Figure 3.

SARS-CoV-2-Reactive CD4⁺ T Cell Subsets Associated with Disease Severity

(A) Unsupervised clustering of COVID-19 patients based on the proportions of SARS-CoV-2-reactive CD4⁺ T cells in different clusters following 6 h peptide stimulation. Clusters with fewer than 5% of the total dataset are not depicted. Gender and hospitalization status per patient are indicated by different color schemes above the heatmap.

(B) Percentage of T_FH cells (clusters 0, 5, and 7) 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. Data are mean ± SEM; significance for comparisons was computed using Mann-Whitney U test; ns, non-significant P value.

(C) Proportion of clusters 5 and 0 cells in SARS-CoV-2-reactive T_FH cells (clusters 0, 5, and 7) in non-hospitalized and hospitalized COVID-19 patients_. Data are mean ± SEM; significance for comparisons was computed using Mann-Whitney U test; ^∗∗∗∗p < 0.0001.

(D) Violin plots showing normalized expression level (log₂(CPM+1)) of ZBTB32 and ZBED2 transcripts in SARS-CoV-2-reactive cells from clusters 0, 5, and 7 (top); color indicates percentage of cells expressing indicated transcript. Plots below show average expression and percent of cells expressing selected transcripts in indicated clusters.

(E) Scatterplot displaying normalized co-expression level (log₂(CPM+1)) between PRF1 and GZMB transcripts in SARS-CoV-2-reactive cells present in clusters 5 (left) and 0 (right). Numbers indicate percentage of cells in each quadrant.

(F) Correlation between percentage of SARS-CoV-2-reactive CD4⁺ T_FH cells and S1/S2 antibody titers in 15 non-hospitalized (left) and 20 hospitalized (right) COVID-19 patients. Correlation coefficient r and the related P value were computed using Spearman correlation; ^∗p < 0.05.

(G) Correlation between percentage of SARS-CoV-2-reactive CD4⁺ T_FH cells form cluster 5 as a frequency of total CD4⁺ T_FH and S1/S2 antibody titers (left two plots) and interval between symptom onset and blood draw (right two plots) in 15 non-hospitalized and 20 hospitalized (left) COVID-19 patients. Correlation coefficient r and the related P value were computed using Spearman correlation; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ns, non-significant P value.

(H) Single-cell trajectory analysis of cells in cluster 5 and 0 showing pseudotime, expression of indicated genes, and IFN response signature score.

See also Figure S3 and Table S3.