Targeting the Retinoid X Receptor Pathway Prevents and Ameliorates Murine Chronic Graft-Versus-Host Disease

Abstract

Most allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients receive peripheral blood stem cell grafts resulting in a 30%-70% incidence of chronic graft-versus-host disease (cGVHD), a major cause of mortality and morbidity in long-term survivors. While systemic steroids remain the standard of care for first-line therapy, patients may require long-term administration, and those with steroid-resistant or refractory cGVHD have a worse prognosis. Although durable and deep responses with second-line therapies can be achieved in some patients, there remains an urgent need for new therapies. In this study, we evaluated the efficacy of IRX4204, a novel agonist that activates RXRs and is in clinical trials for cancer treatment to prevent and treat cGVHD in two complementary murine models. In a major histocompatibility complex mismatched, non-sclerodermatous multiorgan system model with bronchiolitis obliterans, IRX4204 prevented and reversed cGVHD including associated pulmonary dysfunction with restoration of germinal center T-follicular helper: T-follicular regulatory cell balance. In a minor histocompatibility antigen disparate sclerodermatous model, IRX4204 treatment significantly prevented and ameliorated skin cGVHD by reducing Th1 and Th17 differentiation due to anti-inflammatory properties. Together, these results indicate that IRX4204 is a promising therapeutic option to treat cGVHD with bronchiolitis obliterans or sclerodermatous manifestations.

Keywords: IL-17; RXR; TFH; TfR; germinal center B cells.

Figure 1

IRX4204 prevents and reverses established cGVHD-mediated BO. Conditioned B10.BR mice were transplanted with B6 donor BM ± T cells. A cohort of BM + T recipients were treated with IRX4204 either from days 0 to 28, or 28 to 56. Pulmonary tests including lung resistance, elastance, and compliance were performed on (A) day 28 and (B) day 56 post-transplantation n = 4 – 5/group. (C) Histopathology scores of hematoxylin and eosin–stained tissue sections from lung, liver, spleen, and colon on recipient on day 58. n = 5/group. Representative flow plots of (D) Tfh and (E) GC B cells. Frequency of Tfh (F), Ratio of Tfr/Tfh (G) and frequency of GC B cells (H) in recipient spleen on day 58. *p <.05; **p <.01; ***p <.001; ****p <.0001. Error bars represent standard error of the mean (SEM); n = 5 - 10 per group.

Figure 2

IRX4204 impairs GC B cell response in an immunization model but not in cGVHD. (A, B) Conditioned B10.BR mice were transplanted with B6 donor BM ± T cells. A cohort of BM + T recipients were treated with IRX4204 either from days 0 to 28, or days 28 to 56. n = 3 mice/group. (A) GC size and (B) representative splenic GC immunofluorescence images from BM only and BM plus T-cell mice on day 58 showing peanut agglutinin (PNA; Red). CD4 FITC (green) and B220 BV421 (blue). Germinal centers are highlighted in white circle. An Olympus FluoView500 confocal laser scanning microscope was used to acquire images at magnification 200×. (C) Representative images of Masson’s trichrome staining. Collagen was identified as the area stained in blue. EVOS XL Imaging system was used to acquire images at magnification 200×. (D–G) WT mice were immunized with NP-OVA and treated with either vehicle or IRX4204 daily. (D, E) Flow plots of Tfh, GC B cells, and quantification of GC B cells, Tfh, and Tfr/Tfh ratio from inguinal draining lymph nodes 7 days post-immunization. IRX4204 or vehicle was given from day 0 to 7 (prophylactic). (F, G) Flow plots of Tfh, GC B cells, and quantification of GC B cells, Tfh, and Tfr/Tfh ratio from inguinal draining lymph nodes 14 days post-immunization. IRX4204 or vehicle was given from day 7 to 14 (therapeutic). *p < .05. Error bars represent standard error of the mean (SEM); n = 4/group.

Figure 3

IRX4204 inhibits and treats sclerodermatous cGVHD. Lethally irradiated BALB/c mice were transplanted with B10.D2 BM only or with purified T cells (CD4, CD8: 1.8 x 10⁶ and 0.9 x 10⁶, respectively). Recipients were treated with IRX4204 either from day 0-21 (IRX4204 prophylactic) or day 21 (IRX4204 therapeutic). (A) Photographs of mice in BM, cGVHD + vehicle or IRX4204 treated cGVHD groups. (B) Clinical manifestations of cGVHD were assessed by giving scores to weight loss, activity, posture and fur condition. Healthy mice receive score 0. (C) Skin scores were assessed by measuring the area of skin with fur loss or sclerodermatous lesion. Intact skin was given a score of 0. (D–I) Peripheral lymph nodes (LNs) and spleens (SPL) were harvested on day 50 post-transplantation and stimulated with PMA and ionomycin in vitro. (D–G) The percentages and representative flow plots of (D–E) IFN-γ+ and (F, G) IL-17+ producing CD4+ T cells in LNs are shown. (H, I) The percentages of (H) IFN-γ+ and (I) IL-17+ producing CD4+ T cells in SPL are shown. (J) Trichrome staining of skin. Collagen was stained in blue. *p < .05; **p < .01; ***p < .001; ****p < .0001. Error bars represent standard error of the mean (SEM); n = 8 – 10 per group.