Early and Polyantigenic CD4 T Cell Responses Correlate with Mild Disease in Acute COVID-19 Donors

Abstract

We assessed SARS-CoV-2-specific CD4+ and CD8+ T cell responses in samples from 89 acute COVID-19 patients, utilizing blood samples collected during the first wave of COVID-19 in Italy. The goal of the study was to examine correlations between SARS-CoV-2-specific T cell responses in the early phase comparing mild, moderate, or severe COVID-19 disease outcomes. T cell responses to the spike (S) and non-S proteins were measured in a combined activation-induced marker (AIM) and intracellular cytokine staining (ICS) assay. Early CD4+ T cell responses to SARS-CoV-2 S correlated with milder disease by both AIM and IFNγ ICS readouts. The correlation of S-specific CD4+ T cell responses with milder disease severity was most striking within the first two weeks of symptom onset compared to later time points. Furthermore, donors with milder disease were associated with polyantigenic CD4+ T cell responses that recognized more prominently non-S proteins in addition to S, while severe acute COVID-19 was characterized by lower magnitudes of CD4+ T cell responses and a narrower repertoire. In conclusion, this study highlights that both the magnitude and breadth of early SARS-CoV-2-specific CD4+ T cell responses correlated with milder disease outcomes in acute COVID-19 patients.

Keywords: COVID-19; SARS-CoV-2; T cells; acute; breadth; early.

Figure 1

Time from symptom onset, sex, and age distributions of mild, moderate, and severe acute COVID-19 subjects. Peripheral blood mononuclear cells (PBMCs) were collected from 89 acute COVID-19 donors. (A) COVID-19 disease severity composition; mild (n = 21, blue), moderate (n = 35, yellow), and severe (n = 33, red). The p-value listed above the pie chart was calculated by a one sample Wilcoxon t test. (B) Days post-symptom onset (PSO) for the different disease severities. Bars represent the median with 95% confidence intervals. p-value is calculated by Kruskal–Wallis t test. (C) Percentage of female (dark grey) and male (white) subjects by disease severity. p-value is calculated by Friedman one-way ANOVA. (D) Age composition for each disease severity category. Bars represent the median and 95% confidence interval and the Kruskal–Wallis t test p-value is listed above the graph. When the Kruskal–Wallis t test was significant, Mann–Whitney U tests were applied between disease severities. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 2

CD4+ T cell responses to SARS-CoV-2 spike in mild, moderate, and severe COVID-19 donors. PBMCs from mild (n = 21, blue), moderate (n = 35, yellow), and severe (n = 33, red) acute COVID-19 donors were tested for CD4+ T cell responses to SARS-CoV-2 S by AIM + ICS assay. (A) AIM CD4+ T cell responses were measured by the co-expression of OX40 and CD69. The frequency of response is shown by pie charts. (B) The spike-specific cytokine+ CD4+ T cells were measured by the cells expressing CD40L in combination with production of IFNγ, granzyme B, TNFα, IL-2, IL-4, or IL-17. All T cell data shown are background subtracted and SI > 2. Horizontal lines represent the geometric mean and the dotted line represents the limit of sensitivity (LOS), while the y-axis starts at the limit of detection (LOD). p-values are calculated by Kruskal–Wallis One-way ANOVA and, when significant, further Mann–Whitney U tests are applied between disease severities. Chi-square test (χ²) p-values are indicated below the pie charts and are calculated from the frequency of response. * p < 0.05, ** p < 0.01.

Figure 3

CD8+ T cell responses to SARS-CoV-2 spike in acute COVID-19 donors. PBMCs from acute COVID-19 donors were tested in an AIM + ICS assay for CD8+ responses to SARS-CoV-2 S. (A) CD69 + CD137+ AIM + CD8 T cells were quantified in a subset of the total cohort (n = 8 mild, 25 moderate, and 22 severe). The frequency of responding donors is indicated by pie charts. (B) Spike-specific CD8+ T cell responses to IFNγ, granzyme B, TNFα, IL-2, IL-4, or IL-17 were double positive for CD69. (C) AIM+ and IFNγ CD8+ T cell responses were separated by less than 2 weeks or greater than 15 days PSO (3 mild and 1 severe were excluded from the graph because the days PSO was not reported). All data shown is background subtracted and SI > 2. Lines represent the geometric mean and the dotted line represents the limit of sensitivity (LOS), while the y-axis begins at the LOD. p-values are calculated by Kruskal–Wallis one-way ANOVA and, when significant, further Mann–Whitney U tests are applied between disease severities and significant results are indicated by the symbols. Chi-square test (χ²) p-values indicated below the pie charts are calculated from the frequency of response. * p < 0.05.

Figure 4

Early and late CD4+ T cell responses to SARS-CoV-2 S. The cohort of acute donors was divided by ≤14 days PSO (early) and 15+ days PSO (late), and the PBMCs were tested for CD4+ T cell reactivity to SARS-CoV-2 spike by AIM and IFNγ ICS. The magnitude of spike-specific CD4 reactivity is shown for mild (n = 18, blue), moderate (n = 35, yellow), severe (n = 32, red), or all (n = 85, grey) COVID-19 donors (3 mild and 1 severe were excluded from the graph because the days PSO was not reported). The data are shown for the entire cohort and separated for side-by-side comparisons of early and late PSO within disease severities in AIM (A) and IFNγ (B) ICS. The same data are plotted as shown to compare the T cell responses in AIM (C) and IFNγ (D) ICS within disease severities. Additionally, pie charts show the frequency of the response for each level of disease severity. The y-axis starts at the LOD and the dotted line represents the LOS, while the solid lines or bars represent geometric mean. p-values are calculated by Kruskal–Wallis across all disease severities and Mann–Whitney U tests are applied as indicated by the symbols when significant. Chi-squared test is applied to the frequencies of response as indicated by the p-values below the pie charts. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

CD4+ T cell reactivity as a function of donor sex and age. The cohort of 89 acute COVID-19 donors was analyzed for CD4+ T cell responses to SARS-CoV-2 S by AIM + ICS assays. (A) The spike-specific AIM+ and IFNγ+ CD4+ T cell responses are shown for the entire cohort as a function of donor sex (male in white and female in dark grey). The y-axis begins at the LOD for each assay and the dotted line represents the LOS. Mann–Whitney U tests were applied to the data and the results are indicated by symbols when significant. (B) The CD4+ AIM and IFNγ reactivity to S is shown as a function of donor age. Mild (n = 21, blue), moderate (n = 35, yellow), and severe (n = 33, red) disease states are plotted together. R and p-values are calculated by Spearman correlation.

Figure 6

SARS-CoV-2 spike protein and CD4-RE-specific CD4+ T cell reactivity. Mild (n = 6, blue), moderate (n = 6, yellow), and severe (n = 14, red) acute COVID-19 donors were assessed for T cell responses to SARS-CoV-2 S and non-Spike proteins. PBMCs from these donors were assayed with the S peptide pool of overlapping 15-mers by 10 and the CD4-RE pool of experimentally defined non-spike epitopes (Yu et al., Cell Host & Microbe 2022). (A) The magnitude for AIM+ CD4+ T cells specific for spike (circles) and CD4-RE (triangles) is shown for mild/moderate and severe COVID-19 donors. The dotted line indicates the LOS for the AIM assay, while the y-axis starts at the LOD. The frequency of positivity is indicated in pie charts for S and CD4-RE for each disease severity. (B) The ratios of CD4-RE to S AIM+ CD4+ responses are shown for responding donors (mild n = 4, moderate n = 2, and severe n = 3). Mann–Whitney or Wilcoxon tests were applied and p-values are shown for significant values. * p < 0.05.