Flow cytometric analysis of B-lineage cells and transplantation of T_FH-tumor and GCB cells from the spleen of tumor-bearing MxTR. (A) Proportions of B220⁺Cd19⁺/Cd138⁻ cells, Cd138⁺/7AAD⁻ cells, and Cd138⁻B220⁺Cd19⁺Fas⁺Gl-7⁺ (GCB)/B cells in the spleen from mice with the indicated genotypes at 20 and 40 weeks of age. MxTR, n = 6; MxWT, n = 4; CD4TR, n = 4; and CD4WT, n = 5. (B) Representative t-Distributed Stochastic Neighbor Embedding (tSNE) plots of flow cytometric data from B-cell fractions in the spleen of mice of the indicated genotypes at 40 weeks of age. B-lineage markers, including Gl-7, Fas, B220, Cd138, and Cd19. (C) tSNE plots of manually gated and integrated Cd138⁻Cd19⁺B220⁺Fas⁺Gl-7⁺ (red), Cd138⁻Cd19⁺B220⁺Fas⁻Gl-7⁻ (orange), Cd138⁺ (light blue) cells, and others (gray) using data from Figure 2B. (D) Representative flow cytometry plots of Cd138, Cd19, Fas, Gl-7, Cxcr4, and Cd86 in spleen cells from tumor-bearing MxTR and MxWT at ∼50 weeks of age. (E) Histogram of Cd86 or Cxcr4 expression of GCB fractions in spleen cells from tumor-bearing MxTR and MxWT. (F) Workflows of transplant experiments with GCB and T_FH cells sorted from tumors of MxTR transplanted into nude mice. i.p., intraperitoneal injection; no. inj., no injection. (G) Proportions of H2kb⁺ donor-derived cells in spleen cells of the indicated groups on day 7 after injection. Five groups (i–v) represent (i) T_FH + GCB cells, (ii) T_FH + non-GCB cells, (iii) T_FH cells, (iv) no. inj., and (v) whole cells. ∗∗∗P value < .001; n.s., not significant.