Treatment with a rho kinase inhibitor improves survival from graft-versus-host disease in mice after MHC-haploidentical hematopoietic cell transplantation

Abstract

Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) and the main cause of nonrelapse mortality during the first 100 days post-transplant. Although GVHD can be prevented by extensive removal of mature donor T cells from the donor hematopoietic stem cell population, doing so eliminates any potential allogeneic graft-versus-tumor (GVT) effect also mediated by donor T cells and results in unacceptable rates of cancer relapse. One potential solution to this problem of separating GVHD development from a GVT response is to prevent T cell-mediated GVHD in the intestinal tract (IT) while preserving systemic antihost alloreactivity of donor T cells that target residual tumor cells expressing host alloantigens. We examined the ability of the anti-inflammatory rho kinase inhibitor, fasudil, given orally and intraperitoneally, to prevent GVHD in a C3H → B6C3F1 mouse model of MHC-haploidentical bone marrow transplantation. Fasudil-treated recipients of anti-thy-1 mAb + C' treated bone marrow (ATBM) cells plus T cells had a 73% 90-day survival compared with 25% among untreated ATBM + T cell recipients (P < .0001). Severe initial weight loss was similar in the 2 groups, but less diarrhea was observed among treated animals, and fasudil-treated survivors recovered more weight than untreated survivors. Skin inflammation occurred and resolved between weeks 2 and 8 with similar severity and kinetics in both treated and untreated surviving animals, indicating persistent alloreactivity. Day 10 post-transplantation splenocytes from fasudil-treated mice, containing mature donor T cells, and day 98 splenocytes, containing mature donor and de novo thymus-derived T cells, exhibited alloreactivity against host parental antigens, as assessed by in vitro IFN-γ production and rounds of allostimulated proliferation, respectively. These data support the idea that targeted treatment of the IT with rho kinase inhibitors can ameliorate lethal GVHD while preserving systemic alloreactivity. The results also suggest that similar mechanisms of IT-specific tolerance or resistance to GVHD operate in fasudil-treated and untreated long-term survivors of allogeneic ATBM + T cells.

Keywords: Allogeneic; Fasudil; GVHD; Haploidentical; Intestinal tract; Rho kinase.

Conflict of interest statement

Conflict of interest statement: ■■■.

Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Fasudil decreases mortality from GVHD. Fasudil administered i.p. for 10 days and in drinking water for the duration of the experiment starting 1 day before lethal irradiation and transfer of ATBM + donor T cells significantly increased 90-day survival from 25% to 73% (P < .0001). Irradiated mice not receiving donor cells showed different early survival rates, with all deaths occurring within a relatively narrow window of 12 to 16 days. Data were pooled from 3 separate replicate experiments involving total sample sizes of 10 to 26 for the different groups, as indicated.

Figure 2

Fasudil does not fully prevent weight loss during acute GVHD. Despite impressive reduction in mortality, mice receiving fasudil along with ATBM + donor T cells were not spared significant weight loss, comparable with untreated ATBM + T recipients, during the first month post-transplantation. In the first week, these groups lost 2 to 3 times as much weight as did ATBM only recipients or as irradiated mice receiving no donor cells. Fasudil-treated mice surviving the first month of ATBM + T cell transplantation, as well as far fewer untreated surviving mice, gradually gained back more of their lost weight than the fewer surviving untreated mice (P = .001), but never caught up with the ATBM-only group (P < .0001).

Figure 3

Long-term fasudil-treated and untreated survivors of GVHD have minimal inflammation of sampled tissues. Most small bowel sections from fasudil-treated animals showed normal architecture of crypts and villi, without evidence of inflammation (A). In 3 of 5 animals, scattered areas of lamina propria and basilar crypt inflammation were seen (dashed white lines), with no disruption of the crypt or villous architecture (B). No inflammation was observed in multiple samples taken from 3 untreated surviving animals (not shown).

Figure 4

CFSE dilution peak determination of spleen cell alloreactivity in 1-way mixed lymphocyte reactions (responder versus irradiated stimulator). Pooled splenocytes from mice 98 days after receiving C3H donor anti-thy-1 treated bone marrow only (ATBM) made some proliferative response to host B6C3F1 cells (A). Roughly 11% of cells divided once, and 17% divided more than once, which is expected as a consequence of normal responses to self-restricted foreign environmental antigens. More cells (40% versus 17%, P < .001) responded with 2 or more cell divisions when third-party BALB/c cells were used as stimulators (B). By contrast, spleen cells from mice receiving mature donor T cells along with ATBM made a strong response to host B6C3F1 cell stimulation, with ~ 7% dividing once and ~31% more than once (C), not significantly different from their response (8% and 32%, respectively) to BALB/c (D), but significantly greater than (A) ATBM anti-F1 responses (P < .001). Mice receiving fasudil, along with donor T cells and ATBM (E), also made a stronger alloresponse to B6C3F1 host cells than mice receiving only ATBM (A), with 20% dividing once and 21 % dividing more than once. Although these proportions both differ significantly (P < .001) from the respective proportions of cells from the untreated mice (C), they are still greater in both cases than the corresponding peaks in ATBM-only spleen cultures (P < .001) and, compared with (C), also have a population of cells undergoing additional rounds of alloreactive division (lowest signal intensity peak). Fasudil-treated mice also retained strong anti-BALB/c MHC responses (F), with a peak profile roughly similar in distribution to (E). Data are representative of 2 replicate experiments and 4 culture time points.