Combination of intra-bone marrow-bone marrow transplantation and subcutaneous donor splenocyte injection diminishes risk of graft-versus-host disease and enhances survival rate

Abstract

The combination of allogeneic bone marrow transplantation (allo-BMT) and donor lymphocyte infusion (DLI) is a useful method for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, there is a risk of inducing uncontrollable fatal graft-versus-host disease (GVHD). In fact, allo-BMT plus intravenous (IV)-DLI using donor splenocytes induces fatal GVHD in recipient mice. In this study, we examined the effects of the combination of intra-bone marrow (IBM)-BMT and the subcutaneous injection of donor splenocytes (SC-DLI) on the allo-BMT system. Recipient BALB/c mice were conditioned by sublethal irradiation (5 Gy), followed by IBM-BMT plus IV-DLI or SC-DLI in C57BL/6 mice. The IV-DLI group showed better engraftment of donor hemopoietic cells than the control group (without DLI) but showed fatal GVHD. The SC-DLI group, however, showed good reconstitution and mild GVHD. These results suggest that the combination of SC-DLI and IBM-BMT promotes the reconstitution of hemopoiesis and helps reduce the risk of GVHD.

FIG. 1.

Experiment protocol. The days of irradiation, BMT, and DLI are shown. The observed days of T-cell infiltration, reconstitution, and the analyses of histopathological scoring are also shown. BMT, bone marrow transplantation; DLI, donor lymphocyte infusion.

FIG. 2.

IV-DLI shortens the survival but SC-DLI does not. Recipient (BALB/c) mice were irradiated at 6 Gy (A), 5 Gy (B) or 4 Gy (C) on day −1. Bone marrow cells (3 × 10⁷) from donors (B6) were injected by intra-bone marrow–BMT with or without 5 × 10⁷ splenocytes DLI on day 0. The splenocytes were injected intravenously into the tail vein in the IV-DLI group or subcutaneously in the back side in the SC-DLI group. n ≥ 10. IV, intravenous; SC, subcutaneous.

FIG. 3.

Reconstitution in different irradiation doses. Recipient mice were irradiated with 4, 5, or 6 Gy on day −1. BMT and DLI were carried out on day 0. Peripheral blood of 6 Gy (A and D) and 4 Gy (C and F) irradiated mice was analyzed for reconstitution at 1 month after BMT. In the 5 Gy (B and E) experiment, peripheral blood was analyzed on day 17. Each small square (▪) represents the percentage of reconstitution for each mouse in the left panels. The right panels show the percentage of donor CD45⁺ by means ± standard deviation. *P < 0.05.

FIG. 4.

SC-DLI significantly ameliorates GVHD. Recipient mice were irradiated at 6 Gy (A), 5 Gy (B) or 4 Gy (C) on day −1. After BMT and DLI, the body weight of the recipients was recorded and clinical signs of GVHD were assessed every 5 days. The clinical scoring was based on 6 parameters: weight loss, posture, activity, fur texture, skin integrity, and diarrhea. n ≥ 10. GVHD, graft-versus-host disease.

FIG. 5.

Histopathological score and representative photographs of GVHD target tissues: liver, skin, and intestine. Autopsies were performed on mice that had died (IV-DLI group) or had been sacrificed at 3 months after BMT (control group and SC-DLI group). Tissues from GVHD target organs (liver, skin, and intestine) were prepared for histopathological scoring. For the SC-DLI group, the skin from the site of the injection is shown. Representative photographs of 6 Gy (A) and 5 Gy (C) irradiated mice are shown in the left panels. The right panels (B and D) show the pathological scores by means ±; standard deviation: n ≥ 4; *P < 0.05.

FIG. 6.

SC-DLI inhibited donor T-cell infiltration of the GVHD target tissues. Five days after BMT and DLI, the weight of the spleen (A) and the percentage of donor T cells in the spleen (B) and liver (C) were compared between groups. Control group: n = 3; IV-DLI group and SC-DLI group: n = 4; *P < 0.05.