During these analyses, check details it was noticed that there were two forms of cellular mass displaying different histological characteristics (Fig. 2). In one type, cells were confined to a single layer of the skin, surrounded by normal tissue (Fig. 2a,b); however, in the other type,

inflammatory cells were found spread throughout the layers of the skin (Fig. 2c,d). Upon assessment of sections for these characteristics, none of the sections from PC61-treated mice, and around half of the GL113-treated mice, displayed the ‘confined’ phenotype (Fig. 2e). This is noteworthy when compared with the percentage of mice that reject these tumours; approximately 50% in GL113-treated mice and 100% in PC61-treated mice.9 To perform a more quantitative assessment of the differences between cellular masses termed ‘confined’ versus those termed ‘non-confined’, the total volume of each cellular mass within the GL113-treated and PC61-treated groups (> 4 per group),

4 and 24 hr see more after tumour cell inoculation, was calculated. These data, shown in Fig. 3(a), corroborated our previous observation in that at 24 hr larger masses were observed in the PC61 group compared with those treated with GL113. At later time-points (96 hr), larger cellular masses were measured in the latter, control group of mice, coinciding with detection of live tumour cells in this group. Live tumour cells were identified by histological examination of H&E-stained also sections in GL113-treated mice but not in PC61-treated mice. In the former group, within the tumour cell mass, amid cell debris, there are areas of homogeneous healthy cells, forming foci of organized tissue, similar to that seen in large, established tumours (Fig. 3b,c). These data are consistent with the observation that around 50% of mice inoculated with B16FasL develop palpable tumours whereas tumours

are rarely seen in B16FasL-inoculated mice pre-treated with PC61.9 Overall, these data indicate that an inflammatory infiltrate into the tumour creates a disorganized, non-confined mass that is associated with tumour cell death and tumour rejection, favoured by depletion of Treg cells by PC61 mAbs. We were struck by how rapidly Treg-cell depletion affected the accumulation of inflammatory cells at the site of the tumour cell inoculum. The ability of Treg cells to suppress an inflammatory response within hours of an antigenic challenge and at a peripheral site implies that skin-resident Treg cells are rapidly mobilized. To visualize Treg cells at the site of tumour cell challenge, skin sections were stained with Foxp3-specific mAbs. Foxp3+ cells were found in the skin and particularly at the site of tumour cell inoculation (Fig. 4). This is in agreement with other studies reporting Treg-cell identification in the skin of mice16 and humans.17 Stained cells were not observed in sections prepared from PC61-treated mice (data not shown).