These cells were then incubated at a ratio of 40 : 1

with 51Cr-labelled B16 or B16FasL cells for 4 hr at 37°. For minimal and maximal lysis, cells were LY2606368 ic50 incubated with medium or 5% Triton-X-100, respectively. Lytic activity was measured by 51Cr release with the formula: % lysis = [(sample − min)/(max − min)] × 100. B6 mice were injected i.p. with 0·5 mg of PC61 or GL113 antibodies 4 days and 1 day before s.c. injection of 0·5 × 106 B16-FasL cells. Twenty-four hours later, the skin area including the tumour cells was dissected, snap-frozen in liquid nitrogen and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was reverse transcribed using Superscript III (Invitrogen), and subsequently cDNA was amplified in triplicate Selleck LBH589 by real-time PCR using 1 × Platinum SYBR Green qPCR SuperMix (Invitrogen) with primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), CXCL1/KC or CXCL2/MIP-2. Messenger mRNA levels were normalized relative to GAPDH mRNA expression. The average C(t) values were taken from three mice per group and data are presented as gene expression in PC61-treated mice relative to control GL113-treated mice. Primer pairs were as follows: GAPDH, 5′-TGACCTTGCCCACAGCCTTG-3′ (sense) and 5′-GAACGGGAAGCTTGTCATCA-3′ (anti-sense): CXCL1/KC, 5′-CTCAAGAATGGTCGCGAGGCT-3′ (sense) and 5′-GCACAGTGGTTGACACTTAGTGGTCTC-3′ (anti-sense); CXCL2/MIP-2 5′-CCACTCTCAAGGGCGGTCAAA-3′ (sense) and 5′-TACGATCCAGGCTTC-CCGGGT-3′

(anti-sense). We previously found that B16FasL cells are rejected more efficiently by C57BL/6 (B6) mice when Treg cells are partially depleted by in vivo administration of CD25-specific mAbs.9 Furthermore, this effect is attributable to the ability of Treg cells to suppress innate immune responses.9 To characterize the Flavopiridol (Alvocidib) nature of the innate response inhibited by Treg cells, we injected mice partially depleted of Treg cells and control mice with B16FasL cells and assessed the response to this whole cell challenge at early time-points thereafter. We first performed histological analyses to study the cellular

infiltrate at the non-palpable B16FasL inoculation site. B6 mice treated with depleting CD25-specific mAbs (PC61) or non-depleting control mAbs (GL113) were injected s.c. with 105 live B16FasL, then 4, 24 and 96 hr after tumour injection mice were killed and the injected skin was removed for histology. Tissue was embedded in paraffin and 5-μm sections were cut at 300-μm intervals throughout the skin. Sections were stained with H&E to locate the midsection of the tumour inoculation site (Fig. 1a–d). A large amount of cell death was observed at each inoculation site, as indicated by the lack of cellular cohesion and the presence of fragmented nuclei (Fig. 1b,d). Analyses at these early time-points revealed the presence of an inflammatory infiltrate evident within 24 hr of tumour cell inoculation and which was significantly larger in the PC61-treated group (Fig. 1c,d) compared with the GL113-treated group (Fig.