The Role of Regulatory T Cells Expanded by Anti-DR3 Antibody for Alleviation of Acute Graft-versus-Host Disease

Abstract

Introduction: Regulatory T cells (Treg) alleviate acute graft-versus- host disease (aGVHD) while preserving graft-versus-tumor (GVT) effects. However, their low frequency hinders clinical translation. Although a lot of protocols to expand Treg have been reported, they still have limitations. DR3 belongs to the TNF receptor superfamily and is expressed primarily on T cells. Its agonistic antibody (Ab) was reported to expand Treg in vivo and prevent the rejection of a cardiac allograft. Methods: C57BL/6 donor mice were intraperitoneally injected with anti-DR3 Ab or hamster IgG isotype control and conventional T cells (Tcon) were isolated from them four days later. Tcon and T cell- depleted (TCD) bone marrow (BM) cells were transplanted to lethally irradiated Balb/c recipients. Clinical signs of aGVHD and bioluminescence imaging (BLI) were regularly monitored. Recipient mice were sacrificed for flow cytometric analyses and serum cytokine assay at predefined time points. Results: Donor mice treated with anti-DR3 Ab yielded Tcon containing higher proportions of Treg that suppressed Tcon proliferation with lower numbers. Mixed lymphocyte reaction (MLR) showed that these Tcon maintained higher Treg proportions, were less proliferative, had reduced Th1 differentiation and more PD-1 expression on Treg in response to allogeneic stimuli. In vivo studies confirmed that Tcon from anti-DR3-treated mice expanded to a lesser extent and caused the reduced aGVHD. Treg from anti-DR3-treated donors expanded robustly and their proportions within donor CD4+ T cells were maintained higher. CD25 expression and BrdU incorporation of non-Treg CD4+ and CD8+ T cells derived from anti-DR3-treated donors were reduced in vivo. Recipients of Tcon from anti-DR3-treated donors had lower serum IFNγ, IL-1β, and TNFα levels. Tcon from anti-DR3-treated donors also preserved their GVT effects. Conclusions: Together these data suggest that treating donors with anti-DR3 Ab can effectively expand donor Treg, alleviate aGVHD and preserve GVT effects. Conclusively, anti-DR3 Ab could be utilized as one of the strategies for in situ expansion of regulatory T cells.