er, the mechanism(s) by which GITR ligation leads to tumor rejection remain unclear. The direct co-stimulation of tumor- specific effector CD4+ and CD8+ T cells (Teffs) has been demonstrated, particularly in combination with active vaccination [16,17,19]; yet, the in vivo effects of DTA-1 on Tregs have not been well-defined. In fact, prior in vitro studies have suggested that the ability of DTA-1 to ‘‘block’’ Treg suppressive activity is due solely to its co-stimulation of Teffs, with little to no impact on Tregs themselves.[6] In this study, we demonstrate that when used as a monotherapy, DTA-1 modulates both Tregs and Teff during treatment of B16 melanoma. In addition, GITR expression by both Teffs and Tregs was needed for the full effects of DTA-1. We show that while in vivo GITR ligation does not globally abrogate Treg suppressive activity, it does impair Treg tumor infiltration and leads to loss of foxp3 expression within intra-tumor Tregs, suggesting a localized abrogation of suppression. The net result is an augmented intra- tumor Teff:Treg ratio and greater Teff activation and function within the tumor. Results GITR expression is upregulated on tumor-infiltrating Tregs and CD8+ T cells during B16 melanoma growth We have shown previously that in vivo GITR ligation by DTA- 1 can induce rejection of B16 melanoma tumors when Targeting GITR on Tregs administered multiple times starting 1 day after tumor challenge [18]. Although we established that DTA-1 can cure very early melanoma tumors, our prior research did not differentiate its contribution to the priming phase versus the effector phase of the immune response. Therefore, to more fully comprehend the mechanisms of GITR ligation therapy, we examined the effects of a single dose of DTA-1 at different time points post-tumor challenge to understand the consequences of ligation at distinct phases of the immune response. We found that 1 mg of DTA-1 on day 4 of tumor growth led to long-term tumor-free survival in 50– 60% of C57BL/6 mice (Figures 1A, 1B). As in other tumor models [15], DTA-1 was more effective when given after several days of tumor growth, with nearly twice as many mice treated on day 4 rejecting tumors compared with mice treated on the day of tumor inoculation (Figure S1). This suggests that GITR ligation therapy in B16 requires initiation of priming, which correlates with in vitro data showing that upregulation of GITR expression on T cells requires 48– 72 hours after TCR-mediated activation [7,13]. Thus, we hypothesized that the greater efficacy on day 4 was due, in part, to increased GITR expression on tumor-activated T cells, providing a more abundant target for ligation by the agonist antibody. In fact, we found that by day 4 after B16 inoculation, GITR expression was significantly higher on tumor-infiltrating CD8+ and Treg cells than on these populations in the spleen or tumor-draining lymph node (TDLN) (Figure 1C). This increased Figure 1. Upregulation of GITR expression correlates with optimal timing of single dose DTA-1 therapy. A and B. C57BL/6 mice (n = 10/ group) were challenged intradermally with 50,000 B16 melanoma cells and treated with 1 mg DTA-1 or rat IgG i.p. on day 4 after tumor challenge. Tumor survival (A) and mean tumor diameter + SEM over time is depicted (B) C. Untreated mice (n = 3/group) bearing 4 day-old B16 matrigel tumors (500,000 cells) were sacrificed and lymphocytes isolated from spleens (S), tumor-draining lymph nodes (DLN), and tumors (T), were stained for CD4, CD8, foxp3, and GITR. Mean GITR fluorescence intensity (MFI) and SEM within each T cell subset is depicted. doi:10.1371/journal.pone.0010436.g001 PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10436