In selected experiments, rapamycin 1 or 10 ng/mL or CsA 0.1 or 1.0 mcg/mL was added into cultures containing 100 IU/mL human recombinant IL-2. Multiscreen-IP 96-well microtiter plates (Millipore, Bedford, MA) were coated with a mouse anti-human CD3 mAbs (2 μg/mL) check details and mouse anti-human IFN-γ capture mAbs (4 μg/mL). Freshly isolated T cells (1×105 cells/well in 200 μL) were cultured for 36 h, isolated,

washed and incubated with a biotinylated mouse anti-human IFN-γ mAbs (2 μg/mL). After washing, HRP-labeled streptavidin (DAKO, Carpinteria, CA) was added for 1 h and subsequently the spots were developed with AEC substrate (Sigma-Aldrich, St. Louis, MO) and analyzed in an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH). Cytokine secretion is expressed

as spots/well. CD4+ T cells were stained with up to four directly conjugated fluorescent antibodies or control antibodies for 30 min at 4°C. After extensive washing the cells were fixed and permeabilized using the Fixation & Permeabilization kit (eBioscience), and intracellular staining of FOXP3 and CTLA-4 was performed according to the manufacturer’s recommendations. Data were acquired on a FACsCalibur (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR). For cell sorting experiments, CD4+ cells stained for desired cell surface markers were isolated using a FACSAria or FACSVantage (BD Rucaparib clinical trial Biosciences) apparatus. PCR was performed using the TaqMan Gene Expression Assay Kit (TaqMan, MGC probes, Applied Biosystems,

Foster City, CA) and the 7300 real-time PCR system. Gene-specific primers for the analysis of human Tbet and GAPDH by real-time PCR were obtained from Applied Biosystems. Migration of lymphocyte subpopulations in response to IP-10 (CXCL10) was quantified at single-cell resolution using microfluidic devices and time-lapse microscopy, as described previously 46. Briefly, photoresist (SU8, Microchem, Newton, MA), medroxyprogesterone was patterned within silicon wafers, which were used as a mold to produce a PDMS (Fisher Scientific, Fair Lawn, NJ) device, which was then bonded onto standard 1×3 in. glass slides (Fisher Scientific). The microfluidic network inside each device consisted of an array of up to 450 parallel channels (6×6 μm cross-section and 800 μm long) connected to one main channel, (50 μm tall, 400 μm wide and 10 mm long) with inlets and outlets. The devices were first primed with a solution of IP-10 (100 nM) and fibronectin (250 nM) for 15 min. After priming, sorted populations of either CXCR3+ or CXCR3− CD4+CD25+CD127dim/− Tregs (∼1×105/condition) suspended in 15 μL of media were introduced into the main channel through tubing connected to the main inlet. The cells were flushed through the main channel until media was seen to emerge from the main outlet.