(a,b) CD8 depletion of DLI abrogates sustained engraftment of donor cells administered after Cy. BALB/c mice (H-2^d; n=10/group) were conditioned with Cy 200 mg/kg IP on day −1 and received 10⁶ A20 lymphoma cells IV on Day 0. Mice received 5 × 10⁷ spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors on Day 0, either undepleted (■) or depleted of CD4⁺ T cells (●), CD8⁺ T cells (▲), or both (□). Donor CD4⁺ T cell (a) and B220 (b) chimerism in peripheral blood was measured on days 3, 7, 14, and 21 after DLI via staining for H-2K^b and H-2K^d antibody. (c) CD8 depletion abrogates DLI-induced GVHD. BALB/c mice (H-2^d; n=10/group) were conditioned with Cy 200 mg/kg IP on day −1. Mice received 5 × 10⁷ spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors on Day 0, either undepleted (■) or depleted of CD4⁺ T cells (●), CD8⁺ T cells (▲), or both (□). (d) CD8 depletion of DLI does not induce GVHD-associated aplasia. BALB/c mice (H-2^d; n=10/group) were conditioned with Cy 200 mg/kg IP on day −1. Mice received 5 × 10⁷ spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors on Day 0, either undepleted (■) or depleted of CD4⁺ T cells (●), CD8⁺ T cells (▲), or 5 × 10⁷ spleen cells from syngeneic BALB/c mice (H-2^d) (○). ▲ indicates the WBC in a naïve BLAB/c mouse. Peripheral white blood cell count was monitored via TruCOUNT™ on days 3, 7, 14, and 21 after DLI. All experiments were repeated at least once.

(a) CD8⁻ DLI mediates anti-tumor immunity through a GVT effect that requires direct tumor expression of alloantigens. BALB/c mice (H-2^d; n=10/group) received 10⁶ A20 cells IV on Day 0, either alone (■) or followed by treatment with 2.43 antibody on Day 9 and every 3 weeks (□), or Cy 200 mg/kg IP on day 14 (▲), or 2.43 antibody on Day 9 and every 3 weeks, and Cy 200 mg/kg IP on day 14 (△), or Cy 200 mg/kg IP on day 14 and 5 × 10⁷ spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors, depleted of CD8⁺ T cells on Day 15 (●), or 2.43 antibody on Day 9 and every 3 weeks, Cy 200 mg/kg IP on day 14, and 5 × 10⁷ spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors, depleted of CD8⁺ T cells on Day 15 (○). Results show: (▲) v (△) p=.005, (○) v (△) p=.005, (●) v (○) p=.08. (b) Cy + CD8-depleted DLI syngeneic to the tumor is sufficient to provide anti-tumor immunity. CB6F1 mice (H-2^b; n=10/group) received 1.5 ×10⁴ B16 melanoma cells IV on Day 0 either alone (■) or followed by treatment 2.43 antibody on Day 9 and every 3 weeks (□), or Cy 200 mg/kg IP on day 14 (▲), or 2.43 antibody on Day 9 and every 3 weeks, and Cy 200 mg/kg IP on day 14 (△), or Cy 200 mg/kg IP on day 14 and 5 × 10⁷ spleen cells from one haplotype matched C57BL/6 (H-2^b) donors, depleted of CD8⁺ T cells on Day 15 (●), or 2.43 antibody on Day 9 and every 3 weeks, Cy 200 mg/kg IP on day 14, and 5 × 10⁷ spleen cells from one haplotype matched C57BL/6 (H-2^b) donors, depleted of CD8⁺ T cells on Day 15 (○). Results show: (●) v (▲) p=.02, (○) v (△) p=.41. All experiments were repeated at least once.

(a,b) CD8 depletion of DLI abrogates risk of GVHD without compromising anti-tumor immunity. BALB/c mice (H-2^d; n=10/group) either received 10⁶ A20 lymphoma cells IV on Day 0 alone (△) or were conditioned with Cy 200 mg/kg IP on day −1 and received 10⁶ A20 lymphoma cells IV on Day 0 (○). Additional mice were then treated with 5 × 10⁷ whole spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors, either undepleted (■) or depleted of CD4⁺ T cells (●), CD8⁺ T cells (▲), or both (□). Results for 1a: (▲) v (□) p =.04, (●) v (□) p=.03. Results for 1b: (▲) v (○) v (○) p<.0001, (▲) v (□) v p=.0002, (○) v ((□) p=.17. (c) Cy + CD8-depleted allogeneic DLI prolongs survival of animals with established, metastatic lymphoma. BALB/c mice (H-2^d; n=10/group) received 10⁶ A20 cells IV on Day 0 either alone (■), or followed by treatment with Cy 200 mg/kg IP on day 14 (●), or Cy 200 mg/kg IP on day 14 and 5 × 10⁷ spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors, depleted of CD8+ T cells on Day 15 (▲). Results show: (●) v (■) p<.0001, (●) v (▲) p=.02. (d) Cy + CD8-depleted allogeneic DLI prolongs survival of animals with established, metastatic renal cell carcinoma. BALB/c mice (H-2^d; n=10/group) received 10⁶ RENCA cells IV alone on Day 0 either alone (■), or followed by treatment with Cy 200 mg/kg IP on day 14 (●), or Cy 200 mg/kg IP on day 14 and 5 × 10⁷ spleen cells from fully MHC-mismatched C57BL/6 (H-2^b) donors, depleted of CD8+ T cells on Day 15 (▲). Results show: (●) v (■) p=.01, (●) v (▲) p=.001. All experiments were repeated at least once.

CB6 F₁ mice (H-2^dxb; n=10/group) received 3×10⁴ B16 melanoma cells IV on Day 0 followed by treatment with Cy 200 mg/kg IP on day 14 (■), or Cy 200 mg/kg IP on Day 14 and 5 × 10⁷ spleen cells on Day 15 from syngeneic (CB6F1) (□), one haplotype matched (C57Bl/6) (●), or fully MHC mismatched (BALB/c) (○) donors, depleted of CD8⁺ T cells. The table included describes the GVT and GVH relationships between the donor and recipient strains given that B16 melanoma is of B6 background. Results show: (●) v (■) p=.04, (○) v (●) p=.06, (●) v (□) p= .04, (○) v (□) p=.03. All experiments were repeated at least once.