1. The Allogeneic Effect Revisited: Exogenous Help for Endogenous, Tumor-Specific T Cells 
Heather J. Symons, Moshe Y. Levy, Jie Wang, Xiaotao Zhou, Gang Zhou, Sarah E. Cohen, Leo Luznik, Hyam I. Levitsky, Ephraim J. Fuchs 
Biology of Blood and Marrow Transplantation 
Volume 14, Issue 5, Pages (May 2008)
DOI: /j.bbmt
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

2. 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: (▴) 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-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: (●) 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-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: (●) versus (▪) P = .01, (▴) versus (●) P = All experiments were repeated at least once.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

3. 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 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.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

4. 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 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.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
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-2d; 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-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 i.p. 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, C) Cy + CD8-depleted DLI syngeneic to the tumor is sufficient to provide antitumor immunity. CB6F1 mice (H-2b; 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-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 i.p. on day 14, and 5 × 107 spleen cells from 1 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.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions