Targeting GITR on Tregs Figure 7. DTA-1-induces foxp3 loss in intra-tumor Tregs after transfer, and the effects of DTA-1 require GITR expression on effector and regulatory T cells. A. CD4+GFP+ Tregs isolated from spleens and TDLN of B16-bearing CD45.2+ foxp3gfp donor mice on day 8 after tumor challenge were transferred (0.76106 per recipient) into B16-bearing CD45.1+ mice treated with cyclophosphamide and DTA-1 or IgG, as described in Figure 4. Tumor-infiltrating lymphocytes were isolated 2–3 days later and CD45.2+ donor T cells were assessed for their level of foxp3 and GFP expression by FACS. Left: The % + SEM of donor (CD45.2+) Tregs found within the total live CD4+ gate is depicted, along with relative proportions that became foxp3-negative. Donor Tregs within DTA-1-treated tumors showed a significantly greater reduction in foxp3 expression (p = 0.001) Right:. Representative FACS plots gated on live CD45.2+CD4+ TIL. Data are pooled (n = 7 per group) from 2 independent experiments. B RAG12/2 mice (n = 529/group) were reconstituted with indicated combinations of effector (Te: CD8+ and CD4+CD25-) and regulatory (Tr: CD4+CD25+) T cells from GITR2/2 (2) or GITR+/+ (+) littermates (See Figure S5 for schema). After 4 weeks, mice were challenged with B16 and treated with DTA-1 or IgG on day 4. At 18 and 22 days post-challenge, when all tumor-free survival is lost in WT untreated animals (Figure 1A), mean diameter of the Te+/Tr+ (DTA1) group was significantly different from all other groups. (p,0.05; two-tailed student’s t-test). Data are pooled from 2 independent experiments. doi:10.1371/journal.pone.0010436.g007 expression of GITR on both Tregs and Teffs was necessary for the protective effect of DTA-1. Therefore, we reconstituted RAG12/2 mice with different combinations of Teffs and Tregs isolated from either GITR2/2 or GITR+/+ littermates, challenged with B16, and treated with DTA-1 or IgG on day 4 of tumor growth (Figure S5). Although we did not see full protection with DTA-1 in this RAG12/2 reconstitution system, there was a statistically signifi- cant delay in tumor growth seen only when both Teff and Treg were GITR +/+ (Figure 7B). Lack of GITR on either population led to an attenuated effect, while lack of GITR on both populations abrogated any protective effect. Taken together, these findings provide further evidence that agonist anti-GITR mAb is modulating the activity of both Teffs and Tregs to effect tumor immunity. Discussion Although prior studies have demonstrated that in vivo GITR ligation results in anti-tumor effects, the exact mechanisms involved remain elusive. This study sought to determine whether GITR ligation on Teffs, Tregs, or both was primarily responsible for these anti-tumor effects, and explore how these subsets were PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10436