Soluble CTLA-4 expression was compared with that of autologous CD4+CD25− T cells prepared and rested at 37°C 5% CO2 in culture medium for 24 h before coanalysis. SDS PAGE and western blotting analysis were performed with affinity purified sCTLA-4 samples. Samples were mixed with Laemmli’s sample buffer with the reducing agent 2-Mercaptoethanol. The denatured protein was electrophoretically separated

on a NuPAGE 4–12% Bis-Tris Alectinib purchase precast gel (Invitrogen, UK) and subsequently electroblotted onto a polyvinylidene fluoride membrane (GE Healthcare, UK). After blocking, the blot was reacted with biotinylated anti-CTLA-4 mAb (clone: AS32B Ab Solutions), washed, and incubated with alkaline phosphatase conjugated ExtraAvidin (Sigma, UK). The blot was developed with a commercially available chemiluminescence detection kit (BCIP/NBT tablets, Sigma-Aldrich) or enzyme-linked chemiluminescent detection (GE Healthcare, UK) according to the manufacturer’s instructions. Day 5 PBMC cultures were incubated with Brefeldin A, stained

with anti-CD4-allophycocyanin (BD Biosciences), fixed, permeabilized, LY294002 cell line and stained with biotinylated anti-sCTLA-4 Ab conjugated with streptavidin-FITC (Invitrogen). Cytospin samples were mounted with Vectashield mounting medium (Vector Laboratories Ltd., Peterborough, UK) and observed by confocal microscopy (LSM510 META, Carl Zeiss Meditec, Gottingen, Germany). Female BALB/c mice aged between 10 and 14 weeks received two weekly s.c. injections of ovalbumin

in sterile PBS (100 μg/mouse, n = 4) emulsified in Freund’s Complete adjuvant, before sacrifice 2 weeks later. Splenocytes were recovered from pooled spleens and incubated with Ovalbumin Ag in the presence of anti-sCTLA-4 mAb, JMW-3B3 (10 μg/mL), or an IgG1 isotype control for 5 days at 37°C, 5% CO2. Culture cell proliferation and cytokine levels were measured as described above. This work was performed by the Piedmont Research Center contract research organization, Morrisville, North Carolina, USA. Female B6D2F1/Crl mice were 7–8 weeks old and had a body weight range of 18.4–22.1 g on entry to the study. Mice were divided into test groups of 10 animals, and a further group of untreated 15 Clomifene mice was used to monitor progress of disease at intervals throughout the experiment. B16F10 melanoma cells were harvested during exponential growth and resuspended at a concentration of 5 × 105 cells/mL in PBS. Each mouse received an intravenous (i.v.) injection of 1 × 105 B16F10 cells (0.2 mL cell suspension) into the tail vein on day 1 of the study. Group 1 animals received no treatment (vehicle only). Group 2 animals received 5 mg/kg IgG1 isotype control i.p. on day1 and 2.5 mg/kg on day 3, day 5, and day 7. Group 3 animals received 5 mg/kg pan-specific anti-CTLA-4 mAb (clone: 9H10) on day 1 and 2.5 mg/kg on day 3, day 5, and day 7. Group 4 animals received 5 mg/kg JMW-3B3 anti-sCTLA-4 mAb on day 1 and 2.