BackgroundGranulocyte colony-stimulating factor (G-CSF) can increase populations of myeloid-derived suppressor cells, innate immune suppressors that play an immunoregulatory role in antitumor immunity. However, the roles of myeloid-derived suppressor cells and G-CSF in renal ischemia-reperfusion injury remain unclear.MethodsWe used mouse models of ischemia-reperfusion injury to investigate whether G-CSF can attenuate renal injury by increasing infiltration of myeloid-derived suppressor cells into kidney tissue.ResultsG-CSF treatment before ischemia-reperfusion injury subsequently attenuated acute renal dysfunction, tissue injury, and tubular apoptosis. Additionally, G-CSF treatment suppressed renal infiltration of macrophages and T cells as well as renal levels of IL-6, MCP-1, IL-12, TNF-α, and IFN-γ, but it increased levels of IL-10, arginase-1, and reactive oxygen species. Moreover, administering G-CSF after ischemia-reperfusion injury improved the recovery of renal function and attenuated renal fibrosis on day 28. G-CSF treatment increased renal infiltration of myeloid-derived suppressor cells (F4/80−CD11b+Gr-1int), especially the granulocytic myeloid-derived suppressor cell population (CD11b+Ly6GintLy6Clow); splenic F4/80−CD11b+Gr-1+ cells sorted from G-CSF–treated mice displayed higher levels of arginase-1, IL-10, and reactive oxygen species relative to those from control mice. Furthermore, these splenic cells effectively suppressed in vitro T cell activation mainly through arginase-1 and reactive oxygen species, and their adoptive transfer attenuated renal injury. Combined treatment with anti–Gr-1 and G-CSF showed better renoprotective effects than G-CSF alone, whereas preferential depletion of myeloid-derived suppressor cells by pep-G3 or gemcitabine abrogated the beneficial effects of G-CSF against renal injury.ConclusionsG-CSF induced renal myeloid-derived suppressor cells, thereby attenuating acute renal injury and chronic renal fibrosis after ischemia-reperfusion injury. These results suggest therapeutic potential of myeloid-derived suppressor cells and G-CSF in renal ischemia-reperfusion injury.

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Foxp3 stability of vitamin C-treated induced-regulatory T cells (V-iTregs) is superior to that of conventional iTregs (C-iTregs). However, the role of V-iTregs in allograft rejection under vitamin C-deficient conditions, such as those seen in humans, remains unclear. We aimed to elucidate the role of vitamin C treatment on generation and maintenance of iTregs from gulo knockout (Gulo-KO) mice as well as wild type (WT) mice, and in vitro and in vivo suppressive effects of V-iTregs on heart allograft rejection in either Gulo-KO or WT recipient mice. Conversion efficiency of iTregs was similar between C- and V-iTregs in both WT and Gulo-KO mice. V-iTregs from WT or Gulo-KO mice showed better in vitro Foxp3 stability than C-iTregs, although there was no difference between WT V-iTregs and Gulo-KO V-iTregs. Furthermore, V-iTregs from WT or Gulo-KO mice suppressed in vitro T cell proliferation better than C-iTregs. Heterotrophic heart transplantation from BALB/c mice to WT or vitamin C-deficient Gulo-KO C57BL/6J mice was performed following adoptive transfer of C- or V-iTregs. V-iTregs as well as C-iTregs prolonged heart allograft survival in WT and Gulo-KO mice. However, there was no difference between the C- and V-iTreg groups. Supplementation of low- or high-dose vitamin C did not induce significant changes in heart allograft survival in Gulo-KO recipients that had received V-iTregs. In conclusion, V-iTregs do not exert better suppressive effects on heart allograft survival than C-iTregs in either WT or vitamin C-deficient recipients.

Introduction: Antibody-mediated rejection (ABMR) is the main hurdle in ABO blood group-incompatible (ABOi) transplantation. C4d deposition is a marker of ABMR and is also found in most ABOi allograft tissues as a result of accommodation. Previously, we reported that anti-C4d chimeric antigen receptor (CAR) regulatory T cells (Tregs) suppressed ABMR in ABOi allografts. Here, we aimed to improve efficacy of anti-C4d CAR Tregs by modifying CAR structure and increasing dose of CAR Tregs. Methods: Intracellular costimulatory domains of anti-C4d CAR Treg consisted of mouse CD3ζ and CD28. CD62L+CD4+CD25+ T cells were sorted and transduced with retroviral CAR using retronection on two consecutive days. We assessed in vitro suppressive function of anti-C4d CAR Tregs against T cell proliferation in response to polyclonal stimulation. Wild-type C57BL/6J mice were sensitized on day -21 and on day -14 by injecting human blood group A-expressing cells. Hearts from human blood group A-transgenic BALB/c mice were transplanted into the sensitized CD45.2+ C57BL/6J mice to make an anti-ABO antibody-mediated rejection model. CD45.1+ non-transduced, control CAR, or anti-C4d CAR Tregs (1×106) were transferred into recipient mice on day-1 and day 2 after transplantation in combination with prednisolone, tacrolimus, and rapamycin. ABOi heart allograft were analyzed for tissue damage, cellular infiltration, and cytokine expression on day 7. Results: Anti-C4d CAR Tregs express Foxp3, CD25, CTLA-4, LAP, and GITR that are associated with immunosuppressive functions of Tregs, to similar extent as either nontransduced or control CAR Tregs. In vitro suppressive activity of anti-C4d CAR Tregs was also similar as that of nontransduced Tregs and control CAR Tregs. ABOi heart allografts showed typical features of ABMR, such as, peritubular capillitis and diffuse endothelial C4d+ deposition. Adoptive transfer of anti-C4d CAR Tregs suppressed ABMR-related tissue injury and significantly prolonged ABOi heart allograft survival compared to PBS control group, nontransduced Treg group, and control CAR Treg group. Both flow cytometric analysis and immunofluorescence imaging study demonstrated that number of CD45.1+Foxp3+ Treg infiltration into heart allograft were significantly higher in anti-C4d CAR Treg group compared to PBS control, nontransduced CAR Treg group, and control CAR Treg group. Expression of IL-2, TNF-α, and IFN-γ in heart allografts was significantly lower in anti-C4d CAR Treg group than PBS control group. Conclusions: Anti-C4d CAR Tregs infiltrated into ABOi heart allograft with diffuse C4d deposition to more extent and thereby suppressed ABMR in ABOi heart allografts more effectively, compared to nontransduced Tregs and control CAR Tregs.