4BL cells, present in young subjects, accumulate with old age and induce GrB⁺CD8⁺ cells requiring 4-1BBL/4-1BB axis. (A) Antagonist anti-4-1BBL (α4-1BBL), but not isotype control Ab, abrogates the ability of old-mouse B cells (Old-B) to induce GrB in young-mouse CD8⁺ T cells in vitro. (B-C) As such, anti-4-1BBL Ab blocks the antitumor benefit of old-mouse B cells. (B) μMT mice with B16 melanoma were adoptively transferred with naïve mouse B cells (μMT + Young-B) or B cells from old mice pretreated with either anti-4-1BBL Ab (μMT + Old-B + α4-1BBL) or control Ab (μMT + Old-B + IgG). (C) WT C57BL/C old mice with B16 melanoma were i.p. injected (at days 1, 4, 8, and 11) with anti-4-1BBL Ab (Old-α41BBL) or control Ab (Old-IgG). At day 13 post–tumor challenge, the anti-4-1BBL Ab–injected old mice were also divided into 2 groups, and one group received adoptive transfer of B cells from syngeneic naïve old mice (Old-α41BBL + Old-B). Shown are mean tumor size (mm², Bi, C) and tumor weight (g, Bii) ± SEM of a 4 to 5-mice-per-group experiment repeated 3 times. (D) Old-mouse B cells cannot induce GrB expression in 4-1BB–deficient CD8⁺ T cells (4-1BB KO). Shown are mean ± SEM (GrB, %) within proliferating CD8⁺ T cells of WT or congenic 4-1BB KO mice. (E) Antagonistic 4-1BB Ab, but not control Ab, blocks human GrB expression in CD8⁺ T cells induced by old human B cells. This is shown as a fold change (mean ± SEM) of GrB⁺CD8⁺ cells induced with B cells of young people (dotted line). (F) To demonstrate the presence of 4BL cells in young people, 4-1BBL⁺ B cells were sort-purified (>90%) and used to stimulate GrB (mean ± SEM) in proliferating CD8⁺ T cells in triplicate experiments reproduced independently 4 times.