Figure 1. RLI induces the in vivo expansion of recipient splenic T, B and CD4⁻CD8⁻ cells. (A) Experimental design: BALB/c (H2^d) recipient mice were conditioned with anti-CD4 and anti-CD8 depleting mAbs i.p. on Day −5, CTX 200 mg/kg i.p. on Day −1, and 7 Gy thymic irradiation on Day 0. Some mice were transplanted with 25 × 10⁶ B10.BR BMC i.v. on Day 0. Some groups received 3 × 10⁷ recipient spleen cells (RLI) i.v. on Day +42 post-BMT and A20 BALB/c B lymphoma cells (5 × 10⁵) on Day +49 post-BMT. Peripheral WBC and, in some mice, splenocyte chimerism and phenotype were analyzed before and after RLI. (B-G): Mean (±SD) of absolute total spleen cell numbers (B), absolute splenic recipient (H-D^d+) (stippled bars) and donor (H-D^d−) (solid bars) CD4⁺ T cell (C), CD8⁺ T cell (D), B cell (E), monocyte/granulocyte (F), and CD4⁻CD8⁻B220⁻MAC1⁻ (G) cell numbers in conditioned mice that did not receive BMT or RLI (group A: n = 5), or received BMT without RLI (group B: n = 5) or received BMT and RLI (group C: n = 6), 3 weeks post-RLI. Results of two similar experiments are combined. Significant P values of statistical tests performed between group C and groups A and B are shown (*P < 0.01, **P < 0.001).

Figure 2. RLI induces anti-donor proliferative and cytotoxic responses but no anti-tumor proliferative responses. BALB/c (H-2^d) mice were treated as described in Fig. 1A. Some mice were sacrificed at the indicated times, and splenocytes were used in MLR and cytotoxicity assays as described in Materials and methods. (A) Mean (±SEM) stimulation index indicating the relative proliferative responses of splenocytes from chimeras that did not receive RLI (solid bars, n = 3), chimeric recipients of RLI (stippled bars, n = 4), conditioned mice that did not receive BMT or RLI (open bars, n = 4) and naïve third party mice (C57BL/6, H-2^b) (dashed bars, n = 3), in response to donor (B10.BR, H-2^k) splenocytes, recipient (BALB/c, H-2^d) splenocytes and tumor (A20, H-2^d) cells, 1 week before RLI (pre-RLI) and 3 weeks after RLI (post-RLI) (n indicates the number of mice analyzed per time point). (B) Mean (±SEM) percent specific lysis of donor (B10.BR) LPS blasts after 5 days of co-culture with irradiated donor (B10.BR) splenocytes, 1 to 3 weeks post-RLI (left panel) and 12 weeks post-RLI (right panel) by splenocytes from naïve third party (C57BL/6) mice (*, n = 6), conditioned non-BMT, non-RLI mice (○, n = 2 to 3), chimeras that did not receive RLI (, n = 3 to 5) and chimeric recipients of RLI [♦ at 1 week post-RLI, n = 2 and at 3 weeks post-RLI (n = 5) and ▪ at 12 weeks post-RLI (n = 4 to 8)]. Results of two similar experiments are combined in panel A, and results of three similar experiments are combined in panels B. Significant P values of statistical tests performed between each group and that of chimeras not receiving RLI are shown (*P < 0.04).

Figure 3. Anti-tumor cytotoxic responses can be detected following stimulation with A20 and BALB/c lysates. BALB/c (H-2^d) mice were treated as described in Fig. 1A. Animals were sacrificed at the indicated times and splenocytes were used in cytotoxicity assays as described in Materials and methods. Each panel shows the mean (±SEM) percent specific lysis of tumor cells after 5 day-stimulation with the indicated source of sonicates for splenocytes from naïve BALB/c mice (*, n = 2 at each time), chimeric recipients of RLI plus A20 (▪, n = 2 at 3 weeks and n = 6 at 12 weeks post-RLI) or from chimeric recipients of RLI without A20 (□, n = 1 at 3 weeks and n = 2 at 12 weeks post-RLI). Results from three similar experiments are combined.

Figure 4. Detection of specific anti-tumor CD8 or CD4⁻CD8⁻ cell-mediated cytotoxic responses in chimeras receiving RLI. Anti-A20 tumor specific lysis of non-TCD (●), CD4 TCD (), CD8 TCD () and CD4+8 TCD (□) fractions of A20-stimulated splenocytes from two separate long-term (following tumor injection) tumor survivor animals that rejected the donor marrow graft spontaneously (left panel) or after receiving RLI (right panel).

Figure 5. In vitro stimulation of splenocytes with donor sonicates generates specific anti-tumor cytotoxic responses in chimeric recipients of RLI. BALB/c (H-2^d) mice were conditioned as described in Fig. 1A. Animals were sacrificed at the indicated times post-RLI, and splenocytes were used in cytotoxicity assays as described in Materials and methods. (A, B) Mean (±SEM) percent specific lysis of the indicated target after 5 days of co-culture with donor (B10.BR, H-2^k) sonicates at 3 weeks post-RLI (A, left panel) and at 12 weeks post-RLI (A, right panel and B). Responders were naïve BALB/c mice (*, n = 2, at each time), long tumor survivor chimeric recipients of RLI (, n = 2 at 3 weeks and n = 6 at 12 weeks post-RLI), and chimeric recipients of RLI that did not receive A20 (, n = 1 at 3 weeks and n = 2 at 12 weeks post-RLI). Killing of tumor cells (A) and of recipient LPS blasts (B) by naive third party splenocytes stimulated with A20 cells served as a positive control (×, n = 3 in each case). Results from three similar experiments are combined.

Figure 6. RLI induces anti-tumor responses against A20 in a haplotype-mismatched strain combination by similar mechanisms as those involved in the fully mismatched combination model. BALB/c (H-2^d) mice were conditioned as described in Fig. 1A, and some groups were transplanted with 25 × 10⁶ BMC from either B10.BR (H-2^k) or (B10.BR × BALB/c)F1 (H-2^kd) donors. Some mice received RLI (30 × 10⁶ BALB/c splenocytes) on Day 42 post-BMT and A20 (5 × 10⁵ cells) on Day 49 post-BMT. (A) Survival of nontumor controls (*, n = 6) and tumor survival of conditioned mice that did not receive BMT or RLI (, group A, n = 5), haplotype-mismatched chimeras that did not receive RLI (, group B, n = 10), haplotype-mismatched chimeric recipients of RLI (, group C, n = 12), fully mismatched chimeras that did not receive RLI (, group D, n = 8), and fully mismatched chimeric recipients of RLI (●, group E, n = 8). (B) Mean (±SD) of percentages of peripheral blood donor monocytes at the indicated times in groups B to E as described in panel (A). (C) Serum IFN-γ levels measured 7 days post-RLI in groups A to E described in panel (A). (D and E) Some animals were sacrificed 12 weeks post-RLI, and splenocytes were used in cytotoxicity assays as described in Materials and methods. (D) Percent specific lysis of donor (B10.BR × BALB/c)F1 LPS blasts (H-2^kd) (first panel) and of A20 tumor cells (H-2^d) (second panel), after 5 days of co-culture with irradiated donor splenocytes and A20 cells, respectively, by a naïve third party (C57BL/6) responder (●), a conditioned animal that did not receive BMT, RLI or A20 (○), a haplotype-mismatched chimera that did not receive RLI or A20 (), a haplotype-mismatched chimeric recipient of RLI that showed long-term survival after A20 injection (▪) and a haplotype-mismatched chimeric recipient of RLI that did not receive A20 (□). (E) Specific lysis of A20 tumor cells by splenocytes from a haplotype-mismatched chimeric recipient of RLI that showed long-term survival after A20 injection, after 5 days of stimulation with the indicated source of antigens is compared to the anti-tumor cytotoxic responses of a chimeric animal that did not receive RLI after stimulation with the same sources of antigens. Results from one representative experiment of two performed are shown.