1A). A modest increase in the absolute numbers of Tconv cells was also seen (Fig. 1D). A similar enhancement in Treg cells was seen in mice treated with a different preparation of Fc-GITR-L [13], but these authors did not observe any increase in Tconv cells. To determine if GITR stimulation modulated Treg-cell function, we purified CD4+CD25+T cells from Fc-GITR-L and IgG1-injected mice and assessed their suppressive capacity in vitro (Supporting Information Fig. 1B). Treg cells from Fc-GITR-L-treated

mice were as suppressive as Treg cells from control human IgG1-treated mice. The increase in Treg cells was transient and the percentage of Foxp3+ T cells returned to normal by day 9 after treatment (Supporting Information Fig. LBH589 mw 1C). Previous studies suggested that treatment of mice with an agonist anti-GITR mAb, following anti-CD25 depletion of Treg cells, was capable of enhancing the pathogenicity of autoantigen-specific T cells in an experimental autoimmune encephalomyelitis model [18]. One problem with this approach is that Treg-cell depletion is usually incomplete and Treg cells rapidly

repopulate the treated animals [19]. To more directly address the effects of GITR stimulation on Teff cell numbers and function, we used the IBD model [20] and transferred CD4+CD45RBhi Foxp3− T cells into RAG KO mice Metabolism inhibitor followed by weekly treatment with Fc-GITR-L (100 μg/mouse i.v.). Mice treated with Fc-GITR-L exhibited a markedly enhanced loss of weight compared with mice that just received CD4+CD45RBhi T cells (Fig. 2A). The percentage of CD4+ T cells secreting IFN-γ was similar in treated and control animals (Fig. 2B and D), but the absolute number of CD4+ T cells secreting IFN-γ was markedly increased in the mesenteric LN (Fig. 2C). In contrast, we observed no changes in either the percentages or absolute numbers of IL-17-producing T cells (Fig. 2E and F).

Teff-cell expansion was also reflected in enhanced Ki67 staining in the treated mice (Fig. 2G and H). Thus, engagement of the GITR by GITR-L in vivo has no effect on T-cell differentiation, but significantly augments the absolute number of pathogenic IFN-γ producing T cells and disease severity. Our results are similar to the effects of next GITR engagement that have been reported [21] on CD8+ Teff cells in a viral model where GITR engagement increased CD8+ T-cell expansion without enhancing their effector functions. Small percentages of Foxp3+ iTreg cells were observed in mice that received CD4+CD45RBhi Foxp3− T cells, but the percentages were the same in untreated or GITR-L-treated mice (data not shown). The GITR is also expressed on APCs and NK cells at a low levels [2] and it has been suggested [22, 23] that some of the effects of GITR engagement in vivo may be secondary to modulation of innate immune functions. To address this issue, we transferred CD4+CD45RBhi T cells from GITR−/− mice to RAG−/− mice (Supporting Information Fig. 2A).