Figure 5.

Analysis of SARS-CoV-2-Reactive CD4⁺ T Cells from 24 h Stimulation Condition

(A) Single-cell transcriptomes of sorted CD137⁺ CD69⁺ memory CD4⁺ T cells following 24 h stimulation with SARS-CoV-2-specific peptide megapools are displayed by UMAP. Seurat-based clustering of 38,519 cells colored based on cluster type.

(B) Heatmap showing expression of the most significantly enriched transcripts in each cluster (see Table S5C). Seurat marker gene analysis—comparison of cluster of interest versus all other cells—shown are top 200 transcripts with adjusted P value < 0.05, log₂ fold change > 0.25, and > 10% difference in the percentage of cells expressing differentially expressed transcript between two groups compared.

(C) Plot showing average expression (color scale) and percent of expression (size scale) of selected marker gene transcripts in each cluster.

(D) UMAP showing Seurat-normalized expression level of FOXP3 transcripts (left). Percentage of T_REG cells (cluster A) in the total SARS-CoV-2-reactive CD4⁺ T cell pool for non-hospitalized and hospitalized COVID-19 patients; dots indicate data from a single subject (right plot). Data are mean ± SEM; significance for comparisons was computed using Mann-Whitney U test; ^∗∗∗p < 0.001.

(E) Average frequency of cells per cluster from hospitalized and non-hospitalized COVID-19 patients.

(F) UMAP showing CD4-CTL signature score for each cell (left) and percentage of CD4-CTLs (clusters B and F) in the total SARS-CoV-2-reactive CD4⁺ T cell pool for non-hospitalized and hospitalized COVID-19 patients; dots indicate data from a single subject (left plot). Data are mean ± SEM. Significance for comparisons was computed using Mann-Whitney U test; ns, non-significant P value.

(G) Correlation between percentage of SARS-CoV-2-reactive CD4⁺ T_REG and percentage of SARS-CoV-2-reactive CD4-CTLs in 13 non-hospitalized and 17 hospitalized (left) COVID-19 patients. Correlation coefficient r and the related P value were computed using Spearman correlation; ^∗∗∗∗p < 0.0001.

(H) UMAP showing Seurat-normalized expression level of IL1R2 transcripts (left) and percentage of T_FR cells (IL1R2-expressing cells in cluster A) in the total SARS-CoV-2-reactive CD4⁺ T cell pool for non-hospitalized and hospitalized COVID-19 patients; dots indicate data from a single subject (left plot). Data are mean ± SEM; significance for comparisons were computed using Mann-Whitney U test; ^∗∗∗p < 0.001.

(I) Correlation between percentage of SARS-CoV-2-reactive cytotoxic T_FH cells (proportion of T_FH cells in cluster 5, from 6 h stimulation dataset as in Figure 3C) and percentage of T_FR cells (IL1R2-expressing cells in cluster A) in 25 COVID-19 patients (left). Correlation coefficient r was computed using Spearman correlation; ns, non-significant P value..

See also Figure S5 and Table S5.