The allogeneic effect revisited: exogenous help for endogenous, tumor-specific T cells

Abstract

The "allogeneic effect" refers to the induction of host B cell antibody synthesis or host T cell cytotoxicity, including tumoricidal activity, by an infusion of allogeneic lymphocytes. We show that treatment of mice with cyclophosphamide (Cy) followed by CD8(+) T cell-depleted allogeneic donor lymphocyte infusion (Cy + CD8(-) DLI) induces regression of established tumors with minimal toxicity in models of both hematologic and solid cancers, even though the donor cells are eventually rejected by the host immune system. The optimal antitumor effect of Cy + CD8(-) DLI required the presence of donor CD4(+) T cells, host CD8(+) T cells, and alloantigen expression by normal host but not tumor tissue. The results support a model in which a donor CD4(+) T cell-mediated graft-versus-host (GVH) reaction effectively awakens antitumor immunity among Cy-resistant host CD8(+) T cells. These events provide the cellular mechanism of the "allogeneic effect" in antitumor immunity. Cy + CD8(-) DLI may be an effective and minimally toxic strategy for awakening the host immune response to advanced cancers.

Figures

Figure 1. 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-2d; 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-2b) 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-2d; 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-2b) 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-2d; 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-2b) donors, depleted of CD8+ T cells on Day 15 (▲). Results show: (●) v (■) p=.01, (●) v (▲) p=.001. All experiments were repeated at least once.

Figure 2. 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-2d; 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-2b) 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-2Kb and H-2Kd antibody. (c) CD8 depletion abrogates DLI-induced GVHD. BALB/c mice (H-2d; 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-2b) 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-2d; 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-2b) 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-2d) (○). ▲ 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.

Figure 3. Tumor expression of alloantigens is not required for the beneficial effect of Cy plus CD8-depleted DLI

CB6 F1 mice (H-2dxb; 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.

Figure 4. The anti-tumor 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 anti-tumor immunity through a GVT effect that requires direct tumor expression of alloantigens. BALB/c mice (H-2d; 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-2b) 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-2b) 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-2b; 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-2b) 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-2b) donors, depleted of CD8+ T cells on Day 15 (○). Results show: (●) v (▲) p=.02, (○) v (△) p=.41. All experiments were repeated at least once.