there was a 50% reduction in the intra-tumor accumulation of DTA-1-treated donor Tregs, compared to IgG-treated controls (Figure 4A). The effect was even more dramatic when the recipients were treated with DTA-1 following donor Treg transfer, with a .90% reduction in donor Treg frequency within the tumor, despite equal distribution within spleen and TDLN. To determine if this effect was Treg-intrinsic, we repeated this experiment using Tregs from GITR+/+ or GITR2/2 donors. Tregs from GITR2/2 mice did not show a significant block in trafficking when transferred into DTA-1 treated animals, com- pared to IgG controls, demonstrating a requirement for direct GITR ligation on Tregs themselves (Figure 4B). GITR ligation leads to loss of foxp3 expression within intra-tumor Tregs The above experiments suggest that one mechanism of DTA-1’s anti-tumor efficacy may be interfering with Treg infiltration into tumors, and imply a requirement for GITR expression on Tregs. However, these studies examined a small transferred population in a lympho-depleted recipient, which may not fully reflect the biologic effects of GITR ligation under steady-state conditions. We therefore examined other possible mechanisms that could contribute to the DTA-1-induced decrease in intra-tumor Tregs, including impaired proliferation, impaired survival, or loss of foxp3 expression. We found no evidence for impaired prolifera- tion, in fact, in vivo GITR ligation on Tregs increased their proliferation in the spleen, TDLN, and tumor (Figure 5A), consistent with GITR’s known co-stimulatory effects. Likewise, we could not find evidence of impaired survival following in vivo GITR signaling. Tregs within TDLN and tumors from DTA-1- treated mice showed no difference in apoptosis compared with IgG-treated controls, either by immunoflourescence (TUNEL (Figure S4A) or flow cytometric staining (Annexin V, Figure S4B, or activated caspase 3 (data not shown)) We did observe, however, that the mean fluorescence intensity (MFI) of the foxp3-GFP fusion protein was slightly lower within intra-tumor Tregs from DTA-1-treated foxp3GFP mice (Figure 5B), without changes seen in spleen and TDLN (data not shown). Interestingly, there was an inverse correlation between foxp3-GFP MFI and CD8:Treg ratio. Mice that had the lowest expression of foxp3-GFP within intra- Targeting GITR on Tregs tumor Tregs had the greatest corresponding CD8:Treg ratio, while those with normal expression had ratios similar to those seen in IgG-treated mice (Figure 5C). Therefore, to confirm these findings and further characterize tumor-infiltrating Tregs, we performed immunoflourescent histo- logical analysis of B16 tumors in foxp3GFP mice. Mice were treated with DTA-1 on day 4 of tumor growth and tumors and TDLN were isolated on day 10, when Treg tumor infiltration in untreated mice begins to peak (Schaer DA, unpublished results). At this time, the total number of GFP+ cells within the DTA-1 treated tumors showed a ,28% decrease compared to IgG control treated mice (mean 17.8 per high power field (hpf) for DTA-1 vs. 25/hpf for IgG, p = 0.06, Figure 6E). While this decrease was not as dramatic as what was seen with transferred Tregs (Figure 4A), it was on par with the reduced number of Tregs seen by FACS (Figure 3A). More striking, however, was the finding that over 80% of Tregs in DTA-1 treated tumors had an irregular appearance, with weak and aberrant GFP expression that did not co-localize with nuclear DAPI staining (Figure 6A, insert). This is in contrast to IgG tumors where Tregs displayed ‘‘normal’’ overlay of foxp3GFP transcription factor with nuclear DAPI staining. The abnormal phenotype was noted only in DTA-1-treated tumors and not IgG-treated tumors (Figure 6B) or DTA-1-treated TDLN (Figure 6C), demonstrating a specific effect within the tumor microenvironment. Thus, when we quantified the numbers of ‘‘normal’’ Tregs (i.e. co-localizing GFP and nuclear DAPI), we observed a marked decrease within DTA- 1-treated tumors (mean 4/hpf) compared with IgG-treated controls (25/hpf, p,0.0001, Figure 6E). This represents a ,84% reduction in the numbers of ‘‘normal’’ Tregs. Recent reports have demonstrated the plasticity of Tregs and their ability to lose foxp3 expression under inflammatory conditions [25,26]. Therefore, we asked if the discrepancy in our data was due to loss of foxp3 in the irregular Tregs. Co-staining for foxp3 demonstrat- ed that these aberrant cells were all foxp3-negative (Figure 6D), indicating that they had lost expression of the foxp3 protein. While the irregular Tregs still maintained some GFP protein, it now was no longer linked to foxp3 and its nuclear localization signal. The majority of DTA-1-treated tumor sections contained only the aberrant Tregs; however, some sections contained both normal (foxp3+) and aberrant (foxp3-) GFP+ cells (Figure 6F), demon- strating that this was not a staining artifact. Figure 4. DTA-1 reduces Treg tumor trafficking in a cell intrinsic manner. A. B16-bearing Thy1.1+ donor mice were treated on day 5 with DTA-1 (‘‘DTA-1 donor’’) or IgG (‘‘IgG donor’’), and Tregs isolated on day 8 from their spleens and TDLN were transferred (0.76106 per recipient) to B16- bearing Thy1.2+ mice, treated 1 day earlier with cyclophosphamide 250 mg/kg i.p. Some recipients received Tregs from untreated B16-bearing donors and were then treated with DTA-1 (‘‘DTA-1 recipient’’) or IgG (‘‘IgG recipient’’) 12 hours after adoptive transfer. Recipient spleens, TDLN, and tumors were harvested 4 days after transfer. The percentage + SEM of donor (Thy1.1+CD4+foxp3+) Tregs within the total live CD4+ gate is depicted. *p = 0.05 compared with IgG donor, **p = 0.04 compared with IgG recipient. B. Tregs from Day 8 B16-bearing Thy1.2+ GITR2/2 (‘‘KO donor’’) or GITR+/+ (‘‘wt donor’’) donors were transferred into day 8 B16-bearing Thy1.1+ recipients treated 1 day earlier with cyclophosphamide 250 mg/kg i.p. Recipients received DTA-1 or IgG 12 hours post-transfer, and spleens, TDLN, and tumors were harvested 4 days later. The % + SEM of donor (Thy1.2+CD4+foxp3+) Tregs within the total live CD4+ gate is depicted. *p = 0.07 compared with wt donor + IgG, **p = 0.24 compared with wt donor + IgG. doi:10.1371/journal.pone.0010436.g004 PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10436