The effects of thalidomide on the immune cells and identification of the immune-modulatory mechanism

Abstract

Standard immunosuppressants used in transplantation are effective inhibitors of acute rejection but are accompanied by immunodeficiency complications and nonimmune complications. Thus, future immunosuppressive therapy such as immunomodulation and induction of tolerance is targeted at increasing graft survival and reducing immunosuppression-related complications. The immune-modulatory effects of thalidomide (TM) and dexamethasone (DX) on immune cells and their co-stimulatory, co-inhibitory molecules in vitro and in vivo have been previously reported. The current study investigated the effects of TM, and the combination treatment with DX on immune cells using a murine cardiac allograft transplantation model. Intraabdominal transplant of cardiac allografts from BALB/c (H-2d) donors to C57BL/6 (H-2b) recipients or C57BL/6 donors to BALB/c recipients were performed. After transplantation, mice were injected with TM 100 mg/kg or DX 0.1 mg/kg or a combination of both TM and DX daily by intra-peritoneal route until the time of graft loss. CD4+ T cells and subsets in the peripheral blood mononuclear cells and spleen were examined and quantified with flow cytometry. The changes in B cells (CD19+) and dendritic cells (DCs, CD11c+) were also analysed. The expression of co-inhibitory and co-stimulatory markers, glucocorticoid-induced TNF receptor-related protein (GITR), and programmed cell death-1 (PD-1) were also quantified by flow cytometry. Serum IL-6 collected at day 7 was measured by enzyme-linked immunosorbent assay (ELISA). A significant increase in allograft survival was noted in both murine cardiac transplant models. The mean graft survival of the BALB/c donors to C57BL/6 recipients in the untreated group was 6.86 days and 10.0 days in the TM/DX group (p<0.001). The mean graft survival of the C57BL/6 donors to BALB/c recipients was 9.0 days in the untreated group and 22.5 days in the TM/DX group (p<0.001). TM showed immune-modulatory features which were enhanced with the complementary combination of DX. The TM/DX treatment affected the CD4+ T cell subsets without inhibiting the total CD4+ T cell population. The CD4+FOXP3+/CD4+CD44hi T cell ratio was increased which indicates the expansion of Treg cells. The increase in tolerogenic DCs (CD11c+CD85k+) with TM/DX was observed. The inhibition of pro-inflammatory cytokine IL-6 was also observed. TM/DX treatment showed the tendency to suppress co-stimulatory molecule GITR expression while TM-based treatments increased or preserved co-inhibitory molecule PD-1 expression in CD4+CD44+ and CD4+FOXP3+ T cells after transplantation. TM/DX treatment resulted in selective T cell subset changes and the induction of tolerogenic DCs, inhibition of IL-6, and improved allograft survival. These outcomes suggest the immune-modulating effect of the TM/DX combinatorial treatment. In conclusion, TM/DX combination may be a promising immune-modulatory approach for preventing allograft rejection and improving graft survival by inducing tolerance in transplantation.