Enhanced SARS-CoV-2-Specific CD4+ T Cell Activation and Multifunctionality in Late Convalescent COVID-19 Individuals

Abstract

Background: Examination of CD4+ T cell responses during the natural course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection offers useful information for the improvement of vaccination strategies against this virus and the protective effect of these T cells. Methods: We characterized the SARS-CoV-2-specific CD4+ T cell activation marker, multifunctional cytokine and cytotoxic marker expression in recovered coronavirus disease 2019 (COVID-19) individuals. Results: CD4+ T-cell responses in late convalescent (>6 months of diagnosis) individuals are characterized by elevated frequencies of activated as well as mono, dual- and multi-functional Th1 and Th17 CD4+ T cells in comparison to early convalescent (<1 month of diagnosis) individuals following stimulation with SARS-CoV-2-specific antigens. Similarly, the frequencies of cytotoxic marker expressing CD4+ T cells were also enhanced in late convalescent compared to early convalescent individuals. Conclusion: Our findings from a low-to middle-income country suggest protective adaptive immune responses following natural infection of SARS-CoV-2 are elevated even at six months following initial symptoms, indicating the CD4+ T cell mediated immune protection lasts for six months or more in natural infection.

Keywords: CD4+ T cells; COVID-19; SARS-CoV-2; cytokines.

Figure 1

Enhanced frequencies of CD4⁺ T cells expressing activation markers in late convalescent COVID-19 individuals. PBMCs were cultured with media alone or SARS-CoV-2 or control antigens for 12 h and the baseline and antigen-stimulated frequencies of CD69, CD38 and OX-40 were determined in early convalescent (ECV) COVID-19 (n = 20) and late convalescent (LCV) COVID-19 individuals (n = 20). (A) Gating strategy and representative plots for CD4⁺ T cells expressing activation markers. (B) The frequencies of CD4⁺ T cells expressing activation markers in early and late convalescent individuals at baseline (B) as well as in response to stimulation with (C) SARS-CoV-2 WCL, (D) SARS-CoV-2 S+S1 peptide pools, (E) SARS-CoV-2 M+N peptide pools and (F) PMA/Ionomycin were measured by flow cytometry. The bars represent the geometric mean values. p values were calculated using the Mann–Whitney test. Any comparison that is not labelled with a p value is statistically non-significant.

Figure 1

Enhanced frequencies of CD4⁺ T cells expressing activation markers in late convalescent COVID-19 individuals. PBMCs were cultured with media alone or SARS-CoV-2 or control antigens for 12 h and the baseline and antigen-stimulated frequencies of CD69, CD38 and OX-40 were determined in early convalescent (ECV) COVID-19 (n = 20) and late convalescent (LCV) COVID-19 individuals (n = 20). (A) Gating strategy and representative plots for CD4⁺ T cells expressing activation markers. (B) The frequencies of CD4⁺ T cells expressing activation markers in early and late convalescent individuals at baseline (B) as well as in response to stimulation with (C) SARS-CoV-2 WCL, (D) SARS-CoV-2 S+S1 peptide pools, (E) SARS-CoV-2 M+N peptide pools and (F) PMA/Ionomycin were measured by flow cytometry. The bars represent the geometric mean values. p values were calculated using the Mann–Whitney test. Any comparison that is not labelled with a p value is statistically non-significant.

Figure 2

Enhanced frequencies of CD4⁺ T cells expressing multifunctional Th1 and Th17 cytokines in late convalescent COVID-19 individuals. PBMCs were cultured with media alone or SARS-CoV-2 or control antigens for 12 h and the baseline and antigen-stimulated frequencies of multifunctional Th1/Th17 cells were determined in determined in early convalescent (ECV) COVID-19 (n = 20) and late convalescent (LCV) COVID-19 individuals (n = 20). (A) Gating strategy and representative plots for Th1/Th17 CD4⁺ T cell subsets. The frequencies of mono-, dual- and multifunctional CD4⁺ Th1/Th17 cells in early convalescent and late convalescent individuals (B) At baseline as well as in response to stimulation with (C) SARS-CoV-2 WCL, (D) SARS-CoV-2 S+S1 peptide pools, (E) SARS-CoV-2 M+N peptide pools and (F) PMA/Ionomycin were measured by flow cytometry. The bars represent the geometric mean values. Net frequencies were calculated by subtracting baseline frequencies from the antigen- induced frequencies for each individual. p values were calculated using the Mann–Whitney test. Any comparison that is not labelled with a p value is statistically non-significant.

Figure 3

Enhanced frequencies of CD4⁺ T cells expressing cytotoxic markers in late convalescent COVID-19 individuals. PBMCs were cultured with media alone or SARS-CoV-2 or control antigens for 12 h and the baseline and antigen-stimulated frequencies of cytotoxic marker expressing CD4⁺ T cells were determined in early convalescent (ECV) COVID-19 (n = 20) and late convalescent (LCV) COVID-19 individuals (n = 20). The frequencies of cytotoxic marker expressing CD4⁺ T cells in early convalescent and late convalescent individuals (A) Gating strategy and representative plots for CD4⁺ T cells expressing cytotoxic markers. (B) at baseline as well as in response to stimulation with (C) SARS-CoV-2 WCL, (D) SARS-CoV-2 S+S1 peptide pools, (E) SARS-CoV-2 M+N peptide pools and (F) PMA/Ionomycin were measured by flow cytometry. The bars represent the geometric mean values. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. p values were calculated using the Mann–Whitney test. Any comparison that is not labelled with a p value is statistically non-significant.

Figure 3

Enhanced frequencies of CD4⁺ T cells expressing cytotoxic markers in late convalescent COVID-19 individuals. PBMCs were cultured with media alone or SARS-CoV-2 or control antigens for 12 h and the baseline and antigen-stimulated frequencies of cytotoxic marker expressing CD4⁺ T cells were determined in early convalescent (ECV) COVID-19 (n = 20) and late convalescent (LCV) COVID-19 individuals (n = 20). The frequencies of cytotoxic marker expressing CD4⁺ T cells in early convalescent and late convalescent individuals (A) Gating strategy and representative plots for CD4⁺ T cells expressing cytotoxic markers. (B) at baseline as well as in response to stimulation with (C) SARS-CoV-2 WCL, (D) SARS-CoV-2 S+S1 peptide pools, (E) SARS-CoV-2 M+N peptide pools and (F) PMA/Ionomycin were measured by flow cytometry. The bars represent the geometric mean values. Net frequencies were calculated by subtracting baseline frequencies from the antigen-induced frequencies for each individual. p values were calculated using the Mann–Whitney test. Any comparison that is not labelled with a p value is statistically non-significant.

Figure 4

Relationship between Immune markers and SARS-CoV-2 antibodies. (A) Multiparametric matrix correlation plot of immune markers and SARS-CoV-2-specific antibodies in all individuals of early convalescent (ECV) COVID-19 (n = 20) and late convalescent (LCV) COVID-19 (n = 20). (B) Spearman’s correlation coefficients are visualized by color intensity. p values and Spearman r values are ordered by hierarchical clustering.