Fig. 3

Remodeling of CD4 + T cells after chemoimmunotherapy and the spatial distribution of CD4 + T-cell subclusters in NSCLC tumors. A Reclustering of CD4 + T cells into 8 subpopulations shown by a t-SNE plot (left) and the distribution of CD4 + T cells in the treatment-naïve and posttreatment groups (right). B Heatmaps showing the significantly upregulated genes in all the CD4 + T-cell subclusters. C Box plot showing the comparison of the fractions of CD4 + T-cell subtypes between the TN and PT (Wilcoxon, * indicates P < 0.05). D The top 20 KEGG pathways enriched with the upregulated genes in the CD4 + Th17 subtype (adjusted P < 0.05). E Violin plots comparing the average exhausted gene signature of CD4 + T cells between the TN and PT groups. The violin is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to the maximum and minimum value. P values determined by two-sided Wilcoxon rank-sum test. F Violin plots comparing the average exhausted gene signature between CD4 + T-cell subtypes in the PT group. P values determined by two-sided Wilcoxon rank-sum test. G Bubble plot showing the top 20 KEGG pathways enriched by the DEGs from the CD4 + Th17 subtype between the TN and PT groups. Each bubble indicates a signaling pathway (left), and the top 20 pathways associated with the relative bubbles are labeled, as listed in the right panel. Box plot indicating the proportion of Th17 CD4 + T cells (H), Treg CD4 + T cells (I) and ratio of Th17/Treg (J) in responder and non-responder. P values determined by Wilcoxon tests. K UMAP plot showing the cell subtypes identified in Hu’s cohort. L UMAP plot showing the identification of subclusters of CD4 + T cells. UMAP plots demonstrating the expression of maker genes GZMA (M) and FOXP3 (N) in subclusters of CD4 + T cells. O The proportions of Th17 (left), Treg CD4 + T cells (middle) and ratio of Th17/Treg (right) in pre-treatment and post-treatment groups were shown by box plots. P values determined by Wilcoxon tests. P The proportions of Th17 (left), Treg CD4 + T cells (middle) and ratio of Th17/Treg (right) in non-responders and responders were shown by box plots. P values determined by Wilcoxon tests. Q Differentiation trajectory of all CD4 + T-cell subtypes; the subtypes are labeled with different colors. The numbers on the trajectory tree indicate the branch nodes, and the differentiation tree was divided into 5 branches. R Heatmaps showing the signature scores of Th17/Treg in each spot of the tissue sections. The spots were labeled with colors, and red color indicates high score, while blue color indicates low score. S Box plots showing the level of Log2(Th17/Treg) in each section (left) and each group (right). Each spot is considered as a duplicate, and P values were determined by two-sided Wilcoxon rank-sum test