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

Fischer, D. S., Ansari, M., Wagner, K. I., 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-Menhard, K., Koch, I., Stoleriu, M. G., Hilgendorff, A., Behr, J., Pichlmair, A., Schubert, B., Theis, F. J., Busch, D. H., Schiller, H. B., and Schober, K. (2021). Nat Commun 12, 4515. doi: 10.1038/s41467-021-24730-4


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.