Quantitative approaches for profiling the T cell receptor repertoire in human tissues

The study of B and T cell receptor repertoires from high throughput sequencing is a recent development that allows for unprecedented resolution and quantification of the adaptive immune response. The immense diversity and long tailed distribution of these repertoires has up until now limited such studies to expanded clonal signatures or to analysis of imprecise signals with limited dynamic range collected by techniques such as radioactive and fluorescent labeling. This thesis presents a number of quantitative methods to characterize the repertoire and examine the questions of sequence diversity and inter-repertoire divergence of T cell repertoires. These approaches attempt to accurately parametrize the inherent distribution of T cell clones drawing from statistical tools derived from ecological literature and information theory. The methods presented are applied to T cell analyses of various tissue compartments of the human body, including peripheral blood mononucleocytes, thymic tissues, spleen, inguinal lymph nodes, lung lymph nodes and the brain. A number of applications are explored with strong implications for translational use in medicine. Novel insights are made into the mechanism of maintenance and compartmentalization of na{\"i}ve T cells from human donors of many different ages. Diversity and divergence of the tumor infiltrating sequence repertoire is measured in low grade gliomas and glioblastomas from cancer patients, and potential sequence based biomarkers are assessed for studying glioma phenotype progression. A careful investigation of the immune response to allogeneic stimulus reveals the effect of HLA on sequence sharing and diversity of the alloresponse, and quantifies for the first time using sequence data the fraction of T cells in a repertoire that are alloreactive. The use of repertoire sequencing and mathematical models within immunology is a new and emerging concept within the rapidly expanding field of systems immunology and will undoubtedly have a profound impact on the future of immunology research. It is hoped that the tools presented in this thesis will give insight into how to quantitatively explore the breadth and depth of the T cell receptor repertoire, and provide future directions for TCR repertoire analysis

Tumor-associated T cell receptor repertoires in low- and high-grade gliomas

Glioblastoma (GBM) remains prognostically dismal, with care centered on resection, motivating research into novel therapies. Although inducing anti-tumor immunity remains an attractive target for therapeutic and preventative intervention, the interplay between evolving dysregulation of the glioma microenvironment and T cell inefficacy remains poorly understood. In our murine model of proneural glioma, retroviral delivery of PDGF and cre-mediated knockout of PTEN in glial progenitors of adult C57BL/6 gives rise to slow-growing tumors, which were harvested at early- mid- and late-stage progression timepoints following induction, along with peripheral blood. From human patients, tissue from low- and high-grade glioma resections and corresponding peripheral lymphocytes were cryofrozen during surgery at New York Presbyterian-CUMC. For both species, we employed a commercially available primer set (iRepertoire) for nested PCR of the complementarity-determining region 3 (CDR3) of the TCR-alpha and TCR-beta chains from the T cell RNA, followed by next-generation sequencing on an Illumina MiSeq. We developed a computational pipeline for mapping TCR cassettes, in silico translation, pairwise analysis of tissue/periphery per subject, and error analysis. In the murine model, we observe that at late-stage, the intratumoral TCR repertoire diverges significantly from the peripheral, including dramatic expansion of single tumor-associated CDR3s, while the peripheral repertoire itself diverges from those of healthy mice. In both human patients and mice, we observed tumor-associated CDR3s, disproportionately abundant in tumor tissue compared to the corresponding peripheral blood, at both the amino acid and nucleotide level. In human samples we observed tumor-specific TCR expansions that were associated with particular functional subsets (CD8+, CD4+, Treg, NKT). Sequence-level study of the TCR repertoire promises new insight into the scope of glioma immunosuppression, especially systemic effects which remain elusive and the origins of intratumoral suppressive populations, and holds the potential for immunotherapeutic interventions, non-invasive diagnostics, and direct assessment of global responses to immunotherapy

Spatial Map of Human T Cell Compartmentalization and Maintenance over Decades of Life

SummaryMechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid, and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naive, central, and effector memory, and terminal effector subsets is contingent on both their differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is different in CD4+ or CD8+ T cell lineages, varies with their differentiation stage and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting distinct pathways for human T cell fate determination and homeostasis

TCR repertoire divergence reflects micro-environmental immune phenotypes in glioma

Background & significance: Glioblastoma (GBM) remains prognostically dismal, with only modest gains in mean survival time with chemo- and radiotherapy motivating research into reversing its characteristic local and systemic immunosuppression with precision in this high-risk tissue. While whole-repertoire amplification of the TCR repertoire allows unprecedented depth regarding the potentiation of anti-tumor responses, most studies utilize TCRseq for monitoring reactivity to specific tumor antigens, or the identities of particular TCRs as biomarkers. In this study, we have utilized whole-repertoire analysis to describe the relationship between intra-tumoral T cells and peripheral circulation, and leverage mutual information between gene expression and the behavior of the T cell population to characterize glioma-reactive states, driven by the gene expression of the principal resident monocyte population, and perturbable by immunological interventions. Methods & results: From resected tumor tissue and peripheral lymphocytes of low- and high-grade human glioma patients, TCRseq libraries were generated using reverse transcription and nested PCR (iRepertoire 1) of the complementarity-determining region 3 (CDR3) of the TCR-alpha and TCR-beta chains, then sequenced on an Illumina MiSeq. We developed a computational pipeline for mapping TCR cassettes, in silico translation, and sequence error correction from these libraries, enabling sensitive calculation of tumor-infiltrating lymphocyte (TIL) and peripheral TCR diversity (Shannon entropy) 2, as well as the divergence (Jensen-Shannon divergence metric) between the two T cell populations. By integrating amino acid identity and V-J cassette combination, we observed varying levels of divergence between the TIL and peripheral lymphocytes of glioma patients, and changes in this divergence over tumor progression in a PDGF-driven murine model. Correlation of these properties with tumor tissue RNA profiling, by differential gene expression and mutual-information gene ontology, revealed an association between tumor growth and high blood-brain TCR divergence - particularly in amino-acid sequence, suggesting antigen-driven selection - while high expression of inflammatory and certain immune pathway markers computationally attributed to microglia 3 were anti-correlated with divergence. Preliminary murine experiments suggest that TCR divergence can be altered by induction and blockade of cytokine-mediated activation of these pathways. Conclusion: The expression of a subset of microglia-associated genes appears to describe micro-environmental states which are strongly tied to the tumor-specificity of the intra-tumoral TCR repertoire, complementary to the tumor-centric classifications of TCGA. TCRseq-based profiling not only promises to inform tailoring of local and systemic immunotherapy to target the most relevant immunosuppressive mechanisms, but may also provide non-invasive assessment of the intra-tumoral environment for refined diagnosis and monitoring during clinical trials