Anti cd8α

Peripheral blood mononuclear cell (PBMC) isolation was carried out via a differential centrifugation method exactly as described by Kol et al.[45 (link)] PBMCs were harvested and enumerated using an automated cell counter (Coulter ACT diff, Beckman Coulter, Brea, CA). PBMCs were depleted of monocytes, B cells and granulocytes via an LD column (MACS Separation Columns, Miltenyi Biotec, Auburn, CA) using a cocktail of mouse anti-canine antibodies including anti-CD11b (clone CA16.3E10), anti-CD8α (clone CA9.3D3), anti-CD21 (clone CA2.1D6) and goat-anti mouse IgG-microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Flow through cells were collected and treated with an anti-canine CD4 antibody (clone CA13.1E4) followed by goat-anti mouse IgG-microbeads (Miltenyi Biotec) and run on an MS column (MACS Separation Columns, Miltenyi Biotec), according to the manufacturer’s instructions, to yield the final CD4+ T cell enriched fraction. All mouse anti-canine antibodies were purchased from the Leukocytes Antigen Biology Lab, UCD School of Veterinary Medicine.

Spleens from B10.A/SgSnAi TCR–Cyt 5C.C7-1 RAG-2^-/- mice were dissociated into homogenous single cell suspensions and passed through a cell strainer (70-μm Nylon mesh) from BD Falcon^TM. Erythrocytes were lysed using ACK lysis buffer (Biosource). Splenocytes were washed 3x in cold complete medium and adjusted to 1x10⁶/ml, then cultured in complete medium plus 1 μM of the Moth Cytochrome C (MCC) 88–103 peptide at 37°C with 5% CO₂. Fresh medium was added after 3 days of culture. After 5 days, the cultured cells were harvested, washed 2x with medium, and re-cultured in the absence of peptide with 10–20 U/ml of recombinant mouse (rm) IL-2. The medium was replenished every 5–7 days with fresh medium plus rmIL-2. For T-DC coculture experiments, these CD4⁺ T cells were purified with a CD4⁺ T cell isolation kit containing anti-CD8α, CD11b, CD45, DX5, and Ter-119 mAbs for depletion (Miltenyi Biotech.) with the addition of anti-CD11c and anti-class-II (Miltenyi Biotech). We used 3–4 LS columns in tandem instead of using LD columns to deplete the cells. These T cells, which were >95% CD4⁺, were used at various days of culture as a source of in vitro primed/antigen-activated T cells.