Combination of anti-CD4 antibody treatment and donor lymphocyte infusion ameliorates graft-versus-host disease while preserving graft-versus-tumor effects in murine allogeneic hematopoietic stem cell transplantation

Abstract

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not only a well-established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo-HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft-versus-host disease (GVHD). CD4⁺ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8⁺ T-cell responses. In the present study, we investigated clinically applicable allo-HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo-HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti-CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo-HSCT. Late treatment with anti-CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti-CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo-HSCT followed by anti-CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.

Keywords: Allogeneic hematopoietic stem cell transplantation; CD4; donor lymphocyte infusion; graft-versus-host disease; graft-versus-tumor.

Figure 1

Myeloablative allogeneic hematopoietic stem cell transplantation (allo‐HSCT) in combination with anti‐CD4 mAb treatment confers moderate graft‐versus‐tumor effects without graft‐versus‐host disease (GVHD). (a) Schema of the graft‐versus‐tumor model. BDF ₁ mice (H‐2^b×d) were irradiated with myeloablative (9 Gy) or non‐myeloablative (4 Gy) X‐ray irradiation on day −1, s.c. inoculated with colon‐26 (H‐2^d) cells 5 h before allo‐HSCT, and transplanted with T cell‐depleted bone marrow (TCD BM) alone (BMT) or with TCD BM plus T cells (GVHD) from B6 mice (H‐2^b) on day 0. An anti‐CD4 mAb was i.p. injected on day 3 after allo‐HSCT (αCD4). (b) Tumor growth curves and (c) tumor volume at day 22 after allo‐HSCT. *P < 0.05; ***P < 0.001 (4 Gy vs. 9 Gy). †P < 0.05; †††P < 0.001 (vs. BMT); §§P < 0.01 (vs. GVHD). NS, not significant. (d, e) GVHD scores (d) and survival (e). (f) Scatter plot of GVHD score versus tumor volume at day 24 after allo‐HSCT. Dotted lines indicate average values across all groups; numbers to the right of the graph indicate the number of mice from each group that had less‐than‐average GVHD scores and tumor volumes (mice appearing in the lower left‐hand corner of the plot), and the total number in each group. **P < 0.01; ***P < 0.001. Data represent mean ± SEM (a−d; n = 10) from one of two independent experiments.

Figure 2

Timing effects of anti‐CD4 mAb treatment on graft‐versus‐tumor and graft‐versus‐host disease (GVHD) in murine allogeneic hematopoietic stem cell transplantation (allo‐HSCT). BDF ₁ mice receiving myeloablative X‐ray irradiation were s.c. inoculated with colon‐26 cells and transplanted with T cell‐depleted bone marrow (TCD) BM alone (BMT) or with TCD BM plus T cells (GVHD) on day 0. GVHD mice were untreated or treated with anti‐CD4 mAb on day (d) 3, 6, or 17. (a) Kinetics of GVHD score. (b,c) GVHD score (b) and tumor volume (c) at day 27 after allo‐HSCT. (d) Scatter plots of GVHD score versus tumor volume at day 23 after allo‐HSCT. Dotted lines indicate average values across all groups. The numbers to the right of the graph indicate the number of individuals plotted in the lower left box and the total number in each group. *P < 0.05; ***P < 0.001 (vs. BMT). ††P < 0.01; †††P < 0.001 (vs. GVHD). Data represent mean ± SEM (n = 10) from one of two independent experiments.

Figure 3

Combination of anti‐CD4 mAb treatment and donor lymphocyte infusion (DLI) enhances graft‐versus‐tumor effects without exacerbating graft‐versus‐host disease (GVHD) in murine allogeneic hematopoietic stem cell transplantation (allo‐HSCT). (a) Schema of DLI model. BDF ₁ mice receiving myeloablative X‐ray irradiation were s.c. inoculated with colon‐26 cells and transplanted with T cell‐depleted bone marrow (TCD BM) alone (BMT) or with TCD BM plus T cells (GVHD) on day 0. GVHD mice were untreated or treated with anti‐CD4 mAb on day 3. On day 12, DLI group mice received i.v. injection of B6‐derived unfractionated T cells (CD4/8 T cells [“4/8” in subsequent panels]), CD8⁺ T cells (“8”), or CD4⁺ T cells (“4”). (b) Kinetics of GVHD score. (c) GVHD score at day 23 after allo‐HSCT. (d) Kinetics of GVHD score for individual GVHD mice that were treated with anti‐CD4 mAb and received DLI with unfractionated (CD4/8) T cells. (e) Tumor volume at day 23. (f) Scatter plots of GVHD score versus tumor volume at day 24 after allo‐HSCT. Dotted lines indicate average values. Numbers to the right indicate the number of individuals plotted in the lower left box over the total group number. **P < 0.01; ***P < 0.001 (vs. GVHD ⁺/αCD4⁻/DLI ⁻); ††P < 0.01 (vs. GVHD ⁺/αCD4⁺/DLI ⁻). Data represent mean ± SEM (n = 10), from one of two independent experiments.