LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation

Abstract

Acute graft-versus-host disease (GVHD) and leukemic relapse remain the two major obstacles to successful outcomes after allogeneic bone marrow transplantation (BMT). Recent studies have demonstrated that the loss of gastrointestinal tract integrity, and specifically the translocation of LPS into the systemic circulation, is critical to the induction of cytokine dysregulation that contributes to GVHD. Using a mouse BMT model, we studied the effects of direct LPS antagonism on GVHD severity and graft-versus-leukemia (GVL) activity. Administration of B975, a synthetic lipid-A analogue from day 0 to day +6, reduced serum TNF-alpha levels, decreased intestinal histopathology, and resulted in significantly improved survival and a reduction in clinical GVHD, compared with control-treated animals. Importantly, B975 had no effect on donor T cell responses to host antigens in vivo or in vitro. When mice received lethal doses of P815 tumor cells at the time of BMT, administration of B975 did not impair GVL activity and resulted in significantly improved leukemia-free survival. These findings reveal a critical role for LPS in the early inflammatory events contributing to GVHD and suggest that a new class of pharmacologic agents, LPS antagonists, may help to prevent GVHD while preserving T cell responses to host antigens and GVL activity.

Figures

Figure 1

Injection of B975 inhibits in vivo TNF-α production after BMT. Lethally irradiated B6D2F1 received allogeneic BMT from C57BL/6 donors and were treated with B975 (gray bar) or D5W (black bar) as described in Methods. Syngeneic BMT recipients treated with D5W (open bar) served as negative GVHD controls. Serum was harvested between days 4 and 6 after BMT and analyzed for TNF-α by ELISA. Data are expressed as mean ± SEM. AP < 0.05; gray bar vs. black bar. n = 6 to 8 per group. UD, undetectable.

Figure 2

Treatment with B975 results in decreased intestinal histopathology after allogeneic BMT. B6D2F1 animals received syngeneic BMT or allogeneic BMT from C57BL/6 donors as in Figure 1. On days +4 to +6, small-bowel samples from BMT recipient mice were obtained and analyzed microscopically as described in Methods. Syngeneic BMT recipients (a) demonstrate reestablishment of intestinal architecture with villi of near normal length and without significant cellular infiltration into the lamina propria. Regenerative change is evident by focally increased nuclear staining and an increased nuclear/cytoplasmic ratios. Animals treated with B975 (b) exhibit partial restoration of small-intestinal villous architecture, regenerative change, and little inflammatory infiltration. Recipients of allogeneic BMT (c) show severe intestinal toxicity including surface erosion, villous blunting, epithelial attenuation, and an intense cellular infiltration in the lamina propria. Original magnification, ×200.

Figure 3

Treatment with B975 reduces damage to the small (a) and large (b) bowel after allogeneic BMT. B6D2F1 mice received allogeneic BMT from C57BL/6 donors and either B975 (gray bars) or D5W (black bars) as in Figure 1. Syngeneic BMT recipients treated with D5W served as controls (white bars). Samples of small (a) and large (b) bowel were harvested from transplanted animals on days +4 to +6 and analyzed using the semiquantitative scoring system described in Methods. Data are expressed as mean plus or minus SEM. AP < 0.01; gray bar vs. black bar. n = 4 (syngeneic) to 8 (allogeneic) per group.

Figure 4

Treatment with B975 reduces GVHD-related mortality (a) and clinical score (b) after BMT. B6D2F1 mice received syngeneic (syn; n = 12) or allogeneic (allo) BMT from C57BL/6 donors and either B975 (n = 24) or D5W (n = 26) as described in Figure 1. Transplanted animals were monitored daily for survival and assessed weekly for clinical score as described in Methods. (a) Percentage of surviving animals after BMT. Results represent a combination of three similar experiments. AP < 0.01; allogeneic + B975 vs. allogeneic + D5W. (b) GVHD scores of surviving animals. Data are expressed as mean ± SEM and represent a combination of three similar experiments. AP < 0.01, BP < 0.05; allogeneic + B975 vs. allogeneic + D5W.

Figure 5

Treatment with B975 preserves GVL activity and improves leukemia-free survival. As shown, B6D2F1 mice received syngeneic (n = 8) or allogeneic BMT with B6Ly5.2 donors and either B975 (n = 10) or D5W (n = 10) as described in Figure 1. P815 cells (2,000) were added to the BM inoculum at day 0 as described in Methods. Animals were monitored daily for survival, and necropsy was performed on all dying animals to determine whether cause of death was from leukemia or GVHD. AP < 0.05; allogeneic + B975 vs. allogeneic + D5W. Syn no P815, syngeneic BMT without P815 cells.