Preparation of cell suspensions from liver, lung and bone marrow was as described

previously 13. To enumerate cell number, cytometric bead-based counting assays were performed as described previously 13. Intracellular cytokine staining was performed according to standard procedures 4 using stimulation with OVA323-339 (16–18 h) or PMA/ionomycin (3 h, Merck Biosciences, Darmstadt, Germany; Fluka, Switzerland). Cytometric data were collected using a FACSCalibur or FACSCanto cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with CellQuest or FACSDiva software. The total number of IFN-γ-producing OT-II cells was calculated based on the intracellular cytokine staining and absolute OT-II cell number determined using a bead-based counting assay. To assess proliferation in vivo, OVA-specific OT-II T cells were labeled with CFSE as described previously 46 and used for selleck compound culture or injected i.v. Three days later, recipient spleens or LN were harvested and CFSE dilution (CD45.1+/CD4+ gated cells)

assessed by flow cytometry. To determine responsiveness to antigen challenge, mice were immunized s.c. at the tail base with OVA (100μg) emulsified in complete Freund’s adjuvant. In vitro peptide restimulations were performed on splenocyte single-cell suspensions plated at 2×106/mL (1 mL, 24-well plates) in learn more complete RPMI with or without added OVA323–339 (10 μg/mL). Culture

3-mercaptopyruvate sulfurtransferase supernatants were harvested after 3 days and ELISA assays were performed using standard procedures with the following capture and detection antibodies (capture/detection IFN-γ: R4-6A2/XMG1.2, IL-2: JES6-1A12, JES6-5H4, IL-4:11B11/BVD6-24G2) or kits purchased from eBioscience and used in accordance with the manufacturer’s instructions (IL-10 and TGF-β). Comparison of means was performed using Student’s t-test and multiple comparisons were performed using one-way ANOVA followed by Newman–Keuls post-test (GraphPad Prism). This study was supported by the National Health and Medical Research Council (R. J. S.) and Juvenile Diabetes Research Foundation (R. J. S.). Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“Staphylococcal enterotoxin A (SEA) is one of the bacterial products tested for modulation of unwanted immune responses. Of all the staphylococcal enterotoxins, SEA is the most potent stimulator of T cells. When administered orally, SEA acts as a superantigen (SA), producing unspecific stimulation of intra-epithelial lymphocytes (IELs) in the intestinal mucosa. This stimulation results in amplification of the normal local immunologic responses, which are mainly regulatory. This amplification is based on increased local production of IFN-γ by IELs, which acts on the nearby enterocytes.