In addition, the results further support the potential for ��-MSH to convert effector T cells into functional Treg cells. 2. Materials and Methods 2.1. In Vitro sellekchem Simulation and ��-MSH Treatment of Effector T Cells C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were treated with the approval of the Schepens Eye Research Institute and the Boston University School of Medicine Institutional Animal Care and Use Committees. The immunization of the mice and isolation of lymph node T cells were as we have done before [14, 17, 30, 31]. The mice Inhibitors,Modulators,Libraries were injected with 50��L of Complete Freund’s Adjuvant fortified with 10mg/mL desiccated Mycobacterium tuberculosis into the footpad. Seven days later; the draining popliteal lymph node was collected to obtain effector T cells.

The lymph nodes were removed and placed in 5% fetal Inhibitors,Modulators,Libraries bovine serum (FBS) in RPMI-1640 supplemented with 10��g/mL Gentamycin (Sigma, St Louis, MO), 10mM HEPES, 1mM Sodium Pyruvate (BioWhittaker, Walkersville, MD), and 1X Nonessential Amino Acids (NEAA). The lymph nodes were made into a single cell suspension, depleted of red blood cells, Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries and washed with serum free media (SFM), RPMI-1640 supplemented with 0.2% ITS+1-media supplement (Sigma), and 0.1% bovine serum albumin (BSA). The CD4+ T cells were isolated from the cell suspension using negative selection CD4 columns (R&D Systems, Minneapolis, MN). The isolated CD4+ T cells (98% CD4+ by flow cytometry analysis) were plated into the wells of 96-well plate at 1 �� 106 cells per well.

Into each well Inhibitors,Modulators,Libraries was added 1��g anti-CD3 (Tcr) 2C11 antibody (BD Biosciences, San Diego, CA), and ��-MSH (Bachem, Torrance, CA) at a physiological concentration of 30pg/mL [14]. The cultures were incubated at 37��C, 5% CO2 until collected for the assays described below. 2.2. Flow Cytometry Staining Antibodies used for flow cytometry staining were anti-CD25-PerCP-Cy5.5 (BD Biosciences), anti-GITR-FITC (R&D Systems), anti-CTLA4-FITC (R&D Systems), anti-CD127-PE (BD Biosciences), CD44-FITC (BD Biosciences), CD62L-FITC (BD Biosciences), anti-LAP-PerCP (R&D Systems), anti-CD4-AF700 (Biolegend, San Diego, CA), and anti-CD25-APC-Cy7 (Biolegend). The cultured cells were collected at 72 hours or at 48 hours for LAP staining, and washed once in ice cold staining buffer (0.01M PBS with 1% BSA). The cells were resuspend in staining buffer, and all the cells were stained for CD25.

The cells were costained for GITR, CTLA-4, CD44, AV-951 CD62L, or LAP. The cells were incubated with the antibodies for 30 minutes on ice, washed twice with ice cold staining buffer, and resuspended in staining buffer. The cells were filtered through nylon mesh and analyzed by flow cytometry. The flow cytometry data was evaluated using FlowJo software (Tree Star, Inc, Ashland, OR) gating on the CD25+ T cells. All flow cytometry results presented are representative of two independent experiments. 2.