The synthetic triterpenoid, CDDO, suppresses alloreactive T cell responses and reduces murine early acute graft-versus-host disease mortality

Abstract

Acute graft-versus-host disease (aGVHD) still remains one of the life-threatening complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Immunomodulation of alloreactive donor T cell responses, as well as cytokine secretion is a potential therapeutic approach for the prevention of aGVHD. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid), exhibits potent antitumor activity and has also been shown to mediate anti-inflammatory and immunomodulatory effects. We therefore wanted to assess the effects of CDDO on early lethal aGVHD. In this study, we found that CDDO significantly inhibited in vitro mixed lymphocyte responses and preferentially promoted the apoptosis of proliferating but not resting alloreactive T cells. Using a full major histocompatibility complex (MHC)-disparate murine aGVHD model, we found that the administration of CDDO immediately after transplantation significantly decreased liver pathology as determined by histologic assessment and prolonged survival in mice. Importantly, administration of CDDO did not adversely impair donor myeloid reconstitution as determined by peripheral blood cell count and the extent of donor chimerism. These findings indicate that CDDO has a significant immunomodulatory effects in vitro and on early lethal aGVHD development, particularly affecting the liver, in a murine allo-HSCT model.

Figure 1

CDDO inhibits the mitogen and alloantigen-induced T cell proliferation. (A) BALB/c splenocytes were exposed to ConA (2 (μg/mL), plus 4 different concentrations of CDDO or vehicle and cultured for 2 days as described in Materials and Methods. T cell proliferation was significantly inhibited in a dose-dependent manner in the presence of ≥0.4 μM CDDO. (B, D) BALB/c splenocytes were cultured with irradiated B6 splenocytes at a 1:1 ratio for 4 days (B) or 3-7 days (D), then pulsed with [³H] thymidine for 16 hours prior to harvesting as described in Materials and Methods. T cell proliferation was significantly inhibited in a dose-dependent manner in the presence of ≥0.15 μM CDDO (significant differences compared with vehicle control, *P < 0.05 and **P < 0.01). Results from 1 of 4 independent experiments are presented. (C) B6 splenocytes (2 × 10⁵ cells/well) were exposed to ConA (2 μg/mL), with different concentrations of CDDO, bortezomib, or individual vehicles. Plates were incubated at 37°C and 5% CO₂ for 48 hours, then an MTT assay was performed. T cell proliferation was significantly inhibited in the presence of 250 and 500 nM CDDO (P < 0.05 and P < 0.01, respectively) and in the presence of 10 and 25 nM bortezomib (P < 0.05) compared to their vehicle controls (ANOVA with Dunnett's posttest comparison).

Figure 2

Proliferating, and not resting, T cells are highly sensitive to CDDO-mediated apoptosis. CFSE-labeled BALB/c splenocytes were incubated with irradiated T cell-depleted B6 splenocytes at a 1:1 ratio with a dose range of CDDO as described in Materials and Methods. After 3 days in culture, cells were harvested and analyzed by flow cytometry. Cells were first gated on CD4 or CD8 expression, then gated on CFSE^high or CFSE^low staining, and analyzed for annexin V binding in proliferating or resting T cells. (A) Proportionally greater increases in annexin V binding were observed on proliferating (CFSE^low) compared with nonalloreactive (CFSE^high) CD4⁺ T cells with exposure to 0.25 μM, 0.5 μM, and 0.75 μM CDDO. (B) Proportionally greater increases in annexin V binding were observed on proliferating (CFSE^low) compared with nonalloreactive (CFSE^high) CD8⁺ T cells with exposure to 0.25 μM, 0.5 μM, and 0.75 μM CDDO. *Significant differences of annexin V binding on CDDO-exposed T cells compared with control (P < 0.05). Results from 1 of 3 independent experiments are presented.

Figure 3

CDDO administration immediately after transplantation results in delayed development of GVHD and significant improvement in survival. B6 (H2^b) mice received TBI (900 cGy) followed by infusion of 10 × 10⁶ 10 BALB/c (H2^d) BMC and 35 × 10⁶ BALB/c SC. Mice were treated with vehicle control or CDDO (240 μg/dose) twice a day intraperitoneally on day 0 and day +1 for a total of 4 doses. (A) Administration of CDDO protected mice from early GVHD mortality. Significant improvement in survival were observed in CDDO-treated mice (●) compared with GVHD control (vehicle control-treated) mice (■; P < .001). Results from 1 of 3 independent experiments are presented. (B) Significant decrease in serum TNF-α levels of mice on day +5 post-BMT in mice that received CDDO compared to mice treated with vehicle. (C) Significant protection from GVHD associated weight loss on day 6 post-BMT in mice that received CDDO. Mean body weights from 1 of 3 independent experiments are presented.

Figure 4

CDDO administration results in decreased hepatic injury but not intestinal injury in mice with GVHD. B6 (H2^b) mice received TBI (950 cGy), followed by infusion of 10 × 10⁶ BALB/c (H2^d)BMC and 35 × 10⁶ BALB/c SC. Mice were treated with vehicle control or CDDO (240 μg/dose) three times a day intraperitoneally on day 0 and day +1 for a total of 2 doses. (A, B). Pathology scores in the liver (A) and gut (B) of mice that received BMC alone or with splenocytes, followed by administration of vehicle control or CDDO. (C, D) Photomicrographs of representative gut tissue sections. Mice that received BMC with spleen cells followed by vehicle control (C) and mice that received BMC with spleen cells followed by CDDO (D) demonstrated similar prominent tissue damage with numerous apoptotic bodies. (E, F). Photomicrographs of representative liver tissue sections. Mice that received BMC with spleen cells followed by vehicle control (E) demonstrated expensive GVHD with bile duct proliferation, vasculitis, lobular hepatitis, and numerous apoptotic cells. In contrast, mice that received BMC with spleen cells followed by CDDO (F) demonstrated preserved portal triad architecture, no vasculitis, no lobular hepatitis, and virtually no apoptotic cells. H&E staining, original magnification ×500.

Figure 5

CDDO administration does not adversely affect donor engraftment. B6 (H2^b) mice received TBI (900 cGy) followed by infusion of 10 × 10⁶ BALB/c (H2^d) BMC and 40 × 10⁶ BALB/c SC. Mice were treated with vehicle control or CDDO (240 μg/dose) twice a day intraperitoneally on day 0 and day +1 for a total of 4 doses. Blood, bone marrow, and spleen were collected and analyzed for engraftment of donor cells on day 14 posttransplantation (4-6 mice/group). (A) CBC parameters were not significantly affected by CDDO treatment in mice. (B) The frequency of donor cells in the bone marrow and spleen following allogeneic BMT are not affected by CDDO administration. Results from 1 of 2 independent experiments are presented.