Persistent Cellular Immunity to SARS-CoV-2 Infection

Abstract

SARS-CoV-2 is responsible for an ongoing pandemic that affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 months after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen specific memory that could contribute to rapid recall responses. In addition, recovered individuals show enduring immune alterations in relative numbers of CD4 ⁺ and CD8 ⁺ T cells, expression of activation/exhaustion markers, and cell division.

Summary: We show that SARS-CoV-2 infection elicits broadly reactive and highly functional memory T cell responses that persist 6 months after infection. In addition, recovered individuals show enduring immune alterations in CD4 ⁺ and CD8 ⁺ T cells compartments.

Fig. 1.. Persistent longitudinal changes in the phenotypic landscape of T cells in individuals recovered from COVID-19.

(A) Global viSNE projection of pooled T cells for all participants pooled shown in background contour plots, with overlaid projections of concatenated controls, convalescent patients 1.3 months, and convalescent 6.1 months, respectively. (B) viSNE projection of pooled T cells for all participants of T cell clusters identified by FlowSOM clustering. (C) Column-scaled z-scores of median fluorescence intensity (MFI) as indicated by cluster and marker. (D) Frequency of T cells from each group in FlowSOM clusters indicated. Each dot represents an individual with COVID-19 at 1.3 months (dark blue for CD4⁺ T cells and dark red for CD8⁺ T cells) or 6.1 months (light blue for CD4⁺ T cells and orange for CD8⁺ T cells) as well as control individuals (green).

Fig. 2.. T cell abnormalities persist after 6.1 months.

(A) Frequency of CD4⁺ T cells out of total CD3⁺ T cells. (B) PD-1, TIGIT, TIM-3 and CD25 expression of CD4⁺ T cells. (C) Frequency of CD8⁺ T cells out of total CD3⁺ T cells. (D) PD-1, TIGIT, TIM-3 and CD25 expression of CD8⁺ T cells. (E) Percentage of Tscm, Tn, Tcm, Ttm, Tem, and Ttd CD4⁺ T cells. (F) Percentage of Tscm, Tn, Tcm, Ttm, Tem, and Te CD8⁺ T cells. (G) Frequency of cycling CD4⁺ T cells. (H) Frequency of cycling CD8⁺ T cells. Each dot represents an individual with COVID-19 at 1.3 months (dark blue for CD4⁺ T cells and dark red for CD8⁺ T cells) or 6.1 months (light blue for CD4⁺ T cells and orange for CD8⁺ T cells) as well as control individuals (green). Significance determined by paired t test for comparisons between time points within individuals and unpaired t test for comparison between controls and COVID-19 individuals. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 3.. Antigen-specific CD4⁺ T cells dynamics in COVID-19 convalescent individuals.

(A) viSNE representations of CD137⁺ CD154⁺ SARS-CoV-2-stimulated CD4⁺ T cells in unexposed individuals (controls) and COVID-19 convalescent individuals pooled. Density plots from each group concatenated is overlaid on the total contour viSNE plot. (B) viSNE representation of antigen-specific CD4⁺ T cell clusters, identified by FlowSOM clustering. (C) Column-scaled z-scores of median fluorescence intensity (MFI) as indicated by cluster and marker. (D) Percentage of antigen-specific CD4⁺ cells in the indicated FlowSOM clusters. Each bar represents the mean percentage for all COVID-19 convalescent individuals for the indicated SARS-CoV-2 peptide pools. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 4.. SARS-CoV-2-specific CD4⁺ T cells responses in convalescent COVID-19 individuals.

Longitudinal analysis of COVID-19 specific CD4⁺ T cell responses in paired samples obtained 1.3 months and 6.1 months after infection. Cytokine production (IL-2, IFN-γ, and TNF-α) by CD4⁺ T cells analyzed by Intracellular Cytokine Staining (ICS). Spike (Aggregation of responses to Spike peptide pool S1 and S2), Nucleocapsid (NCAP) Membrane (Memb) and non-structural protein 3a (AP3a) peptide pools responses by controls (n=20), and convalescent COVID-19 individuals (n=41). (A) Combined frequency of SARS-CoV-2 specific CD4⁺ T cells that produce either IL-2, IFN-γ, and/or TNF-α. (B) Frequency of SARS-CoV-2 specific memory CD4⁺ T cells (CD45RA⁻ CD27⁺) that produce either IL-2, IFN-γ, and/or TNF-α. (C) Frequency of SARS-CoV-2-specific CD4⁺ T cells that produce either 3 cytokines, 2 cytokines or 1 cytokine. Each dot represents an individual at 1.3 months (dark blue) or 6.1 months (light blue) or control individuals (green). Significance determined by paired t test for comparisons between time points within individuals and unpaired t test for comparison between controls and COVID-19 individuals. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 5.. SARS-CoV-2-specific CD8⁺ T cells responses in convalescent COVID-19 individuals.

Longitudinal analysis of COVID-19 specific CD8⁺ T cell responses in paired samples obtained from the same individual at 1.3 months and 6.1 months after infection. Cytokine production (MIP-1β, CD107a, IL-2, IFN-γ, and TNF-α) by CD8⁺ T cells analyzed by Intracellular Cytokine Staining (ICS). Spike (Aggregation of responses to spike peptide pool S1 and S2), Nucleocapsid (NCAP) Membrane (Memb) and non-structural protein 3a (AP3a) peptide pools responses by controls (n=20), and convalescent COVID-19 individuals (n=41). (A) Frequency of SARS-CoV-2 specific CD8⁺ T cells that produce MIP-1β, CD107a, IL-2, IFN-γ, or TNF-α. (B) Frequency of SARSCoV-2 specific CD8⁺ T cells that produce either 5 cytokines, 4 cytokines or 3 cytokines. Each dot represents an individual at 1.3 months (dark red) or 6.1 months (orange) or control individuals (green). Significance determined by paired t test for comparisons between time points within individuals and unpaired T test for comparison between controls and COVID-19 individuals. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.