Targeting GITR on Tregs Figure 3. GITR ligation by DTA-1 limits Treg accumulation within the tumor and enhances intra-tumor CD8+ T-cell activity. A. and B. B16-bearing mice treated with DTA-1 or IgG on day 4 had spleens, TDLN, and tumors harvested on day 10 and lymphocytes analyzed by FACS. A. Representative FACS plots with gate frequencies, (left) and mean +SEM for frequency of Tregs within live CD4+ TIL gate (right) are shown. B. Ratio of CD8+ to CD4+foxp3+ cells in spleen, TDLN, and tumor. *p = 0.05 compared with IgG tumor. Pooled data from 3 independent experiments are shown. C. Na¨ıve C57BL/6 mice (n = 325/group) received 46106 CFSE-labeled pmel-1 Thy1.1+CD8+ T cells 1 day prior to B16 inoculation. Recipients received DTA-1 or IgG on day 4, and donor pmel-1 CD8+ cells analyzed in spleens, TDLN, and tumors on day 14. Mean frequency +SEM is shown for IFNc+ (left), activated CD44hiCD62Llo phenotype (center) and proliferation by CFSE dilution (right) of transferred pmel-1 T cells is shown. For IFNc recall assay (C, left), lymphocytes from spleen, TDLN, and tumor were re-stimulated for 6 hours with irradiated, gp10025-33 peptide-pulsed EL4 cells. Background IFNc production for lymphocytes cultured with unpulsed EL4 cells was ,1%. Over 85% of IFNc+ cells were also CD107a+ (data not shown). * p,0.05 compared with IgG group. Representative of 3 independent experiments. doi:10.1371/journal.pone.0010436.g003 without major increases in CD8+ T cell numbers/gram of tumor (Figure S3). This demonstrated that the augmented CD8:Treg ratio within tumors from DTA-1 treated mice resulted primarily from changes in the Treg compartment. No significant changes in these relative frequencies were seen in spleen or TDLN, demonstrating a specific effect within the tumor (Figure 3B). Interestingly, the proportion of mice with a .2-fold increase in intra-tumor CD8:Treg ratio over the IgG control (8/13, 61%) mirrored the percentage of mice with long-term tumor-free survival (,60%, Figure 1A and Figure S1). This suggests that achieving a skewed CD8:Treg ratio may be predictive of long- term tumor control, as described recently with other immune- modulating antibodies.[22,23] We next investigated possible functional consequences of this altered intra-tumor Teff:Treg ratio by exploring the effects of GITR ligation on tumor-specific CD8+ effector cells. This was accomplished by adoptively transferring and tracking CD8+ T cells from Thy1.1+ pmel-1 TCR transgenic mice, which are specific for the melanoma antigen gp100, into C57BL/6 (Thy1.2+) mice 1 day prior to B16 challenge. GITR ligation did not consistently increase the frequency or absolute number of donor pmel-1 cells infiltrating the tumor (data not shown). However, DTA-1 significantly augmented the activation of those cells that did traffic to tumor, leading to enhanced effector function (IFN-c secretion following gp100 peptide re-stimulation) activation (CD44hiCD62Llo phenotype), and proliferation (as measured by CFSE dilution) (Figure 3C). IFN-c-secreting pmel-1 T cells also mobilized CD107a (LAMP-1), a surrogate for lytic degranulation [24], demonstrating their cytolytic capacity (data not shown). These data indicate that GITR ligation results in a more effective intra-tumor CTL population, which may be a consequence of both direct CD8+ T-cell co-stimulation and a more favorable local CD8:Treg ratio. Treatment with agonist anti-GITR mAb modulates Treg accumulation in the tumor Because Treg frequency was decreased only in the tumor and not systemically, we explored the hypothesis that GITR ligation was altering the migraton of Tregs to the tumor. Tumor- experienced Tregs isolated from B16-bearing, Thy1.1+ mice treated 72 hours earlier (day 4 of tumor growth) with DTA-1 or IgG were adoptively transferred into B16-bearing Thy1.2+ recipients. Recipient mice were treated 1 day earlier with cyclophosphamide to reduce endogenous lymphocyte populations and aid recovery of donor cells. While DTA-1-treated and IgG- treated Tregs distributed equally within the spleen and TDLN, PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10436