Adoptive transfer of Treg_IL-33 cells ameliorates acute graft-versus-host disease by regulating the HPGD/PGE2 pathway

Adoptive transfer of regulatory T-cells (Tregs) has shown promising therapeutic efficacy in ameliorating graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. However, the underlying mechanisms have not been thoroughly studied.

In this study, we investigated the potential application of adoptive transfer of IL-33-cultured Tregs to prevent acute graft-versus-host disease (aGVHD) and its underlying mechanism.

Tregs were magnetically isolated from the spleen of C57BL/6 mice and subsequently cultured with IL-2 (Treg_IL-2) or IL-2 + IL-33 (Treg_IL-33) for 7d. BALB/c mice received 6 Gy of total body irradiation 24 h prior to transplantation. After irradiation, ex vivo cultured Treg_IL-2 or Treg_IL-33 cells were co-adoptively transferred with splenocytes and T cell-depleted bone marrow cells from C57BL/6 donors. On day 8 post transplantation, splenocytes and liver cells were collected and analyzed by flow cytometry for M1/M2 macrophage populations and T cell subsets. For in vitro experiments, bone marrow derived macrophages (BMDM) were cultured with Treg_IL-33 cells for 48 h. The M1/M2 macrophage population was detected at both mRNA and protein levels. For Treg_IL-33 cells, hydroxyprostaglandin dehydrogenase (HPGD) expression and prostaglandin E2 (PGE2) production were examined. To demonstrate that the regulation of macrophage polarization was mediated through the HPGD/PGE2 axis, the HPGD inhibitor SW033291 and an anti-PGE2 antibody were used in a co-culture setting.

Adoptive transfer of Treg_IL-33 cells was more effective in preventing aGVHD than adoptive transfer of Treg_IL-2. In addition, recipients receiving Treg_IL-33 cells showed increased Tregs, especially ST2⁺Tregs frequencies in the spleen and liver, while frequencies of IFN-γ and IL-17 producing conventional T cells were decreased. Meanwhile, the expression of CD86⁺ type I macrophages was downregulated and that of CD206⁺ type II macrophages was upregulated in mice co-transferred with Treg_IL-33 cells. In in vitro study, Treg_IL-33 cells promoted M2 polarization and inhibited the M1 transition of BMDM compared with BMDM cultured with Treg_IL-2. Mechanistically, IL-33 induced ST2 expression on Tregs, increased HPGD synthesis, and accelerated PGE2 degradation. Using the HPGD inhibitor SW033291, and an anti-PGE2 antibody, we demonstrated that Treg_IL-33 cells regulated M1/M2 macrophage polarization via the HPGD/PGE2 axis. Furthermore, blocking HPGD activity with SW033291 abolished the efficacy of Treg_IL-33 cells in protecting against aGVHD in vivo.

Our data demonstrate that adoptive transfer of Treg_IL-33 cells could ameliorate aGVHD by regulating the HPGD/PGE2 axis, thus regulating M1/M2 paradigm.