Repertoire-based mapping and time-tracking of helper T cell subsets in scRNA-Seq

Dmitry M Chudakov ^1* Daniil K Lukyanov ² Valeriia Kriukova ³ Krisitin Ladell ⁴ Irina Shagina ² Dmitry Staroverov ² Bella E Minasian ⁵ Anna S Fedosova ⁵ Pavel Shelyakin ⁵ Oleg N Suchalko ⁵ Alexander Y Komkov ⁵ Konstantin A. Blagodatskikh ⁶ Kelly Miners ⁴ Olga Britanova ⁵ Andre Franke ³ David A Price ⁴

• ¹ Masaryk University, Brno, Czechia
• ² Institute of Bioorganic Chemistry (RAS), Moscow, Moscow Oblast, Russia
• ³ University of Kiel, Kiel, Schleswig-Holstein, Germany
• ⁴ Cardiff University, Cardiff, United Kingdom
• ⁵ Abu Dhabi Stem Cells Center (ADSCC), Abu Dhabi, United Arab Emirates
• ⁶ Pirogov Russian National Research Medical University, Moscow, Moscow Oblast, Russia

The functional programs of CD4 + helper (Th) T cell clones play a central role in shaping immune responses to different challenges. While advances in scRNA-Seq have significantly improved our understanding of Th cell diversity, the relationship between scRNA-Seq clusters and traditionally characterized Th subsets remains ambiguous. In this study, we introduce TCR-Track, a method leveraging immune repertoire data to map phenotypically sorted Th subsets onto scRNA-Seq profiles. This approach accurately positions Th1, Th1-17, Th17, Th22, Th2a, Th2, Tfh, and Treg subsets, outperforming mapping based on CITE-Seq. Remarkably, the mapping is tightly focused on specific scRNA-Seq clusters despite a four-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 circulating Th1, Th2, Th2a, Th17, Th22, and Treg subsets are clonally independent. However, a significant clonal overlap between Th1 and cytotoxic CD4 + T cell clusters suggests that cytotoxic CD4 + T cells differentiate from Th1 clones. Additionally, our 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 CCR10 low Th22 cells. The clear distinction between Th17 and Th22 subsets should influence vaccine and T cell-based therapies development. Furthermore, we show that SARS-CoV-2 infection induces systemic IFN type 1 activation of naive helper T 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.