Poster Abstracts

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.

VeitBucholz

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: JörgKirberg 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

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.