Fig. 5: P4 memory B cells express functional TIGIT and granzyme B that can directly act on T cells.

a RNA-seq t-SNE plots showing clustering of P1–P6 B cells subsets with all genes (left) or genes related to cell surface markers (right). Individual color indicates each of the B cell populations (P1–P6) and each dot represents an individual donor. Data from experiments performed with cells from different healthy subjects (n = 6) are presented. b RNA-seq volcano plots of differential gene expression between P (2, 4) and P (1, 3, 5, 6) in before (left) and after overnight activation (right) with CpG-B. FC fold change. c, d Representative FACS data (left) and summarized data (right) showing the frequency of surface TIGIT (c) and intracellular granzyme B (d) expression by sort-purified P1–P6 B cells upon 48 h CpG-B stimulation. PMA, ionomycin, brefeldin A, and monensin cocktails were added 5 h before staining them. Following surface staring with CD19 and TIGIT, intracellular granzyme B staining was performed. Data from six independent experiments performed with cells from different healthy subjects (n = 6). Isotype antibody staining was performed using mixture of P1–P6 B cells after CpG-B stimulation. Cells were gated based on isotype antibody staining. e, f Summarized data showing the suppression of CD4⁺ T cell responses (IFNγ expression in upper panels and IL-17 expression in lower panels) by P1, P2, and P4 B cells in presence or absence of anti-TIGIT (e) and granzyme B blocker (f). FACS-sorted P1, P2, and P4 B cells were stimulated for 48 h with CpG-B, preincubated with anti-TIGIT or granzyme B blocker or isotypes, and then cocultured for 4 days with autologous CD4⁺ T cells stimulated with anti-CD3/anti-CD28 beads. PMA, ionomycin, brefeldin A, and monensin cocktails were added 5 h before staining for intracellular cytokines. Data from four independent experiments performed with cells from different healthy subjects (n = 5). Error bars are mean ± SD. P values were determined with one-way ANOVA with Holm–Sidak’s multiple comparisons test (c, d) and with a two-tailed paired t test (e, f).