SARS-CoV-2-specific T cell responses and correlations with COVID-19 patient predisposition

Abstract

Coronavirus disease 2019 (COVID-19) has emerged as a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). So far, viral targets of cellular immunity and factors determining successful mounting of T cell responses are poorly defined. We therefore analyzed cellular responses to membrane, nucleocapsid, and spike proteins in individuals suffering from moderate or severe infection and in individuals who recovered from mild disease. We demonstrate that the CoV-2-specific CD4+ T helper cell response is directed against all 3 proteins with comparable magnitude, ex vivo proliferation, and portions of responding patients. However, individuals who died were more likely to have not mounted a cellular response to the proteins. Higher patient age and comorbidity index correlated with increased frequencies of CoV-2-specific CD4+ T cells, harboring higher portions of IL-2-secreting, but lower portions of IFN-γ-secreting, cells. Diminished frequencies of membrane protein-reactive IFN-γ+ T cells were particularly associated with higher acute physiology and chronic health evaluation II scores in patients admitted to intensive care. CoV-2-specific T cells exhibited elevated PD-1 expression in patients with active disease as compared with those individuals who recovered from previous mild disease. In summary, our data suggest a link between individual patient predisposition with respect to age and comorbidity and impairment of CoV-2-specific Th1-type cellular immunity, thereby supporting a concept of altered T cell function in at-risk patients.

Keywords: Antigen; COVID-19; T cells.

Figure 1. Identification of SARS–CoV-2–specific T cells in patients with COVID-19.

(A) PBMCs were stimulated or not with M, N, or S peptide mix or SEB for 16 hours as indicated. Live single CD14^–CD19^–CD3⁺–specific CD4⁺ Th cells were identified based on coexpression of CD154 and CD137. Specific CD4⁺CD154⁺CD137⁺ T cells were further analyzed for expression of IFN-γ, TNF-α, IL-2, and Ki67, with the latter including a fluorescence-minus-one (FMO) control. (B) Percentage of all patients with a positive CD4⁺ T cell response (M: n = 34; N: n = 34; S: n = 33), frequencies of antigen-reactive CD4⁺ T cells in responding patients (Kruskal-Wallis test), and frequency distribution in males versus females (M: n = 23; N: n = 21; S: n = 23) (Mann-Whitney test). (C) Frequencies of antigen-specific CD4⁺ T cells expressing IFN-γ (Kruskal-Wallis test), IFN-γ + TNF-α (Kruskal-Wallis test), or Ki67 (in comparison with the total CD4⁺ population; Kruskal-Wallis test) (M: n = 23; N: n = 21; S: n = 20). Where applicable, graphs show mean ± SEM.

Figure 2. Features of cellular nonresponders to CoV-2–specific stimulation.

(A–D) All patients were stratified according to their capacity to mount a specific CD4⁺ T cell response or not after M, N, or S protein stimulation with n as in the legend to Figure 1B. In responders and nonresponders, the number of days since symptom onset was analyzed by (A) t test or (B) the percentage of patients showing spike-protein–specific IgG (left; Fisher’s exact test) or IgA (right; Fisher′s exact test) responses. (C) The percentage of individuals who died within 6 weeks after analysis (Fisher’s exact test) and (D) the severity of pneumonia (t test) were examined. Where applicable, graphs show mean ± SEM.

Figure 3. Correlation of the CoV-2–specific CD4⁺ T cell response with patient age and comorbidities.

Frequencies of M, N, or S protein–specific CD4⁺ T cells were correlated with patient age (A) or comorbidities (B) with n as in the legend to Figure 1B. Frequencies of M-, N-, or S-specific IFN-γ–secreting T cells with n as in the legend to Figure 1C were correlated with patient age (C) or comorbidities (D). Simple linear regression analysis was performed throughout.

Figure 4. Characteristics of the CoV-2–specific T cell response in patients admitted to intensive care.

(A) Patients were stratified according to ICU (M, N, S: n = 23, respectively) or non-ICU treatment (M: n = 11; N: n = 11; S: n = 10) and compared with recovered individuals who were not hospitalized (n = 7) for the percentage of donors showing specific CD4 responses (χ² test), (B) frequencies of antigen-specific CD4⁺ T cells in cellular responders (M: n = 17, N: n = 17, S: n = 16 [ICU]; M: n = 6, N: n = 4, S: n = 7 [non-ICU]; M: n = 5, N: n = 5, S: n = 6 [recovered]), (C) frequencies of antigen-specific CD4⁺ T cells secreting IFN-γ, or (D) ex vivo proliferating Ki67⁺ coexpressing IFN-γ and TNF-α (with n as in B and analyzed by Kruskal-Wallis test, respectively). Where applicable, graphs show mean ± SEM.

Figure 5. Correlation of CoV-2–specific IFN-γ secretion and APACHE II scores in patients treated in the ICU.

Frequencies of antigen-specific IFN-γ–expressing CD4⁺ T cells after M, N, or S protein stimulation were determined in responding patients treated in the ICU and correlated with the individual APACHE II score values as indicated (M: n = 17; N: n = 17; S: n = 16) by simple linear regression analysis.

Figure 6. CoV-2–specific IL-2 secretion and its correlation with patient predisposition.

Frequencies of antigen-specific IL-2–expressing CD4⁺ T cells after M, N, or S protein stimulation were (A) determined in patients stratified according to ICU or non-ICU care or in recovered individuals who were not hospitalized (M: n = 10, N: n = 8, S: n = 10 [ICU]; M: n = 5, N: n = 3, S: n = 3 [non-ICU]; M: n = 5, N: n = 5, S: n = 5 [recovered]). Analysis by ANOVA. Frequencies of specific IL-2–secreting T cells were further correlated in patients with active COVID-19 with (B) age or (C) comorbidity (M: n = 16; N: n = 10; S: n = 13) and analyzed by simple linear regression. Bar graphs show mean ± SEM.

Figure 7. Exhaustion and memory phenotype of CoV-2–specific T cells.

(A) Exemplary PD-1 expression in S-specific CD4⁺ T cells in a recovered individual versus a patient in ICU care with FMO control (left) and MFI of PD-1 in M-specific (ANOVA analysis), N-specific (ANOVA-analysis), and S-specific (Kruskal-Wallis analysis) T cells in patients stratified as indicated (right). (B) Memory subset distribution of CoV-2–specific T cells was determined based on CD45RO and CD62L expression. Exemplary subset identification in total (gray) and M-specific (red) CD4⁺ T cells (ICU patient, left) as well as frequencies of M-specific (ANOVA analysis), N-specific (ANOVA analysis), and S-specific (Kruskal-Wallis analysis) CD45RO^–CD62L^– T cells (right) in patients stratified as indicated in A and B (M: n = 10, N: n = 8, S: n = 10 [ICU]; M: n = 5, N: n = 3, S: n = 3 [non-ICU]; M: n = 5, N: n = 5, S: n = 5 [recovered]). Bar graphs show mean ± SEM.