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POSTER ABSTRACTS
Enas Abu Shah
Universtiy of Oxford
T-cells integrate signals from their surrounding to determine their functional output; activation
towards infected tissues or tolerance to avoid self-reactivity. The signal integration spans
several length scales, from the molecular to the population levels. Research has focused on each
one of these scales separately, largely in a qualitative manner. Despite the importance of the
integration mechanism, we are still lacking basic understanding of this process. I aim to study Tcell activation and its modulation by the cells’ milieu. In particular, I plan to investigate the
importance of sequential cell contacts for signal integration by following the interactions
formed between human T-cells and other immune cells, bearing activation and inhibition
signals, in a controlled in vitro environment. Where I am interested in assessing the effect of the
frequency and strength of these signals on T-cell fate. Using state-of-the-art microscopy
techniques combined with molecular manipulation of components of the signalling pathway as
well as the environmental cues, will enable us to obtain the quantitative data needed to analyse
and construct mathematical models that predict the decision making process in immune cells.
The insights obtained will advance our basic understanding of the immune system and guide
the rational design of immuno-therapies.
Katharine Best
University College London
A co-operative model of T cell self-tolerance
The population of T cells in an individual needs to avoid harmful response to self-peptides while
maintaining as broad a range of specificities as possible to allow for effective response to
unknown foreign peptides. Much previous work on self-tolerance has focused on mechanisms
for deletion (or silencing) of individual T cells. However, the enormous possible diversity of
TCRs suggests that each TCR must be able to recognise many peptide-MHC complexes (pMHCs),
and each pMHC is recognised by many different TCRs. This suggests that self-tolerance may
involve co-operative interactions between different clonotypes, in such a way that tolerance
emerges as a property of the overall population rather than at an individual T cell level. We
propose a model where each resting antigen presenting cell integrates signals from many T
cells, and deletes cells in its proximity when the total signal passes a threshold value. We
formulate this model as an optimisation of a set of linear inequalities which can be solved using
classical linear programming techniques. The model produces a repertoire which is tolerant to
self, while maintaining a rich diversity of TCRs with which to respond to future exposures to
pathogens.
Luca Biasco
HSR-TIGET
Upon gene therapy (GT) for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich
Syndrome (WAS), gene-corrected hematopoietic stem/progenitor cells (HSPC) generated a
stable genetically engineered hematopoietic system where each vector-marked cell is univocally
barcoded by a vector integration site (IS). To track human hematopoietic system dynamics, we
collected by LAM-PCR+Illumina sequencing 28.539.414 sequence reads corresponding to
89.373 IS tagging clones belonging to 13 different cell types purified from the bone marrow and
the peripheral blood of 4 WAS patients up to 48 months after GT. We unraveled the nature of
HSPC output showing that distinct waves of populations were observed during the first 6-9
months after GT reaching a homeostatic equilibrium only by 12 months. We exploited IS
similarities to infere/test hematopoietic hierarchies by combining conditional probability
distributions and static/dynamic graphical models of dependencies. We also estimated by markrecapture approaches that few thousands clones are responsible for the long-term maintenance
of the whole genetically engineered hematopoietic system. Tracking of 4.845 clones in ADASCID patients for up to 6 years after GT, we showed that identical IS are consistently detected at
multiple lineages level even many years after GT. Overall our work constitute the first molecular
tracking of hematopoietic system in humans.
Jose Borghans
UMC Utrecht
It is generally thought that lymphocyte homeostasis is maintained through increased
lymphocyte proliferation or survival when lymphopoiesis declines. Although this indeed seems
to be the case in mice, evidence in humans is lacking. Using in vivo 2H2O labeling in healthy
young and elderly individuals, we found that the daily turnover rates of almost all lymphocyte
subsets hardly change during healthy aging. Remarkably, even for naive T cells we found no
evidence for a homeostatic response to a tenfold decline in daily thymic output. The most likely
explanation is that thymic output is already playing such a small role in young adults that its
decline during aging need not be compensated for.
In patients treated with an autologous stem-cell transplantation, on the other hand, we did find
evidence for increased lymphocyte production rates. Despite a reconstitution period of 10-13
months, most lymphocyte counts were still low. 2H2O labeling showed that all lymphocyte
subsets underwent increased turnover, indicating that although lymphocytes reconstitute very
slowly after stem-cell transplantation, they are in fact produced at increased rates. Although
there is little evidence in humans that such homeostatic mechanisms play a role in healthy
aging, they thus do occur in more severe situations of lymphopenia.
Veit Bucholz
TU München
Single T cell fate mapping identifies distinct effects of antigen and inflammation on memory T
cell development
In a vaccine formulation the amount of antigen and the dose of adjuvant – providing
inflammatory signals – are considered as essential modulators of the ensuing T cell immune
response. However, the precise effect of either factor on the differentiation and expansion of
long-lived memory and short-lived effector subsets remains controversial. Here we map
immune responses derived in vivo from single epitope-specific CD8+ T cells, while curtailing
either the presence of antigen or inflammation. This is achieved by timed depletion of
SIINFEKL-pulsed Dendritic cells carrying a diphtheria toxin receptor transgene (curtailed
antigen) or ampicillin-mediated abrogation of the accompanying Listeria monocytogenes
infection (curtailed inflammation). Aided by computational analysis of these two settings, we
predict and experimentally verify that Listeria-associated inflammation is equally important for
expansion of long- and short-lived subsets. In contrast, prolonged antigen presence is chiefly
required for proliferation of memory precursors and largely dispensable for proliferation of
effector T cells. These findings have important implications for the design of vaccine
formulations and suggest that the prolonged availability of antigen in vivo is key to the longterm efficiency of a vaccine.
Judy Cannon
University of New Mexico
T cell search in lymph nodes has been qualitatively described as a random walk; we provide a
precise description of the type of random walk and how motility impacts T cell search efficiency.
We observe the movement patterns of naïve T cells using ex vivo 2-photon microscopy and
describe the statistical distribution of those movements using maximum likelihood methods.
We find that while T cells move with features of a Lévy walk, Brownian and Lévy walks are both
poor descriptors of T cell motion. Instead, distribution fitting and efficiency simulations indicate
that T cells move in lymph nodes using a correlated random walk with a heavy-tailed
distribution of step lengths. We find that a lognormal distribution of step lengths, motion that is
directionally persistent over short time scales, and heterogeneity in movement patterns among
T cells all increase search efficiency. In contrast to Brownian motion and Lévy walks, the
observed T cell pattern of motion balances the need for repeated dendritic cell contact and
discovery of rare dendritic cells bearing cognate antigen.
Benny Chain
University College London
TCR clonal diversity in the response to antigen
The stochastic nature of the recombination machinery giving rise to TCRs ensures that there is a
distinct and largely non-overlapping repertoire of receptors in different genetically identical
individuals. Furthermore, the frequency of each TCR is not uniform even in the resting
repertoire. Charting the evolution of an immune response is therefore a challenging task. We
examine the TCR repertoires of mouse T cells stimulated with a variety of different model
antigens. We demonstrate that responses to individual antigens have a large stochastic
component, and common TCRs identifying the antigen-specific T cells are very rare. Instead, we
investigate the hypothesis that antigen specificity may be defined by small amino acid motifs
within the CDR3 region of the TCR. We use string kernels to quantify the occurrence of possible
motifs (eg. consecutive amino acid triplets) within TCR repertoires. These vectors are then used
to train high dimensional machine learning algorithms. We demonstrate that these algorithms
can partially predict an unknown antigen stimulus for a new repertoire. These experiments
demonstrate that there is no simple one-to-one relationship between antigen and responding
TCR, but local TCR sequence features may define a set of T cells which determine the antigen
specificity of the response.
Hans Diebner
TU Dresden
An Evolutionary Stability Perspective on Oncogenesis Control in Mature T-Cell Populations
(Coauthors: Jörg Kirberg and Ingo Röder)
It is known for roughly two decades that T-cell clones (uniquely defined through their antigenspecific T-cell receptor) maintain homeostasis in the periphery almost independent from new
thymic output through competition for self-peptides presented on MHC molecules (on the
surface of antigen presenting cells). Beyond existing mathematical models, we add leukemic
clone variants to the repertoir of T cells and analyse the system with respect to competitive
exclusion of the oncogenic variants. An analysis known from the studies of evolutionary stability
allows for the derivation of a fitness function that relates systems parameters with clonal
diversity in order to gain conditions under which the leukemic invaders are suppressed. The
model well captures the experimental observation that transgenic clones are outcompeted
under a polyclonal condition whereas monoclonality leads to tumour outgrowth. The
conditional function allows to investigate the system with respect to other dynamical
behaviours as, for example, co-existence of both healthy as well as leukemic clone variants.
Since quality and quantity of available sp-MHC complexes appear to vary over the lymphatic
system (lymph node dependent niche hierarchies), our model may stimulated further
experiments in this direction as, for example, local and temporal niche variations and their
impact on clonal diversity.
Feline Dijkgraaf
NKI
Kinship of skin-resident CD8+ memory T cells
Feline Dijkgraaf1, David Vredevoogd1, Lianne Kok1, Silvia Ariotti2, Leila Perie1 and Ton
Schumacher1
1Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
2 Department of Molecular and Cell Biology, University of California, Berkeley California, The
United States
After local skin infection, CD8+ memory T cells populations are formed in the circulation as well
as at the site of pathogen entry, so-called skin-resident memory T cells (skin-Trm). These
populations play an important role in the control of renewed infections. Despite the fact that
there is an increasing knowledge on the factors important for the development of skin-Trm, it is
unclear how they relate to circulating T cells. For example, are there naïve T cells that yield
progeny that is primarily destined to end up in the skin? Or do skin-Trm share common
precursors with (subsets of) circulating effector or memory T cells?
Therefore, this study aims to address the kinship between circulating T cells and skin-Trm. In
order to do so, ‘memory mice’ are generated by transfer of genetically tagged GFP+ naïve OTI
CD8+ T cells and local intradermal vaccination. Subsequently, barcode PCR is performed on
either single (skin) or bulk sorted GFP+ cells (circulation) isolated in effector- or memory phase.
Eventually, the number of barcodes and the contribution of each family from skin and
circulation can be compared using barcode analysis.
This approach allows us to study the ancestors of skin-Trm using unique and heritable genetic
markers to track single cells in vivo.
Yuval Elhanati
LPTENS
Probabilistic inference of selection in immune receptor repertoires
T and B cells identify foreign pathogens using surface receptors. Receptor diversity enables an
effective response to a wide variety of threats. This receptor diversity is the result of two
different stochastic processes, generation and selection. Random generation, a single cell
recombination event, is followed by functional selection of cells by interactions with self and
foreign peptides, essentially a population level effect. Understanding repertoire diversity, an
essential property of the immune system, starts by analyzing those two processes and their
interplay. We approach this problem by modelling them as probabilistic processes, thus
capturing their fundamental stochastic nature. Analyzing human data, we use maximum
likelihood methods to recover the parameters of the distributions – first generation
probabilities for different recombination events, and then selection on particular elements of
the receptor, acting on the initial generated repertoire. We quantify the global and site-specific
selection pressures and disentangle selection on individual amino acids from amino acid biases
in the generated repertoire. For both B and T cells, we find correlations between generation and
selection of receptors, and a significant reduction of diversity during selection, suggesting
natural evolution anticipates somatic evolution.
Michael Flossdorf
DKFZ
T cell immune responses generate diversity through linear cell-fate progression
Upon infection, naive antigen-specific cytotoxic T cells expand vigorously and give rise to a
population of short-lived effector and long-lived memory cells. Conflicting models have been
proposed that suggest either of these subsets to be a precursor of the other or attribute their
generation to asymmetrically dividing naive cells. To gain insight into the mechanism that
underlies T cell diversification we combine stochastic population modeling with large scale
model discrimination based on single cell in vivo fate mapping data. We developed a
computational framework that efficiently incorporates data on single cell dynamics in addition
to population mean dynamics. This resulted in significant improvements in both model
discrimination and identifiability. Our framework allows for stochastic differentiation and
proliferation decisions of individual cells and incorporates both symmetric and asymmetric cell
division. Building on this, we find, first, that asymmetric cell divisions of the activated naive T
cells play a negligible role and, second, that phenotypic diversity is instead generated through
linear cell-fate progression: Naive cytotoxic T cells give rise to slowly proliferating, long-lived
subsets from which rapidly proliferating, short-lived subsets emerge. Critical predictions of this
linear differentiation model have been validated in subsequent experiments. Third, we find that
recall responses initiated by resting memory T cells recapitulate the primary response.
Matthew Fricke
University of New Mexico
T cell search in lymph nodes has been qualitatively described as a random walk; we provide a
precise description of the type of random walk and how motility impacts T cell search efficiency.
We observe the movement patterns of naïve T cells using ex vivo 2-photon microscopy and
describe the statistical distribution of those movements using maximum likelihood methods.
We find that while T cells move with features of a Lévy walk, Brownian and Lévy walks are both
poor descriptors of T cell motion. Instead, distribution fitting and efficiency simulations indicate
that T cells move in lymph nodes using a correlated random walk with a heavy-tailed
distribution of step lengths. We find that a lognormal distribution of step lengths, motion that is
directionally persistent over short time scales, and heterogeneity in movement patterns among
T cells all increase search efficiency. In contrast to Brownian motion and Lévy walks, the
observed T cell pattern of motion balances the need for repeated dendritic cell contact and
discovery of rare dendritic cells bearing cognate antigen.
Graham Gossel
University of Glasgow
Rates of lymphocyte division and loss are commonly measured by the administration of labels
that are incorporated into the DNA of dividing cells, such as BrDU, or deuterium from heavy
water or deuterated glucose. However, interpretation of these data can be complicated. Death
rates of labelled cells may not be representative of the population as a whole; resolving
heterogeneity in turnover rates within populations can be difficult; and the analyses are
typically performed over the time-scales of days or weeks, presenting only snapshots of
homeostatic dynamics. Here we present a novel method of studying the long-term population
dynamics of naive CD4 and CD8 T cells in mice that also provides insight into population-level
heterogeneity within these compartments.
We use the transplant conditioning drug Busulfan to ablate haematopoetic stem cells in mice
but leaving the peripheral lymphocyte compartments intact. We generate chimeras by
reconstituting with congenically labelled (donor) bone marrow and within 6 weeks the
cellularity and total output of thymi in these animals is normal. By following the dilution of
peripheral host-derived by donor-derived lymphocytes for a year post-treatment we estimate
rates of thymic production, division and death of naive CD4 and CD8 T cells. We consider two
(non-exclusive) models: (1) a variant of the canonical birth-death model which allows for
multiple niches. Rather than random replacement at the level of the whole pool, we find stable,
self-renewing populations of host-derived cells that are resistant to displacement by cells
generated post-treatment. We speculate that these cells are established early in life, possibly
conditioned or selected for increased fitness through homeostatic proliferation in the
lymphopenic neonatal environment. (2) A model in which cells become less susceptible to loss
with time since export from the thymus. Both the host-age (incumbent/displaceable) and cellage (structured population) models are able to describe the data and give similar results for the
kinetic parameters; however we find significantly greater support for the first model. Finally,
our analyses show that, heterogeneity aside, the long-term homestatic dynamics of naive CD4
and CD8 T cells can be described with simple birth-death models, without a need to invoke
density-dependent regulation of rates of division or loss.
Henk-Jan van den Ham
Erasmus MC
Helper T cell differentiation: feedback-driven selection of appropriate immune phenotypes
Henk-Jan van den Ham, Arno C. Andeweg, Rob J. de Boer
Helper T cells are important regulators of the immune system. By the production of a range of
cytokines that are linked to different cellular Th phenotypes, Th cells determine the type of
immune response that is raised against an invading pathogen. By forming memory cells, Th cells
retain a record of both the infectious agent and the type of host response that was raised to
contain it.
The regulation of Th phenotypes has been studied extensively using mathematical models,
which have explored both the role of T cell specificity for antigen, and regulatory mechanisms
including autocrine cytokine signalling and cross-inhibition between self-activating
transcription factors. These choices are made at the single cell level, because cells tend to have a
unique antigen receptor and are exposed to a unique environment. Conversely, the collective of
the cells is important because of the high levels of stochasticity that occur at the single cell level.
This differentiation process is therefore a model for cellular decision making that allows the
immune system choose the appropriate phenotype for a particular challenge.
Sébastien Jaeger
CIML
A dynamical model of TCRβ gene rearrangement: Assessing stochasticity in the initiation of
V(D)J recombination and allelic exclusion.
During T lymphocyte development, V(D)J recombination at the TCRβ locus typically yields a
functional, in-frame rearrangement at only one of the gene's two TCRβ alleles. This
phenomenon of allelic exclusion utilizes feedback inhibition in order to interrupt the
recombination process once a primary VDJ+ is expressed. It also requires asynchronous allele
assembly, for which putative mechanisms are still debated. We devised a model that tracks the
evolution of TCRβ rearrangement dynamics at dual alleles from the stochastic onset of the Dβto-Jβ recombination initiating phase onwards, and thus accounted for the genotypic profiles
typically associated with TCRβ allelic exclusion in an emerging population of differentiated T
cells. Disturbances in the dynamics of recombinational activation at an individual allele have
limited consequences on the incidence of allelically-included cells, a robust feature of the system
that is underscored by our simulations.
Overall, these in silico surveys predict biological systems that would exploit cell-to-cell
stochastic variability to both curtail the unavoidable production of allelically-included cells as
well as to optimize the emergence of their allellicaly-excluded relevant counterparts, at a low
energy cost (compared with additional, currently proposed deterministic regulatory
mechanisms).
Can Kesmir
Utrecht University
The cytotoxic T cell (CTL) response is determined by the peptide repertoire presented by the
HLA class I molecules of an individual. To compare features of peptide repertoires associated
with different HLA class I loci we performed an in-depth analysis of the immunopeptidomes of a
common HLA class I molecules on four B lymphoblastoid cell-lines (BLCL). Peptide elution and
mass spectrometry analysis were utilised to investigate the number, abundance and source of
peptides presented under steady state conditions and viral infection.
Altogether, 7902 unique self peptides, derived of 4354 proteins, were eluted. After viral
infection, the number of eluted unique self peptides significantly decreased compared to
uninfected cells, which was paralleled by a decrease in the number of source proteins. The
number of unique self peptides eluted from HLA-B molecules was larger than from HLA-A
molecules, and they were derived from a larger number of source proteins. HLA-B molecules did
not have a preference to present viral peptides over self peptides.
Our results suggest that HLA-B molecules present a more diverse repertoire compared to their
HLA-A counterparts, which may contribute to their immunodominance. This study provides a
comprehensive and unique data set giving new insights into the complex system of antigen
presentation for a broad panel of HLA molecules, certain of which were never studied as
extensively before.
Trine Ahn Kristiansen
Lund University
Resolving B1a and B2 B cell development by single cell fate mapping
Trine A Kristiansen, Shamit Soneji, David Bryder, Joan Yuan.
Division of Molecular Hematology, Faculty of Medicine, Lund University, Sweden.
We aim to understand how the unique differentiation potential of fetal hematopoietic stem and
progenitor cells (HSPCs) contribute to functionally distinct cell types of the adult immune
system. This phenomenon is clearly exemplified in the B cell lineage where innate-like B1a B
cells emerge during a limited window early in life, while adult HSPCs preferentially generate
follicular B2 B cells. We previously identified the fetal specific RNA binding protein Lin28b as a
‘molecular switch’ capable of reinitiating fetal-like lymphoid potential in adult HSPCs (Yuan et
al, Science, 2012), including the generation of B1a cells. While it is clear that cell intrinsic
features contribute to the apparent difference in differentiation potential, it remains unclear if
B1a and follicular B2 B cells share a common hematopoietic progenitor or if they originate from
distinct hematopoietic lineages. The ability to trace single cell fates in vivo is required to truly
distinguish between these non-mutually exclusive models. To this end, we exploit the
advantages of cellular barcoding to resolve the 25-year-old question surrounding the lineage
relationship between B1a and B2 B cells. These studies will shine new light on fetal
lymphopoiesis and generate fundamental insight into the formation of our complex adaptive
immune system.
Rémi Lasserre
CIML
T cells are central in adaptive immune response. Their activation is initiated in vivo by series of
transient interactions with antigen presenting cells (APCs). To date, the mechanisms allowing T
cell to integrate (or sum) sequential and transient signals is ill understood. This research project
aims at elucidating the molecular bases of this process. We hypothesize that the spatiotemporal
regulation of signaling molecules activity allows T cell to maintain an imprint of the successive
stimulations it perceives.
To explore this process, we are developing a new experimental procedure combining
microfluidics and biophotonics that allows the 4D analysis of individual T cell activation. It is
based on the confinement of T cell activation in microwells containing a collagen I lattice that
mimic the 3D physical constraints a T cell encounters in vivo. Combined with the use of
fluorescent probes allowing to measure signaling molecules activity, this interdisciplinary
program will provide for the first time a description of the whole history of individual T cell
interactions with APCs and the associated signaling events during the activation process. It will
unravel new molecular mechanisms underlying activation signal integration in T cells, as well as
their influence on activation outcomes.
Dawn Lin
The Walter and Eliza Hall Institute
Despite extensive studies on haematopoiesis, understanding of how a single haematopoieitc
stem cell gives rise to the entire system consisting of phenotypical and functional distinct
lineages remains unclear. This is largely due to the heterogeneity of stem and progenitor cell
population. Single cells not only differ in fate commitment but also timing of such decision.
Therefore, investigating how and when fate decision occurs will gain valuable insights into the
complexity and dynamics of haematopoiesis. This project will utilize two novel technologies,
namely cellular barcoding and long-term live cell microscopy, to track haematopoietic cell
development at the single cell level.Preliminary barcoding results revealed that increased clonal
expansion, recruitment of dormant progenitors and lineage divergence could be the potential
explanation.Preliminary long-term imaging results revealed several common or distinct
properties both between and within clones. Importantly, possible asymmetric divisions of fate
were observed at different developmental stages in a single pedigree, which could ultimately be
a source of heterogeneity and diversity.
Edward Lee
A novel computational approach to measuring T cell diversity
Diversity in the T cell receptor repertoire allows the adaptive immune system to respond with
specificity to nearly any foreign antigen. Characterizing T cell diversity within repertoires or in
immune responses to pathogens and self-antigens can inform clinical treatment and diagnostic
procedures. Measuring TCRa and TCRb chain pairings is essential for fully assessing TCR
diversity, but using single-cell sequencing to identify large numbers of clones is costly and risks
undersampling rare TCRs. We present efficient algorithms for pairing TCRa and TCRb chains
and estimating their relative abundances that uses bulk sequencing of cell samples, which are
scalable and able to pair clones with dual TCRs, which comprise up as much as 10% of mature T
cells.
Judith Mandl
McGill University
T cells achieve both exquisite specificity and broad coverage for possible antigens by expressing
αβ T cell receptors (TCR) that are generated by a diversification process termed somatic
recombination. Using a dynamic surface marker (CD5) that tracks with signal intensity obtained
through the TCR from interactions with self-peptide MHC, we have previously shown that naïve
CD4+ T cells are heterogeneous in their reactivity for self-peptide MHC and that there is a direct
relationship between strength of self and foreign peptide-MHC binding. Using TCR sequencing,
we investigated the hypothesis that there are fundamental differences between TCRs from CD5hi compared to CD5-lo CD4 T cells that account for distinct peptide-MHC binding strengths. This
work may shed light on features of the architecture of a TCR repertoire that ensure that it is
effective in control of outgrowth of pathogens.
Lindsay Moore
Technion Medical School
Modeling dynamics of the NK cell repertoire
The development of mass cytometry (CYTOF) to study the cells of the immune system has
yielded a new class of experimental data, in which 40 distinct cell markers can be measured for
each single cell. Recent studies using this technique have revealed a surprising diversity of NK
cells, contrary to the previous view of a small number of NK cell types (Horowitz et al. 2013).
This diversity has implications to both the function of individual cells and the whole organism
phenotype, and is critical for the performance of the immune system. Maintenance of this
diversity depends on a combination of stochastic and deterministic factors in single cell
expression. In homeostasis, NK cells in the blood have a half-life of approximately 2 weeks in
humans, but at the system level the diversity needed to mount an effective immune response is
maintained. We are using computational approaches to analyze the NK cell repertoire in
humans and mice to build a single-cell model for this measured diversity.
Ioana Niculescu
Utrecht University
Upon primary HSV-1 infection, effector CD8+ T cells enter the skin and travel
chemotactically from dermis to epidermis. Here they search in a tightly packed
environment for infection foci. Which strategies T cells employ to find and contain
these foci is still largely unknown. To elucidate these strategies, we have created
a computational model of HSV-1 infection that includes healthy epidermis, an infection focus,
and T cells that realistically squeeze through epidermal cells, can follow chemotactic gradients,
and employ different killing strategies. This model allows us to explore different combinations
of hypotheses on T cell migration directionality and infection containment mechanisms. By
analyzing several thousands of simulations, we stematically identify efficient strategies for
finding and containing the infection, and compare our findings with the in vivo reported
behavior. Surprisingly, our preliminary
results revealed that in some settings, overly aggressive infiltration of the infection by T cells
can be detrimental, as it permits the infection to spread unrestrained at the
outer border.
Benedict Seddon
University College London
Turnover and heterogeneity in naive T lymphocyte populations in mice
Thea Hogan*, Graeme Gossel*, Andrew J. Yates2, and Benedict Seddon
Abstract
Rates of lymphocyte turnover have been measured using CFSE dilution or the frequency of
uptake of BrDU or deuterium. However, interpretation of these data can be complicated: death
rates of labelled cells may not be representative of the population as a whole; resolving
heterogeneity in turnover rates within populations can be difficult; and the analyses are
typically performed over the timescales of days or weeks, presenting only snapshots of
homeostatic dynamics. Here we present a novel method of studying the population dynamics of
thymus-derived lymphocytes. We use the transplant conditioning drug Busulfan to ablate
haematopoetic stem cells (HSC) in CD45.1 mice and generate chimeras by reconstituting with
CD45.2 (donor) bone marrow. Busulfan treatment leaves the peripheral host-derived
lymphocyte compartments intact and by 6 weeks the cellularity and total output of thymi in
these animals is normal. By following the dilution of peripheral host- derived by donor-derived
lymphocytes for a year post-treatment and describing these kinetics with simple mathematical
models, we estimate rates of production, division and death of naive CD4 and CD8 T cells. We
show that the size and turnover of the naive T cell compartments can be modeled between the
ages 6 weeks and 12 months without invoking any cell density-dependent homeostatic feedback
regulation of division or loss. In addition, rather than random replacement, we find
subpopulations of host-derived cells that are resistant to displacement by cells generated posttreatment. We speculate that these are cells established early in life, conditioned for increased
fitness through homeostatic expansion into the lymphopenic neonatal environment.
Maarten Slagter1, Matthew Hellmann2,3, Alexandra Snyder2,3,4, Joost B. Beltman5# & Ton N.
Schumacher1
Affiliations:
1. Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The
Netherlands
2. Dept. of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
3. Weill Cornell School of Medicine, New York, NY, 10065, USA.
4. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New
York
5. Division of Toxicology, Leiden Academic Center for Drug Research, Leiden University, 2333
CC Leiden, The Netherlands
# These authors contributed equally.
A probabilistic Lottery model explains clinical responses to immune checkpoint blockade in
melanoma and non-small cell lung cancer
The blockade of the inhibitory receptors CTLA-4 and PD1 on T-cells has resulted in profound
clinical responses in several human malignancies. However, robust prognostic biomarkers for
treatment success remain elusive. Two recent studies have demonstrated a significant
correlation between the number of exonic non-synonymous mutations and clinical response to
anti-CTLA4 treatment in melanoma and anti-PD1 treatment in non-small cell lung cancer, a
finding consistent with T-cell recognition of mutated antigens (neo-antigens) as a driving factor
in tumor control. Nevertheless, direct usage of mutational load as a predictive biomarker is
precluded by the large overlap in the distributions of mutational load between patients with
and without a durable clinical benefit. Here we explore whether the observed relationship
between mutational load and clinical response can be explained by a probabilistic model, in
which each mutation has a small probability of yielding a relevant neo-antigen to which the
patient develops a clinically relevant immune response. We formulate a mathematical version of
this 'Lottery'-model, in which individual mutations represent tickets to the jackpot prize of a
DCB, and show that there is excellent congruence between the model and the clinical data. In
addition, we use this framework to provide first estimates on the magnitude of other factors
that control T-cell mediated tumor immunity. The quantitative analysis of the Cancer–Immunity
cycle that we propose here has the potential to help predict the clinical value of different
immunotherapeutic interventions for individual patients.
Gregory Vogel
Utah State University
We created a multi-type branching process model representing the development of the derived
CD4+ T cell lineage after antigenic stimulation. The purpose was to better understand the
stochastic variation associated with each step in the development of immune memory, and to
compare results with traditional deterministic models. We ran simulations tracking the progeny
of a single activated CD4+ T cell throughout an immune response, accounting for the number of
effector and memory cells, as well as each individual cell's mitotic level. We estimated the
memory to effector transition time parameter and conducted sensitivity analysis to understand
the influence of variation in that parameter on simulations. In this report, we provide our
simulation results showing the dynamic
evolution of the T cells generated through an immune response along with overlaid curves
showing the mean and variance of all trials. Finally we calculate the distribution at each time
step of the number of cells in each category. We find that a Weibull distribution provides the
best fit for the distribution of cells and present those results. This analysis upholds observations
of different responses to the same pathogen in different individuals, opening the potential to be
used for improving parameter estimates, and vaccines.
Tom Weber
Maynooth University
Site-specific recombinatorics: Cellular barcoding using the Cre-LoxP system
Cellular barcoding is becoming an increasingly popular tool to analyze the fate of single cells
and their progeny in vivo. Applied to Immunology, this technique facilitates tracking of the
progenitors of multiple clones originating each from a single activated B or T cells over one or
several immune reactions. Current approaches typically rely on ex-vivo transfection and
subsequent adoptive cell transfer of barcoded cells into an animal. This is known to work well
for naive or resting lymphocytes but precludes barcoding of activated cells in their native
environment during an immune reaction. We will present theoretical results on a genetic
construct based on the Cre-LoxP system. Similar to existing constructs that rely on Rag or SB
transposase, it is designed to induce random barcodes in cells in situ through site-specific
recombination. The cassette design is shown to be robust to sequencing errors and optimal in
terms of the number of codes that can be generated. Its potential diversity is predicted to be
several orders of magnitude higher than published barcode libraries.
Joan Yuan
Lund University
We aim to understand how the unique differentiation potential of fetal hematopoietic stem and
progenitor cells (HSPCs) contribute to functionally distinct cell types of the adult immune
system. This phenomenon is clearly exemplified in the B cell lineage where innate-like B1a B
cells emerge during a limited window early in life, while adult HSPCs preferentially generate
follicular B2 B cells. We previously identified the fetal specific RNA binding protein Lin28b as a
‘molecular switch’ capable of reinitiating fetal-like lymphoid potential in adult HSPCs (Yuan et
al, Science, 2012), including the generation of B1a cells. While it is clear that cell intrinsic
features contribute to the apparent difference in differentiation potential, it remains unclear if
B1a and follicular B2 B cells share a common hematopoietic progenitor or if they originate from
distinct hematopoietic lineages. The ability to trace single cell fates in vivo is required to truly
distinguish between these non-mutually exclusive models. To this end, we exploit the
advantages of cellular barcoding to resolve the 25-year-old question surrounding the lineage
relationship between B1a and B2 B cells. These studies will shine new light on fetal
lymphopoiesis and generate fundamental insight into the formation of our complex adaptive
immune system.
Irina Zaretsky
The Weizmann Institute of Science
While the mechanisms leading to differentiation of induced Tregs from naïve T-cells have been
largely elucidated, the temporal dynamics of the process as well as heterogeneity in cellular
response have not been yet characterized. In addition, we aim to better understand the
contributions of cell contact during Treg-mediated suppression.
Monitoring the dynamics of expression of Foxp3, a key transcription factor regulating Treg
differentiation, we found that both its onset time and final expression levels are highly
heterogeneous. As heterogeneity in gene expression timing and levels often result in functional
diversity, the observed variability in Foxp3 expression may represent phenotypic differences
among Tregs. We developed a mathematical model that describes the dynamics of Treg
differentiation process, based on stochastic choices for cellular processes (division, death or
differentiation). This model provides accurate predictions of the system dynamics, and explains
behaviour of the population based on single cells measurements.
Studying suppression mechanisms, we found that contact is a major factor in Treg-mediated
suppression. Strikingly, Tregs also maintain significant ability to inhibit effector T cells at short
distances without contact.