Biology subjects

Britanova, O. V.

Publications and source records attributed to Britanova, O. V..

7 recordsLinked to original sources

bioRxiv2025

BCR repertoire analysis and cloning of antibody candidates targeting native and Asp7-isomerized beta-amyloid

Computational approaches are increasingly used to predict monoclonal antibody (mAb) candidates from BCR-seq datasets. However, the reliable identification of B cells encoding antibodies against rare antigenic epitopes remains challenging. We employed a repertoire-guided workflow combining antigen-tetramer sorting of B cells from immunized mice, followed by low-input bulk BCR-seq yielding informative clonal repertoires. Clustering and supporting somatic hypermutation (SHM) lineage analysis allowed us to identify IGH and IGK clonotypes potentially targeting {beta}-amyloid and its isoAsp7 variant (isoD7-A{beta}1-16), implicated in Alzheimers disease. We observed recurrent IGHV8-12 and IGKV1-117 usage, consistent with canonical mouse anti-A{beta} responses. Focusing on isoD7-A{beta} binders, we selected ten candidate IGH-IGK pairs for recombinant expression. One recombinant mAb demonstrated preferential binding to isoD7-A{beta} by microscale thermophoresis, supporting the feasibility of the approach but underscoring the challenge of accurate chain pairing. This highlights the potential of bioinformatic workflows to identify mAbs even under low-input conditions.

immunology↗

bioRxiv2025

Discovery of rare antigen-specific TCRs via replicate profiling

Development of effective vaccines and targeted immunotherapies for cancer, autoimmunity, allergy, and infectious diseases requires comprehensive understanding of functionality and antigenic specificity of involved T cell clones. A major technical challenge remains the high-throughput identification of antigen-specific T cells. Here, we present a rapid cost-efficient TCR discovery assay starting from PBMC that enables ultra-sensitive discovery of clonal alpha-beta paired TCRs responding to individual or pooled peptides. In a small-scale experiment with a single donor, assay identified over 90 SARS-CoV-2-specific CD4+ and CD8+ TCR{beta} clonotypes, validated by clonal tracking and comparison against known SARS-CoV-2-specific TCRs. Positioning within the scRNA-Seq map revealed distinct helper T cell subsets involved in primary and secondary response. Further validation in a cohort of five donors identified nearly 1,000 CD4+ and CD8+ TCR clonotypes specific to viral and fungal peptide antigens. The assay demonstrated exceptional sensitivity in capturing low-frequency clones and allowed accurate TCR/TCR{beta} pairing, validated using single-cell transcriptomics. The ability to capture low-frequency antigen-specific TCRs, combined with detailed scRNA-Seq annotation, establishes an integrated pipeline that links antigen-responsive clones to their precise functional phenotypes. This platform provides a robust foundation for dissecting T cell roles in health and disease and accelerates the development of vaccines and immunotherapies.

immunology↗

bioRxiv2024

Tracking antigen-specific T cell response to cancer immunotherapy

In each human or model animal, T cell receptor (TCR) of each effector/memory T cell clone recognizes from one to several cognate peptide-MHC complexes (pMHC). Limited knowledge on TCR repertoire specificities restricts our capacity to rationally interpret this information, both diagnostically and in preclinical research. Here we 1) develop cost-efficient wet lab and computational pipeline to identify mouse TCRs specific to particular peptides, 2) produce dataset of helper T cells (Th) TCR beta CDR3s specific to B16 melanoma neoantigens in the I-Ab pMHCII context, available in VDJdb, and 3) apply this dataset to track tumor-specific T cell response to the CTLA4 blocking immunotherapy on orthotopic B16 melanoma model. We show that tumor promotes clonally independent Tregs carrying tumor-specific TCRs characteristic for Th cells. We further show that CTLA4 blockade promotes Th clonal expansion and induces general, non-tumor-specific Th-to-Treg plasticity. Altogether, we provide a universal pipeline for the investigation of mouse T cell responses at the antigen-specific level, facilitating development and validation of immunotherapeutic and vaccination approaches.

immunology↗

bioRxiv2023

Sort-Seq: immune repertoire-based scRNA-Seq systematization

The functional programs selected by CD4+ helper (Th) T cell clones fundamentally determine the architecture of the immune response to distinct challenges. Advances in scRNA-Seq have enhanced our understanding of the diversity of these programs, yet the correspondence between scRNA-Seq clusters and previously characterized Th subsets remains unclear. In this study, we use immune repertoires to position phenotypically sorted Th subsets within scRNA-Seq data from three healthy donors. This approach, termed TCR-Track, and accurately maps Th1, Th1-17, Th17, Th22, Th2a, Th2, Tfh, and Treg subsets, outperforming CITE-Seq-based mapping. Remarkably, the mapping is tightly focused on specific scRNA-Seq clusters despite a four-year interval between the sorting of subsets and the effector CD4+ scRNA-Seq experiment. Thus, while transient T cell plasticity is commonly observed in functionally active T cell populations, TCR-Track reveals high intrinsic program sustainability of Th clones circulating in peripheral blood. Repertoire overlap analysis at the scRNA-Seq level confirms that circulating Th1, Th2, Th2a, Th17, Th22, and Treg subsets are clonally independent. However, a prominent clonal overlap between corresponding clusters indicates that cytotoxic CD4+ T cells differentiate from Th1 clones. More specifically, we demonstrate that sorted CCR10+ Th cells correspond to a specific Th22 scRNA-Seq cluster, while CCR10-CCR6+CXCR3-CCR4+ cells, traditionally sorted as the Th17 subset, represent a mixture of bona fide Th17 and clonally unrelated CCR10low Th22 cells, which may have confounded investigators in previous studies. This clear distinction of Th17 and Th22 subsets should influence vaccine and T cell based therapies development. Additionally, we show that SARS-CoV-2 infection is associated with transient IFN type 1 activation of naive CD4+ T cells, and 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.

immunology↗

bioRxiv2023

Convergence, plasticity, and tissue residence of regulatory T cell response via TCR repertoire prism

Suppressive function of regulatory T cells (Treg) is dependent on signaling of their antigen receptors triggered by cognate self, dietary or microbial antigens in the form of peptide-MHC class II complexes. 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 used a fixed TCR{beta} chain FoxP3-GFP mouse model to analyze conventional (eCD4) and regulatory (eTreg) effector TCR repertoires in response to six distinct antigenic challenges to the lung and skin. This model showed highly "digital" repertoire behavior, allowing for easy-to-track challenge-specific TCR CDR3 clusters. For both studied subsets, we observed challenge-specific clonal expansion yielding homologous TCR clusters within and across animals and exposure sites, which were also reflected in the draining lymph nodes but not systemically. Some clusters were shared across cancer challenges, suggesting response to common tumor-associated antigens. For most challenges, eCD4 and eTreg clonal response did not overlap, indicating the distinct origin of the two cell subsets. At the same time, we observed such overlap at the sites of certain tumor challenges. The overlaps included dominant responding TCR motif and characteristic iNKT TCR, suggesting the tumor-induced eCD4-eTreg plasticity. Additionally, our TCR repertoire analysis demonstrated that distinct antigenic specificities are characteristic for eTreg cells residing in particular lymphatic tissues, regardless of the challenge, revealing the homing-specific, antigen-specific resident Treg populations. Altogether, our study highlights both challenge-specific and tissue-specific responses of Treg cells associated with distinct clonal expansions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/544726v4_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@5a8353org.highwire.dtl.DTLVardef@63fef3org.highwire.dtl.DTLVardef@f7390forg.highwire.dtl.DTLVardef@c347e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Inhibitory IL-10-producing CD4+ T cells develop in a T-bet-dependent manner and facilitate cytomegalovirus persistence via coexpression of arginase-1

Inhibitory CD4+ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity, but the mechanisms that underpin the development of such regulatory networks in vivo have remained obscure. To address this knowledge gap, we performed a comprehensive functional, phenotypic, and transcriptomic analysis of IL-10-producing CD4+ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated TH1-like cells that developed at sites of viral persistence in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4+ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4+ lineage suppressed the development of these inhibitory cells and also enabled better immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4+ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the coexpression of Arg1.

immunology↗

bioRxiv2018

T cell immunity does not age in a long-lived rodent species

Numerous studies have demonstrated that the percentage of naive T cells and diversity of T cell receptor (TCR) repertoire decrease with age, with some findings likewise suggesting that increased repertoire diversity may be associated with longer lifespan and healthy aging. In this work, we have analyzed peripheral TCR diversity from humans, mice, and blind mole-rats (Spalax spp.)--long-lived, hypoxia- and cancer-tolerant rodents. We employed a quantitative approach to TCR repertoire profiling based on 5RACE with unique molecular identifiers (UMI) to achieve accurate comparison of repertoire diversity, which also required development of specific wet lab protocol and TCR gene reference for Spalax. Our direct comparison reveals a striking phenomenon. Whereas TCR diversity of mice and humans decreases with age, resulting primarily from the shrinkage of the naive T cell pool, Spalax TCR diversity remains stable even for the animals that reach extreme old age (15-17 years). This indicates that T cell immunity does not meaningfully age in long-lived rodents, at least in terms of the classical understanding of immunosenescence, which is associated with the accumulation of large numbers of memory clones. We suggest that the extraordinary longevity of Spalax may be attributable at least in part to the distinctive organization of their T cell immunity. Our findings should therefore encourage a close re-examination of the contribution of immunosenescence to life span in mammals.

immunology↗