modulated in vivo following GITR ligation. Similar to a study using a fibrosarcoma model [15], we found that GITR ligation was more effective after several days of B16 melanoma growth compared with day 0. This suggested a requirement for initial priming of the immune response in order to induce upregulation of GITR on activated cells. The need for activation before effective GITR ligation is also consistent with our prior study combining DTA-1 with xenogeneic DNA vaccination, in which DTA-1 augmented CD8+ responses and tumor protection only when administered with the second vaccination, and was ineffective when given prior to the initial priming vaccine.[16] During optimal therapy with DTA-1, it became apparent that Treg tumor infiltration was dramatically decreased compared to untreated animals. This resulted in an enhanced intra-tumor CD8:Treg ratio, without significant changes in the spleen or TDLN. A similar skewed ratio has recently been reported with an agonist anti-OX40 mAb, as well as antagonist anti-CTLA4 mAb, suggesting that an altered intra-tumor Teff:Treg ratio may be predictive of anti-tumor activity of these immune-modulating approaches[22,23,30,31]. Regardless of its prognostic implication, the reduced Treg infiltration without major changes in total CD8+ T cell numbers was our initial evidence that a Treg-intrinsic effect was occurring during DTA-1 treatment. In exploring the effects of DTA-1 on Tregs further, we discovered that the altered CD8:Treg ratio following GITR ligation was due both to decreased Treg tumor infiltration and to loss of foxp3 within Tregs that did infiltrate the tumor. Transferring tumor-experienced Tregs into DTA-1-treated, tu- mor-bearing hosts showed that this effect was dependent on Treg GITR expression, as GITR2/2 Tregs did not show significant changes in tumor infiltration. Regulation of Treg trafficking to peripheral sites is complex, with a host of different integrins and chemokine receptors being implicated [32,33]. CD103 (aE), CCR7, and CD62L (L-selectin) are involved in Treg trafficking from lymph nodes to inflamed sites, with CD103+CCR7-CD62L- Tregs most capable of homing to and suppressing peripheral inflammation [34,35]. We did not see significant changes in CD103, CCR7 or CD62L expression on TDLN Tregs 48 or 72 hours following in vivo DTA-1 treatment (Figure S6). In addition, the chemokine receptor CCR4 (specific for CCL17 and CCL22) has been specifically implicated in Treg migration into tumors [36,37], and we are currently examining alterations in the levels of this and other chemokine receptors on Tregs following GITR ligation in vitro and in vivo. By histology we observed that most Tregs that do enter the tumor lose foxp3 expression, with some residual GFP expression now localized to the cytosol. In fact, repeating the transfer experiment with a highly-purified Treg population from foxp3GFP mice confirmed the DTA-1-induced intra-tumor foxp3 loss in this transferred population as well. Whether this loss of foxp3 protein is due to decreased expression, increased degradation, or some combination of the above is not clear. The plasticity of both naturally-arising and induced CD4+foxp3+ Tregs has recently been documented in several studies, with loss of foxp3 expression and suppressive function occurring during conditions of lympho- penia or inflammation/autoimmunity, in some cases even accompanied by gain of effector function [25,26,27,28,38]. Treg lineage stability may be regulated in part through the integration of external stimuli which either promote/maintain (e.g. IL-2, IL- 10, TGF-b) or decrease (e.g. IL-6) foxp3 expression, through modulation of signaling pathways and altered methylation of the foxp3 locus [39,40]. Interestingly, Luo et al recently reported that TGF-b1-mediated induction of foxp3 in CD4+CD25- cells was due to inhibition of phospho-ERK and down-regulation of DNA Targeting GITR on Tregs methyltransferases, leading to demethylation of the foxp3 promoter [41]. Signaling through GITR is known to activate the MAP kinase pathway and induce ERK phosphorylation [4,42,43]. Thus, GITR ligation may antagonize the effects of TGF-b on pERK and inhibit downstream signaling which normally induces foxp3 gene expression. In support of this, we have found that in vitro GITR ligation is able to block TGF-b-mediated up- regulation of foxp3 in activated CD4+foxp3- T cells (Figure S7), suggesting an effect on gene expression. Whether this mechanism, likely in concert with other inflammatory stimuli present within the tumor microenvironment, plays a role in the selective loss of foxp3 observed in tumor-infiltrating Tregs is currently under investigation. Despite this effect in the tumor, DTA-1 did not modulate the ex vivo suppressive capacity of Tregs isolated from TDLN, consistent with prior ex vivo [17,18] and in vitro [6,44] studies. In all of these studies, Treg suppressive ability was maintained during GITR ligation, and collectively these data refute the argument that GITR ligation globally makes Tregs unable to suppress. Still, it remains possible that intra-tumor Tregs, which lose foxp3 expression, have diminished suppressive capacity. This possibility is supported by recent data demonstrating that Tregs require constant foxp3 expression for the continued expression of suppressive genes, and for the repression of inflammatory genes as well.[29] Unfortu- nately, we were unable to test the ex vivo suppressive capacity of these ‘‘former’’ Tregs, as the yield of purified live GFP+ TIL isolated from DTA-1-treated, B16-bearing foxp3GFP mice was always too low after FACS-sorting (data not shown). Thus, this question remains open for now. Somewhat surprisingly, we did not find that in vivo GITR ligation in our B16 model made CD8+ Teffs from TDLN resistant to Treg-mediated suppression ex vivo, in contrast to prior observations with CD4+ [6,17] or CD8+ Teffs [19]. One explanation for this discrepancy may be that we isolated CD8+ T cells from the TDLN 72 hours after DTA-1. Since CD8+ T cells are less responsive in general to GITR signaling than CD4+ T cells [3,6,10,11], it is possible that the level of GITR signaling required for ‘‘resistance’’ to Tregs had waned by the time these cells were activated ex vivo. In addition, we used bulk CD8+ Teffs isolated from TDLN, only a minority of which were likely activated by tumor antigens and therefore amenable to co- stimulation by DTA-1. In contrast, the study by Nishikawa et al demonstrating generation of ‘‘Treg-resistant’’ CD8+ cells used activated, tetramer-purified, antigen-specific CD8+ T cells in their suppression assays.[19] Based on these data, it is possible that a population of activated, tumor-specific CD8+ T cells from DTA-1- treated B16-bearing TDLN might be similarly ‘‘Treg-resistant.’’ While we did not demonstrate that GITR ligation directly modulated suppression or resistance to suppression, we did observe a reproducible increase in the intra-tumor effector function of tumor specific pmel-1 T cells. Therefore, even if Tregs still retain suppressive capacity following GITR ligation, their relative numerical disadvantage in the tumor may render them less effective overall. Considering our evidence that DTA-1 directly modulated Tregs as well as Teffs, we reconstituted RAG12/2 mice with GITR2/2 or GITR+/+ Teffs and Tregs, to confirm which cell types were able to respond to GITR ligation. While the anti-tumor effect of DTA- 1 in this system was modest, the delay in tumor growth was reduced if either Tregs or Teffs lacked GITR, and was non- existent if both subsets were GITR-negative. It is possible that the weakened effect of DTA-1 was due to inadequate peripheral reconstitution. Alternatively, there could be a contributing role for GITR ligation on B cells or NK-T cells, both of which were absent PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10436