The Allogeneic Effect Revisited: Exogenous Help for Endogenous, Tumor-Specific T Cells

Cy + Non-engrafting CD8-depleted DLI abrogates risk of GVHD, induces anti-tumor immunity, and prolongs survival of animals with established, metastatic hematologic and solid tumor malignancies. (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: (▴) versus (□) P =.04, (●) versus (□) P = .3. Results for 1B: (▴) versus (○) versus (○) P < .0001, (▴) versus (□) versus P = .0002, (○) versus (□) 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: (●) versus (▪) P < .0001, (▴) versus (●) 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: (●) versus (▪) P = .01, (▴) versus (●) P = .001. All experiments were repeated at least once.

Cy + CD8 depletion of DLI abrogates sustained engraftment of donor cells, GVHD and GVHD-associated aplasia (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 BALB/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.

Tumor expression of alloantigens is not required for the beneficial effect of Cy plus CD8-depleted DLI. CB6 F₁ mice (H-2^dxb; n = 10/group) received 3 × 10⁴ B16 melanoma cells i.v. on day 0 followed by treatment with Cy 200 mg/kg i.p. on day 14 (▪), or Cy 200 mg/kg i.p. on day 14 and 5 × 10⁷ spleen cells on day 15 from syngeneic (CB6F1) (□), 1 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.

The antitumor effect of Cy plus CD8-depleted allogeneic DLI requires host CD8⁺ T cells and is mediated by both a direct GVT and an indirect GVHD effect. (A) CD8⁻ DLI mediates antitumor 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 i.v. 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 i.p. on day 14 (▴), or 2.43 antibody on day 9 and every 3 weeks, and Cy 200 mg/kg i.p. on day 14 (▵), or Cy 200 mg/kg i.p. 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 i.p. 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, C) Cy + CD8-depleted DLI syngeneic to the tumor is sufficient to provide antitumor immunity. CB6F1 mice (H-2^b; n = 10/group) received 1.5 × 10⁴ B16 melanoma cells i.v. 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 i.p. on day 14 (▴), or 2.43 antibody on day 9 and every 3 weeks, and Cy 200 mg/kg i.p. on day 14 (▵), or Cy 200 mg/kg i.p. on day 14 and 5 × 10⁷ spleen cells from 1 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 i.p. on day 14, and 5 × 10⁷ spleen cells from 1 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.