MBP Ac1–9[4K] and rhIL-2 at a final concentration of 10 μg/mL and 20 U/mL, respectively,

were added to the cell suspension, and cultures were incubated in 6-well plates at 37°C and 5% CO2 humidified atmosphere. After at least 5 days, the cultured splenocytes were washed and CD4+ T cells were isolated by positive selection. Temsirolimus 5×104in vitro expanded CD4+ T cells from peptide-treated Tg4 mice were co-cultured with an equal number of untreated CD4+ T cells, or at ratios from 1:1 to 1:32 of peptide-treated to untreated CD4+ T cells, at 100 μg/mL of MBP Ac1–9[4K] in the presence of 1×105 APC/mL. After 72 h, wells were pulsed with 0.5 μCi [3H] thymidine overnight and the incorporated radioactivity was

measured on a liquid scintillation β-counter (1450 Microbeta; Wallac). click here Staining for intracellular cytokine expression was performed using BD CytoFix/CytoPerm Plus Kit (BD Biosciences). Splenocytes from peptide-treated mice were collected 2 h after the last treatment and restimulated with PMA and ionomycin (Sigma-Aldrich) at a final concentration of 5 ng/mL and 500 ng/mL of culture, respectively, for 4 h in the presence of GolgiStop (BD Biosciences). After the incubation, cells were stained with anti-Vβ8 FITC (BD Biosciences), fixed, permeabilized and stained intracellularly with anti-IL-2 APC, anti-IL-4 PE, anti-IL-10 APC, anti-IL-17 PE and anti-IFN-γ PE antibodies, or Ig isotype controls (BD Biosciences). Fluorescence intensity was measured on a FACSCalibur or BD™ LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). Conventional sandwich ELISA was carried out according to instructions from the manufacturer using paired antibodies to assay the quantity of IL-2, IL-10 and IFN-γ (BD Biosciences) in cell culture supernatants. Optical densities

were measured at 450/595 nm on a SpectraMax 190 microplate reader and the amount of cytokine present quantified with standard curves using SoftMax Pro software (both from Molecular many Devices). Statistical analyses were performed where stated using GraphPad Prism (GraphPad Software) software. The statistical significance of differences between data groups was determined by an unpaired t-test. A p value of ≤0.05 was considered to be significant. We thank Drs. C.A. Sabatos-Peyton and J. Verhagen for critical reading of this manuscript. We also thank Miss L.E.L. Falk and ASU staff for assistance with the breeding of mice. This work was supported by the Wellcome Trust and the MS Society of Great Britain and Northern Ireland. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors.