Antigenic determinants of SARS-CoV-2-specific CD4+ T cell lines reveals M protein-driven dysregulation of interferon signaling

We generated CD4+ T cell lines (TCLs) reactive to either SARSCoV-2 spike (S) or membrane (M) proteins from unexposed naïve T cells from six healthy donor volunteers to understand in fine detail whether the S and M structural proteins have intrinsic differences in driving antigen-specific CD4+ T cell responses. Having shown that each of the TCLs were antigen-specific and antigen-reactive, single cell mRNA analyses demonstrated that SARS-CoV-2 S and M proteins drive strikingly distinct molecular signatures. Whereas the S-specific responses are transcriptionally similar from those responses induced by other viral antigens (e.g. CMV), the M protein-specific CD4+ TCLs have a transcriptomic signature that indicate a marked suppression of interferon signaling, characterized by a downregulation of the genes encoding ISG15, IFITM1, IFI6, MX1, STAT1, OAS1, IFI35, IFIT3 and IRF7 (a molecular signature which is not dissimilar to that found in severe COVID-19). Our study suggests a potential link between the antigen specificity of the SARS-CoV-2-reactive CD4+ T cells and the development of specific sets of adaptive immune responses. Moreover, the balance between T cells of significantly different specificities may be the key to understand how CD4+ T cell dysregulation can determine the clinical outcomes of COVID-19. Overall design: Naïve CD4+ T cells, as well as monocytes were purified from cryopreserved PBMCs of 6 healthy donor individuals unexposed to SARS-CoV-2 (obtained prior to 2019). Purified monocytes were differentiated into dendritic cells (DCs). Matured DCs were separately seeded in 24 wells plate, and then loaded for 2-4 hours with 1µg/peptide of SARS-CoV-2 peptide megapools (MPs) covering either the entire sequence of SARS-CoV-2 spike protein (MP-S), consisted by a 15-mer peptides overlapping by 10-residues (253 peptides) or covering the complete sequence of SARS-CoV-2 membrane protein (MP-M), consisting of 15-mer sequences with 11 amino acids overlap (PepTivator® SARS-CoV-2 Prot_M, Miltenyi Biotec, USA) and 1µg/peptide of CMV peptide megapools (~500 peptides). On the top of the either MP-S, MP-M or CMV pulsed-DCs, 1x10^6 naïve CD4+ T cells (1:10 ratio) were added and incubated for 12 days in complete R10 media. On days 2, 5 and 8, 60units of human rIL-2 (Cetus, USA) were added at each well. At day 12, cells in culture were washed, counted, and fed (1:1 ratio) with irradiated (40GY) autologous PBMCs (feeder cells) in the presence of either 1µg/peptide of MP-S or MP-M for another 12-day round culture in the same conditions, including the IL-2 stimulation. After three 12-day rounds of peptide pool stimulation/expansion in vitro, we generated a total of 18, 6 MP-S-specific, 6 MP-M-specific and 6 CMV-specific CD4+ TCLs, which were profiled for antigen-specificity and reactivity using multiparameter flow cytometry as well as, for single cell transcriptional analysis.