Many unknowns exist about human immune responses to the SARS-CoV-2 virus. SARS-CoV-2 reactive CD4+ T cells have been reported in unexposed individuals, suggesting pre-existing cross-reactive T cell memory in 20-50% of people. However, the source of those T cells has been speculative. Using human blood samples derived before the SARS-CoV-2 virus was discovered in 2019, we mapped 142 T cell epitopes across the SARS-CoV-2 genome to facilitate precise interrogation of the SARS-CoV-2-specific CD4+ T cell repertoire. We demonstrate a range of pre-existing memory CD4+ T cells that are cross-reactive with comparable affinity to SARS-CoV-2 and the common cold coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63, or HCoV-HKU1. Thus, variegated T cell memory to coronaviruses that cause the common cold may underlie at least some of the extensive heterogeneity observed in COVID-19 disease.

We address whether T cell responses induced by different vaccine platforms (mRNA-1273, BNT162b2, Ad26.COV2.S, NVX-CoV2373) cross-recognize early SARS-CoV-2 variants. T cell responses to early variants were preserved across vaccine platforms. By contrast, significant overall decreases were observed for memory B cells and neutralizing antibodies. In subjects ∼6 months post-vaccination, 90% (CD4 + ) and 87% (CD8 + ) of memory T cell responses were preserved against variants on average by AIM assay, and 84% (CD4 + ) and 85% (CD8 + ) preserved against Omicron. Omicron RBD memory B cell recognition was substantially reduced to 42% compared to other variants. T cell epitope repertoire analysis revealed a median of 11 and 10 spike epitopes recognized by CD4 + and CD8 + T cells, with average preservation > 80% for Omicron. Functional preservation of the majority of T cell responses may play an important role as second-level defenses against diverse variants.

Highlights d T cell responses recognize at least 30-40 epitopes in each donor d Immunodominance is correlated with HLA binding d Immunodominant regions for CD4 + T cells have minimal overlap with antibody epitopes d CD8 + T cell responses depend on the repertoire of HLA class I alleles

Highlights d T cells of exposed donors or vaccinees effectively recognize SARS-CoV-2 variants d Effective recognition in AIM and FluoroSPOT assays, for spike and other proteins d 93% and 97% of CD4 and CD8 epitopes are 100% conserved across variants

Detection of antigen-specific CD4+ T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of antigen-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be ‘stingy’ cytokine producers, fundamentally different than Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of antigen-specific cells by intracellular cytokine staining (ICS) relies on the ability of the CD4+ T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation induced marker (AIM) methodology to identify antigen-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus (Strep)-specific GC Tfh cells produced minimal detectable cytokines by ICS, the AIM method identified 85-fold more antigen-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 (PD-L1) upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare antigen-specific CD4+ T cells in human peripheral blood. Dengue-, tuberculosis-, and pertussis-vaccine-specific CD4+ T cells were readily detectable by AIM. In sum, cytokine assays missed 98% of antigen-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by co-expression of TCR-dependent activation markers.