Female BALB/c mice were housed in the Medical Research Facility, University of Aberdeen. The work conformed to the UK Animal (Scientific Procedures) Act (1986) and was carried out with UK Home Office project license approval. Female B6D2F1/Crl mice (Charles River, Morrisville, NC, USA) were housed at the Piedmont Research Center contract research organization, Morrisville, North Carolina, USA. Piedmont specifically complies with the recommendations of the Guide for Care and Use of Laboratory

Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at Piedmont is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, which assures compliance Bortezomib nmr with accepted standards for the care and use of laboratory animals. Anti-CD3 mAb (OKT-3, ECACC, Salisbury, UK), or tuberculin Silmitasertib mouse purified protein derivative PPD (Statens Serum Institut, Copenhagen, Denmark) were each added to cultures at 10 μg/mL, unless stated otherwise. SEB (Sigma-Aldrich, Poole, Dorset, UK) was used to stimulate cultures at 2 μg/mL, unless stated otherwise. Cell proliferation in cultures was measured from 3H thymidine incorporation in triplicate samples using a 1450 Microbeta liquid scintillation counter (LKB Wallac, Turku, Finland). Results are presented as mean cpm ±SD or as stimulation index of autologous unfractionated cells. ELISA for cytokines

produced in cultures were based on previously published methods [51]. The Ab pairs for human cytokines were: anti-IFN-γ (clones NIB42 and 4S.B3 BD Biosciences, Oxford, UK), anti-IL-17A Dolichyl-phosphate-mannose-protein mannosyltransferase (clones eBio64CAP17 and eBio64DEC17, eBiosciences, Hatfield, UK), and for mouse:

anti-IFN-γ (clones AN-18 and R4–6A2) and IL-17A (clones TC11–18H10.1 and TC11–8H4.1, all BD Biosciences). All cytokine standards were from Peprotech EC Ltd. (London, UK). Bound Ab was detected using streptavidin-labeled alkaline phosphatase with a phosphatase substrate (both Sigma Aldrich), and absorbance measured at 450 nm (corrected with a reference reading at 492 nm) with a Multiskan MS microplate photometer (Life and Laboratory Sciences, Basingstoke, UK). Cell culture supernatant levels of cytokine were measured in stimulated vs. nonstimulated control wells following 5 days culture of PBMCs or fractionated T-cell subsets at 37°C, 5% CO2, unless stated otherwise in individual experiments. Ab JMW-3B3 (IgG1λ) specific for the soluble, but not membrane-bound, isoform of human CTLA-4 was produced by standard hybridoma technologies after immunization of BALB/c mice with a peptide, K120-M137, unique to the C terminus of sCTLA-4 (Supporting Information Fig. 1 and 2). Commercially available antibodies that do not discriminate between the isoforms (pan-specific) were obtained from several sources (Human clones: BNI3, AbD Serotec, Kidlington, UK; 14d3, eBioscience; AS-32P, Ab solutions, Mountain View, CA, USA, ANC.