Memory CD4+ T cells are generated in the human fetal intestine.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

Comparative analysis of murine T-cell receptor repertoires.

For understanding the rules and laws of adaptive immunity, high-throughput profiling of T-cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T-cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V-gene and J-gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.

Human liver infiltrating γδ T cells are composed of clonally expanded circulating and tissue-resident populations.

BACKGROUND & AIMS: γδ T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human γδ T cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of γδ T cells in the human liver. METHODS: We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating γδ T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated Vδ2- γδ subset, which is implicated in liver immunopathology. RESULTS: Intrahepatic Vδ2- γδ T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27lo/- effector lymphocytes, whereas naïve CD27hi, TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RAhi Vδ2- γδ effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vδ2- γδ T cell pool also included a phenotypically distinct CD45RAlo effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vδ2- γδ cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli. CONCLUSION: These findings suggest that the ability of Vδ2- γδ T cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues, such as the liver, which results in functionally distinct peripheral and liver-resident memory γδ T cell subsets. They also highlight the inherent functional plasticity within the Vδ2- γδ T cell compartment and provide information that could be used for the design of cellular therapies that suppress liver inflammation or combat liver cancer. LAY SUMMARY: γδ T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic γδ T cells are enriched for clonally expanded effector T cells, whereas naïve γδ T cells are largely excluded. Moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident γδ T cells was also evident. Our findings suggest that factors triggering γδ T cell clonal selection and differentiation, such as infection, can drive enrichment of γδ T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance.

Dynamics of Individual T Cell Repertoires: From Cord Blood to Centenarians.

The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRß repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.

Naïve Regulatory T Cell Subset Is Altered in X-Linked Agammaglobulinemia.

The interplay between T- and B-cell compartments during naïve, effector and memory T cell maturation is critical for a balanced immune response. Primary B-cell immunodeficiency arising from X-linked agammaglobulinemia (XLA) offers a model to explore B cell impact on T cell subsets, starting from the thymic selection. Here we investigated characteristics of naïve and effector T cell subsets in XLA patients, revealing prominent alterations in the corresponding T-cell receptor (TCR) repertoires. We observed immunosenescence in terms of decreased diversity of naïve CD4+ and CD8+ TCR repertoires in XLA donors. The most substantial alterations were found within naïve CD4+ subsets, and we have investigated these in greater detail. In particular, increased clonality and convergence, along with shorter CDR3 regions, suggested narrower focused antigen-specific maturation of thymus-derived naïve Treg (CD4+CD45RA+CD27+CD25+) in the absence of B cells - normally presenting diverse self and commensal antigens. The naïve Treg proportion among naïve CD4 T cells was decreased in XLA patients, supporting the concept of impaired thymic naïve Treg selection. Furthermore, the naïve Treg subset showed prominent differences at the transcriptome level, including increased expression of genes specific for antigen-presenting and myeloid cells. Altogether, our findings suggest active B cell involvement in CD4 T cell subsets maturation, including B cell-dependent expansion of the naïve Treg TCR repertoire that enables better control of self-reactive T cells.

Functionally specialized human CD4+ T-cell subsets express physicochemically distinct TCRs.

The organizational integrity of the adaptive immune system is determined by functionally discrete subsets of CD4+ T cells, but it has remained unclear to what extent lineage choice is influenced by clonotypically expressed T-cell receptors (TCRs). To address this issue, we used a high-throughput approach to profile the αß TCR repertoires of human naive and effector/memory CD4+ T-cell subsets, irrespective of antigen specificity. Highly conserved physicochemical and recombinatorial features were encoded on a subset-specific basis in the effector/memory compartment. Clonal tracking further identified forbidden and permitted transition pathways, mapping effector/memory subsets related by interconversion or ontogeny. Public sequences were largely confined to particular effector/memory subsets, including regulatory T cells (Tregs), which also displayed hardwired repertoire features in the naive compartment. Accordingly, these cumulative repertoire portraits establish a link between clonotype fate decisions in the complex world of CD4+ T cells and the intrinsic properties of somatically rearranged TCRs.

γδ T-cell Receptors Derived from Breast Cancer-Infiltrating T Lymphocytes Mediate Antitumor Reactivity.

γδ T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating γδ T lymphocytes (γδ TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of γδ TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCRγ or TCRδ chains of γδ TILs and observed a higher proportion of Vδ2+ T cells compared with other tumor types. By reconstructing matched Vδ2- TCRγ and TCRδ pairs derived from single-cell sequencing, our data suggest that γδ TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCRγ and TCRδ chains and was independent of additional costimulation through other innate immune receptors. We conclude that γδ TILs can mediate tumor reactivity through their individual γδ TCR pairs and that engineered T cells expressing TCRγ and δ chains derived from γδ TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies.

The Changing Landscape of Naive T Cell Receptor Repertoire With Human Aging.

Human aging is associated with a profound loss of thymus productivity, yet naïve T lymphocytes still maintain their numbers by division in the periphery for many years. The extent of such proliferation may depend on the cytokine environment, including IL-7 and T-cell receptor (TCR) "tonic" signaling mediated by self pMHCs recognition. Additionally, intrinsic properties of distinct subpopulations of naïve T cells could influence the overall dynamics of aging-related changes within the naïve T cell compartment. Here, we investigated the differences in the architecture of TCR beta repertoires for naïve CD4, naïve CD8, naïve CD4+CD25-CD31+ (enriched with recent thymic emigrants, RTE), and mature naïve CD4+CD25-CD31- peripheral blood subsets between young and middle-age/old healthy individuals. In addition to observing the accumulation of clonal expansions (as was shown previously), we reveal several notable changes in the characteristics of T cell repertoire. We observed significant decrease of CDR3 length, NDN insert, and number of non-template added N nucleotides within TCR beta CDR3 with aging, together with a prominent change of physicochemical properties of the central part of CDR3 loop. These changes were similar across CD4, CD8, RTE-enriched, and mature CD4 subsets of naïve T cells, with minimal or no difference observed between the latter two subsets for individuals of the same age group. We also observed an increase in "publicity" (fraction of shared clonotypes) of CD4, but not CD8 naïve T cell repertoires. We propose several explanations for these phenomena built upon previous studies of naïve T-cell homeostasis, and call for further studies of the mechanisms causing the observed changes and of consequences of these changes in respect of the possible holes formed in the landscape of naïve T cell TCR repertoire.

The human Vδ2+ T-cell compartment comprises distinct innate-like Vγ9+ and adaptive Vγ9- subsets.

Vδ2+ T cells form the predominant human γδ T-cell population in peripheral blood and mediate T-cell receptor (TCR)-dependent anti-microbial and anti-tumour immunity. Here we show that the Vδ2+ compartment comprises both innate-like and adaptive subsets. Vγ9+ Vδ2+ T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9- Vδ2+ T-cell subset that typically has a CD27hiCCR7+CD28+IL-7Rα+ naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27loCD45RA+CX3CR1+granzymeA/B+ effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9- Vδ2+ T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2+ T-cell compartment into innate-like (Vγ9+) and adaptive (Vγ9-) subsets, which have distinct functions in microbial immunosurveillance.

Clonal selection in the human Vδ1 T cell repertoire indicates γδ TCR-dependent adaptive immune surveillance.

γδ T cells are considered to be innate-like lymphocytes that respond rapidly to stress without clonal selection and differentiation. Here we use next-generation sequencing to probe how this paradigm relates to human Vδ2neg T cells, implicated in responses to viral infection and cancer. The prevalent Vδ1 T cell receptor (TCR) repertoire is private and initially unfocused in cord blood, typically becoming strongly focused on a few high-frequency clonotypes by adulthood. Clonal expansions have differentiated from a naive to effector phenotype associated with CD27 downregulation, retaining proliferative capacity and TCR sensitivity, displaying increased cytotoxic markers and altered homing capabilities, and remaining relatively stable over time. Contrastingly, Vδ2+ T cells express semi-invariant TCRs, which are present at birth and shared between individuals. Human Vδ1+ T cells have therefore evolved a distinct biology from the Vδ2+ subset, involving a central, personalized role for the γδ TCR in directing a highly adaptive yet unconventional form of immune surveillance.