Fig 2: TIGIT is downregulated on memory B cells in MS.(A–C) Sorted CD20+CD27+ memory B cells from patients with MS (n = 8) and healthy donors (HD) (n = 9) were cultured with CD40L and IL-21 for 2 days, and RNA-Seq was performed. (A) Heatmap of DEGs (|log2 FC| >0.5, FDR < 0.1) in patients with MS and healthy donors. (B) Volcano plot depicting DEGs in memory B cells. Red dots represent significantly upregulated genes in MS-derived memory B cells, and blue dots represent significantly downregulated genes. Genes whose location is categorized as “plasma membrane” by IPA software are labeled. (C) IPA was performed to identify signatures related to altered molecular and cellular functions. Functions whose –log (Benjamini-Hochberg [B-H] P value) values were greater than 1.8 are shown. (D–F) Sorted CD20+CD27+ memory B cells from patients with MS and healthy donors (n = 12 each) were cultured with CD40L and IL-21 for 2 days. Gene expression was measured relative to B2M by qPCR (D and E). Representative flow data for TIGIT expression (F, left) and proportion of TIGIT+ cells (F, right). mB, memory B cells. Data are presented as the mean ± SEM and were evaluated by 2-tailed, unpaired Student’s t test (D–F). FSC-W, forward scatter width.

Fig 3: The CD40/TCF4/TIGIT axis is dysregulated on MS-derived memory B cells.(A–C) CD20+CD27+ memory B cells were transfected with siRNAs targeting ID2 and ID3 (siID2/ID3) or with siCtrl (n = 4). (A) Representative flow data for TIGIT expression. (B) Proportion of TIGIT+ cells by flow cytometric analysis. (C) TIGIT, TCF4, ID2, and ID3 expression levels were measured relative to B2M by qPCR (FC versus the siRNA/siXBP1 condition). (D and E) Sorted CD20+CD27+ memory B cells from healthy donors (n = 12) and patients with MS (n = 12) were cultured with CD40L+IL-21 for 2 days, and ID2, ID3 (D), and TCF4 (E) expression levels were measured relative to B2M by qPCR. All data are presented as the mean ± SEM and were evaluated by 2-tailed, unpaired Student’s t test (B, D, and E).

Fig 4: TIGIT+ B cells suppress the proliferation of CCR6+ Tfh cells.(A–C) Sorted CD4+CD45RA–CXCR5+ Tfh cells and CD4+CD45RA–CXCR5– non-Tfh cells were stimulated with anti-CD3/anti-CD28 antibodies (each 1 µg/mL) for 3 days. Representative flow data for CD155 expression (A, left) and the proportion of CD155+ cells (A, right) (n = 10) are shown. (B) PVR mRNA expression was measured relative to B2M by qPCR (n = 7). (C) Proportion of CD112+ cells (n = 10). (D) Experimental workflow for coculture assays with sorted CD20+CD27+ memory B cells and CD4+CD45RA–CXCR5+ Tfh cells. (E) Representative flow data for Tfh cell proliferation. (F) Proportion of proliferated CellTrace Violet+ (CTV+) cells. (G) IL-17 and IFN-? expression in the supernatants of coculture assays was evaluated by ELISA. (H) Correlation between CD4+CD45RA–CXCR5+CCR6+ Tfh cells (percentage of CD4+CD45RA–CXCR5+ Tfh cells) and TIGIT+ cells (percentage of CD20+CD27+ memory B cells). Data for healthy donors are indicated by blue dots (n = 15) and by red dots for patients with MS (n = 16). Linear regression is shown with a 95% CI (pink area). (I) Proportion of CCR6+ Tfh cells between healthy donors (n = 18) and patients with MS (n = 17). Data are presented as the mean ± SEM and were evaluated by 2-tailed, unpaired Student’s t test (A–C and I) or Wilcoxon matched-pairs, signed-rank test (F and G).

Fig 5: TIGIT is clearly separated from IL-10 expression.(A) Representative flow data of TIGIT and IL-10 expression in CD40L+IL-21–stimulated memory B cells. (B) The proportions of TIGIT+IL-10+, TIGIT+IL-10–, and TIGIT–IL-10+ (n = 23) cells were evaluated by 2-way ANOVA with Dunn’s multiple-comparison test. Data are presented as the mean ± SEM. (C) Experimental workflow for RNA-Seq with DN (TIGIT–IL-10–) cells, TIGIT (TIGIT+IL-10–) cells, and IL-10 (TIGIT–IL-10+) cells. (D) PCA of RNA-Seq transcriptomes (n = 3 healthy donors). (E) Heatmap of representative genes that were differentially expressed (|log2 FC| >0.5, FDR < 0.1) among 3 groups. (F) Representative flow data for PD-1 and CD226 (left) and their frequencies among TIGIT+ and TIGIT– cells (right). (G) Representative flow data for FGL2 (left) and its frequency among TIGIT+ and TIGIT– cells (right). Data are presented as the mean ± SEM. Significance was determined by 2-tailed, unpaired Student’s t test (F and G).