Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans

Abstract

Many unknowns exist about human immune responses to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. SARS-CoV-2-reactive CD4⁺ T cells have been reported in unexposed individuals, suggesting preexisting cross-reactive T cell memory in 20 to 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 preexisting memory CD4⁺ T cells that are cross-reactive with comparable affinity to SARS-CoV-2 and the common cold coronaviruses human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, and 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 coronavirus disease 2019 (COVID-19) disease.

Fig. 1. Characteristics of SARS-CoV-2 epitopes identified in unexposed donors.

Reactivity was determined by FluoroSPOT assay after 17 days of in vitro stimulation of unexposed donor PBMCs (n = 18) with one pool of peptides spanning the entire sequence of the spike protein (CD4-S) or a nonspike “megapool” (CD4-R) of predicted epitopes from the nonspike (i.e., “remainder”) regions of the viral genome. (A) Summary of the responses as a function of the protein of origin. (B) Spearman correlation of positive responses per SARS-CoV-2 protein size. (C) Percent similarity of the identified epitopes with common cold coronavirus peptides as a function of the number of responding donors. (D) Each dot shows the reactivity of a donor-epitope combination derived from either nonspike (CD4-R) or spike (CD4-S) protein. Black bars indicate the geometric mean and geometric SD. Red indicates donor-epitope combinations with sequence identity >67% with common cold coronaviruses, and blue indicates highly reactive donor-epitope combinations (>1000 SFCs*10⁶) with sequence identity ≤67%. In (C) and (D), statistical comparisons were performed with a two-tailed Mann–Whitney test. ***P < 0.001, ****P < 0.0001.

Fig. 2. CD4⁺ T cells in SARS-CoV-2–unexposed and recovered COVID-19 patients against HCoV epitopes homologous to SARS-CoV-2 epitopes.

(A) Example of flow cytometry gating strategy for antigen-specific CD4⁺ T cells based on activation-induced marker assays (OX40⁺ and CD137⁺ double expression) after stimulation of PBMCs with HCoV or SARS-CoV-2 peptides. (B to D) Antigen-specific CD4⁺ T cells measured as the percentage of activation-induced marker assay–positive (OX40⁺CD137⁺) CD4⁺ T cells after stimulation of PBMCs with HCoV epitopes homologous to SARS-CoV-2 epitopes. Samples were derived from SARS-CoV-2–unexposed donors (n = 25) and recovered COVID-19 patients ( n = 20). Black bars indicate the geometric mean and geometric SD. Each dot is representative of an individual subject. Statistical pairwise comparisons [(B) and (C)] were performed with the Wilcoxon test. P values related to comparisons with the DMSO controls are listed at the bottom of the graphs, and any significant P values related to intergroup comparisons are listed on top of the graphs. Statistical comparisons across cohorts were performed with the Mann–Whitney test (D). See also figs. S5 and S6.

Fig. 3. Phenotypes of antigen-specific CD4⁺ T cells from SARS-CoV-2–unexposed and recovered COVID-19 patients responding to HCoV epitopes homologous to SARS-CoV-2 epitopes.

(A) Example of flow cytometry gating strategy for antigen-specific CD4⁺ T cell subsets after overnight stimulation of PBMCs with HCoV or SARS-CoV-2 peptides ex vivo. (B and C) Phenotype of antigen-specific CD4⁺ T cells (OX40⁺CD137⁺) responding to the indicated pools of SARS-CoV-2 and HCoV epitopes in unexposed subjects and recovered COVID-19 patients. Data are shown as mean ± SD. Each dot represents an individual subject. Statistical pairwise comparisons in (B) and (C) were performed with the Wilcoxon test. (D) Overall averages of antigen-specific CD4⁺ T cell subsets detected in unexposed subjects and recovered COVID-19 patients. See also fig. S5.

Fig. 4. Cross-reactivity of SARS-CoV-2 and homologous HCoV peptides.

Twelve short-term cell lines were generated using specific SARS-CoV-2 donor-epitope combinations selected on the basis of the primary screen. After 14 days of in vitro expansion, each T cell line was tested with the SARS-CoV-2 epitope used for stimulation and peptides corresponding to analogous sequences from other HCoVs at six different concentrations (1, 0.1, 0.01, 0.001, 0.0001, and 0.00001 μg/ml). SFCs/10⁶ PBMCs are plotted for T cell lines stimulated with each peptide. See also fig. S7.