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 T-cell 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 mark-recapture 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 ADA-SCID 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 long-term 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 antigen-specific 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 cell-age (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 Dβ-to-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 CD5-hi 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 containthese 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 preliminaryresults 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 theouter 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 post-treatment. 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 Netherlands2. 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 York5. 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.