Single-cell RNA sequencing reveals in vivo signatures of SARS-CoV-2-reactive T cells through 'reverse phenotyping'

Fischer, D. S.; Ansari, M. I.; Wagner, K.; Jarosch, S.; Huang, Y.; Mayr, C. H.; Strunz, M.; Lang, N. J.; D'Ippolito, E.; Hammel, M.; Mateyka, L.; Weber, S.; Wolff, L. S.; Witter, K.; Fernandez, I. E.; Leuschner, G.; Milger, K.; Frankenberger, M.; Nowak, L.; Heinig, K.; Koch, I.; Stoleriu, M. G.; Hilgendorff, A.; Behr, J.; Pichlmair, A.; Schubert, B.; Theis, F. J.; Busch, D. H.; Schiller, H. B.; Schober, K.

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 used 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 induced transcriptional shifts by antigenic stimulation in vitro and took advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allowed identification of SARS-CoV-2-reactive TCRs and revealed phenotypic effects introduced by antigen-specific stimulation. We characterized transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and showed 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.