Magnetic bead chromatography

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).

One-way mixed lymphocyte reactions (MLRs) were performed testing isolated splenic CD4 and CD8 T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA), 0.5 µM per 10⁷ cells at 37 °C for 10 minutes. For stimulator cells, CD11c⁺ cells were purified from spleen cell suspensions using magnetic bead chromatography (Stem Cell Technologies, Seattle, WA) and were treated with mitomycin C. CD4 or CD8 responder T cells (1 – 4 × 10⁵) were co-cultured with 7 × 10⁵ allogeneic stimulator cells for 96 h in 200 µL of complete RPMI 1640 medium supplemented with 10% fetal calf serum in 96-well flat-bottomed plates (Becton Dickinson Labware, Franklin Lakes, NJ). After 72–96 hours, cells were harvested, stained with CD4- or CD8-specific antibodies, and the dilution of CFSE was assessed by flow cytometry.

ELISPOT assays to enumerate donor-reactive T cells in the spleens of cardiac graft recipients were performed as previously detailed (11 (link), 21 (link)). Briefly, splenic responder cells and mitomycin C-treated self, donor and third-party stimulator cell populations 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, Vancouver, Canada) and then aliquots of the purified responder cells were stimulated with T cell-depleted splenocytes. 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).