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. 2023 Jul 10;120(29):e2305764120. doi: 10.1073/pnas.2305764120

Fig. 4.

Fig. 4.

An effectorness gradient underlies CD8+ T cell heterogeneity in human AA skin. (A) UMAP of scRNAseq data obtained from scalp biopsies of 5 AA patients and 2 healthy controls, split across disease condition. Unsupervised clustering of 32,134 cells (21,766 AA; 32,134 CTRL) after quality control and CCA- based alignment uncovered 16 distinct populations. Note the marked increase in T cell infiltration in AA skin. (B) Percentage of T cells relative to the total number of sequenced immune cells, averaged across the replicates for each disease condition, showing a statistically significant (P = 0.002) increase in T cells in AA skin. (C) Percentage of specific T cell subsets relative to the total number of sequenced immune cells, averaged across the replicates for each disease condition. We observed statistically significant increases in the frequency of CD8+ T cells (P = 0.017), CD4+ Treg (P = 0.0051), NK T cells (P = 0.0016) and γδ T cells (P = 0.047) in AA skin. Significance is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001. (D) UMAP of AA CD8+ T cell subsets. Unsupervised reclustering of AA CD8+ T cells uncovered two subpopulations. (E) Heatmap showing enriched expression of IL7R and TCF7 in cluster 1, while cluster 2 cells showed enriched expression of HSPA1ACD69IFNGGZMAGZMKPRF1, and XCL1. (F) Pseudotime trajectory analysis of AA CD8+ T cells, colored by cluster (Top Left), position along pseudotime (Bottom Left), Effector signature scores (Top Right), and Resident signature scores (Bottom Right). Pink dashed arrow denotes general direction of inferred differentiation across pseudotime. (G) Expression of select genes as identified in (B) along pseudotime, colored by cluster. (H) Pseudotime values were used to compute effectorness scores for each cell. Effectorness scores overlaid onto the UMAP of AA-associated CD8+ T cells demonstrated a high degree of correlation with the separation of the two CD8+ T cell subsets via unsupervised clustering of their transcriptional profiles, as shown in (D). Error bars represent SD. DS, dermal sheath; Fib, fibroblasts; Lymph, lymphatic vessel; VSM, vascular smooth muscle.