Targeting GITR on Tregs Figure 5. Intra-tumor Tregs show increased proliferation and lower Foxp3GFP MFI after DTA-1 treatment. A. Representative Ki67 expression within live CD4+foxp3+ cells isolated from day 10 B16-bearing C57BL/6 mice treated with DTA-1 or IgG on day 4. B. and C. TIL were isolated from day 10 B16-bearing foxp3GFP mice (n = 5/group) treated with DTA-1 or IgG on day 4 and examined by FACS. B. Mean fluorescence intensity (MFI) of GFP on live intra-tumor CD4+GFP+ cells. C. Intra-tumor CD8:Treg ratio of DTA-1- and IgG treated mouse in (B) is plotted against its foxp3GFP MFI, demonstrating a significant inverse correlation between Teff:Treg ratio and foxp3GFP MFI in DTA-1 treated mice only (p = 0.03). Data are representative of 3 independent experiments. doi:10.1371/journal.pone.0010436.g005 In order to determine the relative contributions of impaired tumor infiltration and loss of foxp3 expression to the decreased intra-tumor accumulation of Tregs, we repeated the adoptive transfer experiments described in Figure 4 using highly purified (.99% CD4+GFP+, 93% foxp3+ (data not shown)), FACS-sorted donor Tregs from foxp3GFP mice, in an attempt to track Tregs that become foxp3-negative within the tumor. By gating on donor cells using a congenic marker and assessing dual expression of foxp3 and GFP, we found both decreased Treg infiltration and loss of foxp3 expression within the tumors of DTA-1-treated recipients (Figure 7A), implying that both of these mechanisms are playing a role in this phenomenon. Interestingly, a significant minority of CD4+GFP+ donor cells entering control IgG-treated tumors also became foxp3-negative (and GFP-negative), as described previ- ously for Tregs transferred into lymphopenic hosts [27,28], which is likely related to the pre-treatment of recipients with cyclophos- phamide 1 day prior to transfer. This population, however, was significantly increased following in vivo GITR ligation (mean % of foxp3+ donors becoming foxp3- = 78% for DTA-1 vs. 34% for IgG, p = 0.001, Figure 7A), demonstrating an additional effect of GITR ligation. We were unable, at least by FACS analysis, to identify the aberrant or ‘‘transitioning’’ GFP+foxp3- cells in these adoptive transfer experiments. As Tregs require continued nuclear foxp3 expression to remain suppressive [29], it is possible that these foxp3-,‘‘former’’ Tregs have lost regulatory function. Thus, in addition to impairing tumor infiltration, a mechanism by which GITR ligation alters the Teff:Treg balance within the tumor may be alteration of the lineage stability of intra-tumor Tregs. Maximal anti-tumor effect of agonist anti-GITR mAb requires GITR expression by both effector and regulatory T cells Finally, because of these myriad effects on intra-tumor Tregs, as well as current (Figure 3C) and prior [15,16,17] data showing enhancement of Teff function, we attempted to confirm that PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10436