Boris Grinshpun

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

Mechanisms 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.

Long-term maintenance of human naïve T cells through in situ homeostasis in lymphoid tissue sites

Human naïve T cells are maintained in lymph nodes for decades and clonally expand in situ after cessation of thymopoiesis. T cell life doesn’t end at 40 Naïve T cells develop in the thymus. Although thymic function declines with age, T cells are persistent throughout the human life span. Thome et al. examined human lymphoid tissues from donors ranging from 2 months to 73 years in age. They found that, although the number of double-positive thymocytes and recent thymic emigrants dropped in individuals >40 years of age, naïve T cells were functionally maintained in the lymph nodes. There was minimal overlap in clonotype between the lymph tissues, suggesting that lymph nodes may maintain a diverse set of T cell specificities. These data suggest that location really does matter—tissue compartmentalization and homeostasis are critical for maintaining naïve T cells throughout the human life span. Naïve T cells develop in the thymus and coordinate immune responses to new antigens; however, mechanisms for their long-term persistence over the human life span remain undefined. We investigated human naïve T cell development and maintenance in primary and secondary lymphoid tissues obtained from individual organ donors aged 2 months to 73 years. In the thymus, the frequency of double-positive thymocytes declined sharply in donors >40 years of age, coincident with reduced recent thymic emigrants in lymphoid tissues, whereas naïve T cells were functionally maintained predominantly in lymph nodes (LNs). Analysis of T cell receptor clonal distribution by CDR3 sequencing of naïve CD4+ and CD8+ T cells in spleen and LNs reveals site-specific clonal expansions of naïve T cells from individuals >40 years of age, with minimal clonal overlap between lymphoid tissues. We also identified biased naïve T cell clonal distribution within specific LNs on the basis of VJ usage. Together, these results suggest prolonged maintenance of naïve T cells through in situ homeostasis and retention in lymphoid tissue.

Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire

Significance High-throughput sequencing of T-cell receptor (TCR) repertoires provides a high-dimensional biomarker for monitoring the immune system. We applied this approach, measuring the extent to which the TCR repertoires of T-cell populations infiltrating malignant brain tumors diverge from their peripheral blood. Our analytical strategy separates the statistical properties of the repertoire derived from VJ cassette combination usage from the VJ-independent contribution that reflects the antigen-binding component of the receptor. We discovered a TCR signature strongly inversely correlated with the VJ-independent divergence between the peripheral and tissue-infiltrating repertoires of these patients. Importantly, this signature is detectable in peripheral blood and could serve as a means of noninvasively monitoring immune response in patients. Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a “signature” set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy.

Statistical method for revealing form-function relations in biological networks

Over the past decade, a number of researchers in systems biology have sought to relate the function of biological systems to their network-level descriptions—lists of the most important players and the pairwise interactions between them. Both for large networks (in which statistical analysis is often framed in terms of the abundance of repeated small subgraphs) and for small networks which can be analyzed in greater detail (or even synthesized in vivo and subjected to experiment), revealing the relationship between the topology of small subgraphs and their biological function has been a central goal. We here seek to pose this revelation as a statistical task, illustrated using a particular setup which has been constructed experimentally and for which parameterized models of transcriptional regulation have been studied extensively. The question “how does function follow form” is here mathematized by identifying which topological attributes correlate with the diverse possible information-processing tasks which a transcriptional regulatory network can realize. The resulting method reveals one form-function relationship which had earlier been predicted based on analytic results, and reveals a second for which we can provide an analytic interpretation. Resulting source code is distributed via http://formfunction.sourceforge.net.

Analyzing T cell repertoire diversity by high-throughput sequencing

Diversity on a large scale is one of the most striking and powerful features utilized by the mammalian immune system to fight off a vast universe of pathogens. The T-cell driven immune response is characterized by a multitude of distinct receptors capable of antigen recognition with high specificity. Using high-throughput sequencing we are able to investigate the T cell receptor (TCR) repertoire as the collection of its individual receptors, aiming to profile the global properties of the complementarily determining region 3 (CDR3) of the TCR in human immunity. However, analysis of the data is highly sensitive to single nucleotide polymoprhisms, read length and error rate, accuracy in mapping to a genomic reference, and our ability to translate the sequence, in silico, in the appropriate reading frame. We have developed a computational pipeline that performs error correction on overlapping paired-end long (250 nt) reads, and maps the reads unambiguously to V and J cassettes corresponding to TCR-α and -β chains. Our methods were applied to functional T cell receptors from healthy blood tissue and to several patients with low grade glioma (LGG) and glioblastoma multiforme (GBM). We used Shannon entropy to measure levels of diversity in the productive T cell repertoire and observed that greater than 50% of the TCR diversity can be explained by V, J cassette usage.

Quantifying size and diversity of the human T cell alloresponse

Alloreactive T lymphocytes are the primary mediators of immune responses in transplantation, both in the graft-versus-host and host-versus-graft directions. While essentially all clones comprising the human T cell repertoire have been selected on self-peptide presented by self-human leukocyte antigens (self-HLAs), much remains to be understood about the nature of clones capable of responding to allo-HLA molecules. Quantitative tools to study these cells are critical to understand fundamental features of this important response; however, the large size and diversity of the alloreactive T cell repertoire in humans presents a great technical challenge. We have developed a high-throughput T cell receptor (TCR) sequencing approach to characterize the human alloresponse. We present a statistical method to model T cell clonal frequency distribution and quantify repertoire diversity. Using these approaches, we measured the diversity and frequency of distinct alloreactive CD4+ and CD8+ T cell populations in HLA-mismatched responder-stimulator pairs. Our findings indicate that the alloimmune repertoire is highly specific for a given pair of individuals, that most alloreactive clones circulate at low frequencies, and that a high proportion of TCRs is likely able to recognize alloantigens.

Precision immunophenotyping by high-throughput TCR sequencing in human glioma

Meeting abstracts Immunotherapy for glioblastoma (GBM) is the subject of numerous clinical trials, given the potential for the adaptive immune response to combat this diffusely infiltrating tumor. However, rational application of immunotherapy to these tumors is challenging because of the peculiar

TCR repertoire divergence reflects micro-environmental immune phenotypes in glioma

Meeting abstracts 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

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.