Allogeneic T Cell Proliferation Assay

To assess the T cell allostimulatory activity of liver mDC or pDC, freshly-isolated, unstimulated or stimulated DC were used as stimulators of allogeneic BALB/c T cells (2×10⁵/well) in 72 hr MLR using 96-well, round-bottom plates. For ex vivo T cell re-stimulation, or measurement of anti-donor responses, bulk splenocytes of sensitized or transplanted mice were used as responders, and T cell-depleted (CD3ε Microbeads kit; Miltenyi) B6 splenocytes as stimulators. For the final 18 h of culture, 1 μCi of [³H]-thymidine (Perkin Elmer, Waltham, MA) was added to each well. Radioisotope incorporation was determined using a beta scintillation counter (Perkin Elmer) and results expressed as mean cpm ± 1SD of triplicate wells. Alternatively, T cell proliferation was determined by carboxyfluorescein succinimidyl ester (CFSE)-MLR using responder T cells labeled with CFSE (Invitrogen) as described (42 (link)).