Anti cd8 antibody coated microbeads

Mouse CD4⁺ and CD8⁺ T cells were isolated by magnetic activated cell sorting (MACS) selection of splenocytes with anti-CD4 or anti-CD8 antibody-coated microbeads (Miltenyi Biotech, Bergisch Gladbach, NRW), resulting in >98 % purity and >98 % viability in general. Co-cultivation of the DC-T cell system was performed as previously described [10 , 8 (link)]. Briefly, aliquots of 1 × 10⁵ CD4⁺ or CD8⁺ T cells were co-cultured with BMDCs (at a DC/T ratio of 1:10) for 4 days in a final volume of 200 μl medium, and the BMDC-induced T-cell proliferation activity was determined by Cell Proliferation ELISA (Roche Applied Science, Indianapolis, IN).

Fresh (not frozen) PBMCs were isolated with Ficoll-Hypaque plus (Amersham Bioscience, Uppsala, Sweden) and stained with anti-CD8 antibody-coated microbeads (Miltenyi Biotech, Auburn, CA) in accordance with the manufacturer’s recommendations. Positive selection of CD8 lymphocytes was performed with the Miltenyi Biotech MACS system. Short-term (3-week) CD8 T-cell cultures were set up as previously described (48 (link)). Briefly, sorted CD8 lymphocytes were plated at a 5:1 ratio with 1 μM peptide-pulsed (for >1 h), irradiated T2 cells (CRL-1992; ATCC, Manassas, VA) that were washed free of excess peptide before being mixed with CD8 T cells. CD8 T-cell lines were fed with AIM V medium (Gibco) supplemented with 14% human AB serum (Nabi, Miami, FL, or Gemini, Woodland, CA), 16% MLA-144 supernatant (75 (link)), 10 U/ml recombinant IL-2 (BD), 1% l-glutamine (Gibco), 0.5% β-mercaptoethanol (Sigma, St. Louis, MO), 1% HEPES (HyClone, Logan, UT) every 3 to 4 days and restimulated with fresh peptide-pulsed T2 cells weekly for only 3 weeks. This culture method has been optimized so as not to skew the TCR repertoire from that present in vivo (43 (link), 58 (link), 60 (link)).