Single cell suspensions were prepared from the thymus, PaLN, and spleen, and filtered with a 70-μM strainer

(Fisher Scientific). PBL were obtained via submandibular puncture using lancets (Golden Rod) and RBC lysed with ACK solution. Islet infiltrating cells were isolated from purified, hand-picked islets. Briefly, pancreases were digested with 2.0 mg/mL collagenase P (Roche) for 20 min at 37°C, and islets purified on a Ficoll (Sigma-Aldrich) gradient. Lymphocytes infiltrating the islets were harvested by dissociating the islets using an enzyme-free cell dissociation solution (Sigma-Aldrich). Naïve CD4+ T cells were isolated from splenocytes using a bead-based naïve CD4 T-cell kit (Miltenyi Biotec). Briefly, total lymphocytes were incubated with a biotin-labeled Ab cocktail that selectively enriches for CD4+ T cells but depletes CD4+CD25+ cells. Enriched

CD4+CD25− T cells were then incubated check details with CD62L-conjugated micro-beads and isolated using a magnetic column. For general T-cell cultures, 2×105 cells were resuspended in complete RPMI 1640 medium (Gibco) containing 10% heat-inactivated FBS, 100 U/mL penicillin/streptomycin (Gibco), and 50 μM 2-ME (Sigma-Aldrich). T cells were stimulated in 96-well plates coated with varying concentrations of purified anti-CD3 Ab (2C11, BI 2536 ic50 eBioscience) and soluble, functional-grade anti-CD28 Ab at 2 μg/mL (37.51, eBioscience). In some experiments supernatants were collected, diluted 1:3 in 1% BSA in PBS, and IL-2 secretion measured 24 h post

stimulation. An anti-IL-2 Ab set (eBioscience) was used at 2 μg/mL on a high-binding ELISA plate (Costar). Total cells from the respective tissues were stained with a variety of fluorochrome-conjugated monoclonal Ab including: anti-CD3 (2C11), anti-CD4 (L3T4), anti-CD8 (Ly-2), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD62L (MEL14), and anti-FoxP3 (FJK.16 kit) (eBioscience). Fc receptors were blocked with a 1/200 dilution of rat Ig prior to staining. Intracellular Ki67 (B56; BD Biosciences) staining was done using cytofix/cytoperm reagents (BD Biosciences) according to the manufacturer’s specifications. Data were acquired on a Cyan flow cytometer (DakoCytomation), and analyzed using Summit software (DakoCytomation). In addition, CD4+CD25+ T cells (CD62Llo or CD62Lhi) were sorted by a MoFlo Megestrol Acetate high-speed sorter (DakoCytomation). Intracellular cytokine staining was performed on single cell suspensions from PaLN or islet-infiltrating cells as previously described 50. Briefly, lymphocytes were stimulated with 10 ng/mL PMA (Sigma-Aldrich) and 150 ng/mL ionomycin (Sigma-Aldrich) in complete RPMI 1640 medium for 6 h at 37°C; 10 μg/mL of Brefeldin A (Sigma-Aldrich) was added for the final 4 h of incubation. Cells were stained for surface molecules, fixed and permeabilized with cytokfix/cytoperm reagents (BD Biosciences), and stained for intracellular IFN-γ (XMG1.2) (eBioscience).