ELISPOT assays to enumerate donor-reactive T cells in the spleens of cardiac graft recipients were performed as previously detailed (10 (link), 11 (link)). Briefly, splenic responder cells and mitomycin C-treated T cell depleted autologous or donor stimulator spleen cells were cocultured for 24 h at 37°C in serum-free HL-1 media in 96 well plates coated with anti-IFN-γ or anti-IL-2 capture mAb (BD Biosciences). To compare alloreactive CD4+ and CD8+ T cell priming, each cell population was purified from spleen cell suspensions using magnetic bead chromatography (Stem Cell Technologies) and then these purified responder cells were stimulated with T-cell depleted splenocytes. After culture, all cells were washed from the plate and biotinylated anti-IFN-γ or anti-IL-2 detecting mAb (BD Biosciences, San Jose, CA, USA) were added, followed by anti-biotin alkaline phosphatase. After development with the chromagen, the total number of spots per well was quantified using an ImmunoSpot Series 4 Analyzer (Cellular Technology Ltd., Shaker Heights, OH).