expression was maintained within the tumor environment on days 7 and 14 of tumor growth (data not shown). However, in contrast to the fibrosarcoma or colon carcinoma models previously reported [15,17], as well as to our experience in an A20 lymphoma model (Cohen A, unpublished data), starting treatment with DTA-1 on day 7 or later was generally ineffective (Figure S1). This demonstrates that optimal timing of in vivo GITR ligation likely varies according to strain and the underlying immunoge- nicity and aggressiveness of the tumor subtype. In vivo GITR ligation does not systemically alter capacity of Tregs to suppress or Teffs to resist suppression To investigate the cell-intrinsic effects of DTA-1, we next explored how agonist anti-GITR mAb might be modulating Teff and Treg function. Highly purified Tregs were isolated from tumor-draining lymph nodes (TDLN) of B16-bearing foxp3GFP mice [20] after treatment with DTA-1 or IgG, and tested for their ability to suppress proliferation of CD8+ Teffs isolated from the same TDLN. Consistent with prior in vitro [6] and in vivo [17] studies, DTA-1-treated Tregs maintained suppressive capacity (Figure 2). In addition, neither in vitro nor in vivo GITR ligation on Tregs led to a significant change in expression of granzyme B, IL-10, or TGF-b, three molecules reported to play a role in Treg- mediated suppression in vivo [21] (Figure 2). Surprisingly, CD8+ T cells from DTA-1-treated mice remained susceptible to Treg- mediated suppression, without significant difference from those Targeting GITR on Tregs from IgG-treated mice. We observed similar findings when DTA-1 treated CD8+ T cells were co-cultured with na¨ıve (as opposed to tumor-experienced) Tregs, again displaying no acquired resistance to ex vivo suppression (data not shown). Thus, at least within the TDLN, where initial priming of the anti-tumor effector response is likely occurring, in vivo GITR ligation does not appear to systemically alter the capacity of Tregs or CD8+ Teffs to suppress or be suppressed, respectively. GITR ligation leads to enhanced intra-tumor CD8:Treg ratio, and greater CD8+ effector T-cell activity within the tumor To investigate how in vivo GITR ligation modulates T cell subsets within the tumor, we first examined the relative intra- tumor frequencies of Tregs and Teffs in B16-bearing hosts following DTA-1 treatment. DTA-1 led to a significant decrease in intra-tumor Treg frequency as a percentage of CD4+ TILs (40% of CD4+ TIL for IgG, compared with 18% for DTA-1, p = 0.02, Figure 3A). This was accompanied by a modest increase in the total intra-tumor CD8:CD4 T-cell ratio (0.7 for IgG compared with 1.3 for DTA-1-treated mice, p = 0.04). The net result, therefore, was a five-fold increase in the intra-tumor CD8:Treg ratio (13.4 for DTA-1-treated compared with 2.6 for IgG-treated mice, p = 0.05), favoring the effector population (Figure 3B). Evaluation of absolute numbers/gram of tumors for Teffs and Tregs paralleled the frequency data, showing decreased Tregs Figure 2. In vivo GITR ligation does not globally alter capacity of Tregs to suppress or CD8+ Teffs to resist suppression. B16-bearing C57BL/6 mice (n = 6/group) received DTA-1 or IgG on day 4. TDLN were harvested on day 7 and CD8+ (Teff) and CD4+CD25+ (Treg) cells were isolated by MACS beads. CFSE-labeled, IgG- or DTA-1-treated Teff were cultured with IgG- or DTA-1-treated Treg at 2:1 Teff:Treg ratio, with irradiated APCs and anti-CD3 mAb 1 mg/ml for 4 days. A. Representative sample showing CFSE dilution in CD8+DAPI- cells. doi:10.1371/journal.pone.0010436.g002 PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10436