Anti cd3

Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll Histopaque gradient (GE Health Care Bio-Sciences, Piscataway Township, NJ). CD3⁺ T cells were isolated by negative selection using untouched human T cell isolation kit (Life technologies, Carlsbad, CA). Purity of CD3⁺ T cells was confirmed to be above 97% (data not shown). The cells were cultured in RPMI culture media (Corning CellGro, Manassas, VA) with 10% FCS (Gibco, Eugene, OR), 1% Penicillin/Streptomycin, and 1% L-glutamine (all from Corning CellGro) for 3 days either in the presence or absence of plate-bound anti-CD3 (anti TCR ε hybridoma from ATCC, Manassas, VA) and soluble anti-CD28 (BD Biosciences, San Jose, CA) with or without 100 nM rapamycin (Biotica, Cambridge, UK). In some experiments, IL-4 (100 ng/ml) (Peprotech, Rocky Hill, NJ, Catalog # 200-04), IL-17 (10 ng/ml) (eBioscience, San Diego, CA, Catalog# 14-8179), anti-IL-4 (5 µg/ml) (BioLegend, San Diego, CA, Catalog# 500815), or IL-17 (10 µg/ml) (R&D Systems, Minneapolis, MN, Catalog# MAB 317) was added to the culture media.

CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) was used to isolate cells. The median purity of isolation procedures was >90%. CD4+CD25− T cells were stained with 1.5μM CFSE (Molecular Probes/Invitrogen, Carlsbad, CA) for 10 min at room temperature. After quenching by FBS, cells were washed with PBS. A total of 5×10⁴ of CD4+CD25+ cells were cultured in triplicate with 5×10⁴ CFSE-labelled autologous CD4⁺CD25⁻ T cells stimulated with anti-CD3 (1 μg/mL) (ATCC, Rockville, MD) and anti-CD28 Abs (1 μg/mL) (Miltenyi Biotec, Auburn, CA) in AIM V medium supplemented with IL-2 at 150 IU/mL. As negative controls, CFSE-labelled CD4+CD25-cells were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) in the absence of fresh CD4+CD25+ cells. CFSE expression was measured by flow cytometry on day five after stimulation. CFSE data were analyzed using the ModFit software. The percentages of suppression were calculated based on the proliferation index (PI) of responder cells alone compared with the PI of cultures containing responders and Treg. The program determines the percent of cells within each peak, and the sum of all peaks in the control culture is taken as 100% of proliferation and 0% of suppression.