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

The functional programs chosen by B and T cell clones fundamentally determine the architecture of immune response to distinct challenges. Advances in scRNA-Seq have improved our understanding of the diversity and stability of these programs, but it has proven difficult to link this information with known lymphocyte subsets. Here, we introduce Sort-Seq, an immune repertoire-based method that allows exact positioning of phenotypically defined lymphocyte subsets within scRNA-Seq data. Sort-Seq outperformed CITE-Seq for accurate mapping of the classical CD4+ T helper (Th) cell subsets (Th1, Th1-17, Th17, Th22, Th2a, Th2, and Treg), offering a more powerful approach to the surface phenotype-based scRNA-Seq classification of adaptive lymphocyte subpopulations. Using integrated scRNA-Seq, Sort-Seq, and CITE-Seq data from 122 donors, we provide a comprehensive Th cell scRNA-Seq reference map. Exploration of this dataset revealed the low plasticity and extreme sustainability of the Th17, Th22, Th2, and Th2a cell programs over years. We also develop Cultivation-based Antigen-specific T cell identificatoR in Replicates (CultivAToRR), which identified >80 SARS-CoV-2-specific CD4+ TCRβ clonotypes in a single donor across a wide frequency range. We complemented these results with frequency- based capturing of COVID-19-responsive clonotypes and screening against known SARS-CoV-2- specific TCRs. Positioning within the annotated scRNA-Seq map revealed functional subtypes of Th cell clones involved in primary and secondary responses against SARS-CoV-2. The ability to capture low-frequency antigen-specific T cell clones in combination with Sort-Seq-based scRNA- Seq annotation creates an integral pipeline that links challenge-responsive clones with their exact functional subtypes, providing a solid foundation for investigating T cell roles in healthy and pathological immune responses and vaccine development. ### Competing Interest Statement The authors have declared no competing interest.