Thymic selection is crucial for forming a pool of T-cells that can efficiently discriminate self from non-self using their T-cell receptors (TCRs) to develop adaptive immunity. In the present study we analyzed how a diverse set of physicochemical and sequence features of a TCR can affect the chances of successfully passing the selection. On a global scale we identified differences in selection probabilities based on CDR3 loop length, hydrophobicity, and residue sizes depending on variable genes and TCR chain context. We also observed a substantial decrease in N-glycosylation sites and other short sequence motifs for both alpha and beta chains. At the local scale we used dedicated statistical and machine learning methods coupled with a probabilistic model of the V(D)J rearrangement process to infer patterns in the CDR3 region that are either enriched or depleted during the course of selection. While the abundance of patterns containing poly-Glycines can improve CDR3 flexibility in selected TCRs, the "holes" in the TCR repertoire induced by negative selection can be related to Arginines in the (N)-Diversity (D)-N-region (NDN) region. Corresponding patterns were stored by us in a database available online. We demonstrated how TCR sequence composition affects lineage commitment during thymic selection. Structural modeling reveals that TCRs with "flat" and "bulged" CDR3 loops are more likely to commit T-cells to the CD4+ and CD8+ lineage respectively. Finally, we highlighted the effect of an individual MHC haplotype on the selection process, suggesting that those "holes" can be donor-specific. Our results can be further applied to identify potentially self-reactive TCRs in donor repertoires and aid in TCR selection for immunotherapies.

• Klíčová slova
• HLA alleles, T-cell immunity, T-cell receptor repertoire, immune repertoire analysis, immune repertoire sequencing, thymic selection,
• MeSH
• hypervariabilní oblasti genetika   imunologie   chemie   MeSH
• lidé MeSH
• receptory antigenů T-buněk alfa-beta * genetika   imunologie   chemie   MeSH
• thymus * imunologie   cytologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hypervariabilní oblasti MeSH
• receptory antigenů T-buněk alfa-beta * 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.

• Klíčová slova
• T cell memory, Th17, Th22, cytotoxic CD4+ T cells, helper T cell subsets, immune repertoires, scRNA-Seq, scTCR-seq,
• MeSH
• analýza genové exprese jednotlivých buněk MeSH
• analýza jednotlivých buněk * metody   MeSH
• lidé MeSH
• sekvenční analýza RNA MeSH
• sekvenování transkriptomu * metody   MeSH
• T-lymfocyty - podskupiny * imunologie   MeSH
• T-lymfocyty pomocné-indukující * imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Mycobacterium tuberculosis (Mtb) remains a major threat worldwide, although only a fraction of infected individuals develops tuberculosis (TB). TB susceptibility is shaped by multiple genetic factors, and we performed comparative immunological analysis of two mouse strains to uncover relevant mechanisms underlying susceptibility and resistance. C57BL/6 mice are relatively TB-resistant, whereas I/St mice are prone to develop severe TB, partly due to the MHC-II allelic variant that shapes suboptimal CD4+ T cell receptor repertoire. We investigated the repertoires of lung-infiltrating helper T cells and B cells at the progressed stage in both strains. We found that lung CD4+ T cell repertoires of infected C57BL/6 but not I/St mice contained convergent TCR clusters with functionally confirmed Mtb specificity. Transcriptomic analysis revealed a more prominent Th1 signature in C57BL/6, and expression of pro-inflammatory IL-16 in I/St lung-infiltrating helper T cells. The two strains also showed distinct Th2 signatures. Furthermore, the humoral response of I/St mice was delayed, less focused, and dominated by IgG/IgM isotypes, whereas C57BL/6 mice generated more Mtb antigen-focused IgA response. We conclude that the inability of I/St mice to produce a timely and efficient anti-Mtb adaptive immune responses arises from a suboptimal helper T cell landscape that also impacts the humoral response, leading to diffuse inflammation and severe disease.

• Klíčová slova
• B cells, CD4 + T cells, TB-susceptible mouse strain, TCR repertoire, immunoglobulins, transcriptomic signatures, tuberculosis,
• MeSH
• adaptivní imunita * genetika   MeSH
• B-lymfocyty imunologie   MeSH
• genetická predispozice k nemoci * MeSH
• modely nemocí na zvířatech MeSH
• Mycobacterium tuberculosis * imunologie   MeSH
• myši inbrední C57BL * MeSH
• myši MeSH
• plíce imunologie   patologie   MeSH
• receptory antigenů T-buněk genetika   imunologie   MeSH
• tuberkulóza * imunologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH