The CD4+ T cells were stimulated as described previously for 5 days in the primary culture. Some cultures received n-butyrate (0.8 mm) or TGF-β1 (20 ng/ml). TGF-β1 was added to some primary cultures as a positive control for Treg cell generation. Flow cytometry was used to quantify Treg cells in these cultures through determination of the percentage of CD4+ T cells expressing EGFP. The percentage of living CD4+FoxP3+ T cells was determined daily after exclusion of non-viable cells with 7-AAD (BD Via-Probe;
BD Biosciences, San Jose, CA, USA). Analysis of IL-2 production. CD4+ T cells from control and n-butyrate-treated primary cultures were re-stimulated in triplicate wells in 96-well flat-bottom plates with plate-bound anti-CD3 mAb (0, 0.03, 0.1, 0.3 or 1 μg/ml) and soluble anti-CD28 mAb (1 μg/ml) for 3 days. Soluble anti-CD25 Inhibitor Library concentration mAb (2.5 μg/ml) was added to secondary cultures to block adsorption of IL-2 by the CD4+ T cells. The eBioscience Mouse IL-2 ELISA Ready-SET-Go! reagent set quantified IL-2 secretion in the culture supernatants. Secondary culture suppression
assays. CD4+ T cells from control, n-butyrate and TGF-β-treated primary cultures were subjected to Ficoll–Hypaque separation to remove non-viable cells. All primary culture CD4+ T cells will be referred to as TEFF for the suppression assays. To assess all TEFF for suppressor function, CFSE-labelled naïve CD4+ T cells were harvested and CFSE-labelled BIBW2992 in vivo to serve as proliferation responders. These responders will be referred to as TRESP for the suppression assays. TRESP (2.5 × 104 cells/well) were co-cultured with TEFF at ratios of 2:1, 1:1, 0.5:1 and 0:1 (TEFF:TRESP). Proliferation of the TRESP cells was induced in the 96-well flat-bottom plates via plate-bound anti-CD3 mAb (3 μg/ml) and soluble anti-CD28 mAb (1 μg/ml). After 3 days, Mirabegron proliferation of CFSE-labelled TRESP was quantified with flow cytometry. Briefly, CFSE-labelled TRESP were either un-stimulated or stimulated as described previously. Un-stimulated CFSE-labelled
TRESP were used to determine the CFSE signal intensity of non-proliferating CD4+ T cells. The percentage of stimulated CFSE-labelled TRESP that exhibited a diminished CFSE signal intensity when compared with the un-stimulated CFSE-labelled TRESP signal intensity reflected the percentage of TRESP proliferation within the co-cultures. Flow cytometry. All flow cytometry data were obtained on a Partec CyFlow ML (Swedesboro, NJ, USA) and analysed by De Novo Software’s FCS Express (Los Angeles, CA, USA). CD4+ T cell purity following positive selection of splenic and inguinal lymph node cells was checked with APC-conjugated anti-CD4 mAb and averaged 90%. Statistics. The unpaired Student’s t-test was used to analyse data. A P value <0.05 was considered significant. Gilbert et al. previously reported that n-butyrate anergized murine antigen-specific CD4+ Th1 clones [10, 11, 18, 19].