Isolation and Characterization of CD4+ T Cell Subsets

CD4+ T cells, derived from PBMNC of healthy donors by using the CD4 isolation kit II (Miltenyi Biotec, Bergisch Gladbach), were further divided into CD161+ and CD161− T-cell fractions by a staining with an anti-CD161–PE mAb, followed by incubation with an anti-PE microbead mAb (Miltenyi Biotec). Then the two cell subsets CD4+CD161+ and CD4+CD161− were cultured under limiting dilution (0.3 cell/well) in presence of 10⁵ irradiated (9000 rad) allogeneic PBMCs as feeder cells, 1% PHA (vol/vol), and 50 U/ml rIL-2 (Proleukin, Prometheus, Inc. San Diego, USA), in order to obtain T cell clones. Recovered CD4+ T cell clones were classified on the basis of their ability to produce IFN-γ and/or IL-17 and to express surface marker CD161, as previously described [24 (link)]. Briefly, T cells were polyclonally stimulated with PMA plus ionomycin, fixed in formaldehyde and then analyzed for intracellular cytokines production on a BDLSR II flow cytometry (BDBiosciences). Selected T-cell clones of each phenotype were further analysed by flow cytometry for surface expression of CXCR3A and CCR6. Seven Th17, non-classic -Th1 and classic Th1 clones were stimulated at a culture concentration of 10⁶ cells/ml for 72 h in presence of medium or mAbs anti- anti-CD3-CD28 (5 μg/ml each), then cultures supernatants were collected and stored at −30 °C for further analysis.