To confirm this, neutrophils were further identified as polymorphonuclear cells that express IL-8R (Fig. 5a–d). Furthermore, the results show an increased number of neutrophils in PC61-treated mice at 24 hr post-injection (Fig. 5d) reflecting the data on increased cellular mass in PC61-treated mice (Figs 1 and 3). As neutrophils were more abundant in the Treg-depleted animals, we examined relative levels of neutrophil chemoattractants, CXCL1 (KC) and CXCL2 (MIP-2), in the skin of Treg-reduced and control mice 24 hr post-inoculation with B16FasL cells. Elevated levels of both chemokines were observed in the skin of Treg-depleted

animals suggesting that Treg cells inhibit local neutrophil chemoattractant production (Fig. 5e). As detailed phenotypic characterization of neutrophils from tissue sections is difficult, cytospins were generated from the lavage fluid of mice receiving B16FasL Selleck Belnacasan cells i.p., enabling us to compare neutrophils isolated from PC61-treated and GL113-treated mice (Fig. 6). No differences were observed in expression of the neutrophil activation marker, CD11b or ROS (data not DNA Damage inhibitor shown). An effect of Treg cells on neutrophil activation cannot be ruled out, however, because it is possible that only activated

neutrophils would be recovered in the lavage fluid (and similarly the site of tumour cell inoculation) so any impact of Treg cells on neutrophil activation may be difficult to observe in vivo. However, differences were observed between neutrophils isolated from PC61-treated and GL113-treated mice (Fig. 6). Figure 6(a,b) shows examples of neutrophils isolated from GL113-treated and PC61-treated mice, respectively. Examples of segmented nuclei are given in Fig. 6(c), where segments are joined by thin strands of chromatin. Upon enumeration, it was evident that the proportion of neutrophils with a higher number of segments was increased 17-DMAG (Alvespimycin) HCl in PC61-treated mice (Fig. 6d,e), which results in an increase in the average number of segments per neutrophil (Fig. 6d,e). Hypersegmentation of nuclei in neutrophils has long been associated with more mature

neutrophils, and is an indicator of prolonged neutrophil survival.18 Collectively, these data support the premise that Treg cells affect neutrophil accumulation at the site of antigenic challenge not through inhibiting their activation but through influencing local chemokine production and by limiting their survival. To test the relevance of neutrophils in this model, we first determined, in an in vitro assay, whether neutrophils could impinge on tumour rejection through direct lysis of tumour cells. As shown in Fig. 7(a), neutrophils were capable of lysing both B16 and B16FasL cells. To test the hypothesis in vivo, mice were treated with both PC61 and RB6-8C5, to deplete CD25+ cells and neutrophils, respectively, followed by s.c. challenge with B16FasL (Fig. 7b).