Donor non-CD8⁺ cells were required for optimal antitumor immunity induced by combination therapy with gp96-Ig-secreting tumor cell vaccination and IL-2 cytokine-antibody complexes in syngeneic HSCT recipients with lymphoma. Transplants, tumor inoculation, and treatments were performed as in Figure 5; however, some recipients received BM supplemented with purified CD4⁺ (≥88%) or CD8⁺ (≥90%) T cells in the absence of OT-I. (A) CD4⁺ and CD8⁺ T-cell content of purified CD4⁺ or CD8⁺ T cells. (B) Donor CD8⁺ T cells efficiently expanded following vaccination and IL-2_S4B6 in the absence of donor CD4⁺ T cells. Donor CD8⁺ T-cell frequency in the peripheral blood; n = 5; ●, E.G7-gp96-Ig; ○, E.G7-gp96-Ig (purified CD8); ■, E.G7-gp96-Ig + IL-2_S4B6; □, E.G7-gp96-Ig + IL-2_S4B6 (purified CD8). The results for E.G7-gp96-Ig + IL-2_S4B6 and E.G7-gp96-Ig + IL-2_S4B6 (purified CD8) were repeated in an independent experiment. (C) In the absence of donor CD4⁺ T cells, donor CD8⁺ T-effector cells were not efficiently generated following vaccine and IL-2_S4B6. Donor CD8⁺ T_eff (CD62L⁻ CD44⁺) cell frequency in the peripheral blood; n = 5 from a representative of 2 experiments; ●, E.G7-gp96-Ig; ■, E.G7-gp96-Ig + IL-2_S4B6; □, E.G7-gp96-Ig + IL-2_S4B6 (purified CD8). The results for E.G7-gp96-Ig + IL-2_S4B6 and E.G7-gp96-Ig + IL-2_S4B6 (purified CD8) were repeated in an independent experiment. (D) Survival benefit of vaccination and IL-2_S4B6 therapy is reduced in lymphoma-bearing HSCT recipients receiving TCD-BM supplemented with purified CD8⁺ T cells and abolished in recipients of purified CD4⁺ T cells; n = 5 to 10 from a pool of 3 experiments; x, no vaccine; ●, E.G7-gp96-Ig; ■, E.G7-gp96-Ig + IL-2_S4B6; □, E.G7-gp96-Ig + IL-2_S4B6 (purified CD8); ◊, E.G7-gp96-Ig + IL-2_S4B6 (purified CD4).