Figure 3.

CD34-lineage cells give rise to effector T cells and are essential for tertiary lymphoid organ formation. A, Uniform manifold approximation and projection plot displaying the major natural killer and T cell subtypes (including CD4⁻CD8⁻ T, CD4⁺CD8⁺ T, CD4⁺ T, CD8⁺ toxicity T, naïve T, memory T, regulatory T, T helper 17, and T follicular helper [Tfh] cells) and 10 color-coded cell clusters in the 2-week (AG2w) and 4-week (AG4w) remodeled artery after allograft. There were 357 cells in the AG2w group and 7688 cells in the AG4w group. B, Bar plots showing the percentage distribution of various T cell subsets in arteries of indicated groups analyzed by single-cell RNA sequencing (left) and cytometry by time-of-flight (right). The subsets include CD4⁻CD8⁻ T, CD4⁺CD8⁺ T, CD4⁺ T, CD8⁺ toxicity T, natural killer, naïve T, memory T, regulatory T, T helper 1, T helper 17, and Tfh cells. C, Quantification of the ratio of ICOS⁺ cells in total tdTomato⁺CD3⁺ cells by FC (left) and cytometry by time-of-flight (right) in the indicated groups (n=5 per group). D, Representative FC plots (left) and quantification (right), showing the percentage of tdTomato⁺ and tdTomato⁻ cells among total Tfh cells (CD4⁺ICOS⁺) of a mouse AG4w remodeled artery (n=6). E, Quantification of the percentage of tdTomato⁺ and tdTomato⁻ cells in memory T, regulatory T, and T helper 17 cells of a mouse AG4w remodeled artery by cytometry by time-of-flight (n=6). F, Representative FAC plots (left) and quantification (right), showing the percentage of tdTomato⁺ cells in total germinal center B cells (B220⁺GL7⁺) of a mouse AG4w remodeled artery (n=6). G, quantification of the percentage of tdTomato⁺ and tdTomato⁻ cells in memory B, plasma B, and follicular B cells of a mouse AG4w remodeled artery by cytometry by time-of-flight (n=6). H, Heatmap showing the interaction score of T cell subtypes (columns) and B cell subtypes (rows), as determined by single-cell RNA sequencing. I, Quantification of the ratio of Tfh (left) and germinal center B (right) cells among total cells of remodeled arteries via FC in the indicated groups (n=5 per group). J, Quantification of the ratio of memory T, regulatory T and T helper 17 cells among total CD3⁺ cells of remodeled arteries via FC in the indicated groups (n=5 per group). K, Quantification of the ratio of memory B, plasma B, and follicular B cells among total B220⁺ cells of remodeled arteries via FC in the indicated groups (n=5 per group). L, Schematic illustrating the conclusion that CD34-lineage CD4⁺T cells differentiate into Tfh cells, which then interact with B cells to promote lymphocyte recruitment, proliferation, and tertiary lymphoid organ formation. Data shown are mean±SD (C through G and I through K). For D through G and I through K, normality of the data was analyzed by D’Agostino-Pearson omnibus test, and data that passed the normality tests were tested with standard unpaired Student t test with Welch’s correction. For C, data were first analyzed for normality test by a Shapiro-Wilk test, followed by 1-way ANOVA with Tukey test. P<0.05 was considered to be statistically significant. APB indicates antigen-presenting B cell; CTL, toxicity T cell; Cytof, cytometry by time-of-flight; DNT, double-negative T cell; DPT, double-positive T cell; DZ B, dark zone B cell; FC, flow cytometry; GC, germinal center; NK, natural killer; LZ B, light zone B cell Th1, T helper 1; Th17, T helper 17Tm, memory T; Tn, naïve T; Treg, regulatory T; TLO, tertiary lymphoid organ; and UMAP, uniform manifold approximation and projection.