Analysis of antigen specificity of Treg clonotypes expanded upon SARS-CoV-2 infection

The pandemic outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has threatened human health worldwide. Among protective immune reactions, T cell responses are diverse among individuals, which is related to the differences in severity. A T cell subset, regulatory T (Treg) cells, is crucial for limiting excessive immune responses. If SARS-CoV-2-specific Tregs are developed during infection, they may counteract antiviral immunity and cause severe symptoms. To address this possibility, we conducted single-cell TCR-RNA-sequencing of peripheral blood mononuclear cells from convalescent Coronavirus disease 2019 (COVID-19) patients. Among 13 donors, one with severe symptoms had substantially more FOXP3-expressing Treg clonotypes activated in the presence of SARS-CoV-2 virion or other major antigen proteins. To define the reactivity of these Treg clonotypes, 15 highly expanded Treg clonotypes were reconstituted into reporter cells and stimulated with 27 distinct peptide pools that cover all SARS-CoV-2 proteins. However, none of these clonotypes react to any SARS-CoV-2 antigens. Instead, the reporter cells expressing one TCR clonotype (23599) were activated in the presence of Epstein-Barr virus-transformed B cells without adding exogenous antigens. Furthermore, 23599 TCR-expressing cells were activated by non-transformed naïve syngeneic B cells in a DQA1*03:03-DQB1*04:01-dependent manner, suggesting that clonotype 23599 may be autoreactive. This Treg clonotype, 23599, was also detected in a public TCR database and significantly expanded in COVID-19 patients compared to healthy donors. These results suggest that SARS-CoV-2 is not the dominant antigen inducing Treg cells during infection.