Isolation and Staining of Tetramer+ T Cells and Cytokine Assays The procedures have been previously described [16], [17], [18]. Briefly, splenocytes isolated from p286-immunized NOR mice were pooled and cultured in Click’s medium (Life Technologies, Grand Island, NY) with peptide for 3 days. Cells were further incubated in RPMI medium supplemented with 5% fetal bovine serum and IL-2 before being separated into tetramer+ and tetramer2 cells. CD4+, tetramer+ T cells were isolated using FACS and magnetic beads (Miltenyi Biotech, Auburn CA). Purified tetramer+ cells were maintained in complete medium (CM: RPMI medium supplemented with 5% FBS, plus IL-2). For longer cell cultures, T cells were restimulated with peptide and irradiated APCs (3,000 rads). Cell staining using tetramer has been previously described [16], [22], [23]. Briefly, T cells were stained with PE-labeled tetramer plus the anti-TCR b chain antibody, H57, (37oC, 1–2 h) and analyzed by FACS using FACSCaliber (Becton-Dickinson, San Jose, CA). All antibodies used for FACS and for in vitro inhibition assays were purchased from eBioscience or B-D Pharmingen (San Diego, CA). The IL-2 bioassay has been previously described [16], [22], [23]. For ELISA, cell culture supernatant was harvested after incubating cells with peptides for 24 h. Capture ELISA (B-D PharMingen) was used, according to the manufacturer’s instruction, to measure the amount of cytokines produced in the cell culture. Adoptive Transfer of T Cells into NOD/scid Mice For adoptive transfer experiments, NOD/scid mice received a single i.v. injection of NR286 T cells (16107/mouse), NR286 T cells plus an equal number of NOD mouse splenocytes, or NOD mouse splenocytes alone. Recipient mice were monitored for up to 22 wk after cell transfer (when mice were 30 wk old) and were considered diabetic after two consecutive weeks of glycosuria $2% and blood glucose level $250 mg/dL. Assessment of NO Levels NO production was measured using the Greiss assay [39]. Briefly, supernatant (100 ml) from cells cultured for 24 h or 4 days was added to 100 ml of a 1:1 mixture of 1% sulfanilamide dihydrochloride in 5% H3PO4 buffer and 0.1% naphthylethyle- nediamine dihydrochloride (Sigma-Aldrich). The reaction was incubated (room temperature, 10 min) then measured at OD550 nm using an ELISA reader. Nitrate contents in the reaction were calculated using a sodium nitrite standard curve, with a detection threshold of 1 mM. In Vitro Inhibition Assays Using Co-Cultures CD4+ T cells from BDC2.5 TCR transgenic mouse splenocytes were purified using magnetic beads and labeled with CFSE References 1. Sakaguchi S (2004) Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 22: 531–562. 2. Shevach EM (2004) Regulatory/suppressor T cells in health and disease. Arthritis Rheum 50: 2721–2724. 3. Ziegler SF (2006) FOXP3: of mice and men. Annu Rev Immunol 24: 209–226. 4. O’Garra A, Vieira P (2004) Regulatory T cells and mechanisms of immune system control. Nature Medicine 10: 801–805. 5. Pop SM, Wong CP, Culton DA, Clarke SH, Tisch R (2005) Single cell analysis shows decreasing FoxP3 and TGFbeta1 coexpressing CD4+CD25+ regulatory T cells during autoimmune diabetes. J Exp Med 201: 1333–1346. 6. You S, Belghith M, Cobbold S, Alyanakian MA, Gouarin C, et al. (2005) Autoimmune diabetes onset results from qualitative rather than quantitative age- dependent changes in pathogenic T-cells. Diabetes 54: 1415–1422. Antigen-Specific Foxp32 Treg (carboxy-fluorescein diacetate succinimidyl ester). CD4+ T cells (16107 cells/ml) in serum-free PBS were incubated (37uC, 10 min) with CFSE (0.8 mM), washed, and used for further analyses. CFSE-labeled CD4+ T cells from BDC2.5 mice (the target cells), cultured in the absence of NR286 T cells with or without p79 (0.1 mg/ml) were used as controls. CFSE-labeled cells were also cultured in the presence of NR286 T cells and p79, with or without GAD p286 (15 mg/ml). Irradiated (3,000 rad) CD4+ T cell-depleted NOD mouse splenocytes were used as the APCs. Cells were cultured in RPMI medium containing 5% FCS for 4 days, washed and stained with the tetAg7/p79 tetramer and an anti-CD4 antibody to identify BDC2.5 T cells. The effect of NR286 T cells on BDC2.5 T cell proliferation was analyzed using FACS to determine the intensity of the CFSE in the cells and whether the cell division was altered. To determine the effect of different cytokines on target cell proliferation, a saturating amount of antibodies against IL-10 or IFN2c (24 mg/ml) was added to cell cultures of in vitro inhibition assays; cells were incubated for 4 days before being harvested for analyses. To determine the effect of NO on target cell proliferation, NO inhibitors were added to cell cultures, with procedures similar to those described above in the in vitro inhibition assays, except that a saturating amount (0.8 mM) of L-NMMA (an active NO inhibitor) or D-NMMA (an inactive NO inhibitor analogue) was added to cell culture. Transwell Assays NOD mouse splenocytes (56105 cells/well) were cultured in the lower well of a 24-well transwell plate (Corning Inc., Corning, NY). NR286 T cells were cultured in the upper well in the presence of irradiated CD4+ T cell-depleted NOD mouse APCs. CFSE-labeled CD4+ BDC 2.5 T cells (56105 cells/well) were cultured in the lower well in the presence of irradiated APCs, with NR286 T cells cultured in the upper well. BDC2.5 T cells were activated with p79 (0.1 mg/ml). After 4 days, cells were washed and stained with tetAg7/p79 and an anti-CD4 antibody. Statistical Analyses The Student’s t test, Wilcoxon test, and x2 test for statistical analyses were used to calculate the statistical significance for differences between experimental groups; p,0.05 was considered statistically significant. Author Contributions Conceived and designed the experiments: CC CPL. Performed the experiments: CC. Analyzed the data: CC. Wrote the paper: CC CPL. 7. Brusko TM, Wasserfall CH, Clare-Salzler MJ, Schatz DA, Atkinson MA (2005) Functional defects and the influence of age on the frequency of CD4+ CD25+ T- cells in T1D. Diabetes 54: 1407–1414. 8. Tarbell KV, Yamazaki S, Olson K, Toy P, Steinman RM (2004) CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J Exp Med 199: 1467–1477. 9. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, et al. (2004) In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 199: 1455–1465. 10. Sakaguchi S (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6: 345–352. 11. Piccirillo CA, Shevach EM (2004) Naturally-occurring CD4+CD25+ immuno- regulatory T cells: central players in the arena of peripheral tolerance. Semin Immunol 16: 81–88. PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7863