The paradoxical effect of glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) activation on alloreactive CD4 and CD8 T cells and the development of GVHD

Abstract

GITR is a member of the tumor necrosis factor receptor family and is involved in regulatory T cell function and TCR activation. GITR is expressed at low levels on resting T cells, B cells and macrophages. Upon activation, CD4+ and CD8+ T cells upregulate GITR expression while CD4+CD25+ regulatory T cells (Treg) constitutively express GITR. Treg stimulation through GITR inhibits their suppressor function and in vivo administration of GITR agonistic antibodies induce autoimmune disease. Here we show that GITR is a relevant molecule for immune responses in graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo BMT). We used a MHC class I/II disparate model for murine allo BMT (C57BL/6 (B6) into BALB/c) to determine the effects of a GITR activating antibody (GITR Ab) on alloreactive T cells during the development of GVHD. We found that addition of GITR Ab to a mixed lymphocyte reaction (MLR) with irradiated BALB/c splenic stimulators, CD4+CD25− B6 effectors and B6 Tregs induced the blockade of Treg suppressor function, as previously described. Interestingly, GITR Ab also had a direct effect on CD4+CD25− and CD8+CD25− T cell populations. Addition of GITR Ab in an MLR showed a 2-fold decrease in CD4+CD25− proliferation while CD8+CD25− proliferation increased by 3.5-fold when compared to the control. Fas deficient CD4+CD25− T cells had an intact alloreactive proliferative response, suggesting that GITR can induce Fas-mediated apoptosis of CD4+CD25− alloreactive T cells. We used the adoptive transfer of CFSE-labeled donor CD3+ T cells into irradiated allogeneic recipients to assess the expression of GITR in vivo and found increased levels of GITR on both CD4+ and CD8+ dividing alloreactive T cells. When we administered the GITR Ab to allogeneic recipients of CD4+CD25− or CD8+CD25− CFSE-labeled T cells, we observed a 45% reduction in the number of donor CD4+CD25− CFSE-labeled T cells recovered at day 3 after infusion. In contrast, there was a 54% increase in the recovery of CFSE-labeled CD8+CD25− T cells. More importantly, mouse recipients of an allo BMT accompanied by CD8+CD25− donor T cells had increased morbidity and mortality in the presence of GITR Ab. Conversely, mice treated with GITR antibody that received allogeneic CD4+CD25− T cells in the allograft showed a significant decrease in GVHD. Our findings identify a novel and paradoxical effect of GITR stimulation on alloreactive T cells during the development of GVHD.