Anti cd8a

Spleen cells were purified according to standard protocols as follows. CD4⁺ T cells were negatively selected using a cocktail of anti-CD8a, anti-CD11b, anti-CD45R, anti-DX5, anti-ter 119 antibody-coated magnetic beads, yielding CD4⁺ cells with >95% purity (Miltenyi Biotech). CD11c⁺ cells were positively selected with biotin-conjugated anti-CD11c mAb (7D4, PharMingen) and captured with streptavidin microbeads (Miltenyi Biotec) followed by 2 consecutive magnetic cell separations using LS columns (Miltenyi Biotec), yielding CD11c⁺ cells with >80% purity.

Single cell suspensions of splenocytes or peripheral blood were prepared using the standard techniques. Fc receptors were blocked by using 2.4G2 mAb prior to surface staining with the indicated antibodies. The negative selection of NK cells was determined by staining with cell surface monoclonal antibodies (mAbs), including anti-CD19, anti-CD4, anti-CD8a, anti-CD5, anti-Gr1, and anti-Ter-119 (Miltenyi Biotech). The surface phenotype of NK cells was determined by staining with mAbs, including anti-CD3-PE and anti-NK1.1-APC (Biolegend, San Diego, CA). Intracellular IFNγ was measured by pacific blue-conjugated anti-IFNγ (Biolegend) as described previously [47 (link)]. Dead cells were excluded by propidium iodine (PI) staining and live cells were gated on PI-negative cells. The NK cells of donors or recipients were distinguished by using anti-CD45.1 mAb (A20, BD Biosciences, San Jose, CA), FITC-34-2-12 (H-2D^d mAb, BD Biosciences), or Bio-KH95 (anti-H-2D^b mAb, BD Biosciences). Data acquisition was performed with FACS Calibur flow cytometer (BD Pharmingen, San Jose, CA) using a CellQuest software (BD Biosciences). Data analysis was performed using a FlowJo software [48 (link)].