Understanding T-cell receptor (TCR) specificity is not only essential for fundamental research, but could open up novel avenues for diagnostics, cancer immunotherapy, and the targeted treatment of autoimmune diseases. The immune system responds to challenges through groups of T-cells with similar TCR sequences. In recent years, searching for TCRs with an enrichment of similar sequences - neighbors - in a TCR repertoire has become a standard procedure for antigen-specific TCR identification. This study provides a systematic comparison of computational algorithms-ALICE, TCRNET, GLIPH2, and tcrdist3-that leverage neighborhood enrichment for antigen-specific TCR identification. Using published murine datasets from Lymphocytic choriomeningitis virus (LCMV) infection and novel datasets from Sputnik V vaccination and Mycobacterium tuberculosis (Mtb) infection, we evaluated the performance of these algorithms. To facilitate reproducible analysis, we developed TCRgrapher, an R library that integrates these pipelines into a user-friendly framework. TCRgrapher enables efficient identification of antigen-specific TCRs from single repertoire snapshots and supports flexible parameter customization. Our comparative analysis revealed that ALICE and TCRNET consistently outperformed GLIPH2 and tcrdist3 across most datasets, achieving higher area under precision-recall curve. While murine datasets provide valuable insights into algorithm performance, caution is advised when extrapolating these results to other species or different experimental conditions. TCRgrapher is freely available on GitHub (https://github.com/KseniaMIPT/tcrgrapher), offering researchers a robust tool for investigating TCR specificity and advancing immunological studies.

• Keywords
• TCR repertoire, TCR specificity, immunoinformatics, software,
• MeSH
• Algorithms * MeSH
• Antigens * immunology   MeSH
• Humans MeSH
• Mycobacterium tuberculosis immunology   MeSH
• Mice MeSH
• Receptors, Antigen, T-Cell * immunology   genetics   MeSH
• Lymphocytic choriomeningitis virus immunology   MeSH
• Computational Biology * methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens * MeSH
• Receptors, Antigen, T-Cell * MeSH

INTRODUCTION: The functional programs of CD4+ T helper (Th) cell clones play a central role in shaping immune responses to different challenges. While advances in single-cell RNA sequencing (scRNA-Seq) have significantly improved our understanding of the diversity of Th cells, the relationship between scRNA-Seq clusters and the traditionally characterized Th subsets remains ambiguous. METHODS: In this study, we introduce TCR-Track, a method leveraging immune repertoire data to map phenotypically sorted Th subsets onto scRNA-Seq profiles. RESULTS AND DISCUSSION: This approach accurately positions the Th1, Th1-17, Th17, Th22, Th2a, Th2, T follicular helper (Tfh), and regulatory T-cell (Treg) subsets, outperforming mapping based on CITE-Seq. Remarkably, the mapping is tightly focused on specific scRNA-Seq clusters, despite 4-year interval between subset sorting and the effector CD4+ scRNA-Seq experiment. These findings highlight the intrinsic program stability of Th clones circulating in peripheral blood. Repertoire overlap analysis at the scRNA-Seq level confirms that the circulating Th1, Th2, Th2a, Th17, Th22, and Treg subsets are clonally independent. However, a significant clonal overlap between the Th1 and cytotoxic CD4+ T-cell clusters suggests that cytotoxic CD4+ T cells differentiate from Th1 clones. In addition, this study resolves a longstanding ambiguity: we demonstrate that, while CCR10+ Th cells align with a specific Th22 scRNA-Seq cluster, CCR10-CCR6+CXCR3-CCR4+ cells, typically classified as Th17, represent a mixture of bona fide Th17 cells and clonally unrelated CCR10low Th22 cells. The clear distinction between the Th17 and Th22 subsets should influence the development of vaccine- and T-cell-based therapies. Furthermore, we show that severe acute SARS-CoV-2 infection induces systemic type 1 interferon (IFN) activation of naive Th cells. An increased proportion of effector IFN-induced Th cells is associated with a moderate course of the disease but remains low in critical COVID-19 cases. Using integrated scRNA-Seq, TCR-Track, and CITE-Seq data from 122 donors, we provide a comprehensive Th scRNA-Seq reference that should facilitate further investigation of Th subsets in fundamental and clinical studies.

• Keywords
• T cell memory, Th17, Th22, cytotoxic CD4+ T cells, helper T cell subsets, immune repertoires, scRNA-Seq, scTCR-seq,
• MeSH
• Single-Cell Gene Expression Analysis MeSH
• Single-Cell Analysis * methods   MeSH
• Humans MeSH
• Sequence Analysis, RNA MeSH
• RNA-Seq * methods   MeSH
• T-Lymphocyte Subsets * immunology   MeSH
• T-Lymphocytes, Helper-Inducer * immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Suppressive function of regulatory T cells (Treg) is dependent on signaling of their antigen receptors triggered by cognate self, dietary, or microbial peptides presented on MHC II. However, it remains largely unknown whether distinct or shared repertoires of Treg TCRs are mobilized in response to different challenges in the same tissue or the same challenge in different tissues. Here we use a fixed TCRβ chain FoxP3-GFP mouse model to analyze conventional (eCD4) and regulatory (eTreg) effector TCRα repertoires in response to six distinct antigenic challenges to the lung and skin. This model shows highly 'digital' repertoire behavior with easy-to-track challenge-specific TCRα CDR3 clusters. For both eCD4 and eTreg subsets, we observe challenge-specific clonal expansions yielding homologous TCRα clusters within and across animals and exposure sites, which are also reflected in the draining lymph nodes but not systemically. Some CDR3 clusters are shared across cancer challenges, suggesting a response to common tumor-associated antigens. For most challenges, eCD4 and eTreg clonal response does not overlap. Such overlap is exclusively observed at the sites of certain tumor challenges, and not systematically, suggesting transient and local tumor-induced eCD4=>eTreg plasticity. This transition includes a dominant tumor-responding eCD4 CDR3 motif, as well as characteristic iNKT TCRα CDR3. In addition, we examine the homeostatic tissue residency of clonal eTreg populations by excluding the site of challenge from our analysis. We demonstrate that distinct CDR3 motifs are characteristic of eTreg cells residing in particular lymphatic tissues, regardless of the challenge. This observation reveals the tissue-resident, antigen-specific clonal Treg populations.

• Keywords
• CD4+ T cells, TCR repertoire, antigenic specificity, homing, immunology, inflammation, lung challenges, mouse, treg,
• MeSH
• Clone Cells MeSH
• CD4-Positive T-Lymphocytes * MeSH
• Mice MeSH
• Peptides MeSH
• Receptors, Antigen, T-Cell genetics   MeSH
• T-Lymphocytes, Regulatory * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides MeSH
• Receptors, Antigen, T-Cell MeSH

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones-a unique organizational strategy that potentially underlies this animal's extraordinary longevity and healthy aging.

• MeSH
• Adaptive Immunity MeSH
• Immunoglobulins metabolism   MeSH
• Interleukin-7 metabolism   MeSH
• Humans MeSH
• Mole Rats MeSH
• Mice MeSH
• Spalax * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunoglobulins MeSH
• Interleukin-7 MeSH

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 αβ 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.

• Keywords
• CDR3 properties, TCR repertoire, helper CD4+ subsets, human, immunology, inflammation, plasticity of CD4+ subsets,
• MeSH
• Cell Lineage immunology   MeSH
• CD4-Positive T-Lymphocytes immunology   MeSH
• Humans MeSH
• Receptors, Antigen, T-Cell, alpha-beta immunology   MeSH
• T-Lymphocyte Subsets immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, Antigen, T-Cell, alpha-beta MeSH

There is considerable clinical and fundamental value in measuring the clonal heterogeneity of T and B cell expansions in tumors and tumor-associated lymphoid structures-along with the associated heterogeneity of the tumor neoantigen landscape-but such analyses remain challenging to perform. Here, we propose a straightforward approach to analyze the heterogeneity of immune repertoires between different tissue sections in a quantitative and controlled way, based on a beta-binomial noise model trained on control replicates obtained at the level of single-cell suspensions. This approach allows to identify local clonal expansions with high accuracy. We reveal in situ proliferation of clonal T cells in a mouse model of melanoma, and analyze heterogeneity of immunoglobulin repertoires between sections of a metastatically-infiltrated lymph node in human melanoma and primary human colon tumor. On the latter example, we demonstrate the importance of training the noise model on datasets with depth and content that is comparable to the samples being studied. Altogether, we describe here the crucial basic instrumentarium needed to facilitate proper experimental setup planning in the rapidly evolving field of intratumoral immune repertoires, from the wet lab to bioinformatics analysis.

• Keywords
• TCR repertoire, clonal expansions, immunoglobulin repertoire, tumour clonality, tumour heterogeneity,
• Publication type
• Journal Article MeSH

T follicular helper (Tfh) cells are fundamental for B cell selection and antibody maturation in germinal centers. Circulating Tfh (cTfh) cells constitute a minor proportion of the CD4+ T cells in peripheral blood, but their clonotypic relationship to Tfh populations resident in lymph nodes and the extent to which they differ from non-Tfh CD4+ cells have been unclear. Using donor-matched blood and tonsil samples, we investigate T cell receptor (TCR) sharing between tonsillar Tfh cells and peripheral Tfh and non-Tfh cell populations. TCR transcript sequencing reveals considerable clonal overlap between peripheral and tonsillar Tfh cell subsets as well as a clear distinction between Tfh and non-Tfh cells. Furthermore, influenza-specific cTfh cell clones derived from blood can be found in the repertoire of tonsillar Tfh cells. Therefore, human blood samples can be used to gain insight into the specificity of Tfh responses occurring in lymphoid tissues, provided that cTfh subsets are studied.

• Keywords
• T follicular helper cells, TCR repertoire, blood, influenza, tonsil,
• MeSH
• Clone Cells cytology   MeSH
• CD4-Positive T-Lymphocytes immunology   MeSH
• Tissue Donors MeSH
• Adult MeSH
• T Follicular Helper Cells immunology   MeSH
• Hemagglutinin Glycoproteins, Influenza Virus immunology   MeSH
• Palatine Tonsil immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Computer Simulation MeSH
• Lymphocyte Subsets immunology   MeSH
• Receptors, CXCR3 metabolism   MeSH
• Cell Size MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Hemagglutinin Glycoproteins, Influenza Virus MeSH
• Receptors, CXCR3 MeSH

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

• MeSH
• Lymphocyte Activation immunology   MeSH
• Biomarkers MeSH
• Cell Differentiation immunology   MeSH
• CD8-Positive T-Lymphocytes immunology   metabolism   MeSH
• Adult MeSH
• Immunophenotyping MeSH
• Immunologic Memory MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Mice MeSH
• Receptors, CXCR3 metabolism   MeSH
• Aged MeSH
• T-Lymphocyte Subsets immunology   metabolism   MeSH
• Age Factors MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Mice MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Biomarkers MeSH
• CXCR3 protein, human MeSH Browser
• Receptors, CXCR3 MeSH

• MeSH
• Gene Regulatory Networks * MeSH
• RNA * MeSH
• Sequence Analysis, RNA MeSH
• Publication type
• Journal Article MeSH
• Comment MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA * MeSH

Regulatory T (Treg) cells prevent autoimmunity by limiting immune responses and inflammation in the secondary lymphoid organs and nonlymphoid tissues. While unique subsets of Treg cells have been described in some nonlymphoid tissues, their relationship to Treg cells in secondary lymphoid organs and circulation remains unclear. Furthermore, it is possible that Treg cells from similar tissue types share largely similar properties. We have identified a short-lived effector Treg cell subset that expresses the α2 integrin, CD49b, and exhibits a unique tissue distribution, being abundant in peripheral blood, vasculature, skin, and skin-draining lymph nodes, but uncommon in the intestines and in viscera-draining lymph nodes. CD49b+ Treg cells, which display superior functionality revealed by in vitro and in vivo assays, appear to develop after multiple rounds of cell division and TCR-dependent activation. Accordingly, single-cell RNA-seq analysis placed these cells at the apex of the Treg developmental trajectory. These results shed light on the identity and development of a functionally potent subset of mature effector Treg cells that recirculate through and survey peripheral tissues.

• MeSH
• Blood Vessels immunology   MeSH
• Immunologic Surveillance * MeSH
• Integrin alpha2 genetics   immunology   MeSH
• Skin blood supply   cytology   immunology   MeSH
• Lymph Nodes blood supply   cytology   immunology   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• T-Lymphocytes, Regulatory cytology   immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Integrin alpha2 MeSH