A Selective TNFR2 Agonist Expands Host T_reg Cells in Vivo to Protect from Acute Graft-Versus-Host Disease

Donor CD4+Foxp3+ regulatory T cells (T_regs) suppress graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) while maintaining the anti-tumoral effect of transplanted conventional T cells in preclinical mouse models. Current clinical study protocols with donor T_regs for treatment or prophylaxis of GVHD rely on their ex vivo expansion and infusion in high numbers. Here we present a fundamentally novel strategy for inhibiting GVHD that is based on the in vivo expansion of recipient T_regs prior to allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T_reg biology.

To this end we constructed a recombinant nonameric TNFR2-specific agonist, mimicking the activity of murine membrane-bound TNF on TNFR2 without TNFR1 stimulation, thereby avoiding the inflammatory side effects observed with conventional TNF. In vitro, this TNFR2-agonist expanded natural T_regs from wild type but not from TNFR2 KO mice. Accordingly, a human variant of this TNFR2-specific agonist expanded human T_regsin vitro. In vivo treatment of healthy mice with the murine TNFR2-agonist significantly increased T_reg numbers in secondary lymphoid organs and peripheral tissues, particularly in the gastrointestinal tract, a prime target of acute GVHD.

Next, we pre-treated recipient mice with this novel TNFR2-agonist to expand host-type radiation resistant T_regs prior to of allo-HCT in two models across MHC barriers (C57BL/6, H-2b->Balb/c, H-2d and FVB/N, H-2q->C57BL/6, H-2b). TNFR2-agonist pre-treatment resulted in significantly prolonged survival and reduced GVHD severity when compared to TNFR2-deficient recipients or untreated allo-HCT recipients. This was accompanied by reduced donor T cell proliferation and infiltration into GVHD target organs as assessed by in vivo and ex vivo bioluminescence imaging, flow cytometry and immunofluorescence microscopy. While in vivo TNFR2-agonist pre-treatment protected allo-HCT recipients from GVHD, anti-tumor effects of transplanted T cells remained unaffected in two different murine B cell leukemia models. In vivo depletion of host derived T_regs completely abrogated the protective effect of TNFR2-agonist pre-treatment.

Our study shows that the expansion of host T_regs by selective in vivo TNFR2-activation significantly improves the outcome after allo-HCT and results in prolonged tumor-free survival.