4BL cells induce GrB⁺CD8⁺ T cells. (A) Old human B cells induce higher GrB expression in target CD8⁺ T cells from young donors in mixed lymphocyte reaction (MLR). Shown GrB and IFNγ induction in CD8⁺ T cells (i, representative dot plot, %) and its mean (ii, % after deducting background response without B cells, No B) ± SEM examined in triplicate experiments and reproduced at least 3 times. (B) Positive correlation (P < .0001) between the induction of GrB (y-axis) in young CD8⁺ T cells and 4-1BBL expression levels on B cells (x-axis). (C) Murine 4BL cells induce GrB in TCR transgenic pmel CD8⁺ T cells by presenting cognate antigen (gp100_25-32 peptide). eFluor670-labeled pmel CD8⁺ T cells were in vitro stimulated with B cells from young, Old-IgG, and Old-restored mice (as in Figure 2D) pulsed with gp100_25-32 (gray bars) or control SPANX peptide (open bars). Shown is the mean ± SEM (%) of GrB within proliferating pmel CD8⁺ T cells in triplicate experiments reproduced 3 times. (D) ABCs (sort-purified, open bars) from young and old mice (as in Figure 2C) were compared with total B cells (gray bars) for the ability to induce GrB⁺CD8⁺ T cells stimulated with anti-CD3 Ab. (E) To demonstrate the 4BL cell-induced in vivo expansion of GrB⁺CD8⁺ T cells, naïve old mouse B cells and eFluor670-labeled CD8⁺ T cells from naïve congenic mice were i.v. injected into μMT mice bearing B16 melanoma (schema). One week later, GrB was quantified within proliferating LN CD8⁺ T cells using gp100_25-32 dextramer. Shown are a representative dot plot (i-ii, %) and mean ± SEM (ii, %) of a 4 to 5-mice-per-group experiment independently reproduced 2 times.