 90% of transplanted individuals remain free of severe
hypoglycaemic and life-threatening events 5 years post-transplant150. While insulin14
General Introduction
independence is as yet suboptimal the improvements seen in blood glucose control
and absence of hypoglycaemia in individuals who had unmanageable glucose prior to
transplant, significantly improves their quality of life and manageability of their
diabetes101,102,155,156.
Current Immunosuppressants
Immunosuppressant therapies target the 3 main steps in T cell activation; TCR/MHC
interaction (signal 1), co-stimulation (signal 2) and cytokine & chemokine signalling
(signal 3), while others exert their function via the depletion of T cells15,119.
Firstly, Tacrolimus and Cyclosporine A function through blockade of nuclear factor
of activated T-cells (NFAT), impairing T cell activation via inhibition of calcineurin
(signal 1). However these drugs, also known as CNIs, cause significant
nephrotoxicity, hypertension and impaired beta cell function15,157,158. Sirolimus
(rapamycin) is an mTOR inhibitor, which also impairs cell cycle, proliferation and
activation, although mediates this in a calcineurin independent manner159.
Co-
stimulatory blockade (signal 2) is targeted by CTLA-4-Ig (Abatacept and Belatacept)
and anti-CD40 (5C8), which inhibits interactions of CD80/CD86 with CD28 and
CD40 with CD40L, respectively160-162.
Cytokine signalling (signal 3) is also targeted by a number of drugs. Etanercept
antagonises TNF signalling, reducing the pro-inflammatory effects of this cytokine
on the immune system163. Basiliximab & Dacilizumab are directed against IL-2Rα
and impair T cell proliferation and activation164,165.
Finally drugs which deplete T cells include; OKT3, an anti-CD3 antibody and
aletuzumab, a CD52 targeting antibody. Anti-thymocyte globulin (ATG), a
polyclonal-immunoglobulin preparation with multiple antibody specificities is raised
in rabbits or horses against human thymocytes for T cell depletion via complementdependent cell lysis119,166-168. Importantly use of T-cell depleting antibodies are
associated with increased cancer risk167.
Each of these drugs have adverse side effects that limit their use, as outlined briefly
in table 1.115.
15
General Introduction
In addition to inhibiting these stages of T cell activation, the targeting of immune cell
migration is another attractive strategy. Recruitment of immune cells into sites of
inflammation is essential in allograft and autoimmune destruction, with local
production of chemokines causing migration of cells expressing their cognate
receptors. Therapeutic strategies directed against chemokine receptors preferentially
expressed by the alloreactive and autoreactive T cells mediating disease may prove
useful in the design of novel anti-inflammatory therapies.
Table 1.1: Immunosuppressant Side-effects
Drug
Molecular Target
(pathway/receptor)
Cyclosporine
TCR activation/Calcineurindependent deposphorylation
of NFAT
TCR activation /
Calcineurin-dependent
deposphorylation of NFAT
mTOR/complexes with
FKBP blocking mTOR
activation
Apoptosis of CD3
expressing cells via Ab
binding, prevents T cell
activation
Binds to CD52 causing
antibody-dependent cellularmediated lysis
Tacrolimus
Sirolimus
OKT3
Alemtuzumab
Basiliximab
T cell differentiation/
proliferation/Binds IL-2
receptor α chain (CD25) and
competes with IL-2 for
binding
Primary
Side-effects
Target Cell
Population
T lymphocytes Nephrotoxic,
hypertension,
T lymphocytes Nephrotoxic,
diabetogenic,
hypertension
T lymphocytes Hyperlipidaemia,
peripheral oedema,
myelosuppression
T lymphocytes Cytokine storm,
(mature)
pulmonary oedema,
thrombocytopenia
(mature B & T Thrombocytopenia,
cells, NK
fever, hypotension
cells,
monocytes,
dendritic cells
T lymphocytes Fever and chills
(activated)
16
General Introduction
Chemokines
Chemokines are a family of 8-12 kDa peptides, which induce directional migration
of leukocytes in both physiological and pathological settings. These molecules are
released by many different cells, both of immune and non-immune origin, in order to
recruit immune cells into sites of inflammation. Chemokines can be subdivided into
4 different classes based on cysteine number and spacing. The largest group,
cysteine-cysteine (CC) chemokines, have the first 2 cysteines in adjacent positions
and cluster on chromosome 17q11 & q12. The second largest group, CXC
chemokines, have the first 2 cysteines separated by an intervening amino acid,
clustering on chromosome 4q12-4q13 and 4q21. Finally the last two groups,
containing either two highly related or a single unique chemokine, are the C and
CX3C chemokines respectively. These chemokines consist of either 2 cysteine
residues (C) or two cysteine residues separated by 3 amino acids (CX3C)8,169.
Each of these chemokines also bind to G protein coupled chemokine receptors,
which show redundancy with a single receptor able to bind multiple different
chemokines, although this does seem to be restricted by subclass i.e. CC chemokines
typically don’t bind CXC receptors. Chemokine receptors have 7 transmembrane
spaning domains flanked by an acidic extracellular N-terminal and an intracellular Cterminal8,170-173.
Within the chemokine-chemokine receptor system, in addition to redundancy and
cross-over between chemokine-receptor targets there is also overlap in the functional
targets of each chemokine with multiple chemokine-chemokine receptors responsible
for migration of a single cell type e.g. T cells. Broadly CC chemokines target
monocytes and granulocytes while CXC chemokines target neutrophils, however
both families have particular chemokines responsible for directing T cell migration.
This T cell effect can also be broken down into those chemokines targeting Th1
(CCR5 and CXCR3) and those targeting Th2 (CCR3 and CCR4) responses8,131,169.
Mechanistically chemokines mediate migration of different cells expressing their
cognate receptor along a concentration gradient. Upon inflammation, endothelial
cells of blood vessels up-regulate cellular adhesion molecules (e.g. ICAM-1, VCAM1), these adhesion molecules are able to bind to molecules on the leukocytes
travelling within the circulatory system, slowing leukocytes, which roll across
17
General Introduction
endothelial surfaces. In addition exposure to inflammatory stimuli results in
endothelial cell expression of proteoglycan bound chemokines, causing activation of
firm adhesion by integrins and high affinity binding of chemokine receptor
expressing leukocytes. This synergistic binding both activates leukocytes and causes
extravasation out of the circulatory system, where cells may then travel along a
chemokine gradient towards the site of inflammation (figure 1.3)8,174-176. In addition
chemokines are important in spacial and temporal localisation of immune cells
within lymphoid organs, allowing appropriate T and B cell activation13.
Figure 1.3: Leukocyte Chemotaxis
1) Endothelial cells and leukocytes within the circulation become activated by
inflammation and chemokine exposure up-regulating adhesion molecules (e.g. ICAM-1)
and their ligands (e.g. integrin)
2) Fast moving leukocytes are slowed by binding of adhesion molecules and roll along the
endothelial surface
3) Slow moving leukocytes firmly adhere to the endothelial surface
4) Leukocytes transmigrate out of the circulation and migrate along chemokine gradients
towards inflammation and injury.
T cells are critical immune cells in the inflammatory reaction. Therefore studying the
chemokines and receptors which govern their recruitment is important in
understanding lymphocyte infiltration and also for the development of T cell targeted
therapies. One of the chemokine receptors shown to be highly expressed in a number
of cancers, autoimmune diseases and in allogeneic transplantation is CXCR3, a
chemokine receptor primarily upregulated on activated CTL and Th1 cells21,177-182.
Manipulating the ability of cells to migrate into the islet allograft, or potentially the
pancreas in pre-diabetic individuals via inhibition of this chemokine receptor is an
appealing therapeutic concept.
18
General Introduction
CXCR3
CXCR3 is a G-protein coupled receptor (GPCR) composed of a 7 transmembrane
domain flanked by an intracellular carboxyl (C)-terminal and an extracellular Nterminus both of which are involved in chemokine mediated receptor activation2,183.
CXCR3 is located on the X chromosome (Xq13)184. Up-regulation of CXCR3
expression occurs in the presence of inflammatory signals with T cell expression
dependent on the presence of T-bet and IFN-γ signalling and non-persistent T cell
receptor (TCR) triggering184-186.
CXCR3 exists as 3 alternatively spliced isoforms in humans, the most studied
CXCR3-A consists of a 368 amino acid sequence of 40kDa, which binds the 3
classic CXCR3 chemokines; monokine induced by gamma interferon (MIG or
CXCL9), interferon-induced protein of 10kDa, (IP-10 or CXCL10) and interferoninducible T cell alpha chemoattractant, (I-TAC or CXCL11). The CXCR3-B isoform
has a longer C-terminal domain and in addition to binding the 3 classic chemokines,
binds platelet factor 4 (CXCL4) with high affinity183,187,188. The affinity of binding
differs for the CXCR3-A and CXCR3-B isoforms. CXCL11 has the highest affinity
for CXCR3-A with CXCL10 followed by CXCL9 having lower affinity. However
CXCL4 has highest affinity for CXCR3-B followed by CXCL11, CXCL10 and
CXCL9187-190. Finally, the alternate isoform (CXCR3-alt) differs from CXCR3-A in
its C-terminus, lacking several transmembrane spanning domains and binding only
CXCL11191.
CXCR3 Expression
These CXCR3 isoforms perform differential functions with CXCR3-A expression on
immune cells important in recruitment to sites of inflammation, as well as for their
activation and proliferation2,187,192-194. However, interestingly CXCR3-B seems to be
predominantly expressed on cells of the vascular endothelium facilitating angiostatic,
apoptotic and anti-proliferative functions5,187,195-197. CXCR3 is also implicated in
cancer with higher expression of CXCR3-A and reduced expression of CXCR3-B
thought to predict poor overall survival due to greater proliferative and migratory and
reduced apoptotic and angiostatic action193,195,198. In mice only the CXCR3-A
isoform has been identified with high sequence homology (86%) to human
CXCR3199. It is responsible for facilitating chemotactic, proliferative, angiostatic and
19
General Introduction
apoptotic functions of CXCR3, supporting an important biological role for this
receptor5,199.
CXCR3 is expressed on immune cells with a particularly high expression on
activated and memory CD4+ Th1 and CD8+ CTL with expression also seen on
Natural Killer (NK), Natural killer T cells (NKT), B cells and DC21,177,200-202. This
has also been shown in C57BL/6 mice using an anti-CXCR3 monoclonal antibody
with 23% of CD3+CD4+, 43% of CD3+CD8+ and 34% of CD4+Foxp3+ T cells and
on 59% of CD4+CD44+ T cells and 85% of CD8+CD44+ T cells200.
CXCR3 Chemokines
The 3 characteristic CXCR3 chemokines are transcribed in response to IFN-γ, IFN-α,
IFN-β and TNF-α, triggering calcium mobilisation, and activation of signalling
pathways involving; phosphatidylinositide 3-kinases (PI3K)/Protein kinase B (Akt)
as well as p65 nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)
or c-Jun N-terminal kinase/ Janus kinase (JAK)/STAT-1189,203-208.
Importantly the expression of these chemokines relies on different cytokines and
signalling through different promoters, resulting in temporally and spatially different
expression patterns for the 3 chemokines and accounting for the various roles of
CXCR3. CXCL9 production is dependent only on IFN-γ mediated signalling, while
CXCL10 and CXCL11 production are induced by IFN-γ, IFN-α, IFN-β and TNF-α4.
All of the CXCR3 chemokines can be produced through the common promoter NFκB. Additionally the CXCL9 promoter contains an IFN-response element (γIRE1)208, the CXCL10 promoter contains an IFN-stimulated response element (IRSE)205
and the CXCL11 promoter contains an IFN-regulatory factor 1 (IRF1)209 each of
which can also induce these CXCR3 chemokines.
While IFN-γ is responsible for the transcription of these chemokines it is also
essential in up-regulation of CXCR3 expression185. This results in recruitment of
CXCR3 expressing immune cells, CD4+ Th1, CD8+ cytotoxic T cells and NK cells,
in response to CXCL9, CXCL10 and CXCL11 release. These cells in turn secrete
further IFN-γ increasing chemokine secretion and immune cell recruitment resulting
in an inflammatory amplification loop and exacerbation of inflammation5 (Figure
1.4).
20
General Introduction
Figure 1.4: CXCR3 in immune cell migration
1. Upon insult/injury/infection inflammatory cytokines (IFN-α, IFN-β and TNF-α) are
produced locally. 2. This triggers CXCL10 release fromendothelial and other tissue resident
cells 3. CXCL10 forms a concentration gradient and recruits circulating Natural Killer (NK)
and CD4+ Th1 cells into the target tissue. 4. NK and Th1 cells secrete IFN-γ, stimulating
resident cells, including local dendritic cells (DCs) to 5. increase chemokine (CXCL9 &
CXCL10) release. 6. These chemokines recruit CD8+ CTLs into the target tissue, 7. which also
produce IFN-γ, stimulating chemokine production and amplifying the inflammatory response.
Adapted from4,5
Receptor Structure
CXCR3 signalling and internalisation are activated by chemokine binding through
alternate receptor domains. For this reason a number of studies have investigated the
importance of these domains in activation and internalisation of the human CXCR3
receptor with absence resulting in impaired or abrogated ligand binding and
consequently
reduced
downstream
signalling,
calcium
mobilisation,
actin
polymerisation and chemotaxis as well as receptor internalisation2,183.
Briefly, using a pre-B cell leukaemia cell line transfected to express human CXCR3
a number of regions of the extracellular N-terminus have been shown to play an
essential role in chemokine binding and receptor activation, while the intracellular C-
21
General Introduction
terminal domain and 3 cytoplasmic loops are involved in CXCR3 internalisation
(figure 1.5, human CXCR3-A).
Figure 1.5: Snakeplot structure of Human CXCR3-A amino acid sequence
Including transmembrane conformation and residues of importance to receptor activation
and signalling as well as internalisation:
Red: location of anti-CXCR3-9C5 binding, also contains Y27 and Y29, essential in
chemokine binding and receptor activation
Green: S245 necessary for CXCL11 migration, actin polymerisation and integrin
dependent adhesion, LLL motif necessary for CXCL11 receptor internalisation and
migration
Blue: D112 & D278 for binding and activation by CXCL9, CXCL10 and CXCL11, D282
and E293 for binding and activation by CXCL9 and CXL10. Adapted from6
CXCL9, CXCL10 and CXCL11 require conservation of N-terminal residue R216 of
the second extracellular loop, tyrosine phosphorylation of Y29 and Y27 and charged
residues D112 and D278 for optimal binding. Charged residues D282 and E293 are
also required for CXCL9 and CXCL10, while the proximal 16 amino acids and
residues R197 and R212 of the second extracellular loop are necessary for CXCL10
and CXCL11 binding but non-essential for CXCL9.
22
General Introduction
The intracellular C-terminus of human CXCR3 is necessary for CXCL9 and
CXCL10 internalisation, dependent on serine/threonine phosphorylation of the Cterminal and for CXCL11 internalisation, dependent on conservation of the third
intracellular loop. A DRY (aspartate-arginine-tyrosine) motif in the third
transmembrane domain is necessary for CXCL9, CXCL10 and CXCL11 induced
chemotaxis and calcium mobilisation, while the C-terminal LLL motif is also
necessary for chemotaxis in response to CXCL9, CXCL10 and CXCL11 but not
calcium mobilisation or receptor internalisation183.
Signalling Pathways
Activation of signalling pathways by CXCR3 depends on the ligand as well as the
receptor isoform, G-protein and cell type involved leading to primarily
chemotactic/proliferative or apoptotic/angiostatic effects187,203. Upon ligand binding,
GPCRs signal through G proteins α, β and γ causing activation of the regulatory α
subunit and dissociation of the catalytic β, γ subunits210.
Using a number of in vitro studies it has been identified that human CXCR3 signals
primarily through the pertussis toxin (PTX) sensitive G-protein, Gαi. However the
actions of CXCR3-B have been found in some cases to facilitate PTX insensitive
activation, with proliferation of CXCR3-B transfected cells unaffected by PTX187,203.
Conversely the CXCR3-B selective chemokine CXCL4 was shown to mediate PTX
dependent signalling in T cells203. For this reason it is thought that signalling of the
CXCR3-A isoform occurs through Gαi, while CXCR3-B generally signals through
Gαs187,194,203. Ligand driven human CXCR3 receptor activation stimulates a number
of signalling pathways with all 4 CXCR3 ligands able to activate calcium
mobilisation and signalling through Extracellular signal-regulated kinases (ERK)1/2
(Ras/Raf/Mek),
Mitogen-activated
protein
kinase
(MAPK)
and
PI3K/Akt
pathways194,203. Ligation with CXCL11 stimulates the most rapid and sustained
response for CXCR3-A and similarly CXCL4 is the dominant stimulator of CXCR3B187,194,203. However CXCL4-CXCR3B signalling shows significantly lower calcium
mobilisation and does not result in internalisation or chemotaxis203.
In human derived T cells it was found that although CXCL11-CXCR3 ligation
induced MAPK and PI3K/Akt signalling neither pathway was responsible for
23
General Introduction
chemotaxis, instead this occurred through Phospholipase C (PLC) and F-actin
polymerisation194. It was also found that while ERK signalling was in part
responsible for human CXCR3 mediated chemotaxis it also displayed a pattern of
biphasic activation associated with mitogenic and proliferative activity196. CXCR3-B
mediates apoptotic and anti-proliferative functions though a number of pathways
including inhibition of the anti-apoptotic gene heme-oxygenase 1 (HO-1), via
MAPK, and blockade of ERK1/2 signalling. Increased survival and proliferation of
human breast cancer cells was seen upon inhibition of CXCR3-B195. Human CXCR3
ligation has also been shown to phosphorylate a number of molecules downstream of
the TCR (ZAP-70, LAT and PLCγ1), which suggests cross-talk between these two
molecules and possibly a co-stimulatory role of CXCR3 in T cell activation203,211.
Studies conducted in mice have identified only the CXCR3-A isoform, which
therefore must regulate the opposing functions seen for CXCR3-A and CXCR3-B in
humans. In vivo and in vivo studies using C57BL/6 wild type (WT) and CXCR3-/mice have shown CXCR3-B-like functions with CXCL9 found to inhibit VEGF
induced angiogenesis, proliferation and migration. This occurred via reduced PLCγ
and ERK phosphorylation reducing angiogenesis and fibrosis in endothelial and
stellate cells from fibrotic mouse livers212. In contrast CXCL10 expression in cardiac
allografts, of WT and CXCL10-/- B6/129 (C57BL/6x129Sv/J (H-2b)) mice, was
essential for immune infiltration of allograft, mediating CXCR3-A-like functions213.
Due to the varied roles of CXCR3 it has been identified as a potential therapeutic
target in a number of disease processes, most importantly cancer, transplantation and
autoimmunity178-182,214,215. Due to the potential benefits of reduced immune cell
trafficking to sites of inflammation in autoimmune and allogeneic conditions, such as
Type 1 diabetes and islet transplantation, investigation of CXCR3 mediated immune
cell migration is of great interest. A number of studies have investigated CXCR3 as a
therapeutic target with deletion or use of antibodies against CXCR3 and its
chemokines showing beneficial effects for some but not all studies216,217.
24
General Introduction
Experimental Models
CXCR3 in Inflammation
The role of CXCR3 in health and disease is highly complex, orchestrating a number
of processes from recruitment of immune cells22 to wound healing218 and viral
clearance219,220. Under normal immune conditions CXCR3 is important in the antiviral immune response. One in vivo study investigated this effect using vaginal
infection of C57/BL6 WT and CXCR3-/- mice with HSV-2 revealing a role for
CXCR3 in CD8+ T cell cytotoxicity, T-bet, perforin and granzyme B expression219.
Further, in vivo models using OT-I TCR transgenic WT and CXCR3-/- mice on a
STOCK Tg(CAGDsRed*MST)1Nagy/J background were infected with vaccinia
virus revealing impaired ability of immune cell invasion and contact-dependent
interactions with virally infected cells, leading to poor viral clearance in CXCR3-/mice220.
A model using adoptive transfer of P14 LCMV-specific T cells, comprised of WT &
CXCR3-/- cells, into C57BL/6 recipient mice followed by infection with LCMV lead
to reduced development of short-lived effector T cells when transfected with
CXCR3-/- cells. Further, co-localisation of CD8+ virus specific T cells and antigen
presenting cells in the splenic marginal zone of irradiated C57BL/6 mice was
impaired when mice were adoptively transferred with C57BL/6 WT &
Rag2−/−CXCR3−/−OT-I bone marrow and infection with vaccinia virus (VV)-OVA.
Together these models suggest an import role for CXCR3 in T cell-APC colocalisation and subsequent T cell development221,222. In addition in vitro studies
using OVA specific CD8+ T cells from IRF-9-/- C57BL/6 mice suggest that CXCR3
signalling via a type 1 IFN dependent pathway preferentially affects proliferation and
activation (CD25 and CD69 expression) of naïve (CD44low) CD8+ T cells192.
Activated Th1 and CD8+ CTL cells highly express CXCR322, which is necessary for
their recruitment to sites of infection but is also involved in deleterious effects seen
under conditions of disease and uncontrolled inflammation, e.g. autoimmunity. For
this reason the roles of CXCR3 signalling in numerous models of inflammatory
disease have been investigated using either antibody blockade or deletion of CXCR3
signalling, via administration of anti-CXCR3 antibodies or gene deletion.
25
General Introduction
Inhibition of CXCR3 signalling reduces immune cell chemotaxis to inflamed sites,
such as; the dermis, lymph nodes and prostate and is also involved in migration in
response to renal ischemia reperfusion injury, arthritic pathology and experimental
allergic encephalomyelitis (EAE)3,22-25.
Lymphocyte recruitment is reduced in rats following anti-CXCR3 antibody
administration in a Mycobacterium butyricum induced arthritis model3 as well as in
Ischemia reperfusion injury in Sprague–Dawley rats, using temporary left renal
occlusion, following treatment with the CXCR3 antagonist TAK-77924. The
antagonist TAK-779 also reduces immune cell infiltration of the prostate in a
NOD/LtJ mouse model of Experimental autoimmune prostatitis, using immunization
with a mixture of prostate antigens in CFA23. Further antagonism of CXCR3 using
SCH546738 in experimental autoimmune encephalomyelitis (EAE), induced by
immunization of C57BL/6 mice with pertussis toxin and myelin peptide (MOG 3555) in CFA, lead to reduced disease severity and delayed onset25. Disease severity
and immune cell recruitment to the colon are also reduced in an experimental IL-10/- C57BL/6 mouse model of colitis following administration of anti-CXCL10 Ab223.
Finally, CXCR3-/- C57BL/6 mice had reduced recruitment to sites of inflammation
in models of collagen-induced arthritis, Complete Freunds adjuvant (CFA) induced
LN inflammation and TLR agonist (poly I:C and LPS), Con A and OVA induced
dermal inflammation22. In addition within this model of dermal inflammation over
90% of Th1 and CD8+ CTL expressed CXCR322.
In contrast to the beneficial effect seen in these studies, a further study looking at the
effects of an anti-CXCL10 antibody found no significant difference in recruitment or
disease severity in multiple models of inflammatory disease. These models included;
collagen induced arthritis induced in DBA/1 mice, EAE induced in SJL/J mice using
immunization with a myelin protein (proteolipid protein) in incomplete Freunds
adjuvant and inflammatory bowel disease (IBD) induced by CD4þ CD45RBHi T cell
transfer from female CB6 F1 mice to female CB17 scid/scid mice. Of note however,
initial in vivo studies on CD8+ T cell chemotaxis found inhibitory effects of the
antibody occurred only at high doses (1000ug i.p.), while all further experimental
studies used significantly lower doses (8-500ug i.p.)224.
26
General Introduction
In addition to expression on effector T cells, CXCR3 is expressed on a subset of Th1
Tregs. These Tregs are induced in response to Th1 conditions and upregulate
CXCR3, under the control of T-bet, and regulatory markers (IL-10, TGF-β,
Granzyme, CTLA-4, GITR, CD103). These Treg show superior ability to suppress
Th1 mediated inflammatory disease225. In models of CXCR3 deficiency in both the
NOD and C57BL/6 mouse CD25+Foxp3+Treg were found to accumulate in the
spleen and draining lymph nodes with reduced ability to infiltrate the inflamed
pancreas an liver, respectively217,226. However other studies have indicated no impact
of CXCR3 blockade or deficiency on Treg migration to sites of inflammation with
improved disease outcomes124,200,227. One study found impaired migration of Th1 and
CTLs in CXCR3 deficient mice, while migration of Treg to the inflamed dermis was
unaffected22.
Taken together the role of CXCR3 on regulatory T cells appears to be variable with
an important role for migration of Th1 specific Treg and suggesting redundancy or
use of alternative chemokine receptors for recruitment of other Treg subsets. Given
the number of studies showing beneficial results for inhibition of CXCR3 in
inflammatory diseases, it is likely that other subsets of Treg, which are less
dependent on CXCR3, are involved in Th1 mediated diseases.
27
General Introduction
CXCR3 in Diabetes
A number of human studies of patients with T1D show increased expression of
CXCR3 and its ligand CXCL10, with elevated expression correlating with recent
onset of overt diabetes214,228-234. Elevated expression of CXCR3 has also been
suggested to be a good predictor of diabetes development in high risk first degree
relatives178.
CXCR3 and its’ ligands, particularly CXCL10 are involved in both immune cell
recruitment to the pancreas and in impaired beta cell survival or proliferation66,124,235.
The beta cells, originally thought of as innocent bystanders in the immune mediated
attack, are now known to express CXCR3 and CXCL10 following initial
inflammatory insult. CXCL10 acts in an autocrine/paracrine manner through toll-like
receptor (TLR)-4, rather than CXCR3, to enhance Akt driven apoptotic signals,
impair insulin secretion and reduce beta cell viability resulting in decreased beta cell
mass, independent of insulitis66,124,235. CXCL9 is also expressed by islet vascular
endothelial cells rather than beta cells and contributes to immune cell recruitment235.
Investigation into the role of these chemokines in in vivo models of diabetes using
virally induced RIP-LCMV transgenic mice revealed a role for CXCR3 in the
recruitment of T cells into the pancreas. Following infection with LCMV,
development of LCMV specific CD8+ T cells occurs, which are recruited to the
pancreas where LCMV-GP is expressed. In CXCR3 deficient C57BL/6 mice this
recruitment of immune cells into the pancreas was significantly impaired and
consequently diabetes onset was also significantly delayed in this model124. However
these results were not replicable in further studies in RIP-LCMV transgenic mice.
Little to no delay in onset of diabetes or insulitis was evident in C57BL/6 RIPLCMV mice deficient in CXCR3 (B6.129P2-Cxcr3tm1Dgen/J) or CXCL10
(B6.129S4-Cxcl10tm1Adl/J) or in C57BL/6 RIP-LCMV mice following treatment
with an anti-CXCL10 mAb or CXCR3 antagonist NIBR2130236,237.
Within the NOD mouse CD4+ T cells and CD4+Foxp3+ regulatory T cell migration
are dependent on CXCR3 expression217. In CXCR3 deficient NOD mice
accumulation of CD4+Foxp3+ Treg in the pancreatic lymph node and loss of
pancreatic infiltration were evident, resulting in enhanced diabetes incidence217. Of
note disease penetrance and diabetes incidence in this model was quite low.
28
General Introduction
Additional studies in the NOD mouse used deletion of the inflammasome component
NLRP3 (nucleotide-binding oligomerisation domain leucine rich repeat and pyrin
domain-containing protein), which is involved in Th1 cell activation, proliferation
and migration into the pancreatic lymph node and pancreas. This study found that
NLRP3 deficiency altered chemokine-chemokine receptor expression with increased
CXCR3 and CCR5 in the spleen but reduced expression of the chemokine-receptors
and their chemokines in the pancreas. Together this lead to impaired migration of
activated Th1 cells into the pancreas, though no change in the migratory ability of
Tregs was noted238.
Overall, data on the effect of CXCR3 blockade or deletion within the diabetes
literature is variable. Some studies show that reduced CXCR3 expression provides an
inhibitory effect on autoreactive infiltrating T cells, reducing disease incidence. In
contrast other studies show that CXCR3+ Tregs are essential in prevention of the
disease and loss of this chemokine receptor prevents their migration, increasing
diabetes incidence upon CXCR3 gene deletion.
29
General Introduction
CXCR3 in Allograft
Within the context of allograft transplantation the chemokine receptor CXCR3 and
its chemokines are upregulated in kidney, heart and lung donor organs and are also
expressed by pancreatic islets66,179,180,182,239,240. Xenograft models of kidney or islet
transplant from pig to non-human primate also indicate an upregulation of CXCR3
and its chemokines during allograft rejection241-243.
Using a model of BALB/c (H-2d) murine cardiac allograft into C57BL/6 or
C57BL/6x129Sv/J (H-2b) mice expression profiles of CXCR3 and its ligands show
upregulation of CXCR3 and CXCL10 by cardiac endothelial cells within 24hrs213,216.
Donor derived CXCL9 expression has also been shown to increase at 24 hours upon
exposure to IFN-γ secreting memory CD8+ T cells following A/J (H-2a) cardiac
allograft into C57BL/6 (H-2b) mice244. This is followed by expression of CXCL9,
CXCL10 and CXCL11 by the cardiac endothelium and infiltrating macrophages and
lymphocytes at day 3 and persisting to day 7 when rejection typically occurs. mRNA
expression of chemokines; CCL3, CCL4, CCL5, CXCL2 CXCL9, CXCL10 and
CXCL11, as well as their receptors; CCR1, CCR2, CCR5, CXCR4 and CXCR3 were
all increased in wild type BALB/c (H-2d) cardiac allografts into C57BL/6 or
C57BL/6x129Sv/J (H-2b) recipients213,216. For cardiac allograft from BALB/c (H-2d)
mice into C57BL/6x129Sv/J (H-2b) mice the number of CXCR3 expressing cells was
shown to increase steadily over the course of allograft rejection, with NK cell
infiltrate peaking at day 3 and CD3+ cell infiltrate progressively increasing until
graft loss at day 7213. Importantly models using cardiac allograft of WT BALB/c
hearts into C57BL/6 CXCR3 deficient recipient mice or transplantation
C57BL/6x129Sv/J CXCL10 deficient donor hearts into WT BALB/c hosts show
significant prolongation of graft survival (~60 and 40 days, respectively) and reduced
immune cell infiltration of cardiac allografts. This indicates that in cardiac allografts
the initial release of donor derived CXCL10 chemokine drives chemotaxis of
recipient immune cells expressing CXCR3 to the graft213,216,245. Monoclonal
antibodies against CXCR3 or CXCL10 also prolonged BALB/c cardiac allograft
survival when transplanted into C57BL/6 or C57BL/6x129Sv/J recipients. This was
characterised by intact myocardium and vasculature and reduced CD4+ and CD8+ T
cell infiltration, albeit to a lesser extent than seen in knockout models (~20 and 14
days, respectively)200,213,216.
30
General Introduction
In addition to cardiac allografts, skin allografts from C56BL/6 (H-2b) donors were
also shown to upregulate CXCR3 and its chemokines CXCL9, CXCL10 and
CXCL11 when transplanted into MHC mismatched BALB/c (H-2d) recipients,
compared to isografts. Treatment of skin allograft recipients with an anti-CXCL11
antibody did not alter pro-inflammatory cytokine expression (INF-γ, TNF-α) or
proliferation within the allograft. However a reduction in immune cell infiltration of
the graft and prolonged survival was seen in anti-CXCL11 treated compared to
untreated mice246. C57BL/6 CXCR3 deficient MHC mismatched skin allografted
into BALB/c (H-2d) recipients also resulted in prolonged allograft survival compared
to wild type allograft recipients245.
Biopsies from rejecting human lung allografts also showed high expression of
CXCL10 by infiltrating and alveolar macrophages, as well as epithelial cells, which
were obtained from patient bronchiolar lavage fluid (BAL) samples. BAL was highly
chemotactic for CXCR3 expressing, IFN-γ+ T cells (Th1 and CTL), which was
inhibited by anti-CXCR3 or anti-CXCL10 antibodies240. A model of lung allograft
rejection in mice, used transfer of a C57BL/6 CXCR3-/- or WT CD8 T cell clone
specific for OVA antigen (OT-I), into C57BL/6 CC10-OVA mice expressing OVA
antigens on the cells lining their airway. The mice transferred with CXCR3-/- CD8 T
cells displayed significantly improved graft survival and reduced lung infiltration
compared to those transferred with WT CD8 T cells. Interestingly a significantly
increased number of Tregs were recruited to the lung, in a CCR6 and CCR7
dependent but CXCR3 independent manner. The study proposed that under normal
circumstances both CD8+ T cells and Tregs are recruited to the lung, however the
ratio of Teff:Treg is insufficient to prevent rejection. The reduction in Teff cells
recruited to the lung in this CXCR3-/- model, paired with increased Treg
accumulation, appear sufficient for the prevention of rejection247.
The role of CXCR3+ cells were also characterised in a model of graft versus host
disease (GVHD) with CD8+ T cell transfer from C3H.SW (H-2Db CD45.2+) mice
into irradiated B6/SJL(H-2Db CD45.1+) recipients Disease was inducible by both
CXCR3+ and CXCR3- cell transfer, although CXCR3- cells were found to
upregulate CXCR3 upon APC stimulation to mediate this effect. These
CXCR3+CD8+ T cells showed higher expression of cytotoxic markers (TNF-α, IFNγ, Granzyme B, FasL and Annexin B) with an increased ability to lyse target cells
31
General Introduction
and mediate GVHD, compared to CXCR3- CD8+ T cells. GVHD was prevented in
this model upon anti-CXCR3 mAb administration for 21 days, but not when
administered for 7 days. This is thought to be due to the ability of CXCR3-CD8+ T
cells to upregulate CXCR3 at the earlier time point but not later in disease
progression248.
Although CXCR3 has been found to have an important role in allograft rejection,
redundancy within the chemokine system exists and as such loss of CXCR3 does not
confer complete protection on its own. However, in combination with inhibition of
other chemokine receptors, such as CCR5, which is also responsible for T cell
chemotaxis, improved allograft survival was evident. WT BALB/c (H-2d) cardiac
allograft into B6;129P-Cmkbr5tm1Kuz (H-2b) CCR5-/- recipient mice or WT
B6;129PF2/J recipient mice treated with an anti-CXCR3 mAb resulted in allograft
survival of 29-34 days. However when CCR5-/- mice were treated in combination
with an anti-CXCR3 mAb, 100% of WT BALB/c (H-2d) cardiac allografts showed
long term survival for over 100 days249. Interestingly, while graft infiltration was
significantly reduced, an increased frequency of graft infiltrating Tregs were present.
These cells play a crucial role in long-term graft survival, as indicated by graft loss at
post-operative day (POD) 22 upon depletion of these cells249. Use of a small
molecule antagonist, TAK-779, which targets CCR5 and CXCR3, was also shown to
improve cardiac (C57BL/6 H-2b > BALB/c H-2d) and islet (C57BL/6 H-2b >
C3H/HeJ H-2k) allograft survival, although mean survival times were increased only
by ~5days compared to controls, rather than long-term survival seen with the
previous study. Immune cell recruitment and expression of inflammatory
chemokines (CCL3, CCL2 and CCL5) and cytokines (TNF-α, IFN-γ, IL-4 and IL10) were all reduced with preserved cardiac allograft morphology250.
A role for islet allograft survival has also been shown in a number of studies.
However, typically these studies focus on cardiac allograft, with minimal data on the
islet allograft. It is important to note that although similarities within allograft
rejection exist, tissue specific differences, such as for vascularised vs. nonvascularised grafts are important considerations. In support of this, differential
functions of the chemokine receptor CCR2 have been shown, via MHC mismatched
allograft from WT BALB/c (H-2d) donors into WT or CCR2-/- C57BL/6 (H-2b)
recipients. Using these models CCR2 was shown to have an important early role in
32
General Introduction
islet allograft rejection but no significant role was seen for cardiac allograft
rejection251. Studies on BALB/c (H-2d) or C3H/HeJ (H-2k) donor islet allograft into
C57BL/6 (H-2b) recipients show promising results with administration of an antiCXCR3 mAb or the TAK-779 antagonist, found to prolong mean islet allograft
survival from 12.5 to 25 days or 22 to 28 days, respectively. Further, histology from
treated mice indicates a reduced immune cell infiltration of the islet allograft 200,250.
The study in 2003 by Baker et al. on the role of CXCR3 and its ligand CXCL10 in
islet allograft represents a more in depth investigation of the role of this signalling
pathway in islet allograft. Using either CXCR3 deficient or anti-CXCL10 antibody
treated C57BL/6 (H2b) recipients of A/J strain (H2Ka) islets grafted under the kidney
capsule, Baker et al. found a significant prolongation of graft survival for both
treatment groups (~20days) compared to controls (~12.7days). Further investigation
revealed that although graft chemokine expression (CCL5, CCL3 and CXCL10) was
not different for treated compared to controls a significant reduction in CD8 T cells
was evident (both mRNA and graft histology) as well as reduced markers of NK
cells, CD4 T cells and macrophages (CD19, CD4, F4/80)252.
Fully MHC-mismatched BALB/c (H-2d) to C57BL/6 (H-2b) cardiac allograft
survival is also further enhanced by synergistic effects of CXCR3 inhibition and low
dose immunosuppressive therapy (rapamycin or cyclosporine A) resulting in
permanent engraftment of 100% of cardiac and 80% of islet allografts200,216.
Taken together this indicates that although the role of CXCR3 in islet allograft
rejection has not been extensively studied, its roles in immune cell migration into
sites of allograft are well characterised. In particular inhibition of CXCR3 signalling
has been shown to significantly prolong islet allograft survival.
33
General Introduction
Thesis Aims
Inflammation is a naturally occurring process, responsible for clearance of infections
and dead or damaged tissues and initiation of tissue repair. However an inflammatory
disease represents a condition where inflammation is not controlled, characterised by
continuous recruitment of immune cells to target sites resulting in collateral damage
to local tissues. In the context of autoimmune disease this inflammation is
misdirected specifically at self-tissue, while in allogeneic transplantation
inflammation occurs against non-self-tissue. In both circumstances T cells are
recruited to sites of inflammation and play a critical and unwanted role in tissue
damage. Chemokines and their receptors are necessary for recruitment of T cells, as
well as other cells involved in the autoimmune and alloimmune inflammatory
processes, making them an appealing therapeutic target to intervene in these
inflammatory responses.
The current landscape of available immunosuppressants is limited by their broad
action, impairing the response against auto- and allo-antigens, as well as those
responses to viral infection and cancer. In addition many of these drugs display
adverse side effects or toxicity towards tissues such as the kidney16-20. For this reason
much research is currently being conducted to identify new therapeutic targets as
alternatives to current immunosuppressive therapy.
One such approach would be to impair lymphocyte migration to sites of
inflammation via chemokine receptor blockade. To test this idea we have developed
a novel anti-CXCR3 monoclonal antibody, targeting a conserved epitope found in
mice, non-human primates and humans. Due to the long-term risks associated with
current immunosuppressant regimens, which limit the use of islet transplantation to
individuals with severe, unmanageable T1D, we determined the efficacy of this
antibody in mouse models in the context of islet allograft and T1D, for use clinically
in islet transplant into patients with type 1 diabetes.
In addition to a clinical application in islet transplantation we were interested in
targeting CXCR3 broadly in inflammatory responses as a T cell targeting therapy3,23.
Historically these therapies directed at T cells, such as tacrolimus and ATG15, have
shown promise in the clinic and due to the importance of CXCR3 in regulating
34
General Introduction
proliferation, activation and migration of T cells we believe this will be another
encouraging target15,188,192,197,253-255. Mouse models of islet allograft transplantation
and diabetes will also offer insight into the efficacy of our antibody broadly in
inflammation. The conserved nature of our antibody in mice and humans also makes
results generated in the mouse model more readily translatable for clinical
application by humanising the antibody254.
The chemokine receptor CXCR3 is highly expressed on Th1 and CTLs and has been
shown to play an important role in recruitment of these cells into sites of
inflammation21,177,187,194. Previous literature has also shown that expression of
CXCR3
rejection
correlates
with
66,179,180,182,239,240
both
onset
of
diabetes214,228-234
and
allograft
. CXCR3s’ involvement in allograft rejection is well
established in mouse models200,216. However the role of CXCR3 signalling in the
development of diabetes is less clear124,217,235,236. Importantly CXCR3 signalling has
been found to promote allograft loss in a number of mouse models200,216. In contrast
the incidence of diabetes in the NOD mouse following deletion of CXCR3 was
increased. This was attributed to an impaired ability of regulatory T cells to migrate
into the diabetic NOD pancreas217. As yet studies on temporal CXCR3 blockade in
the NOD mouse have not been performed, although the possibility of differential
effects for deletion versus antibody blockade have been highlighted in studies of
other molecules256-259. Finally, as islet transplantation is a treatment for patients with
T1D, it is of interest to test the efficacy of targeting CXCR3 for islet transplantation
in the NOD model. We note that no previous literature has investigated a role for
CXCR3 in this context.
Therefore the aims of this thesis were;
 To characterise the in vivo effects of our novel anti-CXCR3 monoclonal
antibody, particularly on T cell chemotaxis.
 To characterise the efficacy of this anti-CXCR3 antibody in a model of
BALB/c to C57BL/6 MHC mismatched islet allograft.
 To characterise the efficacy of this anti-CXCR3 antibody in a model of
autoimmune type 1 diabetes using the NOD mouse model.

To characterise the efficacy of this anti-CXCR3 antibody in a model of
mismatched islet allograft into NOD autoimmune diabetic recipients.
35
Chapter 2. Materials and Methods
Materials and Methods
Materials
Table 2.1. Buffers/Reagents
Buffer
Components
Manufacturer
FACS buffer
0.5% bovine serum albumin
Gibco BRL
(BSA) 0.2% Sodium azide
Sigma-Aldrich
1x Dulbeccos Phosphate
Buffered Saline (DPBS) to 1L
0.5% BSA,
MACS Buffer
2mM
Gibco BRL
ethylenediamin
tetra Sigma-Aldrich
acetic acid (EDTA)
DPBS to 1L
Invitrogen
Red Blood Cell Lysis 0.15M Ammonium chloride
Buffer
Merck
10mM potassium bicarbonate
Merck
0.1mM Disodium EDTA
Merck
Triple distilled water to 1L
pH 7.2-7.4
Chemotaxis buffer
49% HBSS (phenol red)
49% RPMI (L-glut, Pen/strep) Invitrogen
2% FBS
Serum
Isolation
(M199-)
Free
Islet 10.6g
Medium
199
(with Sigma-Aldrich
Media Hanks salts and L-glutamine)
0.35g sodium bicarbonate
Sigma-Aldrich
dH2O to 1L
Islet isolation media 100mL heat inactivated bovine Gibco
(M199+)
calf serum (BCS)
M199- to 900mL
Liberase
Liberase-Enzyme Blend-RI
Roche T-Flex,
30μL Liberase + 0.5μL
Indianapolis, IN, USA
Thermolysin
Sterile M199- to 3mL per
mouse
37
Materials and Methods
Avertin 2.5%
Alloxan
2-2-2 tribromoethanol
Sigma-Aldrich
2-methyl-2-butanol
Fisher
Saline 0.9%
Baxter
0.04g alloxan tetrahydrate
Sigma-Aldrich
2mL sterile water for injection
Streptozotocin (STZ)
0.02g streptozotocin
0.1% TBS-T
0.12% Tris Base
Ajax Finechem
0.87% Sodium Chloride
Ajax Finechem
0.1% Tween20
Ajax Finechem
Millique Water to 1L
CFSE culture media
10% FCS, 1:100 L-glutamine
Invitrogen
1:100 Pen-strep
Invitrogen
1:2000 β-mercaptoethanol
GIBCO
(@1000x 55mM)
1:100 Sodium pyruvate
Invitrogen
(@100x 100mM)
CFSE staining buffer
CFSE
1:100 HEPES (@1M)
Invitrogen
RPMI-1640 to 500mL
Invitrogen
1% FCS
Invitrogen
DPBS
Invitrogen
quenching 20% FCS
Invitrogen
buffer
DPBS
Invitrogen
Rapamycin Vehicle
0.2% Carboxymethyl cellulose Sigma-Aldrich
(used with rapamycin
0.25% Polysorbate 80 (tween)
stock in DMSO)
0.9% NaCl solution to 100ml
Sigma-Aldrich
38
Materials and Methods
Methods
Mice
6-10 week old C57BL/6 mice and BALB/c mice were obtained from Australian
BioResources (Moss Vale, NSW Australia) for use as allograft recipients and donors,
respectively or for use in in vivo MLR experiments. C57BL/6 mice were also used
for antibody characterisation experiments.
C57BL/6 recipient mice selected for islet allograft were treated with Alloxan
(110mg/kg) two days prior or Streptozotocin (STZ)(180mg/kg) five days prior to
islet cell transplantation, to chemically induce diabetes, and blood glucose levels
monitored for onset of hyperglycaemia (BGL>15mmol/L). Following transplant both
C57BL/6 and NOD allograft recipients were monitored daily for elevated BGL for
the first week followed by monitoring 2-3x/week. BGL >15mmol/L for 2
consecutive days indicated rejection of transplanted islet grafts and mice were
euthanised.
6-8 week old female non-obese diabetic (NOD/Lt or NOD/ShiLtJ) mice were
obtained from Australian BioResources (Moss Vale, NSW Australia) or Walter and
Eliza Hall Institute (Parkville, Victoria Australia). Mice were monitored for weight
and blood glucose level (BGL) using a Free Style Lite Blood Glucose Monitoring
System, once per week until BGL reached above 8mmol/L at which point twice
weekly monitoring was employed. Mice were monitored for onset of diabetes,
classified as blood glucose level (BGL) >15mmol/L for 2 consecutive days, at which
point mice were euthanised. For studies using pre-diabetic mice, NOD mice were
euthanised at 12 weeks of age. NOD islet allograft experiments used NOD mice
with BGL>8mmol/L as recipients of BALB/c islet allograft (4 donors:1 recipient).
RAG-/- mice were obtained from Animal Resource Centre (ARC, Perth Australia)
for use in cell transfer experiments.
All animal experiments were performed with approval from Garvan/St Vincents
Hospital Animal Ethics Committee (AEC) and mice housed in static micro-isolator
cages.
39
Materials and Methods
Islet Isolation and Transplantation
Islet Isolation
Cannulations were performed by S. Walters or N. Zammit. Donor mice were injected
with 400μL avertin solution and a midline incision performed to expose the
peritoneal cavity (pancreas and liver) and heart cut to stop blood flow. Distension of
the pancreas was performed by clamping the pancreatic/bile duct followed by careful
insertion of a 30G needle into the bile duct and perfusion with 3mL LiberaseEnzyme Blend into the pancreas. Pancreas was carefully excised and put into
prepared 50mL Falcon tubes on ice. This was repeated for each donor mouse to give
a ratio of 3 donor mice per 1 recipient mouse for C57BL/6 and RAG-/- recipients or
4 donor mice per 1 recipient mouse for NOD recipients with 1 50mL Falcon tube per
recipient mouse.
Pancreata were digested at 37°C in a water bath for 10 minutes. Tubes were then
removed directly onto ice, resuspended in 30mL islet isolation media (M199+),
shaken and vortexed to dislodge acinar tissue from islets followed by centrifugation
at 1250rpm for 3min. The supernatant was discarded and cells washed twice more.
Cell pellets were resuspended again, filtered through a 425 micron sieve (US
standard sieve, A.S.T.E. E.11 specifications dial MFG, Co. Chicago, II, USA) and
centrifuged as above. Supernatant was discarded and tubes containing pellet left
upside down briefly to drain excess supernatant. Pellets were then resuspended in
10mL Ficoll-paque plus (Amersham) followed by addition of a further 10mL Ficoll.
Ficoll was then overlayed with a further 10mL serum free islet isolation media
(M199-) and centrifuged on a density gradient at 2830rpm for 22min with no
acceleration or brake. Supernatant, containing separated islets, was then poured into
clean tubes, divided in two and topped up to 50mL before centrifuging at 1250rpm
for 3min. Here supernatant was discarded and cell pellets transferred to a single
Falcon tube. The cell pellet was then divided between 1.5mL Eppendorf tubes
depending on the number of recipients and pellet aspirated into a section of thin
tubing in preparation for transplantation. All prior steps were done on ice at 4°C
unless otherwise stated.
40
Materials and Methods
Islet Transplantation
S. Walters or N. Zammit conducted all islet transplants. Following isolation of islets
recipient mice were prepared for transplantation and maintained under light
isoflurane anaesthesia (1.5-2% isoflurane in O2) for the duration of the transplant
using a Bains paediatric circuit. Briefly mice were placed on a heat mat to maintain
body temperature and the area over the left kidney shaved and sterilised using
alcohol wipes. The tubing containing the islet pellet was attached to a Hamilton
syringe and tubing trimmed leaving pellet close to the end of the tubing. A small
incision was made over the kidney and kidney brought into the wound through gentle
blunt dissection, allowing access to the capsule. A small nick was made at the
inferior renal pole of the kidney capsule allowing the tubing to be inserted and pellet
gently released towards the superior renal pole. Mice were administered 0.2mL
ketoprofen analgesic (0.5mg/kg) and 0.5-1mL saline for rehydration into the surgical
site. The inner and outer skin layers of the incision were then sutured, wound closed
with surgical staples and betadine antiseptic applied. Mice were left to recover in a
fresh cage with heat lamp and heat pad till fully awake. Blood glucose was
monitored on days 1-7 and every second/third day thereafter until rejection occurred
(BGL>15mmol/L).
Antibody
The anti-CXCR3 antibody (clone 9C5) was supplied by the Mackay Laboratory
(Monash University), produced by Dr Remy Robert. Briefly the mouse IgG1
antibody was generated through immunisation of mice deficient for the CXCR3
receptor (CXCR3-/- mice) with a murine pre-B cell line (L1.2) transfected with the
receptor of interest (CXCR3 transfected cells). Following immunisation, antibody
clones were isolated and selected for strong reactivity against mouse CXCR3
(mCXCR3). The clone used in this study recognises a highly conserved region of the
CXCR3 N-terminus and efficiently binds human, mouse and marmoset lymphocytes.
Fluorescent conjugation of αCXCR3-9C5 antibody was performed for use in
antibody characterisation assays (internalisation and competitive binding). For this
an Allophycocyanin (APC) Conjugation Kit (Abcam) was used and conjugation
performed according to protocol provided. Briefly 6μL APC modifier reagent was
added to 60μL αCXCR3-9C5 at 2μg/μL antibody and resuspended gently. This mix
41
Materials and Methods
was then transferred into the lyophilized APC conjugation mix, gently resuspended
and left standing in the dark for 3hrs at room temperature (20-25°C). Finally APC
conjugate was resuspended with 6μL APC Quencher reagent and stored at 4°C until
required. This procedure was also performed for IgG Isotype control antibody.
Antibody Treatments
Allograft
C57BL/6 mice received 2mg/kg αCXCR3-9C5 monoclonal antibody intravenously
(i.v.), unless otherwise stated. Allograft recipient mice, C57BL/6 or NOD, received
2mg/kg αCXCR3-9C5 once per week from the day of allograft for 9 weeks or until
loss of allograft, respectively. Alternatively allograft recipient mice received 2mg/kg
i.v. Isotype (IgG) control antibody once per week from the day of allograft until loss
of allograft, as a control for αCXCR3-9C5 treated mice. Additional groups received
2mg/kg i.v. αCXCR3-9C5 or Isotype in combination with sub-therapeutic
rapamycin, administered i.p. at 0.1mg/kg for 7 days in C57BL/6 allografts260 or
0.5mg/kg for 7 or 14 days in NOD allografts. An untreated control group was also
used in NOD allograft experiments. 10x Rapamycin stock (LC laboratories, Woburn,
MA, USA) (in DMSO) was diluted in vehicle solution for 50μL i.p.
Type 1 Diabetes
NOD mice were treated with 1mg/kg αCXCR3-9C5 monoclonal antibody i.v. unless
otherwise stated. Mice received 1mg/kg i.v. αCXCR3-9C5 once every 4 days from 6
weeks of age (6W) or 8 weeks of age (8W) until study end point was reached, at
12weeks of age or onset of overt diabetes. An Isotype (IgG) antibody was
administered to an additional control group using the same regimen or mice were
untreated as controls.
CD25 Depletion – PC61
C57BL/6 islet allograft recipient mice, receiving 2mg/kg αCXCR3-9C5, were also
treated with 200μg purified rat anti-mouse CD25 IgG mAb (clone PC61, Bioexpress)
via i.v. tail vein injection. The antibody was confirmed via flow cytometry to deplete
the CD4+CD25+ T cell compartment, containing the CD25+Foxp3+ regulatory T
cell subset261.
42
Materials and Methods
Cell Population Analysis
Lymphocyte Isolation
Lymphocytes were isolated from spleen, pooled lymph nodes (axillary and inguinal),
pancreatic lymph node and blood. Splenocytes were homogenised in a 50x9mm petri
dish using two frosted glass slides (Menzel-Glaser, Braunschweig, Germany) and
centrifuged at 1250rpm for 3min. Supernatant was discarded and pellet resuspended
in 2mL red blood cell lysis buffer (RCLB) for 2min to deplete erythrocytes. Lysis
was quenched by addition of FACS buffer and cells washed twice. Blood samples
were collected, through tail bleed in live mice or cardiac puncture in euthanised
mice, in 200μL or 2mL Heparinised saline (10UI/mL Pfizer), respectively. Blood
samples underwent red blood cell lysis to deplete erythrocytes leaving lymphocytes
and washed as above. Lymph nodes and thymus were homogenised as above with no
red blood cell lysis and cells washed twice to remove fat. All cell suspensions were
filtered using a 70μm nylon mesh filter (BD), counted using typan blue (0.4%
Sigma) and a haemocytometer (Blood samples excluded) and resuspended at
1x107cells/mL, unless otherwise stated. All steps were performed on ice at 4°C
unless otherwise stated.
Splenocyte isolation for culture and antibody characterisation were performed as
above in azide free MACS buffer rather than FACS buffer under aseptic conditions
and used a 2mL syringe plunger to homogenise cells through a 70μm nylon mesh
filter (BD).
Flow Cytometry
Isolated lymphocytes were resuspended at a concentration of 4x107cells/mL and
2x106 cells per 50μL plated out per well in FACS buffer for each staining condition
in a 96 well-V bottom plate (Greiner Bio-One)(Table 2.2). Antibody staining was
performed at a final dilution of 1:200 unless otherwise specified (Table 2.3).
Following incubation in the dark at 4°C for 30min the plate was centrifuged and
supernatant removed. Cells stained for Natural killer and B cell markers as well as
those from internalisation, competitive binding or CFSE assays were resuspended in
200μL FACS buffer and passed through a 70μm filter into 5mL microtubes (BD
Biosciences).
Cells
stained
for
T
cell
markers
were
resuspended
in
43
Materials and Methods
fixation/permeabilisation buffer (fixation/permeabilisation concentrate and diluent
1:3 dilution, eBioscience) for at least 2 hours followed by incubation with Foxp3
antibody resuspended in 1x permeabilisation buffer (eBioscience) in the dark at 4°C
for 30min. Following the final antibody incubation T cells were washed and filtered
as above into 5mL microtubes before flow cytometry was performed on a LSRII
SORP flow cytometer with FACSDiva software (BD Bioscience). Data was analysed
using FlowJo (TreeStar Inc, Ashland Oregon USA). Light scatter gating was
performed on all samples to include live lymphocytes and exclude doublet cells, dead
cells and debris unless otherwise specified.
CFSE Proliferation Assay/Mixed Lymphocyte Reaction
Total lymphocytes were isolated from the spleen as above in MACS buffer under
sterile conditions and cells washed twice and resuspended at 1x107cells/mL in warm
CFSE staining buffer. The cell suspension was transferred to a fresh 15mL Falcon
tube wrapped in aluminium foil. CFSE (carboxyfluorescein succinimidyl ester;
Sigma-Aldrich) was prepared from stock (10μM) for in vivo (1μL of 10μM CFSE
stock) studies. On a dry portion of a fresh 15mL tube 1μL CFSE per 1x107cells/mL
was placed and cells quickly inverted and vortexed to ensure even distribution of
CFSE stain before incubation at 37°C for 10min. Staining was quenched by adding
5mL ice cold CFSE quenching buffer and incubating for 3 min. Cells were
centrifuged and washed twice more with ice cold CFSE quenching buffer before
performing a final cell count using trypan blue exclusion.
In vivo cell transfer experiments were performed using cells from C57BL/6 mice and
transferred into sub-lethally irradiated BALB/c mice. BALB/c mice were irradiated
with a single irradiation dose of 2.7Gy using an X-RAD 320 Biologic Irradiator
(Precision X-ray). 48 hours later mice were intravenously injected with 2x107 CFSE
labelled whole spleen cells. Mice also received either 2mg/kg anti-CXCR3-9C5 i.v.
immediately following cells or were untreated as controls. Mice were euthanised and
splenocytes isolated 72hrs later for flow cytometry.
44
Materials and Methods
Antibody Characterisation
Chemotaxis Assay
This was carried out by the Mackay Laboratory, briefly a murine pre-B cell line
transfected with human CXCR3 (L1.2 hCXCR3) was incubated with a range of
concentrations of αCXCR3-9C5, from 0μg/mL to 20μg/mL, for 10min at 37°C.
Following incubation with αCXCR3-9C5, 500,000 cells of each antibody
concentration were suspended in 75μL chemotaxis buffer and loaded into the top
chamber of individual HTS transwell plates (Corning; 5μm pore size, 96 well #33878EA). Human chemokine; CXCL9, CXCL10 or CXCL11 (Prepotech) diluted to
30nM in 240μL chemotaxis buffer were added to the bottom chamber of each HTS
transwell plate and incubated at 37°C for 4 hours. Following incubation 200μL of
cells from the bottom chamber were resuspended in 100μL FACS buffer + 2%PFA
and left overnight. Cells were read on the HTS plate reader on BD LSR flow
cytometer and the degree to which αCXCR3-9C5 inhibited migration expressed as a
percentage inhibition, compared to 0μg/mL αCXCR3-9C5, for each concentration of
αCXCR3-9C5 for hCXCL9, hCXCL10 and hCXCL11.
Receptor Internalisation Assay
Following isolation of splenocytes in MACS buffer, cells were resuspended at 2x106
cells/50μL in duplicate for each time point (0, 10, 20, 30, 60, 120 and 180 minutes)
of the internalisation assay. Cells were resuspended with 1μl (2μg/μL) APCconjugated αCXCR3-9C5 for each time point or APC-conjugated IgG Isotype in
50μL MACS buffer and incubated at 4°C for 30min to allow for antibody-receptor
binding. Isotype treated cells and αCXCR3-9C5 treated 0 time point cells remained
at 4°C for all further steps. For all other time points cells were incubated at 37°C for
the allocated time (0, 10, 20, 30, 60, 120 and 180 minutes) followed by removal onto
ice and quenching with 500μL ice cold PBS. Cells were washed twice followed by
fixation in 1:1 PBS and 4% formaldehyde. Once all time points were collected cells
were washed with cold PBS twice and stained for flow cytometry (table 2.2). All
steps were performed in azide free conditions.
45
Materials and Methods
Competitive Binding Assay
Following isolation of splenocytes in MACS buffer, cells were resuspended at
2x106cells/50μL in duplicate. Cells were incubated for 30 minutes at 4°C in azide
free conditions with 0.2μg APC-conjugated αCXCR3-9C5, APC-conjugated Isotype,
PerCpCy5.5-conjugated CXCR3-173 or PerCpCy5.5-conjugated Isotype antibody.
Cells were then washed twice with MACS buffer followed by a further round of
staining with 0.2μg antibody for 30 minutes at 4°C. Cells stained initially with APCconjugated
αCXCR3-9C5
or
APC-conjugated
Isotype
were
stained
with
PerCpCy5.5-conjugated CXCR3-173 or PerCpCy5.5-conjugated Isotype antibody.
Cells stained initially with PerCpCy5.5-conjugated CXCR3-173 or PerCpCy5.5conjugated Isotype were stained with APC-conjugated αCXCR3-9C5 or APCconjugated Isotype antibody. Cells were then stained for flow cytometry (table 2.2).
Antibody Titrations
In order to determine frequency and dose of αCXCR3-9C5 appropriate for the NOD
and C57BL/6 mouse strains antibody titration experiments were conducted. C57BL/6
mice were injected with a range of doses of αCXCR3 9C5 (0, 1mg/kg, 2mg/kg,
5mg/kg and 10mg/kg) on day 0 and followed for 10 days with tail vein blood
collected throughout the 10 days. NOD mice were injected with 1mg/kg αCXCR39C5, 1mg/kg Isotype or untreated as controls on day 0 and followed for 6 days with
blood taken throughout the 6 days. Blood samples were processed and analysed by
flow cytometry to determine CXCR3 expression on T and B cells for each
concentration and time point (table 2.2).
Adoptive Transfer
Adoptive transfer experiments were performed on RAG-/- recipients of BALB/c islet
allografts 14 days post allograft. Transferred splenocytes were harvested from long
term surviving (>100days) C57BL/6 recipients of BALB/c islet allografts, treated
with a combination of αCXCR3-9C5 (2mg/kg i.v. 1/week for 9 weeks) and subtherapeutic rapamycin (0.1mg/kg i.p for 7 days). Alternatively, spleens from
untouched C57BL/6 mice were used as controls.
Splenocytes were isolated under aseptic conditions and total T cells isolated via
magnetic separation using a Pan T cell kit (Miltenyi Biotec, Auburn, CA).
46
Materials and Methods
Subsequently CD25- effector T cells were isolated through positive depletion of
CD25+ cells using a CD25 Microbead kit (Miltenyi Biotec, Auburn, CA) following
manufacturers instructions. Magnetic separations were performed to a purity of
>95% using AUTOMACS (Miltenyi Biotec, Auburn CA), assessed by flow
cytometric analysis.
Whole T cells or CD25 depleted effector T cells (2x106 cells) were then adoptively
transferred into Rag-/- recipient mice via tail vein i.v.
Histology
At designated end points mice were euthanized using CO2 and tissues collected.
Pancreata were collected from NOD mice. Kidneys were collected from long term
surviving islet transplant recipient mice.
Tissues were collected into labelled cassettes stored in 10% formaldehyde for 2 days
followed by transfer into 70% ethanol and paraffin embedding (performed by TKCC
Histopathology Facility). Sections of 5μm were cut using a Microtome (Leica,
Wentzler, Germany), mounted onto slides and allowed to air dry overnight. Kidney
sections were mounted on 20 slides with sections 1-20 at the top of each slide
followed by sections 21-40 at the base of each slide allowing better visualisation of
islet graft site. Pancreas sections were mounted with sections on slides 1-8
contiguous, 9-16 contiguous and 17-24 contiguous (2-3 sections per slide) with a
~150μm section discarded between each set of 8 slides, to prevent recounting the
same islet.
H&E
For kidney sections slides 1, 10 and 20 were stained using H&E to identify the
optimum slide to visualise remaining islet grafts. Pancreas sections slides 1, 9 and 17
were stained for H&E to allow grading of insulitis. H&E was performed using an
autostainer (Leica autostainer XL) and coverslipper (Leica CV5030) provided by
TKCC Histopathology Facility.
Briefly insulitis in the NOD sections was graded on a 5 point scale using Leica
microscope (Leica DC 200 camera, Leica Microsystems) and software to visualise
each islet at 100-200x magnification. Degree of insulitis was graded as; 0; no
47
Materials and Methods
infiltration, 1; periductal/peri-insulitis, within the pancreas, localised around but not
within the islet, 2; circumferential, <25% of the islet infiltrated, 3; invasive, 25%50% of the islet infiltrated, 4; destructive, >75% of the islet infiltrated262. A
minimum of 70 islets were counted per mouse over the 3 histological levels and
expressed for each grade as a percentage of total islets scored for each group.
Insulin Staining
In order to determine degree of insulin production by islets remaining within the islet
allograft, slides contiguous with H&E stained sections were deparaffinised using the
autostainer (Leica autostainer XL). Insulin staining was performed using the DAKO
EnVision + System-HRP (DAKOCytomation) with 100μL reagent per tissue section
and incubations at room temperature in the dark maintaining sections under moist
conditions.
Once deparaffinised, tissue sections were treated with 0.3% H2O2 (DAKO;
Peroxidase block) for 5min and washed twice with 1x PBS. Tissue sections were
incubated for 1 hour with insulin primary antibody (Promega #4590; 1:100 rabbit
anti-mouse insulin antibody in DAKO antibody diluent; Cell signalling). Slides were
washed twice with 1x PBS and incubated for 30min with secondary, polymer-HRP
anti-rabbit antibody (DAKO). Slides were washed three times followed by
development with DAB and substrate buffer (DAKO; 1 drop DAB/mL substrate) for
2min keeping timing consistent for all slides. Slides were washed thoroughly with
dH2O and counterstained (Leica autostainer XL) and cover slipped (Leica CV5030).
Foxp3+ Staining
In order to determine infiltration of pancreata and islet grafts by Foxp3+ regulatory T
cells, Foxp3 staining was performed on sections contiguous with those used to stain
for H&E and/or insulin. Slides were deparaffinised using the autostainer (Leica
autostainer XL) followed by antigen retrieval using a pressure cooker (DAKO
Cytomation, Pascal, California Inc. S2800). Briefly, slides were placed in an antigen
retrieval box containing citrate buffer (DAKO; 10mM, pH 6, 1:10 with dH2O) and
heated in a pressure cooker (125°C for 30sec, 95°C for 10sec) followed by cooling
the antigen retrieval box with cold running water. Following antigen retrieval tissue
sections were washed with dH2O, treated with 0.3% H2O2 (DAKO; Peroxidase
48
Materials and Methods
block) for 10min and washed twice with TBS-T. Tissue sections were treated with
protein block (DAKO) and incubated for 1 hour. Protein block was flicked off and
briefly washed with TBS-T followed by incubation for 2 hours with Foxp3 primary
antibody (eBioscience; rat anti-mouse purified Foxp3, clone FJK-16s, 1:100 in
DAKO antibody diluent; Cell signalling). Slides were washed three times with TBST and incubated for 30min with secondary, biotin anti-rat antibody (Jackson
Laboratories; biotin SP-conjugated affinity purified F(ab’) fragment goat anti-rat
IgG, Fc with spacer, 1:1000 in DAKO antibody diluent; Cell signalling). Slides were
washed three times with TBS-T and incubated for 30min with the ABC Vectastain
kit (Elite PK6100) in PBS. Slides were washed thoroughly with TBS-T followed by
development with DAB and substrate buffer (DAKO; 1 drop DAB/mL substrate) for
30sec keeping timing consistent for all slides. Slides were washed thoroughly with
dH2O, counterstained (Leica autostainer XL) and cover slipped (Leica CV5030).
Statistics
Data was analysed using GraphPad Prism version 6.04 for Windows (GraphPad
Software, La Jolla California USA, www.graphpad.com). Kaplan-Meir survival
curve analysis using a Log-rank (Mantel Cox) test was performed to determine
changes in allograft survival or diabetes onset. Holm-Sidak multiple comparison t
tests were perfomed followed by further analysis using unpaired t test (MannWhitney) or one-way ANOVA on select results. Results were considered statistically
significant when the p value was less than 0.05
Table 2.2: Flow Cytometry Staining Conditions;
Stains
T
cells
Antibodies
(C57BL/6
and CD4, CD8, CD25, Foxp3, CD44, CD62L, CXCR3
NOD)
NK & B cells (NOD only)
B220, CD21, CD23, CD3, NKp46, CXCR3
CFSE Staining
CD4, CD8, CD44, CD62L, CXCR3, CFSE (521nm)
Antibody Titration
B220, CD4, CD8, CD25, Foxp3, CXCR3
Internalisation Assay
CD4, CD8, APC-CXCR3-9C5/Isotype, PerCpCy5.5CXCR3-173/Isotype
Competitive
Assay
Binding CD4, CD8, APC-CXCR3-9C5/Isotype, PerCpCy5.5CXCR3-173/Isotype
49
Materials and Methods
Table 2.3: Antibodies for Flow Cytometry
Antibody
Clone
Flurochrome
Final Dilution
Manufacturer
CD4
RM4-5
Pacific Blue
1:200
BD Pharmingen
CD8
53-6.7
Pe-Cy7
1:200
eBioscience
CD44
IM7
APC-Cy7/FITC
1:200
BD Pharmingen
CD25
7D4
FITC
1:200
BD Pharmingen
CD62L
MEL-14
PE
1:200
BD Pharmingen
Foxp3
FJK-16s
APC
1:200
eBioscience
B220
RA3-6B2
APC-Cy7
1:200
Biolegend
CD21
7G6
FITC
1:200
BD Pharmingen
CD23
B3B4
PE
1:200
BD Pharmingen
CD3
145-2C11
APC
1:200
eBioscience
NKp46
29A1.4
Pe-Cy7
1:200
eBioscience
NK1.1
PK136
FITC
1:200
BD Pharmingen
IgM
II/41
APC
1:200
BD Pharmingen
CXCR3
CXCR3-173
PerCp-Cy5.5
1:100
Biolegend
PerCp-Cy5.5
1:100
Biolegend
APC
1:100
Abcam kit
APC
1:100
Abcam kit
IgG Isotype (for HTK888
CXCR3-173)
Armenian
hamster IgG
CXCR3
αCXCR3-9C5
IgG Isotype (for IgG
CXCR3-9C5)
50
Characterising αCXCR3-9C5
Chapter 3. Characterising αCXCR3-9C5
51
Characterising αCXCR3-9C5
Chapter 3. Characterising αCXCR3-9C5
The chemokine receptor CXCR3 is involved in the recruitment of immune cells to
sites of inflammation3,24,200,263. Targeted blockade of immune cell recruitment to sites
of inflammation has clinical potential for preventing conditions like autoimmunity
and transplant rejection. For this thesis I will explore the efficacy of a novel
monoclonal antibody against CXCR3 in animal models of autoimmunity and
transplant rejection.
αCXCR3-9C5 Impairs Chemotaxis of CXCR3 transfected cells
This antibody was generated by Remy Robert in the Mackay lab. The CXCR3
antibody was raised in a CXCR3-/- mouse against a highly conserved epitope found
in the mouse, marmoset and human CXCR3 sequence (figure 3.1a). The antibody
clone, CXCR3-9C5, binds a region of the CXCR3 sequence critical for chemokine
binding2, as shown for the human CXCR3-A sequence (Figure 3.1b). As a result it
was expected that administration of the anti-CXCR3-9C5 (αCXCR3-9C5) antibody
would inhibit chemotaxis of a murine pre-B cell line transfected with human
CXCR3-A (L1.2 hCXCR3) to express the CXCR3 receptor. The efficacy of this
antibody in inhibition of CXCR3 induced chemotaxis was determined by incubating
CXCR3 transfected cells with αCXCR3-9C5 (0-100μg/mL) and measuring the
change in the ability of these cells to migrate across a membrane towards human
CXCR3 chemokines; hCXCL9, hCXCL10 and hCXCL11. A strong inhibitory effect
on chemotaxis of transfected cells in response to increasing doses of αCXCR3-9C5
for human (h)CXCL9 was seen, with ~90% inhibition at a concentration of 20μg/mL.
In addition chemotaxis in response to hCXCL10 and hCXCL11 showed 50-60%
inhibition at a concentration of 20μg/mL αCXCR3-9C5 (figure 3.1c). These data
show the efficacy of the mAb αCXCR3-9C5 in blockade of CXCR3 dependent
chemotaxis.
52
Characterising αCXCR3-9C5
Figure 3.1: αCXCR3-9C5 antibody binds a conserved epitope and impairs
Lymphocyte Chemotaxis
(a) Amino acid sequence of CXCR3 with a highly conserved region of the extracellular N
terminal across a number of species. Figure includes the highly conserved region against
which the αCXCR3-9C5 mAb was raised (red). (b) Snakeplot structure of human CXCR3-A
amino acid sequence including transmembrane conformation and location of anti-CXCR39C5 binding on the N terminal (red). This region contains Y27 and Y29 (black), essential
in chemokine binding and receptor activation2,3. (c) Chemotaxis assay showing inhibition
of Murine pre-B cell line transfected with Human CXCR3-A (L1.2 hCXCR3) chemotaxis for
increasing doses of αCXCR3-9C5 from 0μg/mL to 20μg/mL for human (h)CXCL9, hCXCL10
and hCXCL11.
Effects of αCXCR3-9C5 on CXCR3 expression and cell stability
We next investigated CXCR3 receptor expression and internalisation following
αCXCR3-9C5 treatment of target cells to determine the effects of αCXCR3-9C5
ligation on cell stability. For this we investigated if αCXCR3-9C5 ligation mediated
neutralisation of the receptor or depletion of bound cells or internalisation of the
chemokine receptor.
In order to examine receptor expression on splenocytes the αCXCR3-9C5 antibody
was fluorescently conjugated followed by incubation with isolated splenocytes to
determine CXCR3 expression by flow cytometry. A second flurochrome conjugated
53
Characterising αCXCR3-9C5
CXCR3 antibody sold commercially (CXCR3-173) was used to validate CXCR3
expression. Upon treatment of splenocytes with αCXCR3-9C5 or CXCR3-173,
binding to the CXCR3 receptor on CD4 and CD8 T cells was found to be comparable
for both antibodies (figure 3.2a). This CXCR3-173 monoclonal antibody was raised
against a similar epitope of CXCR3 as αCXCR3-9C5 (figure 3.2b)200.
Figure 3.2: Equivalent Binding of CXCR3+ T cells by αCXCR3-9C5 and commercially
available CXCR3-173 antibody.
(a) Lymphocytes incubated with either CXCR3-173 (commercially available) or αCXCR3-9C5
at 4°C were found to bind comparable frequencies of CXCR3+CD4+ and CXCR3+CD8+ T cells,
by flow cytometry. Mean+/-SEM data from 2 replicate experiments performed in duplicate.
(b) Both antibodies were raised against the extracellular N-terminal domain of the CXCR3
receptor, targeting similar and overlapping epitopes of the CXCR3 receptor.
Receptor internalisation and competitive binding assays
Receptor internalisation and competitive binding assays were then performed (see
figure 3.3 for schematic representation) in order to determine the mechanism by
which αCXCR3-9C5 elicits its inhibitory effect on ligand binding and chemotaxis.
Receptor internalisation was examined through incubation of splenocytes with
αCXCR3-9C5 antibody for varying times (0, 10, 20, 30, 60, 120, 180 minutes) at
37°C, in azide free conditions, followed by fixation and staining for flow cytometry
(figure 3.4a). In comparison to isotype treated cells, those incubated with αCXCR39C5 showed binding for both CD4+ and CD8+ T cells indicating specificity of
binding to CXCR3. No significant difference in the degree of binding was seen
following incubation with αCXCR3-9C5 at any time, suggesting the αCXCR3-9C5
antibody clone was not affecting basal CXCR3 expression on these cells and was not
54
Characterising αCXCR3-9C5
inducing receptor internalisation. Due to the use of fixation however, the possibility
of detecting internalised receptor poses a potential caveat.
Following incubation with αCXCR3-9C5 at 37°C, cells were also treated with the
alternate CXCR3-173 antibody to validate this result. However CXCR3-173 was
unable to bind those cells pre-treated with αCXCR3-9C5 (figure 3.4b). Binding of
CXCR3-173 to cells pre-treated with isotype rather than αCXCR3-9C5 showed a
similar frequency of binding to CD4+ and CD8+ T cells compared to binding by
αCXCR3-9C5. This result, combined with the overlapping peptides against which
the two antibodies were raised, suggested competitive binding between the
antibodies for a similar epitope on the CXCR3 receptor and as such competitive
binding was investigated further.
Figure 3.3: Internalisation and competitive binding assay.
Schematic representation; order of antibody application for each in vitro assay.
Splenocytes, represented by blue cells, αCXCR3-9C5 fluorescently labelled (red) and
CXCR3-173 fluorescently labelled (grey).
In order to clarify the ability of CXCR3 to be bound by the two antibodies
splenocytes were incubated at 4°C in azide free conditions with either antibody alone
(αCXCR3-9C5 or CXCR3-173 alone) or sequential incubation of cells with
αCXCR3-9C5 followed by CXCR3-173 or incubation with CXCR3-173 followed by
αCXCR3-9C5. Firstly this did not affect the frequency of CD4+ or CD8+ T cells
55
Characterising αCXCR3-9C5
detected through flow cytometry (figure 3.4c) suggesting that the antibody is not
causing T cell depletion. Incubation with each antibody alone showed binding of
significantly more CXCR3 expressing cells compared to isotype, as expected, with a
slightly lower frequency of CD4+ T cells bound by CXCR3-173 than αCXCR3-9C5
(figure 3.4d).
Figure 3.4: αCXCR3-9C5 competitively binds CXCR3 receptor but does not cause
internalisation or lymphocyte depletion.
Internalisation Assay; Incubation of splenocytes at 37°C, azide free, with CXCR3-9C5
antibody for 0, 10, 20, 30, 60, 120 and 180 minutes or isotype control antibody and analysis
of CXCR3+CD4+ and CXCR3+CD8+ T cell binding by flow cytometry for (a) CXCR3-9C5
binding (b) or CXCR3-173 binding post incubation with CXCR3-9C5.
Competitive binding assay; Incubation of splenocytes with αCXCR3-9C5 or CXCR3-173
alone or sequentially with CXCR3-173 staining prior to or followed by αCXCR3-9C5
staining, or isotype control expression. (c) The frequency of CD4+ and CD8+ T cells. (d)
Expression of CXCR3 for isotype, αCXCR3-9C5 and CXCR3-173 treated CD4+ and CD8+ T
cells, (e) binding of αCXCR3-9C5 or (f) binding of CXCR3-173 to CD4+ and CD8+ T cells
treated with either antibody alone or sequentially.
Mean+/-SEM p<0.05*, p<0.01**, p<0.001***, data from 2-3 replicate experiments
performed in duplicate.
56
Characterising αCXCR3-9C5
Incubation of cells with CXCR3-173 did not affect binding of cells by αCXCR39C5, regardless of whether CXCR3-173 was applied prior to or post αCXCR3-9C5
(figure 3.4e). However binding of cells by CXCR3-173 was impaired when cells
were incubated with αCXCR3-9C5 following initial treatment with CXCR3-173 and
to an even greater extent when stained with αCXCR3-9C5 prior to incubation with
CXCR3-173 (figure 3.4f).
57
Characterising αCXCR3-9C5
Summary & Conclusions
CXCR3 is an important chemokine receptor, particularly for activated CD4+ and
CD8+ T cells and natural killer cells21,22,188,200,201. Here we characterise a novel
αCXCR3-9C5 antibody raised against a conserved epitope of CXCR3 found in a
number of species including mice and humans. This monoclonal antibody targets an
epitope containing residues critical for chemokine binding and as a result impairs
chemotaxis of CXCR3 transfected cells in a concentration dependent manner to the
three CXCR3 ligands, CXCL9, CXCL10 and CXCL112. In addition ligands with
higher affinity to CXCR3, CXCL10 and CXCL11, showed a lower level of
chemotactic inhibition compared to the lower affinity chemokine CXCL9189,190.
These results show no change in CXCR3 expression in internalisation assays with
αCXCR3-9C5,
suggesting
that
αCXCR3-9C5
does
not
induce
receptor
internalisation. However, due to the fixation of cells following αCXCR3-9C5
treatment this data may represent surface and internal CXCR3 expression. Future
experiments using biotinylated αCXCR3-9C5 followed by staining post incubation
with fluorochrome conjugated streptavidin and with no fixation would be expected to
clarify this.
Results also did not indicate depletion of T cells with no change in the frequency of
CD4+ of CD8+ T cells following αCXCR3-9C5 treatment compared to isotype
control, as well as compared to CXCR3-173, known to neutralise but not deplete
CXCR3 expressing cells200. This result is in agreement with previous literature
showing minimal effector function of mouse IgG1 antibodies for both antibodydependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity
(CDC)264. However, further in vivo investigation will be required to clarify cell
depletion through these pathways. This can be achieved briefly by incubating
effector cells with the αCXCR3-9C5 mAb followed by addition of complement
(CDC) or NK92 (a cell line with characteristics of NK cells)(ADCC) which lyse the
target effector cells. Lysis can then be measured using absorbance to determine
lactate dehydrogenase release.
When looking at our αCXCR3-9C5 antibody in relation to the commercially
available CXCR3-173 we found that the degree of chemotactic inhibition for
CXCL10 found with αCXCR3-9C5 showed a similar efficacy, ~50-60% inhibition at
58
Characterising αCXCR3-9C5
20μg/mL, as the CXCR3-173 antibody200. In contrast, CXCR3-173 showed only
minor (~25%) inhibition for CXCL11 and no effect on chemotaxis in response to
CXCL9, while αCXCR3-9C5 resulted in a higher level of chemotaxis inhibition for
CXCL11 (~50%) and CXCL9 (~90%) at concentrations of 20μg/mL. Both
antibodies were found to neutralise rather than deplete T cells upon binding200.
Studies of the CXCR3 receptor structure by Colvin et al. revealed that
phosphorylation of residues Y29 and Y27 is essential for chemokine binding by
CXCL9, CXCL10 and CXCL112. The region targeted by αCXCR3-9C5
encompasses these residues (figure 3.1 & 3.2). Further the proximal 16 amino acids
of the CXCR3 N-terminal, which are targeted by CXCR3-173 but not αCXCR3-9C5,
are important for CXCL10 and CXCL11 but not CXCL9 binding, this difference in
binding sites may account for the distinct effects of the two antibodies2.
Finally these results strongly suggest competitive binding between the αCXCR3-9C5
and CXCR3-173 antibodies. These antibodies were raised against overlapping
peptides of the CXCR3 receptor with binding assays showing impaired binding of
CXCR3-173 in the presence of our αCXCR3-9C5 antibody. The ability of αCXCR39C5 to bind to T cells was not significantly affected by the presence of CXCR3-173,
either administered prior to or post incubation with αCXCR3-9C5. In contrast the
ability of CXCR3-173 to bind T cells, both pre-incubated with αCXCR3-9C5 or
where αCXCR3-9C5 was administered following CXCR3-173 was significantly
impaired. This suggests that CXCR3-173 was displaced by αCXCR3-9C5, due to
steric hindrance or a higher binding affinity for this shared epitope, resulting in
competitive binding265,266. Due to the competitive binding of αCXCR3-9C5 and
CXCR3-173 for this shared epitope of the CXCR3 receptor, fluorescently labelled
CXCR3-173 expression represents CXCR3 receptor not bound by αCXCR3-9C5.
Thus comparisons between αCXCR3-9C5 treated and untreated cells for expression
of CXCR3-173 could be used to determine the frequency of cells bound by
αCXCR3-9C5. This also allows the expression of free, functionally available
CXCR3 to be determined and was used in further experiments as a measure of
αCXCR3-9C5 binding and free CXCR3 expression.
59
Chapter 4. αCXCR3-9C5 in the Islet Allograft
Model
αCXCR3-9C5 in the Islet Allograft Model
Chapter 4. αCXCR3-9C5 in the Islet Allograft Model
The chemokine receptor CXCR3 has been shown to be highly upregulated in a
number of inflammatory conditions in humans including; cancer, autoimmunity and
allograft rejection178,179,181,182,239. Due to its known role in recruitment of
lymphocytes we sought to investigate the role of CXCR3 in a mouse model of islet
allograft transplantation. Islet allograft rejection is both a well-established and wellstudied model for human transplant rejection267-269. This model has also gained
further importance due to the emergence of clinical islet transplantation as a
treatment
for
individuals
with
brittle
diabetes
and
hypoglycaemic
unawareness102,149,155.
A number of studies have investigated the effects of CXCR3 blockade and deletion
in mouse models of allograft transplantation (skin, cardiac, GVHD and also in islets)
showing improved allograft survival due to impaired recruitment of immune cells to
the foreign tissue44,200,216,246,252. This prompted us to investigate the role of αCXCR39C5 antibody in a BALB/c (H2d) to C57BL/6 (H2b) MHC mismatched islet allograft
model allowing us to determine the efficacy of this new antibody in an established
model.
Peripheral Immune cell characterisation of CXCR3 Expression
Initial studies of the efficacy of different doses of αCXCR3-9C5 antibody in
C57BL/6 mice were conducted to determine the effect of αCXCR3-9C5 treatment on
the peripheral lymphocyte populations. Using this information we also determined
the ideal dose and timing regimen of αCXCR3-9C5 treatment for mice receiving an
islet allograft transplant or for untouched mice.
These experiments were conducted on 10 week old C57BL/6 mice, intravenously
(i.v.) administered 1mg/kg, 2mg/kg, 5mg/kg or 10mg/kg doses of αCXCR3-9C5 on
day 0 followed by tail bleed on days 2, 4, 8 and 10 post i.v. Untouched C57BL/6
mice were used as controls. The peripheral blood frequency of B cells and CD4+,
CD4+Foxp3+ (Treg) and CD8+ T cells and their respective binding by CXCR3-173,
as an indication of free CXCR3 unbound by αCXCR3-9C5, were determined via
flow cytometry. No significant changes in peripheral lymphocyte (B or T cell)
61
αCXCR3-9C5 in the Islet Allograft Model
frequencies were evident with administration of αCXCR3-9C5 regardless of dose or
time post administration (figure 4.1 & 4.2).
The frequency of CXCR3-173 binding to CXCR3 was significantly decreased in
mice treated with αCXCR3-9C5 with a similar reduction in binding seen for all doses
of αCXCR3-9C5 (1mg/kg-10mg/kg) administered. For this reason statistical
comparisons were performed between the lowest dose (1mg/kg) of αCXCR3-9C5
and controls, unless otherwise stated. Administration of CXCR3-173 showed
minimal binding to B cells regardless of αCXCR3-9C5 administration for all doses
and time points examined, indicating a very low expression of CXCR3 on B cells,
unaffected by αCXCR3-9C5 (figure 4.1a). CXCR3 expression on T cell subsets
showed reduced binding by CXCR3-173 compared to control expression for
CXCR3+CD4 (figure 4.1b), CXCR3+CD4Foxp3+ (figure 4.2a) and CXCR3+CD8
(figure 4.2b) T cells, This indicates a reduced frequency of free CXCR3, unbound by
αCXCR3-9C5.
A significant reduction in CXCR3-173 binding was evident for CD4+ T cells at day
2 (figure 4.1bi) and day 8 (figure 4.1biii) between control and all αCXCR3-9C5
doses. However due to variability at day 4 only control and the 2mg/kg αCXCR39C5 dose were found to differ significantly (figure 4.1bii). On day 10 no significant
difference was found between control and 1mg/kg αCXCR3-9C5 treated peripheral
blood CD4+ T cell CXCR3-173 binding, indicating a loss of efficacy at this time
point (figure 4.1biv).
Analysis of CXCR3-173 binding for CD4+Foxp3+ (figure 4.2a) and CD8+ T cells
(figure 4.2b) revealed significant reductions at all time points compared to controls.
This lead us to consider all doses equally effective at blocking CXCR3 function in
vivo for up to 8 days, with loss of efficacy for CD4+ T cells at this point. For this
reason administration of multiple doses of αCXCR3-9C5 antibody at 2mg/kg on a
weekly basis (every 7 days) to consistently block the CXCR3 receptor was
considered optimal for further experiments.
62
αCXCR3-9C5 in the Islet Allograft Model
Figure 4.1: αCXCR3-9C5 Antibody Titrations – C57BL/6
Analysis of peripheral blood lymphocyte expression of CXCR3, determined using CXCR3173 by flow cytometry. Mice were treated with 1mg/kg, 2mg/kg, 5mg/kg or 10mg/kg
αCXCR3-9C5 i.v. on day 0 or untouched as controls for (a) B cells and (b) CD4+ T cells on
day (i) 2, (ii) 4, (iii) 8 and (iv) 10. Expressed as percent lymphocytes (n=3 mice/group).
Mean+/-SEM p<0.05*, p<0.001***.
63
αCXCR3-9C5 in the Islet Allograft Model
Figure 4.2: αCXCR3-9C5 Antibody Titrations – C57BL/6
Analysis of peripheral blood lymphocyte expression of CXCR3, determined using
CXCR3-173 by flow cytometry. Mice were treated with 1mg/kg, 2mg/kg, 5mg/kg or
10mg/kg αCXCR3-9C5 i.v. on day 0 or untouched as controls for (a) CD4+Foxp3+
Tregs and (b) CD8+ T cells on day (i) 2, (ii) 4, (iii) 8 and (iv) 10. Expressed as percent
lymphocytes (n=3 mice/group). Mean+/-SEM p<0.05*, p<0.01**, p<0.001***,
p<0.0001****
64
αCXCR3-9C5 in the Islet Allograft Model
αCXCR3-9C5 in C57BL/6 Islet Allografts
Once an appropriate timing and dosage had been determined in C57BL/6 mice we
investigated the expression of CXCR3 in the spleens of untouched control C57BL/6
and control C57BL/6 recipients of an islet allograft.
Islet allograft survival follows a typical pattern of initially elevated blood glucose,
due to chemical treatment with alloxan/STZ to induce diabetes. This is followed by a
sharp decline in blood glucose back to euglycaemia upon islet allograft. Mice were
monitored for an initial reduction in blood glucose, back to euglycaemic levels,
followed by a rise in blood glucose i.e. hyperglycaemia. This model is able to
physiologically mimic what would happen clinically. In the islet allograft model
euglycaemia indicates that the islet allograft is functionally able to produce insulin
and restore blood glucose. This is then followed by a gradual loss of function,
measured by a rise in blood glucose level (BGL), over the course of immune
mediated destruction and allograft loss (see figure 4.3d). Islet allograft survival was
determined as the number of post-operative days maintaining euglycaemia before
onset of hyperglycaemia (BGL>15mmol/L) indicating loss of allograft.
Islet allograft was performed using diabetic C57BL/6 (H2b) recipient mice,
chemically induced using alloxan (110mg/kg) or STZ (180mg/kg), transplanted with
MHC mismatched BALB/c (H2d) islets under the kidney capsule (figure 4.3). These
mice were then either left untreated as controls or administered 2mg/kg i.v.
αCXCR3-9C5. An additional group of C57BL/6 mice, which did not receive islet
allograft transplant, remained untouched, with the exception of αCXCR3-9C5
administration (2mg/kg i.v) as indicated.
Initial experiments aimed to characterise the expression of CXCR3 and effects of
administration of 2mg/kg i.v. αCXCR3-9C5 on levels of free CXCR3 and changes in
the frequency of T cell populations. These experiments used either αCXCR3-9C5
treated C57BL/6 mice (αCXCR3-9C5 treated untouched recipients) which were
untouched or islet allograft recipients (αCXCR3-9C5 treated islet allograft
recipients) compared to untreated control mice (untouched controls or control islet
allograft recipients).
65
αCXCR3-9C5 in the Islet Allograft Model
Further investigation of the effects of αCXCR3-9C5 on islet allograft survival
employed 4 treatment regimens from the day of transplant. These included; once
weekly i.v. injections of 2mg/kg Isotype control antibody or αCXCR3-9C5 antibody
with no other treatment or Isotype or αCXCR3-9C5 antibody in combination with
sub-therapeutic doses of the immunosuppressant rapamycin, at 0.1mg/kg, i.p., every
day for the first week following transplant260 (see figure 4.3c).
Figure 4.3: Allogeneic Experimental Model
Overview of C57BL/6 experimental model including a) BALB/c to C57BL/6 Islet allograft
schematic, b) timing of treatments and endpoints, chemical induction of diabetes with
alloxan 48 hours prior to first treatment dose with isotype or αCXCR3-9C5 mAb with or
without rapamycin and receipt of MHC mismatched BALB/c islet allograft c) treatments
received by Islet allograft recipients for determination of efficacy of αCXCR3-9C5 mAb on
graft survival, d) schematic of typical BGL over the course of rejection.
Basal and Islet Allograft CXCR3 Expression
In order to determine the proportion of cells expressing CXCR3 in untouched and
islet allograft recipient mice, flow cytometry was performed on isolated splenic T
cells.
66
αCXCR3-9C5 in the Islet Allograft Model
Basal expression of CXCR3 on T cell subsets within the spleens of control
untouched mice (unfilled black) was determined based on isotype antibody
expression (grey line with gating based on grey dashed line) (figure 4.4). The median
of the values for proportion of CXCR3 positive cells were determined for untouched
control mice with 8.5% of CD4+, 21.9% of Treg and 24.7% of CD8+ T cells found
to express CXCR3. The median of the values for proportion of CXCR3 positive cells
for control islet allograft recipient mice (unfilled black) was also determined at day 4
post-transplant compared to isotype antibody expression (grey line with gating based
on grey dashed line) (figure 4.4). This revealed upregulation of CXCR3 expression
on T cells isolated from the spleens of control islet allograft recipients compared to
untouched control mice with 15.5% of CD4+, 33.4% of CD4+Foxp3+ and 28.1% of
CD8+ T cells expressing CXCR3. CXCR3 expression for CD4+, Tregs and CD8+ T
cells were also determined for αCXCR3-9C5 treated untouched and islet allograft
recipient mice on POD 4, determined via CXCR3-173 binding. CXCR3 expression
was detected on each T cell subset following treatment with 2mg/kg αCXCR3-9C5
(grey shaded, dashed line) at a reduced level compared to control treated mice.
Mean, median and range values are given for each group in Table 4.1.
Figure 4.4: C57BL/6 CXCR3 Expression Profiles
Representative histograms of C57BL/6 splenic T cell subsets (CD4+, CD4+Foxp3+Treg
and CD8+) showing median for proportion of CXCR3+ staining for untouched C57BL/6
and islet allograft recipient C57BL/6 mice at 4 days post-transplant. Isotype mAb
(grey line) compared to untreated control (unfilled black) or αCXCR3-9C5 treated
(grey shaded, dashed line) CXCR3 expression (n=2-6 mice/group).
67
αCXCR3-9C5 in the Islet Allograft Model
CD4+CXCR3+
CD4+Foxp3+
CXCR3+
CD8+CXCR3+
Untouched
Control
Untouched
αCXCR3
Islet Allograft
Transplant
Control
Islet Allograft
Transplant
αCXCR3
Mean
Median
Range
8.8225
8.545
11.6-6.6
4.48
3.855
9.79-1.86
19.52
15.5
38.9-4.16
5.64
5.64
6.16-5.12
Mean
25.975
5.76833333
28.5
18.35
Median
Range
Mean
Median
Range
n=
21.95
49.3-10.7
25.075
24.7
29.1-21.8
4
3.47
18.6-1.7
3.455
1.94
10.7-1.55
6
33.4
39.4-12.7
23.49666667
28.1
34.2-8.19
3
18.35
22.3-14.4
15.025
15.025
20.6-9.45
2
Table 4.1: C57BL/6 CXCR3 Expression Profiles
Mean and median values as well as range of C57BL/6 splenic T cell subsets (CD4+,
CD4+Foxp3+Treg and CD8+) proportion of CXCR3+ staining for untouched C57BL/6 and
islet allograft recipient C57BL/6 mice at 4 days post-transplant (n=2-6 mice/group).
αCXCR3-9C5 Binds CXCR3 and Reduces Memory and Effector CD8+ T cells
The proportion of cells expressing free unbound CXCR3 on CD4+, Treg and CD8+
T cell subsets was next determined for C57BL/6 mice, which received 2mg/kg, IV,
αCXCR3-9C5 or were left untreated as controls, at day 4 and day 8 post treatment.
This was also performed for an additional group of mice which received an islet
allograft transplant followed by treatment with 2mg/kg, IV, αCXCR3-9C5 or left
untreated as controls and expression determined at day 4 and day 8 post treatment.
Initial analysis revealed little change in raw cell counts at day 4 or between
untouched groups. Initial raw counts of the total number of splenocytes in control
transplant recipients showed a large but non-significant decrease at day 8 (figure
4.5). Of note these mice were treated with STZ to chemically induce diabetes prior to
receipt of an allograft (see summary & conclusions). Although this change was nonsignificant compared to αCXCR3-9C5 treated islet allograft recipients it did impact
upon determination of changes in cell populations by flow cytometry. As a result
both total number and frequency were assessed for allograft recipients (figure 4.94.12), while only total splenocyte number was analysed for untouched mice (figure
4.6-4.9).
68
αCXCR3-9C5 in the Islet Allograft Model
The total number of CD4+, CD4+Foxp3+ or CD8+ T cells found in untouched
C57BL/6 mice either treated with αCXCR3-9C5 or untreated controls was not
different between groups at either day 4 or day 8 post-treatment (figure 4.6a, 4.7a,
# s p le n o c y te s
4.8a).
8 .0  1 0
7
6 .0  1 0
7
4 .0  1 0
7
2 .0  1 0
7
U n to u c h e d C o n tro l
U n to u c h e d  C X C R 3 -9 C 5
T ra n s p la n t c o n tro l
T ra n s p la n t  C X C R 3 -9 C 5
y
a
d
8
4
d
a
y
s
s
0
Figure 4.5: Untouched and transplant recipient mice total splenocyte counts
C57BL/6 mice were treated i.v. with 2mg/kg αCXCR3-9C5 or untreated as controls, of these
a cohort of mice were received a BALB/c islet allograft under the kidney capsule
(transplant) while another group did not (untreated). Mice were then assessed for total
splenic lymphocyte counts at day 4 and 8 post i.v. (n=2-6 mice/group)
b
# C D 4 + S p le n o c y te s
1 .0  1 0 7
8 .0  1 0 6
6 .0  1 0 6
4 .0  1 0 6
2 .0  1 0 6
0
# C D 4 + C X C R 3 + S p le n o c y t e s
a
500000
*
400000
300000
200000
100000
0
U n to u c h e d C o n tr o l 4 d a y
U n to u c h e d  C X C R 3 - 9 C 5 4 d a y
U n to u c h e d C o n tr o l 8 d a y
U n to u c h e d  C X C R 3 - 9 C 5 8 d a y
Figure 4.6: αCXCR3-9C5 treatment in untouched C57BL/6 mice – CD4+ T cells
Untouched C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5, were assessed by flow
cytometry for splenic lymphocyte populations at day 4 and 8 post i.v. Splenocytes were
expressed as cell number for (a) CD4+ T cells and (b) CD4+CXCR3+ showing binding of
free CXCR3, determined via CXCR3-173 binding. Mean+/- SEM p<0.05* (n=26mice/group).
Analysis of the number of cells expressing free unbound CXCR3, via CXCR3-173
binding, for each T cell subset suggested a trend toward reduced expression for
69
αCXCR3-9C5 in the Islet Allograft Model
CD4+, CD4+Foxp3+ and CD8+ T cells at day 4 compared to untreated controls,
although small group sizes prevent statistical analysis (figure 4.6b, 4.7b, 4.8b). When
the number of cells expressing free CXCR3 was determined at day 8 post αCXCR39C5 administration a significant decrease was evident for CD8+ T cells but not
CD4+ or CD4+Foxp3+ T cells, compared to untreated controls (figure 4.8b).
b
80000
1000000
500000
S p le n o c y te s
1500000
# C D4+Foxp3+C XCR 3+
# C D 4 + F o x p 3 + S p le n o c y te s
a
**
60000
40000
20000
0
0
U n to u c h e d C o n tr o l 4 d a y
U n to u c h e d  C X C R 3 - 9 C 5 4 d a y
U n to u c h e d C o n tro l 8 d a y
U n to u c h e d  C X C R 3 - 9 C 5 8 d a y
Figure 4.7: αCXCR3-9C5 treatment in untouched C57BL/6 mice – CD4+ Foxp3+ T
cells
Untouched C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5, were assessed by flow
cytometry for splenic lymphocyte populations at day 4 and 8 post i.v. Splenocytes were
expressed as cell number for (a) CD4+Foxp3+ T cells and (b) CD4+Foxp3+CXCR3+
showing binding of free CXCR3, determined via CXCR3-173 binding. Mean+/- SEM
p<0.01** (n=2-6mice/group).
8 .0  1 0 6
# C D 8 + S p le n o c y te s
*
b
6 .0  1 0 6
4 .0  1 0
6
2 .0  1 0 6
0
# C D 8 + C X C R 3 + S p le n o c y t e s
a
*
600000
400000
200000
0
U n to u c h e d C o n tro l4 d a y
U n to u c h e d  C X C R 3 - 9 C 5 4 d a y
U n to u c h e d C o n tr o l 8 d a y
U n to u c h e d  C X C R 3 - 9 C 5 8 d a y
Figure 4.8: αCXCR3-9C5 treatment in untouched C57BL/6 mice – CD8+ T cells
Untouched C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5, were assessed by flow
cytometry for splenic lymphocyte populations at day 4 and 8 post i.v. Splenocytes were
expressed as cell number for (a) CD8+ T cells and (b) CD8+CXCR3+ T cells showing
binding of free CXCR3, determined via CXCR3-173 binding. Mean+/- SEM p<0.05* (n=26mice/group).
70
αCXCR3-9C5 in the Islet Allograft Model
The number of cells expressing free unbound CXCR3 was also found to differ
significantly for CD4+, CD4+Foxp3+ and CD8+ T cell subsets at day 8 post
αCXCR3-9C5 administration compared to day 4 post treatment. The number of cells
expressing free CXCR3 was found to be significantly decreased at day 4 compared to
day 8 post treatment, suggesting loss of αCXCR3-9C5 binding and recovery of free
unbound CXCR3 at 8 days post treatment (figure 4.6b, 4.7b, 4.8b).
Analysis of CD4+ and CD8+ effector (CD44high, CD62Llow), memory (CD44high,
CD62Lhigh) and naïve (CD44low, CD62Lhigh) T cell subsets at day 8 following
treatment with αCXCR3-9C5 was also performed compared to untreated controls
(figure 4.9a & b). No difference was seen between groups for CD4+ T cells. Of note,
changes in CD4+CXCR3+ frequency seen at day 4 were not maintained to day 8
post-treatment (figure 4.6b) and as such although no change in CD4+ T cell subsets
are evident at day 8 controls (figure 4.9a), changes may still exist at day 4 posttreatment. At day 8 following treatment with αCXCR3-9C5 a decrease in the number
of CD8+ memory T cells was evident compared to untreated controls, although this
was found to be non-significant. Of note analysis of percentage CD8+ memory T
cells did show a significant decrease in CD8+ memory T cells for αCXCR3-9C5
compared to controls.
b
8 .0  1 0 6
5000000
6 .0  1 0 6
4000000
# C D 8 + S p le n o c y te s
4 .0  1 0 6
2 .0  1 0 6
1000000
800000
600000
400000
200000
0
p = 0 .0 6
3000000
2000000
1000000
*
**
400000
200000
3
e
R
C
a
C
X
n
X
a
n
m
e
iv
m
e
C
C
ff
e
ïv
3
R
C
e
m
X
m
3
R
C
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C
X
C
e
iv
n
a
e
m
ff
3
e
a
n
C
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m
ff
e
ïv
3
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m
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m
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C
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R
3
ff
0
e
# C D 4 + S p le n o c y te s
a
U n to u c h e d C o n tr o l 8 d a y
U n to u c h e d  C X C R 3 - 9 C 5 8 d a y
Figure 4.9: αCXCR3-9C5 treatment in untouched C57BL/6 mice – T cell activation
Untouched C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5, were assessed by flow
cytometry for splenic lymphocyte populations at day 8 post i.v. Splenocytes were expressed
as cell number for (a) CD4+ and (b) CD8+ effector (CD44highCD62Llow), memory
(CD44highCD62Lhigh), and naïve (CD44lowCD62Lhigh) subsets and their respective levels of free
CXCR3 determined via CXCR3-173 binding. Mean+/- SEM p<0.05*, p<0.01**, (n=56mice/group).
71
αCXCR3-9C5 in the Islet Allograft Model
Analysis of the number of cells expressing free unbound CXCR3, via CXCR3-173
binding for effector and memory CD8+ T cells showed significantly reduced free
CXCR3 following αCXCR3-9C5 treatment compared to controls (figure 4.9b).
These same analyses were then performed in islet allograft recipients treated with
αCXCR3-9C5 compared to control islet allograft transplant recipient mice.
Splenocytes were examined for changes in both number and frequency of CD4+,
CD4+Foxp3+ and CD8+ T cells and their respective number and frequency of cells
expressing free CXCR3 at day 4 and 8 post injection and transplant in these mice
(figure 4.10-4.13).
As no change in raw cell counts was evident for islet allograft recipient splenocytes
collected at day 4 post-transplant and αCXCR3-9C5 treatment, changes in number
and frequency of T cell populations and their respective CXCR3 expression were
consistent. No change in the number or frequency of CD4+, CD4+Foxp3+ or CD8+
T cells was evident at day 4 following αCXCR3-9C5 treatment compared to control
islet allograft recipients (figure 4.10a, 4.11a and 4.12a).
The proportion of cells expressing free unbound CXCR3 were reduced for both
number and frequency of CD4+, CD4+Foxp3+ and CD8+ T cells at day 4 post
αCXCR3-9C5 administration compared to controls, although due to small group size
this was non-significant (figure 4.10c & d, 4.11c & d, 4.12c & d).
Differences in 8 day raw cell counts for islet allograft recipients (figure 4.5) resulted
in inconsistent changes between groups for number and frequency of splenocyte T
cell subsets. Due to the decreased raw cell counts for control islet allograft recipients
the number of CD4+, CD4+Foxp3+ and CD8+ T cells were decreased compared to
αCXCR3-9C5 treated islet allograft recipients (figure 4.10-4.12a). In comparison the
frequency of CD4+, CD4+Foxp3+ and CD8+ T cells were not different (figure 4.104.12b) for αCXCR3-9C5 treated islet allograft recipients compared to controls.
72
αCXCR3-9C5 in the Islet Allograft Model
c
4000000
3000000
2000000
1000000
0
% C D 4 + S p le n o c y t e s
# C D 4 + S p le n o c y te s
12
5000000
10
8
6
4
2
0
d
500000
400000
300000
200000
100000
0
% C D 4 + C X C R 3 + S p le n o c y t e s
b
# C D 4 + C X C R 3 + S p le n o c y t e s
a
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
T ra n s p la n t c o n tro l 4 d a y
T ra n s p la n t  C X C R 3 - 9 C 5 4 d a y
T ra n s p la n t c o n tro l 8 d a y
T ra n s p la n t  C X C R 3 - 9 C 5 8 d a y
Figure 4.10: αCXCR3-9C5 treatment in transplanted C57BL/6 mice – CD4+ T cells
C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5 received a BALB/c islet allograft under
the kidney capsule followed by flow cytometry for splenic lymphocyte populations at day 4
and 8 post i.v. and allograft. Controls did not receive αCXCR3-9C5 treatment. Splenocytes
were expressed as cell number (a & c) or frequency (b & d) for CD4+ T cells (a & b) and
showing their respective binding of free CXCR3 (c & d), determined via CXCR3-173 binding.
Mean+/-SEM (n=2-4mice/group)
This difference in raw cell counts also obscured any change in the proportion of cells
compared to controls. While analysis of cell number for CD4+, CD4+Foxp3+ and
CD8+ T cells showed little change in the number of cells expressing free CXCR3
(figure 4.10c, 4.11c, 4.12c) a decreased frequency of cells expressing free CXCR3 is
evident for αCXCR3-9C5 treated islet allograft recipients compared to controls for
each subset (figure 4.10d, 4.11d, 4.12d). Due to small group size these changes were
non-significant.
Control islet allograft recipient mice showed an upregulation in the frequency of
cells expressing free CXCR3 from day 4 to day 8 post-transplant for CD4+,
CD4+Foxp3+ and CD8+ T cell frequency, as seen in Figure 4.4 (figure 4.10d, 4.11d,
4.12d). This same trend was evident for αCXCR3-9C5 treated transplant recipients
from day 4 to day 8 with increased free unbound CXCR3 for CD4+, CD4+Foxp3+
and CD8+ T cells for both number and frequency, compared to controls (figure 4.10c
& d, 4.11c & d, 4.12c & d). This trend was similar to that seen in naïve mice (figure
4.1, 4.2, 4.6, 4.7 & 4.8), suggesting a partial loss of αCXCR3-9C5 binding to
CXCR3 by day 8 post αCXCR3-9C5 administration.
73
αCXCR3-9C5 in the Islet Allograft Model
c
0
0 .5
60000
40000
20000
0 .0
0 .4
S p le n o c y te s
1 .0
S p le n o c y te s
200000
# C D4+Foxp3+C XCR 3+
400000
d
80000
1 .5
% CD4+Foxp3+CXCR3+
b
600000
% C D 4 + F o x p 3 + S p le n o c y t e s
# C D 4 + F o x p 3 + S p le n o c y te s
a
0 .3
0 .2
0 .1
0 .0
0
T ra n s p la n t c o n tro l 4 d a y
T ra n s p la n t  C X C R 3 - 9 C 5 4 d a y
T ra n s p la n t c o n tro l 8 d a y
T ra n s p la n t  C X C R 3 - 9 C 5 8 d a y
Figure 4.11: αCXCR3-9C5 treatment in transplanted C57BL/6 mice – CD4+Foxp3+ T
cells
C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5 received a BALB/c islet allograft under
the kidney capsule followed by flow cytometry for splenic lymphocyte populations at day 4
and 8 post i.v. and allograft. Controls did not receive αCXCR3-9C5 treatment. Splenocytes
were expressed as cell number (a & c) or frequency (b & d) for CD4+Foxp3+ T cells (a & b)
and showing their respective binding of free CXCR3 (c & d), determined via CXCR3-173
binding. Mean+/-SEM (n=2-4mice/group)
c
2000000
1000000
0
% C D 8 + S p le n o c y t e s
# C D 8 + S p le n o c y te s
10
3000000
8
6
4
2
0
d
400000
300000
200000
100000
0
% C D 8 + C X C R 3 + S p le n o c y t e s
b
# C D 8 + C X C R 3 + S p le n o c y t e s
a
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
T ra n s p la n t c o n tro l 4 d a y
T ra n s p la n t  C X C R 3 - 9 C 5 4 d a y
T ra n s p la n t c o n tro l 8 d a y
T ra n s p la n t  C X C R 3 - 9 C 5 8 d a y
Figure 4.12: αCXCR3-9C5 treatment in transplanted C57BL/6 mice – CD8+ T cells
C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5 received a BALB/c islet allograft
under the kidney capsule followed by flow cytometry for splenic lymphocyte populations
at day 4 and 8 post i.v. and allograft. Controls did not receive αCXCR3-9C5 treatment.
Splenocytes were expressed as cell number (a & c) or frequency (b & d) for CD8+ T cells (a
& b) and showing their respective binding of free CXCR3 (c & d), determined via CXCR3173 binding. Mean+/-SEM (n=2-4mice/group)
CD4+ and CD8+ effector (CD44high, CD62Llow), memory (CD44high, CD62Lhigh) and
naïve (CD44low, CD62Lhigh) T cell subsets were also analysed at day 8 following islet
transplant and treatment with αCXCR3-9C5 compared to control islet transplant
74
αCXCR3-9C5 in the Islet Allograft Model
recipients (figure 4.13). A reduced number of CD4 and CD8 effector, memory and
naïve cell subsets were evident due to reduced absolute raw cell counts in untreated 8
day allograft transplant recipients (figure 4.13a & c). However, these changes were
not reflected in cell frequencies, where a non-significant reduction in total CD8
effector and memory T cells was evident in αCXCR3-9C5 treated mice compared to
control islet allograft recipients (figure 4.13b & d). In addition following αCXCR39C5 treatment a trend towards a reduced frequency of free CXCR3 on CD4 effector
and memory and CD8 effector, memory and naïve T cells was also evident,
compared to control expression (figure 4.13b & d). Due to small group size these
changes were non-significant.
b
a
8
% C D 4 + S p le n o c y t e s
2500000
2000000
1500000
1000000
500000
2
2 .0
1 .5
1 .0
0 .5
R
C
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ïv
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C
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C
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C
ff
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c
d
2000000
6
% C D 8 + S p le n o c y t e s
1500000
1000000
400000
200000
0
5
4
3
2
1
R
C
a
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ïv
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ff
# C D 8 + S p le n o c y te s
4
0 .0
ff
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6
e
# C D 4 + S p le n o c y te s
3000000
T ra n s p la n t c o n tro l 8 d a y
T ra n s p la n t C X C R 3 -9 C 5 8 d a y
Figure 4.13: αCXCR3-9C5 treatment in transplanted C57BL/6 mice – T cell
activation
C57BL/6 mice, treated i.v. with 2mg/kg αCXCR3-9C5 received a BALB/c islet allograft
under the kidney capsule followed by flow cytometry for splenic lymphocyte populations
at day 8 post i.v. and allograft. Splenocytes were expressed as cell number (a & c) or
frequency (b & d) for CD4+ or CD8+ effector (CD44highCD62Llow), memory
(CD44highCD62Lhigh), and naïve (CD44lowCD62Llow) T cell subsets and showing their
respective binding of free CXCR3, determined via CXCR3-173 binding. Mean+/- SEM
(n=2-4mice/group)
75
αCXCR3-9C5 in the Islet Allograft Model
Effector and memory T cell subsets are important mediators of inflammation and
allograft rejection21,270-272. Due to the reductions in CD8+ effector and memory
splenocyte populations of untouched and allograft recipients at POD 8 we assessed
changes in markers of T cell activation, CD44 and CD62L. Representative FACS
plots of CD8+ T cells from the spleens of untouched or islet allograft transplant
recipients (figure 4.14ai & bi) at POD 8 show increased memory and effector subsets
for control mice compared to mice treated with 2mg/kg αCXCR3-9C5 (figure 4.14aii
& bii).
Representative histograms of the activation markers CD44 (figure 4.14a & biii) and
CD62L (figure 4.14a & b iv) are also shown for control mice (black unfilled) or
those treated with αCXCR3-9C5 (grey shaded). The addition of αCXCR3-9C5
resulted in no change in CD62L expression, however a substantial decrease in the
expression of CD44high CD8+ T cells compared to untreated controls was observed
for both untouched and islet allograft recipient mice.
Figure 4.14: Altered activation of naïve and allogeneic C57BL/6 splenocytes
Representative FACS plots of CD8+ effector (CD44highCD62Llow), memory
(CD44highCD62Lhigh), and naïve (CD44lowCD62Llow) subsets from either (a)
untouched/untransplanted or (b) islet allograft recipient mice for (i) untreated
controls or (ii) αCXCR3-9C5 treated (2mg/kg i.v.) mice at 8 days post-transplant
and/or αCXCR3-9C5. Representative histograms of (iii) CD44 and (iv) CD62L are also
shown for mice treated with αCXCR3-9C5 (grey shaded) or untreated controls
(unfilled black) (n=2-4mice/group)
76
αCXCR3-9C5 in the Islet Allograft Model
The decreased CD8+CD44high expression found in these mice resulted in a reduction
in CD44+CD8+ T cells at day 8 for both untouched and islet allograft recipient mice
following treatment with αCXCR3-9C5 when compared to untreated controls (figure
4.15b). This change was non-significant due to small group size. No change was seen
for CD4+ CD44 expression. When frequency of CD8+ T cells were analysed a slight
decrease was evident for both untouched and allograft recipients treated with
αCXCR3-9C5 compared to controls (figure 4.15a).
a
b
30
5
10
0
C
C
D
D
8
4
+
0
20
+
10
% C D 4 4 + T c e lls
% C D 8 + T c e lls
15
U n to u c h e d c o n tro l
U n to u c h e d  C X C R 3 - 9 C 5
T ra n s p la n t c o n tr o l
T r a n s p la n t  C X C R 3 - 9 C 5
Figure 4.15: Altered CD44 expression of untouched and transplanted C57BL/6
splenocytes
Untouched or islet allograft recipient mice were treated with 2mg/kg αCXCR3-9C5 i.v.
or untreated as controls and analysed by flow cytometry at day 8 post-transplant/i.v.
for frequency of (a) CD8+ T cells or (b) expression of CD44 high CD4+ and CD8+ T cells.
Mean+/- SEM (n=2-4 mice/group)
αCXCR3-9C5 Prolongs C57BL/6 Islet Allograft Survival
Using this initial characterisation it was determined that regular administration of
αCXCR3-9C5 once per 7 days at a concentration of 2mg/mL would maintain
expression of free unbound CXCR3 at a low level. We then further investigated the
effect this antagonism of the CXCR3 receptor had on islet allograft survival.
Transplantation of BALB/c to C57BL/6 islet allografts were performed and mice
were treated with one of 4 regimens. These were either; 2mg/kg i.v. Isotype control
antibody or 2mg/kg i.v. αCXCR3-9C5 antibody once per week or a combination of
Isotype
or
αCXCR3-9C5
antibody
with
sub-therapeutic
doses
of
the
immunosuppressant rapamycin, at 0.1mg/kg, i.p., every day for the first week
77
αCXCR3-9C5 in the Islet Allograft Model
following transplant260 (see figure 4.3). Mice were then monitored for the onset of
hyperglycaemia, indicative of allograft rejection.
Isotype vs. αCXCR3 p= 0.0291*
Isotype+Rapa vs. αCXCR3+Rapa p=0.087
αCXCR3 vs. αCXCR3+Rapa p=0.3309
Figure 4.16: Allogeneic C57BL/6 Islet Transplant
Kaplan-Meier survival curve of C57BL/6 (H2b) recipient mice, transplanted under the
kidney capsule with BALB/c (H2d) islet allografts. Mice received either; isotype antibody
(1/week, 2mg/kg, i.v. for 9 weeks)(●), αCXCR3-9C5 mAb (1/week, 2mg/kg, i.v. for 9
weeks)(■), or combined Isotype + rapamycin (1x/day, 0.1mg/kg, i.p. for 7 days)(▼) or
αCXCR3-9C5 + rapamycin (1x/day, 0.1mg/kg, i.p. for 7 days)(♦) from the morning of
allograft . Mice were monitored a loss of euglycaemia indicating loss of allograft. Mean+/SEM p<0.05* (n=4-7 mice/group).
Rejection of islet allografts occurred in all recipients treated with isotype control
antibody by POD 24 with a median survival time (MST) of 22 days. However,
treatment of mice with αCXCR3-9C5 resulted in a significant prolongation of
allograft survival with MST of 31 days and permanent engraftment (>100days) in 1
out of 7 mice. When rapamycin was given in combination with αCXCR3-9C5 50%
of mice showed long term islet allograft survival with a MST of 81.5 days (figure
4.16). In contrast to those mice treated with a combination of αCXCR3-9C5 and
rapamycin, treatment of mice with isotype and rapamycin resulted in a MST of 40
days and did not show any long-term allograft survival. Although treatment with
rapamycin improved allograft survival compared to mice treated with isotype alone
this change was not significant.
78
αCXCR3-9C5 in the Islet Allograft Model
αCXCR3-9C5 and Rapamycin Therapy Preserve Islet Allograft Architecture
Examination of islet allograft pathology taken from long term (>POD 100) surviving
grafts from mice treated with a combination of αCXCR3-9C5 and rapamycin, was
then performed. Staining for H&E and insulin revealed a preserved islet architecture
and strong insulin labelling with isolated pockets of mononuclear cell infiltration
restricted to the edges of the graft (figure 4.17a). Due to the role of regulatory T cells
in allograft survival144,273-275, characterised by their expression of the Foxp3
transcription factor, we next examined Foxp3+ T cells within the graft. Consequently
we found robust staining (brown) within the observed pockets of immune infiltrate at
the edge of the long term surviving islet allografts (figure 4.17b).
Figure 4.17: Long-term surviving allograft pathology
Representative histology of long-term surviving (POD>100) islet allografts from mice
treated with 2mg/kg αCXCR3-9C5 mAb (i.v. 1x/week for 9 weeks) + 0.1mg/kg rapamycin
(i.p. 1x/day for 7 days) POD>100. (a) Hematoxylin & Eosin staining (H&E), Insulin
staining (INS) (brown) and (b) Foxp3+ cell staining (brown). Flow cytometric analysis of
CD4+Foxp3+ T cells as % of total lymphocytes for untouched controls (o), αCXCR3-9C5 +
rapamycin (as above, POD>100) (♦) and αCXCR3-9C5 alone (POD>100) (■) measured in
spleen, thymus and blood. Individual values of each mouse represented. p<0.05 *
79
αCXCR3-9C5 in the Islet Allograft Model
In addition to the presence of Foxp3+ cells at the graft site, we found an increased
frequency of CD4+Foxp3+ Tregs within the spleens of long term surviving islet
allograft recipients (Figure 4.17c). The frequency of Foxp3+ regulatory T cells
isolated from the spleens of long-term surviving mice treated with αCXCR3-9C5 and
rapamycin was significantly greater, ~1.05% of total lymphocytes, than that seen for
untreated control C57BL/6 mice, which had not received an allograft, ~0.625% of
total lymphocytes. The individual long-term surviving αCXCR3-9C5 treated mouse
showed a similarly elevated regulatory T cell population as that seen in αCXCR39C5 and rapamycin treated mice. No change was seen in the thymus or blood of
these mice compared to controls (figure 4.17c).
A role for Regulatory T cells in αCXCR3-9C5 and Rapamycin treated Islet
Allograft survival
Next to establish whether regulatory T cells played a role in promoting allograft
survival in this model we employed strategies to deplete T cells from our allograft
models. Firstly we performed islet allografts, as previously described, followed by
weekly treatments with isotype or αCXCR3-9C5 antibody. In addition one group
received αCXCR3-9C5 in combination with an anti-CD25 depleting antibody
(PC61). This effectively removes the CD4+CD25+Foxp3+ T cell fraction by starving
them of IL-2, necessary for Treg survival261. As previously described mice treated
with isotype had a MST of 22 days, while αCXCR3-9C5 treatment resulted in a MST
31 days. However, in mice treated with PC61 the beneficial effects of αCXCR3-9C5
on graft survival were lost, resulting in a MST of 26 days (figure 4.18a).
Next, to further elucidate this role of Tregs, an in vivo cell transfer model was used
(figure 4.18b). RAG-/- recipient mice firstly received a BALB/c islet allograft
followed by transfer of T cells from either untreated control mice or long term
surviving islet allograft recipient mice (treated with αCXCR3-9C5 and rapamycin).
Transferred T cells were either whole T cells (figure 4.18c) or CD25 depleted T cells
(CD25-), lacking CD4+CD25+Foxp3+ T cells (figure 4.18d). In support of previous
findings the transfer of whole T cells from long-term surviving mice resulted in
prolongation of allograft survival in RAG-/- mice compared to T cells from controls.
These results were non-significant due to small group size (figure 4.18c). Transfer of
80
αCXCR3-9C5 in the Islet Allograft Model
CD25 depleted cells on the other hand resulted in a complete loss of this
prolongation compared to control T cell transfer (figure 4.18d). This supports a role
for CD4+CD25+Foxp3+ Tregs in the maintenance of αCXCR3-9C5 induced long
term allograft survival.
Figure 4.18: A role for Regulatory T cells in αCXCR3-9C5 allograft survival
(a) Kaplan-Meier survival curve of BALB/c islet (H2d) allografts transplanted under the
kidney capsule of C57BL/6 (H2b) recipient mice treated i.v. with 2mg/kg Isotype mAb
(●), 2mg/kg αCXCR3-9C5 mAb (■)(1x/week for 9 weeks) or 2mg/kg αCXCR3-9C5 mAb
in combination with the αCD25 mAb PC61 (depletes CD25+ cells) ( ). (n>3mice/group)
(b) Schematic of T cell transfer experiments for RAG-/- mice which received BALB/c islet
allograft followed by adoptive T cell transfer from long-term surviving αCXCR3-9C5 and
rapamycin treated mice or untouched controls. Transfer experiment mice received T cell
transfer of either (c) whole T cells or (d) CD25- T cells. (n=2-3mice/group)
81
αCXCR3-9C5 in the Islet Allograft Model
Summary & Conclusions
The chemokine receptor CXCR3 has a well-established role in mediating chemotaxis
of T cells to sites of inflammation and as such has been shown to undergo
upregulation under conditions of inflammation22,185,194. Here we show that in
agreement with previous literature, upon receipt of an islet allograft, stimulating an
inflammatory response, the proportion of cells expressing CXCR3 was upregulated
from basal levels on CD4+, Treg and CD8+ T cell subsets (figure 4.4). In addition
administration of our novel αCXCR3-9C5 antibody resulted in a reduced ability of
CXCR3-173 mAb to bind to the CXCR3 receptor. We interpret this to indicate
receptor occupancy by αCXCR3-9C5, acting as a receptor antagonist, and possibly
affecting the ability of chemokines, CXCL9, CXCL10 and CXCL11 to bind CXCR3
and mediate biological functions e.g. chemotaxis.
Doses of αCXCR3-9C5 between 1-10mg/kg were found to be equally efficacious at
blocking CXCR3-173 binding for up to 8 days for CD4+ T cells and for over 10days
for CD4+Foxp3+ Treg and CD8+ T cells (figure 4.1 & 4.2). From this, a once
weekly 2mg/kg αCXCR3-9C5 dose was chosen for use in further experiments. In the
spleens of both un-transplanted and allograft recipient mice the proportion of cells
expressing free CXCR3 on T cell subsets was reduced at day 4 following treatment
with αCXCR3-9C5 with partial or complete recovery of free, unbound CXCR3 by
day 8, validating findings in peripheral blood (figure 4.6-4.13).
Importantly a reduction in absolute number of whole splenocytes for islet allograft
recipient mice at day 8 was noted, which affected determination of real changes in
the lymphocyte populations in the spleens (figure 4.5) For this reason both cell
number and frequencies were displayed for islet allograft splenocyte analyses (figure
4.10-4.13). A number of possible explanations for this exist, such as immune cell
migration from the spleen to the graft site. However, this experiment was performed
using STZ to induce diabetes with a previous paper showing lymphopenia post STZ
administration276. The difference between groups then may result from slightly
longer time from STZ administration to transplant or differential effects between
mice and for this reason and due to small group size future studies will involve
82
αCXCR3-9C5 in the Islet Allograft Model
repetition of this experiment using alloxan rather than STZ to eliminate this
possibility.
Investigation of the effect of αCXCR3-9C5 on activation of T cells revealed that
both the proportion of CD4+ and CD8+ naïve, memory and effector T cells
expressing free CXCR3 were reduced. Although only CD8+ T cells showed a
marked decrease in the total memory and effector T cell populations compared to
untreated mice (figure 4.9 & 4.13). This reduction, seen in both un-transplanted and
allograft recipients, resulted from a decrease in CD44high CD8+ T cells (figure 4.14).
No significant change in the frequency of CD8+ T cells between αCXCR3-9C5
treated and control groups were evident (figure 4.15). It is difficult to determine if in
this case depletion is occurring as this population accounts for only a 1-1.5% change
in total CD8+ T cells as a frequency of lymphocytes with variability and a small
group size. This may indicate depletion or natural variability and as such repetition
with larger group number may assist in determination if the change in CD44high
CD8+ T cells was a result of depletion of memory/effector subsets, or a reduction in
expression of the activation marker CD44.
The role of CXCR3 in allograft rejection is well-established in humans, xenografts
and in mouse models180,182,200,216,239-243,252. Here we provide further evidence of the
essential role of CXCR3 in allograft rejection and for the efficacy of αCXCR3-9C5
treatment of mice. Treatment of mice with αCXCR3-9C5 alone or in combination
with low doses of the immunosuppressant rapamycin resulted in a significant
prolongation of functioning islet allograft survival (figure 4.16). In addition 15% of
αCXCR3-9C5 treated and 50% of αCXCR3-9C5 and rapamycin treated mice
exhibited functional islet allografts for over 100 days. Islet allograft pathology also
displayed preserved islet morphology with no lymphocytes found within the graft
site and concurrently showed a robust staining with insulin, indicating wellfunctioning grafts (figure 4.17). The limited immune infiltrate present at these grafts
was encircled by Foxp3+ regulatory T cells. These Treg were found in significant
frequencies in the spleens of long term surviving mice and were found to be partially
responsible for the prolonged allograft survival in αCXCR3-9C5 treated mice, as
determined by PC61 treatment and T cell transfer experiments (figure 4.18a). For
controls tissue from younger 6-8 week old un-manipulated C57BL/6 mice were
83
αCXCR3-9C5 in the Islet Allograft Model
compared to treated long-term surviving allograft recipient mice (figure 4.17c).
These controls were chosen due to the logistical and ethical issues involving use of
aged controls. Of note previous literature indicates that an age related increase in
Treg frequency occurs between 16 and 40 weeks of age in C57BL/6 mice, which
may account for this change277,278. However, while literature suggests that this
increase can be seen throughout the periphery, our data shows a significant increase
in Treg frequency only in the spleens of treated mice and no significant change
within the blood. In order to rule out this age related increase in Treg as a potential
confounding factor, it would be interesting to repeat this experiment using age
matched controls.
Interestingly a similar pattern of infiltration was shown in a recent article focusing on
the role of CXCR3 in viral infection. This paper indicated that while CXCR3
knockout CD8+ T cells were able to migrate to sites of infection they were less likely
to penetrate regions of heavy infection, hesitating at the perimeter of the infected
area. CXCR3 knockout CD8+ T cells were also significantly more motile with an
impaired ability to maintain contact with infected cells220. It is possible that
αCXCR3-9C5 treatment may also impair contact dependent interactions resulting in
reduced allograft destruction and greater peripheral lymphocyte localisation,
restricted to small pockets at the periphery of the graft but not within the allograft.
From this we conclude that αCXCR3-9C5 is able to antagonise the CXCR3 receptor
resulting in reduced free CXCR3 chemokine receptor and a reduction in activation
marker CD44. Reduced frequencies of CD44high effector and memory CD8+ T cell
subsets were also evident as a result of altered CD44 expression. In addition
αCXCR3-9C5 appears to work synergistically with rapamycin to enhance islet
allograft survival, likely through promoting regulatory and inhibiting effector T
cells279.
84
αCXCR3-9C5 in the Islet Allograft Model
85
Chapter 5. αCXCR3-9C5 in the NOD
Autoimmune Diabetic Model
αCXCR3-9C5 in the NOD Islet Allograft Model
Chapter 5. αCXCR3-9C5 in the NOD Autoimmune
Diabetic Model
Type 1 diabetes is an autoimmune disease characterised by progressive loss of islet
beta cells until onset of overt diabetes. For a number of individuals maintenance of
euglycaemia with insulin administration is inadequate to prevent hyper- and
hypoglycaemic episodes. In these individuals with variable blood glucose or with
hypoglycaemic unawareness islet or pancreas transplant may be recommended98,100102
. For this reason we next investigated the role of αCXCR3-9C5 mAb in models of
type 1 diabetes.
To do this we determined the effect of αCXCR3-9C5 in the NOD mouse model of
diabetes. The NOD mouse is a model of spontaneous diabetes caused by the
development of self-directed B and T cells that target and destroy insulin producing
beta cells. The NOD mouse is a useful model due to the natural development of
autoimmunity, pancreatic islet infiltration and loss of euglycaemia, similar to that
seen in the human disease. However it differs from human T1D as discussed
previously within the introduction61,71,72,77.
We were interested in this model for a number of reasons; firstly no previous
literature looks at both the islet allograft and NOD model in the context of
chemokine therapies within a single study. This will be advantageous if we wish to
pursue αCXCR3-9C5 in a clinical context for islet transplantation in individuals with
type 1 diabetes. Moreover such a study may highlight a clinically relevant role of
αCXCR3-9C5 for use in human islet transplantation. Additionally other models
looking at CXCR3 in the context of diabetes have given variable results with studies
in the RIP-LCMV virally induced model showing reduced immune infiltration and
delayed diabetes onset, while CXCR3-/- NOD mice were shown to have accelerated
diabetes onset resulting from impaired Treg trafficking into the pancreas124,217. Of
particular interest to us was the result of Yamada et al. who found accelerated
development of diabetes in CXCR3 deficient NOD mice217. A role for CXCR3 in the
migration of Treg cells into sites of inflammation has been shown for Th1 type
Tregs, which express CXCR3 under the control of the T-bet transcription factor in a
number of autoimmune conditions217,225,226. Acceleration of diabetes driven by our
87
αCXCR3-9C5 in the NOD Islet Allograft Model
antibody would make further studies into the efficacy of our antibody in a clinical,
type 1 diabetes transplantation context unfounded.
As such it is important to determine whether αCXCR3-9C5 confers protection, with
delayed diabetes incidence/onset or prolonged islet allograft survival, in the NOD
mouse model of diabetes. Deleterious effects of αCXCR3-9C5 treatment, such as
impaired pancreatic Treg infiltration were also investigated.
Peripheral Immune cell characterisation of CXCR3 Expression
In order to determine the appropriate dosing regimen in the NOD mouse model
initial antibody titration experiments were performed, as in C57BL/6 mice. Due to
the previous finding in chapter 4 that doses of 1-10mg/kg were equally efficacious
we performed further NOD experiments with 1mg/kg αCXCR3-9C5. NOD mice
were administered a single i.v. of αCXCR3-9C5 or isotype at 8 weeks of age or
untouched as controls, followed by FACS analysis of peripheral blood post injection
on days 2, 4 and 6 to determine changes in peripheral lymphocytes and their
respective frequency of free CXCR3.
Flow cytometry revealed minimal binding of CXCR3 by CXCR3-173 on B cells, as
seen in C57BL/6 samples (figure 4.1a), indicating very low levels of CXCR3
expression which was not affected by treatment with αCXCR3-9C5(figure 5.1a). No
difference in the frequency of total CD4+, Tregs and CD8+ T cells was evident
between αCXCR3-9C5 treated and isotype or control groups for all time points
(figure 5.1). However administration of αCXCR3-9C5 was found to significantly
reduce the frequency of cells expressing free unbound CXCR3, as determined by
CXCR3-173 binding, for all T cell subsets at day 2 post injection. Reduced CXCR3173 binding of CD8+ T cells was also evident at day 4 for αCXCR3-9C5 treated,
compared to isotype treated T cells. This reduction in the frequency of cells
expressing free unbound CXCR3 was absent for all T cell subsets by day 6,
suggesting loss of αCXCR3-9C5 binding (figure 5.1b-d).
88
αCXCR3-9C5 in the NOD Islet Allograft Model
Figure 5.1: αCXCR3-9C5 Antibody Titrations - NOD
Analysis of peripheral blood lymphocyte expression of CXCR3, determined using CXCR3173 by flow cytometry. Mice were treated with 1mg/kg αCXCR3-9C5 or Isotype antibody
i.v. on day 0 or untouched as controls for total (a) B cells, (b) CD4+ T cells, (c) Treg and
(d) CD8+ T cells and on day (i) 2 (n=5-10mice/group) (ii) 4 (n=5-10mice/group), and (iii)
6 (n=2-3mice/group). Expressed as percent of lymphocytes. Mean+/-SEM p<0.05*,
p<0.001***
89
αCXCR3-9C5 in the NOD Islet Allograft Model
Basal CXCR3 Expression
Analysis of baseline frequency of free unbound CXCR3 in the spleens of untreated
NOD mice (figure 5.2; black histogram) compared to isotype stained cells (grey
histogram) was also performed by flow cytometry for CD4+, CD4+Foxp3+ and
CD8+ T cells as well as for NK cells (figure 5.2).
This was performed at 3 different ages: 6 weeks (figure 5.2a), initial onset of mild
pancreatic infiltration; 12 weeks (figure 5.2b) at which point a significant increase in
islet infiltration occurs and at 15-25 weeks of age, onset of overt diabetes for
females, with heavily infiltrated islets and loss of insulin producing beta cells (figure
5.2c)77.
Figure 5.2: NOD CXCR3 Expression Profiles
Representative histograms of NOD splenic T (CD4+, CD4+Foxp3+ and CD8+) and NK
(CD3-NKp46+) cell subsets, showing median for proportion of CXCR3+ staining (%) for
NOD mice at (a) 6 and (b) 12 weeks of age and (c) at onset of overt diabetes. Isotype
(grey) compared to Control (black) CXCR3 expression (n=3-7 mice/ group).
90
αCXCR3-9C5 in the NOD Islet Allograft Model
The median value for the proportion of cells positively staining for CXCR3 on T
cells, including CD4+, CD4+Foxp3+ Treg and CD8+ T cells, increased from 6
weeks to 12 weeks of age, peaking at 12 weeks where they either stabilised or
decreased until the onset of diabetes at 20-25 weeks of age. In contrast proportion of
cells positively staining for CXCR3 on NK cells was highest at 6 weeks of age with
reduced expression at 12 weeks and at onset of diabetes at 20-25 weeks of age
(figure 5.2). This profile would agree with an early innate response by NK cells at 6
weeks, followed by a more acquired, T cell, response at 12 weeks of age. Mean,
median and range values are provided in Table 5.1.
CD4+CXCR3+
NOD 6W
NOD
12W
Median
4.13
9.98
6.46
Mean
4.033333
3.174.13
9.71
6.6812.2
8.928571
4.8619.3
Median
8.47
14.65
11.5
Mean
Median
9.076667
8.4610.3
10.5
14.8
14.115.8
13.3
14.78143
8.7231.4
13.3
Mean
10.31667
13.75
15.65
Range
8.6511.8
12.116.43
7.05-26
Median
51.2
40.85
35
Mean
51.83333
41.263.1
3
39.15
26.848.1
4
39.48571
Range
CD4+Foxp3+
CXCR3+
Range
CD8+CXCR3+
NK+CXCR3+
Range
n=
NOD
Diabetic
28.7-56
7
Table 5.1: NOD CXCR3 Expression Profiles
Mean and median values as well as range of NOD splenic T (CD4+, CD4+Foxp3+ and CD8+)
and NK (CD3-NKp46+) cell subsets, showing median for proportion of CXCR3+ staining
(%) for NOD mice at (a) 6 and (b) 12 weeks of age and (c) at onset of overt diabetes (n=37 mice/ group).
NOD Insulitis Scoring
Baseline insulitis was determined in the NOD pancreas using formalin fixed, H&E
stained sections from 4-6, 9 and 15-16 (diabetic BGL >15mmol/L) week old mice. In
order to do this we employed an established 0-4 grading system, where grade 0 islets
have no infiltrate, grade 1 peri-insulitis, grade 2 <25% infiltrate, grade 3 >25% and
91
αCXCR3-9C5 in the NOD Islet Allograft Model
grade 4 >75% infiltrate262. Quantification of insulitis for each age expressed as a
percent of total islets scored for each group, revealed a progressive increase in
severity of insulitis as mice aged, as expected from previous literature77(figure 5.3).
Figure 5.3: NOD Insulitis Grading
Representative histological images of H&E stained NOD pancreata, grade 0-4 insulitis scoring;
grade 0 no infiltrate, grade 1 peri-insulitis, grade 2 <25% infiltrate, grade 3 >25% infiltrate
and grade 4 >75%/complete infiltration
Assessment of baseline insulitis in 4-6 week old, 9 week old and 15-16 week old diabetic
(diabetic BGL >15mmol/L) NOD mice using H&E stained histological sections, expressed as
percentage of islets scored per grade (>50 Islets scored/group n=3 mice/group).
NOD Diabetes Incidence
Cumulative diabetes incidence data for our laboratory was also obtained to validate
NOD diabetes incidence data generated within these studies (figure 5.4). This is
necessary as the incidence of diabetes within NOD colonies is strongly influenced by
environmental factors such as diet and microbial infections and is known to vary
between facilities75. Figure 5.4 shows the fraction of normoglycaemic NOD female
mice over time from birth to 50 weeks of age. Diabetes incidence rates of 60% at 25
weeks have been previously documented by our facility280.
92
αCXCR3-9C5 in the NOD Islet Allograft Model
Figure 5.4: NOD Diabetes incidence
Cumulative diabetes incidence data was obtained showing diabetes incidence for NOD
colonies housed at our facility monitored for BGL>15mmol/L for 2 consecutive days
which was considered diabetic. Graph shows fraction of mice maintaining
euglycaemia at each age in weeks. Courtesy of S. T. Grey and E. Marino1
αCXCR3-9C5 does not alter Diabetes Incidence in the NOD mouse
Following determination of basal expression of CXCR3 (figure 5.2), degree of
insulitis at different ages (figure 5.3) and the efficacy of a 1mg/kg dose of αCXCR39C5 to bind CXCR3 expressed by NOD peripheral blood lymphocytes (figure 5.1),
we investigated the effect of long term treatment with αCXCR3-9C5 in the NOD
mouse.
For this we established a model involving treatment of mice every 4 days with
1mg/kg i.v. αCXCR3-9C5 antibody or isotype control antibody, alternatively mice
were untreated as controls. Mice began treatments from either; 6 weeks of age, where
6 weeks of age is early in the initial phase of immune infiltration or 8 weeks of age,
the age at which a 7 fold increase in infiltration of leukocytes occurs in the NOD
mouse77 (figure 5.3). Treatments were continued until euthanasia at 12 weeks of age
for analysis of pancreatic pathology and changes in lymphocyte populations.
Alternatively treatment was continued until onset of overt diabetes or 25 weeks of
age, allowing incidence of diabetes in our model to be determined. Diabetes was
determined as a blood glucose level (BGL) >15mmol/L on 2 consecutive days
(figure 5.5).
93
αCXCR3-9C5 in the NOD Islet Allograft Model
Figure 5.5: Autoimmune NOD Experimental Model
Overview of experimental model including initial age of treatment (6 or 8 weeks of age)
with isotype or αCXCR3-9C5 mAb administered 1mg/kg, every 4 days. Mice were
monitored for onset of hyperglycaemia/diabetes (Incidence Study) or euthanised at 12
weeks of age to examine pathology (Pathology Study).
Mice were treated until onset of overt diabetes, with initial onset for each group at
approximately 15 weeks of age. Mice treated from 6 weeks of age with αCXCR39C5 (6W start) were the exception to this trend with a single mouse becoming
diabetic at 10 weeks of age. A similar incidence of diabetes was also seen for all
groups with 50-60% of mice diabetic at 25 weeks of age. This revealed no significant
effect of 1mg/kg αCXCR3-9C5 on the kinetic of diabetes development in the NOD
mice, regardless of age of initial treatment (figure 5.6).
To ensure the lack of effect on diabetes incidence was not due to the lower 1mg/kg
αCXCR3-9C5 dose, compared to that used in allograft experiments, we also
determined the efficacy of 2mg/kg αCXCR3-9C5 given from 6 weeks of age until
onset of overt diabetes. No difference in diabetes incidence between mice treated
with 1mg/kg or 2mg/kg was evident (figure 5.7).
94
αCXCR3-9C5 in the NOD Islet Allograft Model
Figure 5.6: αCXCR3-9C5 does not alter NOD Diabetes incidence
NOD mice were treated from 6 weeks with 1mg/kg Isotype ( ) or αCXCR3-9C5 antibody
( ) or from 8 weeks with Isotype (■) or αCXCR3-9C5 antibody (■) every 4 days or
untouched as controls (o). Blood glucose levels were monitored for a loss of euglycaemia
and onset of diabetes, designated as BGL>15mmol/L for 2 consecutive days. Rates of
diabetes in each group were monitored until 25 weeks of age and expressed as percent
of normoglycaemic mice per group from 6-25 weeks of age. (n=7-18mice/group)
Figure 5.7: Equivalent diabetes incidence for 1mg/kg or 2mg/kg αCXCR3-9C5
NOD mice were treated every 4 days from 6 weeks with 1mg/kg ( ) or 2mg/kg ( ) αCXCR3-9C5
antibody. Blood glucose levels were monitored for a loss of euglycaemia and onset of diabetes,
designated as BGL>15mmol/L for 2 consecutive days. Rates of diabetes in each group were
monitored until 25 weeks of age and expressed as percent of normoglycaemic mice per group from
6-25 weeks of age. (n=5-13mice/group)
95
αCXCR3-9C5 in the NOD Islet Allograft Model
αCXCR3-9C5 does not alter Insulitis or Regulatory T cell Migration in the
NOD mouse
Although little difference was evident in diabetes incidence for αCXCR3-9C5 treated
mice, markers of pathology including insulitis and changes in immune cell
populations were examined due to the role of αCXCR3-9C5 in inhibiting
chemotaxis.
In mice treated with 1mg/kg αCXCR3-9C5 from 6 or 8 weeks until 12 weeks of age
(pre-diabetic) (figure 5.8a), no significant difference in insulitis was seen compared
to isotype treated mice. This was determined by insulitis grading as shown
previously (figure 5.3), statistical analysis was performed by comparing the
percentage of islets with grade 3 and 4 insulitis for each group. Although severity of
insulitis in pre-diabetic mice was reduced for untreated controls compared to all
other groups, this change was not significant when comparing severe insulitis
(combined percent grade 3 and 4) between isotype or αCXCR3-9C5 treated and
control groups (figure 5.8a).
Figure 5.8: αCXCR3-9C5 does not affect NOD pancreatic insulitis
Insulitis grading of H&E stained pancreata from NOD mice treated from 6 weeks or 8
weeks of age with 1mg/kg αCXCR3-9C5 or isotype every 4 days or untreated as controls.
(a) Insulitis at 12 weeks of age or (b) in diabetic mice graded as; grade 0 no infiltrate,
grade 1 peri-insulitis, grade 2 <25% infiltrate, grade 3 >25% and grade 4 >75% infiltrate,
expressed as percentage of islets scored per grade (>40 islets/group, n=3-7mice/group).
Significance determined for % grade 3+4 insulitis for αCXCR3-9C5 vs. isotype for each age
matched group was found to be non-significant (paired t-Test). 6-12 W = 0.5490, 8-12W
=0.0894, 6-diabetic = 0.2823, 8-diabetic = 0.6792.
96
αCXCR3-9C5 in the NOD Islet Allograft Model
Further, mice treated with 1mg/kg αCXCR3-9C5 from either 6 or 8 weeks of age
until onset of overt diabetes showed no significant difference in insulitis compared to
isotype treated or control mice (figure 5.8b).
Regulatory T cells play an important role in restraining progression of effector cell
mediated destruction in the NOD pancreas281. Due to the previously described role of
CXCR3 in recruitment of Foxp3+ Treg into the pancreas during diabetes in the NOD
model217 we examined Treg infiltration into the pre-diabetic NOD pancreas.
Foxp3 positively stained cells within the NOD pancreas were enumerated from
histological sections of pancreata harvested from mice treated with 1mg/kg
αCXCR3-9C5 or isotype antibody from 6 or 8 weeks until 12 weeks of age or
untreated controls. Representative histological images of pre-diabetic NOD islets,
grade 3, are shown with Foxp3+ stained cells in brown for Isotype and αCXCR39C5 treated mice (figure 5.9a).
Few Foxp3+ T cells were found within the exocrine tissue and as such only Foxp3
positively stained cells associated with islet infiltrate were counted. Cells were
enumerated for 3 non-contiguous sections of pancreas and the average number of
Foxp3+ cells counted per islet for each mouse were expressed as individual data
points (figure 5.9b). Infiltration of Foxp3 positively stained cells did not show
significant differences when comparing αCXCR3-9C5 and respective isotype
antibody treated groups or untreated controls for mice treated either from 6 or 8
weeks of age. The frequency of Foxp3+ staining was also found to generally be
higher for islets with more severe insulitis. However, considerable variation in the
average number of Foxp3+ cells within mice from each group exists.
97
αCXCR3-9C5 in the NOD Islet Allograft Model
Figure 5.9: αCXCR3-9C5 does not affect NOD pancreatic regulatory T cell Infiltrate
(a) Representative Immunohistochemistry of Foxp3+ stained sections of Isotype and
αCXCR3-9C5 treated pre-diabetic (12 week old) NOD pancreas. Foxp3+ stained cells, shown
in brown.
(b) Foxp3+ regulatory T cell quantification as average Foxp3+ cells per islet for 3 noncontiguous sections of pancreas for each NOD mouse, treated from 6 weeks or 8 weeks of
age with 1mg/kg αCXCR3-9C5 or isotype every 4 days until 12 weeks of age or untreated
as controls. Each data point represents average number of Foxp3+ cells per islet for each
mouse. (n=4-5 mice/group)
αCXCR3-9C5 reduces CXCR3 but not CD44 expression in the NOD mouse
Due to the well characterised role of CXCR3 in mediating immune cell migration,
lymphocyte populations in these NOD mice were also investigated3,22,23. Assessment
of the frequency of cells expressing free CXCR3, through binding of CXCR3-173
antibody, and changes in overall cell populations within the spleen, distal lymph
node (dLN) and pancreatic LN (pLN) were performed using flow cytometry. Prediabetic NOD mice treated from 6 or 8 weeks of age with 1mg/kg αCXCR3-9C5 or
98
αCXCR3-9C5 in the NOD Islet Allograft Model
Isotype every 4 days, until 12 weeks of age or untreated as controls were euthanised
and tissues harvested for flow cytometry.
Lymphocytes were analysed for T cell subsets; CD4+, CD4+CD25+Foxp3+,
CD4+CD25-Foxp3+ or CD8+ T cells and for NK cells. No difference in the total
number of any cell population for mice treated with αCXCR3-9C5 was evident in the
spleen, dLN and pLN compared to controls (figure 5.10 & 5.11).
Pre-diabetic mice treated with αCXCR3-9C5 from 6 weeks of age showed no
apparent difference in expression of free CXCR3 for CD4+, CD4+CD25+Foxp3+
(Treg) T cells or for NK cells in any of the tissues examined, compared to isotype
controls. Differences in free unbound CXCR3 were evident in the spleen and pLN
for CD4+CD25-Foxp3+ Treg (figure 5.10b & f) and in the spleen for CD8+ T cells
(figure 5.11a) for mice treated from 6 weeks with αCXCR3-9C5 compared to isotype
controls although due to low group number these were not significant.
99
αCXCR3-9C5 in the NOD Islet Allograft Model
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Figure 5.10: Pre-diabetic NOD αCXCR3 Treatment – CD4+ T cell & CD4+Foxp3+ T cell
analysis
NOD mice treated from 6 or 8 weeks of age with 1mg/kg Isotype (grey triangles 6weeks or
squares 8 weeks) or αCXCR3-9C5 antibody (black triangles 6weeks or squares 8 weeks) every 4
days or untouched as controls (white circles) until 12 weeks of age. CD4 T cells and their
respective CXCR3 expression, determined via CXCR3-173 binding, were determined in the (a)
spleen, (b) distal LN and (c) pancreatic LN. Treg (Foxp3+CD25+ and Foxp3+CD25-) cells and
their respective CXCR3 expression, determined via CXCR3-173 binding, was assessed in the (d)
spleen, (e) distal LN and (f) pancreatic LN. Results are expressed as total number of
lymphocytes (n=2-4mice/group)
100
αCXCR3-9C5 in the NOD Islet Allograft Model
N K c e ll a n a ly s is
C D 8 T c e ll a n a ly s is
( S p le e n )
a
500000
T o ta l L y m p h o c y te s
T o ta l L y m p h o c y te s
( S p le e n )
d
8 .0  1 0 6
6 .0  1 0 6
4 .0  1 0 6
2 .0  1 0 6
600000
400000
200000
400000
300000
200000
100000
0
0
C
+
D8
CD
C
8+
XC
NK
+
R3
NK
C D 8 T c e ll a n a ly s is
12000
T o ta l L y m p h o c y te s
T o ta l L y m p h o c y te s
1500000
1000000
500000
400000
300000
200000
100000
0
10000
8000
6000
4000
2000
0
8
CD
+
CD
8
X
+C
3
CR
+
NK
NK
C D 8 T c e ll a n a ly s is
500000
200000
150000
100000
50000
0
3+
7000
T o ta l L y m p h o c y te s
1000000
CR
( P a n c r e a tic L N )
f
1500000
CX
N K c e ll a n a ly s is
( P a n c r e a tic L N )
T o ta l L y m p h o c y te s
3+
( D is ta l L N )
e
2000000
c
CR
N K c e ll a n a ly s is
( D is ta l L N )
b
CX
6000
5000
4000
3000
2000
1000
0
C
+
D8
CD
8
X
+C
C
+
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NK
NK
CX
CR
3+
C o n tro l
Is o ty p e 8 w e e k s ta rt 1 m g /k g
a C X C R 3 8 w e e k s ta rt 1 m g /k g
Is o ty p e 6 w e e k s ta rt 1 m g /k g
a C X C R 3 6 w e e k s ta rt 1 m g /k g
Figure 5.11: Pre-diabetic NOD αCXCR3 Treatment – CD8+ T cell & NK cell analysis
NOD mice treated from 6 or 8 weeks of age with 1mg/kg grey triangles 6weeks or squares 8
weeks) or αCXCR3-9C5 antibody (black triangles 6weeks or squares 8 weeks) every 4 days or
untouched as controls (white circles) until 12 weeks of age. CD8 T cells and their respective
CXCR3 expression, determined via CXCR3-173 binding, were determined in the (a) spleen, (b)
distal LN and (c) pancreatic LN. NK cells and their respective CXCR3 expression, determined
via CXCR3-173 binding, was determined in the (d) spleen, (e) distal LN and (f) pancreatic LN.
Results are expressed as total number of lymphocytes (n=2-4mice/group)
101
αCXCR3-9C5 in the NOD Islet Allograft Model
Mice treated from 8 weeks of age with αCXCR3-9C5 showed reduced free CXCR3
on CD4+ and CD4+CD25+Foxp3+ cells in the spleen (figure 5.10a & b) and for
CD4+CD25-Foxp3+ cells in the spleen and dLN (figure 5.10b & d), compared to
respective isotype controls. In addition free, unbound CXCR3 was reduced for CD8+
T cells in the spleen (figure 5.11a) and for NK cells in the spleen and dLN (figure
5.11d & e) for mice treated from 8 weeks of age with αCXCR3-9C5 compared to
isotype controls. These changes were non-significant due to small group size.
Finally, due to changes in CD44 expression seen in C57BL/6 naïve mice and
BALB/c to C57BL/6 islet allograft model, we tested if αCXCR3-9C5 treatment in
the NOD model would produce a similar change. Analysis of CD44 expression and
expression of free CXCR3 in the spleens of NOD mice treated with 1mg/kg
αCXCR3-9C5 (grey shaded, figure 5.12) from 6 weeks of age was compared to
splenocytes from both isotype and control (black figure 5.12) treated mice. As shown
previously (chapter 4, figure 4.4) a reduction in CXCR3-173 binding was evident for
αCXCR3-9C5 treated mice compared to control or isotype treated mice, however
their respective CD44 expression was not different. This indicates a differential
effect of αCXCR3-9C5 in the C57BL/6 allograft model compared to that seen in the
NOD model and may offer an explanation as to the beneficial effects seen in the
allograft but not the autoimmune NOD model.
Figure 5.12: Unaltered activation of NOD
splenocytes
Representative histograms of CD4+ and
CD8+ T cell CXCR3 and CD44 expression
for pre-diabetic NOD mice treated from 6
weeks of age with 1mg/kg αCXCR3-9C5 i.v.
(grey shaded) or isotype controls (black).
102
αCXCR3-9C5 in the NOD Islet Allograft Model
Summary & Conclusions
Here we studied the effect of αCXCR3-9C5 in a mouse model of autoimmune
diabetes due to the role of CXCR3 in migration of immune cells to sites of
inflammation3,22. CXCR3 and its ligands are also upregulated within the serum of
individuals with type 1 diabetes214,228-231,233,282. Finally we were interested in the
effect of our anti-CXCR3 antibody in this model due to the variable effects of
inhibiting CXCR3 in previous literature124,217.
In NOD mice validation of diabetes incidence rates is important as diabetes
incidence is strongly influenced by environment (diet and microbial infections) and
thus diabetes incidence in the NOD is known to vary between facilities75. Diabetes
incidence in our NOD colony was consistent with previous literature with 50-60% of
our NOD cohort showing overt diabetes by 25 weeks of age. Incidence rates of 60%
at 25 weeks have been previously documented by our facility280.
Here we found that treatment with αCXCR3-9C5 had no ameliorating or detrimental
effect on the incidence or onset of diabetes in NOD mice. This is in contrast to
previous studies in the NOD mouse showing an accelerated onset of diabetes
following genetic deletion of CXCR3.
Within the NOD mouse we have shown that αCXCR3-9C5 does not alter disease
outcomes with respect to the ability of cells to migrate into the pancreas, pancreatic
LN, distal LN or spleen of treated compared to control mice. This was evident
through unaltered insulitis for pre-diabetic and diabetic NOD mice, as well as a lack
of changes in T and NK cell populations within primary and secondary lymphoid
tissues. In particular no change in the ability of Foxp3+ regulatory T cells to infiltrate
the pancreas was evident. This is in stark contrast to results of Yamada et al., which
showed the CXCR3-/- NOD mice had impaired pancreatic infiltration by Foxp3+
regulatory T cells. This difference in Foxp3+ cell accumulation may explain the
differential effects seen between our study and results for CXCR3-/- NOD mice217.
Of note, studies by Yamada et al. showed a very low diabetes incidence of 20-25% at
25 weeks of age indicating a low disease penetrance within these transgenic mice.
This may indicate that the breeding of this transgenic line altered the genetics of the
NOD mice which predispose to diabetes development and as such may not be a good
103
αCXCR3-9C5 in the NOD Islet Allograft Model
model of diabetes. Together our results show treatment with αCXCR3-9C5is safe for
use in the autoimmune NOD mouse model of type 1 diabetes.
Interestingly although basal expression of free CXCR3 in T cell subsets were seen to
increase from 6 to 12 weeks of age, expression remained below that seen in C57BL/6
islet allograft recipient mice. These results, as well as the lack of effect of αCXCR39C5 administration on immune cell infiltration of the pancreas or CD44 expression
suggest that CXCR3 may be dispensable for development of autoimmune diabetes in
the NOD mouse model. Additionally these results may indicate involvement of other
chemokine signalling pathways in the pathogenesis of diabetes in the NOD model,
such as CCR2 and CCR5283,284.
104
αCXCR3-9C5 in the NOD Islet Allograft Model
Chapter 6. αCXCR3-9C5 in the NOD Islet
Allograft Model
105
αCXCR3-9C5 in the NOD Islet Allograft Model
Chapter 6. αCXCR3-9C5 in the NOD Islet Allograft Model
Unlike results found in a previous study showing an acceleration of diabetes in a
CXCR3 knockout NOD mouse217, we showed no deleterious or beneficial effect of
our αCXCR3-9C5 antibody in the autoimmune NOD mouse model. However we
have shown a clear prolongation of MHC mismatched islet allograft survival upon
treatment with our αCXCR3-9C5 antibody. For this reason we proceeded to test the
efficacy of the antibody in a model of MHC mismatched islet allograft (BALB/c,
H2d) into diabetic NOD mice (H2g7, BGL 8-19mmol/L). Islet allograft is used
clinically to treat patients with unmanageable autoimmune diabetes, which involves a
combined autoimmune and allogeneic immune response, both of which are present in
this mouse model.
Figure 6.1: NOD Allogeneic Experimental Model
Overview of experimental model including monitoring of diabetic NOD mice for
BGL>7mmol/L followed by receipt of a MHC mismatched BALB/c islet allograft and
treatment with isotype or αCXCR3-9C5 mAb +/- rapamycin. Mice were monitored for
onset of hyperglycaemia indicating loss of graft function, see graph of typical BGL over
the course of rejection.
106
αCXCR3-9C5 in the NOD Islet Allograft Model
Mice were treated with one of 5 regimens including; 2mg/kg isotype (2) or
αCXCR3-9C5
(3)
antibody
once
per
week,
sub-therapeutic
Rapamycin
(0.5mg/kg)(4) once per day for the first 7 days of transplant or a combination of
2mg/kg αCXCR3-9C5 (1/week) with sub-therapeutic Rapamycin (0.5mg/kg, 1/day
for 7 days)(5). An untreated control group (1) was also included. Rapamycin was
administered i.p. and αCXCR3-9C5 or isotype antibody was administered i.v. (figure
6.1).
αCXCR3-9C5 Prolongs autoimmune NOD Islet Allograft Survival
Following transplantation of diabetic NOD mice, untreated control and isotype
treated mice showed rapid islet allograft rejection, with a MST of 4 and 5 days
respectively. In comparison allograft survival for mice treated with 2mg/kg
αCXCR3-9C5 was significantly prolonged with a MST of 8 days and one mouse
surviving to day 28 post-transplant (figure 6.2).
Mice treated with rapamycin also showed prolonged allograft survival with a MST of
11 days compared to control mice. However when low dose rapamycin and
αCXCR3-9C5 antibody treatments were combined, similarly to that seen for the
C57BL/6 allografts (chapter 4, figure 4.15), graft survival was substantially
improved. Mice receiving once weekly αCXCR3-9C5 combined with rapamycin
once per day for the first week following transplant showed a MST of 35 days with
long-term survival in 1 out of 5 mice, surviving to day 90 post-transplant (figure 6.2).
Allograft survival was determined as the number of post-operative days maintaining
euglycaemia before onset of hyperglycaemia (BGL>15mmol/L for 2 consecutive
days) indicating loss of the islet allograft (figure 6.3).
An additional group of pre-diabetic NOD mice received a variation of the combined
αCXCR3-9C5 and rapamycin therapy to determine if this altered regimen could
improve graft survival. These mice received a BALB/c islet allograft, as above, and
treatment from the day of allograft with 2mg/kg αCXCR3-9C5 1/week and 0.5mg/kg
rapamycin, 1 per day for 1 week followed by treatment every 2nd day for 1 week (2
weeks total).
107
Is le t A llo g r a f t s u r v iv a l ( % )
αCXCR3-9C5 in the NOD Islet Allograft Model
100
c o n tro l
 C X C R 3 -9 C 5 2 m g /k g
Is o ty p e 2 m g /k g
*
R a p a m y c in 0 .5 m g /k g
50
R a p a m y c in 0 .5 m g /k g (7 d ) +  C X C R 3 -9 C 5
2 m g /k g (1 /w e e k )
*
0
0
20
40
60
80
100
P o s t O p p e r a tiv e D a y s
Treatment Group
Median Survival (days)
Control (n=4)
4
Isotype (2mg/kg) (n=4)
5
αCXCR3-9C5 (2mg/kg) (n=5)
8
Rapamycin (0.5mg/kg 7days) (n=4)
11
p = 0 .2 3 8 9 v s Is o
Rapamycin (0.5mg/kg 7days) + αCXCR3-9C5
35
p = 0 .1 3 5 6 v s r a p a
p = 0 .7 8 6 9 v s r a p a
* p = 0 .0 2
(2mg/kg) (n=5)
v s Is o
* p = 0 .0 1 6 2
v s  C X C R 3 -9 C 5
Figure 6.2: αCXCR3-9C5 Prolongs NOD Islet Allograft Survival
Kaplan-Meier survival curves of BALB/c (H2d) islet graft transplant into diabetic NOD
(H2g7) recipients (BGL 8-19mmol/L). Recipients were left untreated or treated with
Isotype (2mg/kg, 1/week, i.v.), αCXCR3-9C5 antibody (2mg/kg, 1/week, i.v.), rapamycin
(0.5mg/kg, 1/day for 7 days i.p.) or a combination of αCXCR3-9C5 (2mg/kg, 1/week)
with sub-therapeutic rapamycin (0.5mg/kg, 1/day for 7 days). Allograft survival was
determined as the number of post-operative days maintaining euglycaemia before onset
of hyperglycaemia (BGL>15mmol/L, >2 days) indicating loss of allograft. p<0.05* for
αCXCR3-9C5 vs. Isotype (n=4-6mice/group).
Islet allografts were lost, as determined by onset of hyperglycaemia, at day 6, 7 and
17 for these mice (figure 6.4, black diamonds). However two mice treated with this
regimen survived >75days, but upon nephrectomy failed to become hyperglycaemic
(figure 6.4, white diamonds). This indicates that the graft was not the source of
insulin in these mice suggesting maintenance of pancreatic beta cell function. Mice
were followed for 10 days post nephrectomy to confirm this result followed by
euthanasia and collection of tissues for histology and flow cytometry to discern the
mechanism behind this allograft independent long-term survival. Lack of residual
islet mass in the kidney was confirmed by H&E staining with only mild infiltrate and
no islet structures remaining (figure 6.5).
108
αCXCR3-9C5 in the NOD Islet Allograft Model
Figure 6.3: αCXCR3-9C5 NOD Islet Allograft
Blood glucose levels of NOD recipients of BALB/c (H2d) islet allograft from the day of
transplant until loss of allograft (BGL> 15mmol/L) for mice treated with (a) untreated
control, (b) Isotype (2mg/kg, 1/week, i.v.), (c) αCXCR3-9C5 antibody (2mg/kg, 1/week,
i.v.), (d) rapamycin (0.5mg/kg for 7 days i.p.) (e) αCXCR3-9C5 (2mg/kg, 1/week) +
rapamycin (0.5mg/kg, 1/day, 7days) (n=3-5mice/group)
109
αCXCR3-9C5 in the NOD Islet Allograft Model
Figure 6.4: αCXCR3-9C5 NOD Islet Allograft Long-term survival
Blood glucose levels of NOD recipients of BALB/c (H2d) islet allograft from the day of
transplant for mice treated with αCXCR3-9C5 (2mg/kg, 1/week) + rapamycin (0.5mg/kg,
1/day, 7days + 1 every 2 days for 7days). Islet allograft recipients monitored until loss of
allograft (BGL> 15mmol/L)(black) or maintained euglycaemia and received nephrectomy
at day >75 (white) (n=5mice)
Figure 6.5. αCXCR3-9C5 NOD Islet Allograft Long-term survival shows minimal islet
insulin staining
Kidney and pancreas histology from 2 NOD recipients of BALB/c (H2d) islet allograft,
treated with αCXCR3-9C5 (2mg/kg, 1/week) + rapamycin (0.5mg/kg, 1/day, 7days + 1
every 2 days for 7days) which maintained euglycaemia following nephrectomy. H&E
stained sections of kidney, H&E and Insulin stained sections of pancreatic islets and insulin
staining of acinar tissue containing insulin positively stained cells.
110
αCXCR3-9C5 in the NOD Islet Allograft Model
Curiously no significant change in the proportions of T cell or NK cell subsets were
detected through flow cytometry in the spleen, dLN or pLN (data not shown). In
addition, although islets were found within the pancreas of these mice showing minor
if any infiltrate, islets showed very little insulin staining (figure 6.5). However we
did identify regions of acinar tissue, which stained positive for insulin and appeared
to have residual beta cells post islet destruction. This pattern of recovery of
euglycaemia in NOD mice post combined immunomodulation and islet allograft has
been shown previously and may offer an explanation as to these results, see summary
& conclusions285-288.
111
αCXCR3-9C5 in the NOD Islet Allograft Model
Summary & Conclusions
Previous literature looking at the role of CXCR3 in models of islet allograft rejection
have yet to examine the effects of deletion or antibody blockade in a combined
model of islet allograft transplant into diabetic NOD mice. As a result of the efficacy
and safety shown for our αCXCR3-9C5 mAb in islet allograft (chapter 4) and
diabetes models (chapter 5), respectively, we examined the effect of our antibody in
a model of islet allograft into diabetic NOD mice.
Here we show for the first time efficacy of an anti-CXCR3 antibody therapy in a
mouse model of diabetic islet allograft. Similarly to that seen in the C57BL/6 islet
allograft recipients (chapter 4, figure 4.15), NOD mice showed prolonged graft
survival upon treatment with αCXCR3-9C5 with a synergistic effect observed when
treated in combination with low-dose rapamycin. Further both αCXCR3-9C5
treatments, alone or in combination with low-dose rapamycin, lead to a small
proportion of islet allograft recipients exhibiting significantly longer survival.
Together we show the potential of αCXCR3-9C5 treatment for use as part of a
combination immunosuppressive therapy for use in islet allograft recipients with type
1 diabetes.
Of note we have also shown recovery of euglycaemia for two NOD islet allograft
recipients, which received a combination of αCXCR3-9C5 and rapamycin. Although
BGLs of these mice were below 15mmol/L at transplant, NOD mice would be
expected to proceed to onset of overt diabetes due to autoimmune destruction of
residual pancreatic beta cell mass. However here we show that these mice, upon
receipt of an islet allograft, maintained euglycaemia for over 75 days but did not
become hyperglycaemic upon islet graft removal via nephrectomy. Previous papers
showing immunomodulation with complete Freunds adjuvant (CFA) and islet
allograft transplantation of diabetic NOD recipients have reported a similar
phenomenon. These papers attributed euglycaemia post nephrectomy to regeneration
or maintenance of residual beta cell mass with one paper suggesting CFA and islet
allograft transplant skewed the balance of beta cell autoimmune destruction and beta
cell replication toward replication resulting in recovery of euglycaemia285-288. Further
as with results found in our mice Suri et al. indicated that although mice maintained
112
αCXCR3-9C5 in the NOD Islet Allograft Model
euglycaemia, few islets were present (6% or less) and only some were insulin
positive286.
This phenomenon has also been found to occur in humans. In a single case study of
an individual with type 1 diabetes who had received a whole pancreas transplant,
islets were found to be insulin negative, however single insulin positive cells were
found within the native pancreas suggestive of beta cell regeneration289. Further a
previous study by Liu et al. investigated the role of immunosuppression combined
with islet or whole pancreas transplant in patients with type 1 diabetes, showing a
small but detectable amount of C-peptide was produced by the native pancreas a
number of years after transplant290.
Although this occurred in only a small subset of mice in our study it suggests that in
addition to prolongation of survival of islet allografts, combined immunomodulation
with low dose rapamycin and αCXCR3-9C5 antibody may promote survival and
replication of residual beta cell mass allowing maintenance of euglycaemia and this
may also be applicable in humans although further investigation of this result is
required285-288.
113
Chapter 7. Clarifying the Role of αCXCR3-9C5
Clarifying the Role of αCXCR3-9C5
Chapter 7. Clarifying the Role of αCXCR3-9C5
Finally of interest to us was the mechanism behind the effects of αCXCR3-9C5 in
models of islet transplant. Previous studies on CXCR3 mAbs have focused primarily
on disease outcomes with little examination of molecular mechanism. In vivo mixed
lymphocyte reactions (MLR) were performed using transfer of CFSE labelled whole
splenocytes from naïve C57BL/6 mice into sub-lethally irradiated BALB/c mice.
Mice were also administered 2mg/kg i.v. αCXCR3-9C5 or untreated as controls. The
spleens of these mice were then harvested 72 hours later and analysed by flow
cytometry for changes in proliferation and activation markers, particularly for CD44
expression. CD44 is a marker of T cell development and activation with a high
expression on effector and memory T cell subsets. CD44 is also involved in cell-cell
interactions, adhesion and migration of activated cells291.
Firstly proliferation of both CD4 and CD8 T cells was evident from CFSE dilution
with the right-most peak showing un-proliferated cells and successive peaks showing
generation of proliferated cells (figure 7.1a & 7.2a). Activation of proliferated cells,
indicated by up-regulation of CD44 expression and downregulation of CD62L
expression in proliferated compared to un-proliferated cells, was also evident. No
difference was evident in CD4+ or CD8+ T cell proliferation or expression of
CXCR3 or CD62L for both proliferated and non-proliferated cells for αCXCR3-9C5
treated (grey shaded histogram) compared to cells from control mice (black unfilled
histogram) (figure 7.1a & 7.2a). Expression of CD4+CD44, CD44 MFI and CD4+ T
cell subsets (CD44-CD62L+, CD44+CD62L+, CD44+CD62L) was also not different
for αCXCR3-9C5 treated cells compared to controls (black unfilled histogram), for
proliferated and non-proliferated T cells (figure 7.1a, b, c).
In contrast CD8+ T cells treated with αCXCR3-9C5 (grey shaded histogram, figure
7.2a), showed a significantly reduced expression of CD44, similar to that seen in the
C57BL/6 allograft model (chapter 4, figure 4.13), compared to untreated controls
(black unfilled histogram, figure 7.2a). This was particularly evident in nonproliferated cells with reduced CD44 MFI compared to controls (figure 7.2b).
Further when the frequency of CD8+ memory (CD44+CD62L+) T cells was
examined following αCXCR3-9C5 treatment, a reduced frequency of non115
Clarifying the Role of αCXCR3-9C5
proliferated memory CD8+ T cells was evident compared to untreated control cells
(figure 7.2d).
Figure 7.1: αCXCR3-9C5 treatment does not alter CD4+ T cell proliferation or
activation
116
Clarifying the Role of αCXCR3-9C5
Figure 7.1: αCXCR3-9C5 treatment does not alter CD4+ T cell proliferation or
activation
Analysis of CD4+ T cell proliferation and activation from sublethally irradiated BALB/c
recipients of C57BL/6 CFSE labelled splenocytes, treated with 2mg/kg αCXCR3-9C5 or
controls at 72hours post cell transfer. a) Representative histograms of CFSE proliferation
and activation markers (CXCR3, CD62L and CD44) and b) Mean fluorescence intensity
(MFI) for CD44 expression for proliferated and non-proliferated cells: αCXCR3-9C5 treated
(grey shaded histogram) or control mice (black, unfilled histogram). c) Representative
FACS plots of mean frequencies for CD4+ T cell subsets (CD44-CD62L+, CD44+CD62L+,
CD44+CD62L-) for non-proliferated and proliferated αCXCR3-9C5 treated and control T
cells and d) frequencies of CD4+ T cell subsets from c) (n=4/group for 2 repeat
experiments).
Figure 7.2: αCXCR3-9C5 treatment alters expression of CD8+ CD44high T cells
Analysis of CD8+ T cell proliferation and activation from sublethally irradiated BALB/c
recipients of C57BL/6 CFSE labelled splenocytes, treated with 2mg/kg αCXCR3-9C5 or
controls at 72hours post cell transfer. a) Representative histograms of CFSE proliferation
and activation markers (CXCR3, CD62L and CD44) and b) Mean fluorescence intensity
(MFI) for CD44 expression for proliferated and non-proliferated cells from αCXCR3-9C5
treated (grey shaded) or control mice (black, unfilled). c) Representative FACS plots
displaying mean frequencies for CD8+ T cell subsets (CD44-CD62L+, CD44+CD62L+,
CD44+CD62L-) for non-proliferated and proliferated αCXCR3-9C5 treated and control T
cells and d) frequencies of CD8+ T cell subsets from c) (n=4/group for 2 repeat
experiments).
117
Clarifying the Role of αCXCR3-9C5
Figure 7.2: αCXCR3-9C5 treatment alters expression of CD8+ CD44high T cells
118
Clarifying the Role of αCXCR3-9C5
Summary & Conclusions
Here we show a role of CXCR3 in regulating expression of CD44 on CD8+ but not
CD4+ T cells. Use of in vivo MLR revealed reduced CD44 expression without
affecting CD62L expression or the ability of these cells to proliferate following
αCXCR3-9C5 treatment. Interestingly mice administered with αCXCR3-9C5
antibody showed no difference in CXCR3 expression at 72hours post i.v. compared
to untreated control expression. This may result from a high turnover of the CXCR3
receptor under conditions of alloantigen stimulation, which involves receptor
degradation and de novo synthesis of new receptor for surface expression292.
While a greater proportion of cells which have proliferated express CD44, compared
to those cells which are undivided, the effect of blockade of CXCR3 via αCXCR39C5 treatment appears to preferentially affect CD44 expression on non-proliferated
CD8+ T cells (figure 7.2a). These untreated, undivided CD44 high CD8+ T cells
may represent a subset of undivided, naive cells, which are becoming activated and
will go on to proliferate and differentiate into memory and effector T cell subsets.
The effect on CD8+ T cell CD44 expression appears to particularly affect nonproliferated CD8+CD44+CD62L+ memory T cells. The reduction in nonproliferated CD44 high CD8+ T cells also appears to cause a non-significant
decrease in the frequency of CD8+ memory (CD44+CD62L+) cells. This may result
through a reduced frequency of activated parent cells, which subsequently divide into
this memory subset. In the context of allograft immune response this reduction in
CD44 expression resulted in decreased frequencies of effector and memory CD8+ T
cells. CD44 expression also plays an important role in T cell migration to sites of
infection or injury and reduced expression may impede migration and activation of
these cells. Together this reduction in CD8+CD44+ T cells, presumably memory or
effector CD8 T cells, likely contributed to the prolonged islet allograft survival seen
upon treatment with αCXCR3-9C5 (figure 4.16).
This may highlight an important clinical application for αCXCR3-9C5 antibody
targeting CD8+ memory T cells, which are poorly targeted by current
immunosuppressive therapies244,293-296. These cells are instrumental in the allograft
rejection response with CD8+ memory T cells found to infiltrate and produce IFN-γ
119
Clarifying the Role of αCXCR3-9C5
within the first 24 hours following transplant. IFN-γ then stimulates donor
chemokine release, CXCL9 and CXCL10, recruiting effector T cells to the allograft
and promoting rejection213,244,272. Although this memory subset plays a critical role in
allograft rejection, these cells display unique cell surface markers and costimulatory
requirements, as well as distinct trafficking and activation profiles297. As such naïve
T cells are effectively depleted using ATG or aletuzumab and their activation and
proliferation blocked by co-stimulation blockade therapies, such as anti-CTLA4,
anti-CD40L and anti-CD154 antibodies, while memory T cells are resistant to these
immunosuppressive therapies244,293-296. Additionally, non-allograft specific, viral
memory responses have been found to impede allograft tolerance induction, which
were also co-stimulation blockade resistant298.
Interestingly a marker common to CD8+ effector, central memory and effector
memory T cells is the up-regulated expression of CXCR3, which presents an
appealing therapeutic target for each of these cell types involved in mediating the
allograft rejection response299-301.
120
Chapter 8. Discussion
Discussion
Chapter 8. Discussion
Overview
The major aim of our studies was to delineate the function of a novel anti-CXCR3
monoclonal antibody, which targets a conserved epitope of CXCR3 found in a
number of species including mice and humans. Due to prior studies showing efficacy
of CXCR3 deletion and blockade in models of allograft transplantation200,216 we
investigated the role of our αCXCR3-9C5 mAb in a MHC mismatched islet
transplant. In addition islet allograft transplantation is used clinically in individuals
with type 1 diabetes. This application warranted further investigation into the
efficacy of αCXCR3-9C5 as a rejection prophylaxis in mouse models of islet
allograft transplantation into diabetic autoimmune NOD recipients, as well as in a
model of spontaneous autoimmune diabetes alone.
This thesis explores the efficacy of αCXCR3-9C5 mediated blockade of CXCR3
signalling for the prolongation of allograft survival in both autoimmune and nonautoimmune mouse models. However within the NOD model of autoimmune type 1
diabetes, we found no effect of antagonism of CXCR3 using our αCXCR3-9C5 mAb
suggesting the effect is specific to the allogeneic, but not the autoimmune response.
Allograft Transplantation
Several studies have highlighted a strong correlation between increased expression of
CXCR3 and its chemokine ligands with processes of human allograft rejection, as
well as T1D178-180,182,214,228,229,302. In support of those findings our data also highlight
an important role for CXCR3 mediated signalling in the allograft rejection response.
This has been show previously in heart, lung, skin and islet allograft models. In all
those studies, including ours, blockade of CXCR3 reduced the recruitment of
immune cells to the graft site, resulting in a significant delay in allograft loss (figure
4.16, 4.17)200,213,216,245-247,252.
Here we show that similarly to other islet allograft studies antagonism of CXCR3
signalling resulted in a prolonged islet allograft survival216,252. We also found that
within long-term surviving islet allografts, which showed preserved islet architecture
and strong insulin staining, only minimal immune infiltrate was present. This
122
Discussion
infiltrate was restricted to the graft periphery, suggesting impaired immune cell
infiltration. Previous literature has highlighted the importance of CXCR3 signalling
in immune cell migration, with inhibition of this signalling pathway by antibody
blockade or gene silencing, decreasing infiltration and inflammation within the graft
and resulting in enhanced allograft survival200,213,216. Further investigation of the
ability of immune cells to infiltrate the graft in the acute response following
treatment with αCXCR3-9C5 will provide further evidence to confirm a role for
αCXCR3-9C5 in impaired immune cell recruitment to the graft.
T cell Activation & Regulatory T Cells
While the majority of previous studies exclusively investigated the impact of CXCR3
blockade on chemotaxis200,216,252, we have also examined a possible role for altered T
cell activation or regulatory T cell accumulation in the enhanced survival of islet
allografts. Interestingly, although a role for CXCR3 mediated T cell activation has
been described previously it has yet to be investigated within models of allograft
rejection192,219,299. CXCR3 was previously shown to be involved in immune cell
activation and proliferation in a model of type 1 IFN induced T cell stimulation.
CXCR3 mediated signals induced the up-regulation of activation markers CD25 and
CD69 on CD8 T cells, as well as promoting CD8 T cell proliferation, particularly for
CD44low CD8+ T cells192.
We show here that αCXCR3-9C5 mediated antagonism of CXCR3 signalling in untransplanted and islet allograft recipients as well as under conditions of in vivo MLR,
resulted in a decreased proportion of CD8+ T cells with high CD44 expression. This
resulted in a significantly reduced number of CD8 effector and memory T cells, both
important contributors to the allograft rejection response244,270-272. Although a role for
CXCR3 in CD44 expression has not yet been shown it is possible that CXCR3
signalling may directly or indirectly affect T cell activation and CD44 expression.
Moreover other studies have shown a critical role for CXCR3 in the co-localisation
of T cells and DC within the splenic marginal zone, as well as the ability to form
long-lasting cell-cell interactions with virally infected cells220,303. Antagonism of
CXCR3 may therefore result in altered T cell activation that could account for the
decreased CD44 expression seen. This co-localisation and activation has been shown
to favour CD8+ T cell expression of cytotoxic markers, perforin and granzyme
123
Discussion
B219,221. Although we have yet to investigate the phenotype or function of our
αCXCR3-9C5 treated CD8+ T cells it is possible that they may also show reduced
cytotoxicity, contributing to graft survival.
In addition to a role in T cell activation our results suggest that the improved survival
of αCXCR3-9C5 treated islet allograft recipient mice is also dependent on
CD4+CD25+Foxp3+ regulatory T cells. These regulatory T cells have been well
characterised within the allograft immune responses to confer protection and limit
graft destruction144,273,275, and as such altered function of these cells was of interest.
We found high numbers of Foxp3+ cells within the spleen and graft site of long-term
surviving allograft recipients treated with αCXCR3-9C5 alone or in combination
with low dose rapamycin. The involvement of CD25+Foxp3+ T cells is further
supported by the loss of prolonged islet allograft survival in αCXCR3-9C5 treated
mice upon co-treatment with a CD25 depleting antibody (PC61). Finally, cell
transfer experiments from long-term surviving mice show improved islet allograft
survival in the presence of CD25+ T cells compared to those mice transferred CD25
depleted T cells.
Use of the CD25 depleting antibody (PC61) has been previously shown to effectively
deplete the CD4+CD25+Foxp3+ population of regulatory T cells, with up to 90%
depletion for 2-3 weeks post-injection261. This allows the protective role of Treg in
the allograft immune response to be determined. A limitation to this method
however, is the elimination of CD25+ cells, which are not Tregs and likely to play
roles within the allograft immune response. This is particularly true for recently
activated T cells expressing CD25304. An alternative method of eliminating Treg
cells uses the transgenic DEREG mouse (depletion of regulatory T cells), expressing
the diphtheria toxin (DT) receptor under the control of the Foxp3 locus, and
providing selective depletion of Foxp3+ Treg305,306. Future studies may further
clarify the roles of Tregs in this model using a more specific approach, such as the
DREG mouse.
Of note the clinically used immunosuppressive agent used in these studies,
rapamycin, has been shown to differentially affect effector and regulatory T cells.
Rapamycin has been shown to promote the induction of regulatory T cells while
124
Discussion
blocking activation and proliferation of effector T cells279,307. As a result, we propose
that the effects of rapamycin synergise with the impaired chemotaxis and activation
of T cells driven by αCXCR3-9C5, leading to enhanced regulatory and impaired
effector and memory T cell responses; resulting in enhanced islet allograft survival.
We also show that treatment of mice with αCXCR3-9C5 alone is able to
significantly prolong islet allograft survival and cause long-term survival in a small
proportion of these mice, with a notable increase in regulatory T cells. This
represents a novel mechanism by which CXCR3 may affect activation of the
allograft immune response and will be investigated further in future experiments.
Allograft transplantation in the NOD mouse
The role of CXCR3 blockade in the clinically applicable model of islet allograft
transplantation into a recipient with pre-established autoimmune T1D has not yet be
characterised in the literature. To do this we employed islet allograft into the NOD
mouse model of type 1 diabetes. Due to the involvement of both autoimmune and
alloimmune response within this model the kinetic of rejection in isotype or control
treated mice was significantly faster than that of the non-autoimmune allograft
model. Upon investigation of the efficacy of our novel αCXCR3-9C5 antibody
within this model, a single antibody administration was found to significantly
prolong survival of the allograft. Additionally, as seen in the non-autoimmune
allograft model, a synergistic effect was seen when αCXCR3-9C5 was administered
in combination with low dose rapamycin, further enhancing islet allograft survival.
Together this shows promise for clinical application of αCXCR3-9C5 within the
context of islet allograft transplantation into autoimmune recipients with T1D.
The NOD Mouse
Given the combined autoimmune and alloimmune nature of this NOD islet allograft
transplant model and previous literature on accelerated disease in CXCR3-/- NOD
mice, the effect of αCXCR3-9C5 within an autoimmune diabetes model alone was of
interest. Within autoimmune models of type 1 diabetes the role of CXCR3 is less
well characterised. Virally induced models using the RIP-LCMV system (Chapter 1,
animal models) initially showed a substantial role of CXCR3 for the recruitment of
125
Discussion
immune cells into the pancreas124. However, further characterisation of this model
using both deletion and antibody blockade of CXCR3 and its chemokine CXCL10
though suggest that the CXCR3 signalling axis contributes only minimally to viral
induced diabetes and suggests redundancy within the chemokine system may account
for this difference124,236. The spontaneous onset model of diabetes using the NOD
mouse has also shown differential effects when targeting the CXCR3 signalling
pathway. Firstly CXCL10, which is produced by the islets themselves, has been
shown to inhibit beta cell proliferation through CXCR3 independent auto/paracrine
signalling66,235. As such, inhibition of CXCL10 was found to reduce diabetes
incidence. However, upon CXCR3 deletion in a NOD mouse model, diabetes onset
was found to be accelerated. This was attributed to an inability of CD4+Foxp3+
regulatory T cells to traffic from the pancreatic lymph node into the pancreas217.
Investigation using antibody blockade rather than complete deletion of the CXCR3
receptor has not previously been examined. We show here for the first time that use
of antibody mediated blockade of CXCR3 signalling within the NOD mouse model
has no effect on the onset or incidence of diabetes in our model. Furthermore no
change in the frequency of immune cells, including Foxp3+ Treg cells were seen in
the spleen, distal or pancreatic lymph nodes; in αCXCR3-9C5 treated mice compared
to controls. Nor was the ability of immune cells or Foxp3+ T cells to infiltrate the
pancreas different.
Results seen for Yamada et al. using CXCR3 deficient NOD mice differ significantly
from those found using our αCXCR3-9C5 antibody. This differential effect of
transient antibody blockade compared to complete deletion is not unique, as seen in
studies on CTLA-4, a critical negative regulator of T cell activation. Mouse studies
using genetic deletion of the CTLA-4 gene showed high levels of lymphocyte
accumulation within lymphoid and non-lymphoid tissues including the heart, which
showed myocardial lesions. These mice died at 3-4weeks of age, likely due to this
myocarditis and uncontrolled T cell activation259. In contrast use of CTLA-4-Ig
antibody blockade has shown considerable benefit for the treatment of tumours and
is now used in human trials. CTLA-4 antibodies (e.g. ipilimumab) used in cancer,
function through loss of the CTLA-4 co-inhibitory signal, enhancing co-stimulatory
signalling of T cells and the resulting anti-tumour response. This antibody mediated
126
Discussion
blockade has relatively minor symptoms compared to those seen for CTLA-4
knockout mice256-258. From this it is evident that although previous literature shows a
deleterious role for CXCR3 deletion in the NOD mouse this does not preclude the
therapeutic potential of an anti-CXCR3 antibody. In this case we show safety of
αCXCR3-9C5 in a mouse model of diabetes with potential for use in an islet
transplant context.
Conclusion
In conclusion we have characterised a novel αCXCR3-9C5 monoclonal antibody
within models of islet allograft and autoimmune type 1 diabetes. Our findings
support a role for CXCR3 signalling within the allograft immune response as
previously described.
We also establish that CXCR3 signalling in this model
participates in additional, previously uncharacterised, functions involved in T cell
activation and regulatory T cell activity. We have also shown safety and efficacy for
use of αCXCR3-9C5 in models of autoimmune diabetes and islet allograft,
respectively, in the NOD mouse model. This is important due to inconsistency and
potentially detrimental effects seen within previous literature as to the effects of
CXCR3 inhibition within this model of T1D. Further, prior to this study investigation
of the role of CXCR3 blockade in a combined autoimmune islet allograft model had
not been performed.
We propose that αCXCR3-9C5 may be a viable option for use clinically in islet
transplantation into individuals with type 1 diabetes. Following islet allograft, in both
the non-autoimmune C57BL/6 and in the autoimmune NOD mouse, αCXCR3-9C5
treatment shows a considerable benefit in prolonging allograft survival. Further, in
contrast to previous studies, blockade of CXCR3 signalling using this antibody does
not aggravate the autoimmune pathophysiology of the NOD mouse. Additionally due
to the conserved nature of the epitope of CXCR3 which αCXCR3-9C5 targets, there
is potential for efficient translation of the antibody into the clinic, by humanising the
antibody. This study supports further investigation into the use of αCXCR3-9C5 as a
more targeted therapeutic strategy, with potentially reduced toxicity or side effects
than conventional immunosuppression, for use as part of a combination therapy in
clinical islet allograft.
127
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