4BL cells induce GrB⁺CD8⁺ T cells and retard tumor growth in old mice. The retarded B16 melanoma growth in old syngeneic mice (A) is lost in Old-restored mice (B). Young, Old-IgG, and Old-restored C57BL/6 mice (as in Figure 2B,D) were s.c. challenged with 10⁵ B16 melanoma cells. (C) 4BL cells are responsible for the retarded tumor growth. μMT mice with B16 melanoma were adoptively transferred with B cells from young mice (Young-B), Old-IgG mice (Old-IgG-B), or Old-restored mice (Old-restored-B). B16 melanoma growth in (C) correlates with the expansion of GrB⁺CD8⁺ T cells in PB (i), spleen (ii), and tumor (iii). Shown are mean tumor size (mm²) ± SEM (A-Bi), final tumor weight (g) ± SEM (Bii-C), and GrB⁺CD8⁺ T cells ± SEM (number ×10⁵, D) of 4 to 5-mice-per-group experiments independently reproduced at least 2 times. (E) Shown are a representative result showing differences in 4-1BBL⁺ B cells between auto-HSCT patient samples used, such as high (18%, Pat #1B) and low (4%, Pat #2, i) and their in vitro ability to induce GrB expression within healthy young people CD8⁺ T cells (mean ± SEM of triplicate experiment, ii). Control healthy donor B cells (HD, i-ii) contained <6% 4-1BBL⁺ B cells (as in Figure 1B). The GrB induction was completely blocked in the presence of antagonistic anti-4-1BB Ab (α4-1BB), but in not control Ab (ii).