Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

Fischer, David S.; Ansari, Meshal; Wagner, Karolin I.; Jarosch, Sebastian; Huang, Yiqi; Mayr, Christoph H.; Strunz, Maximilian; Lang, Niklas J.; D’Ippolito, Elvira; Hammel, Monika; Mateyka, Laura; Weber, Simone; Wolff, Lisa S.; Witter, Klaus; Fernandez, Isis E.; Leuschner, Gabriela; Milger, Katrin; Frankenberger, Marion; Nowak, Lorenz; Heinig-Menhard, Katharina; Koch, Ina; Stoleriu, Mircea G.; Hilgendorff, Anne; Behr, Jürgen; Pichlmair, Andreas; Schubert, Benjamin; Theis, Fabian J.; Busch, Dirk H.; Schiller, Herbert B.; Schober, Kilian

The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.