Fig 2: Recipients display suppressed changes in pre- to posttransplant IFN-γ-secreting cell numbers, anti-donor MHC antibodies, and CD4+ and CD8+ T cell MLR to donor and third-party stimulators(A–C) ELISpot IFN-γ counts per one million PBMCs for the donor (red bar) and third-party (blue bar) stimulation for all subjects (n = 2 technical replicates). Animal 1 showed a decrease in the frequency of IFN-γ-secreting cells in circulation between pre- and posttransplant time points (A). Animals 2 (B) and 3 (C) showed a slight increase of IFN-γ-secreting cells posttransplant, but all within the normal range of naive or tolerated animals, which can be up to 1,000 spots per million PBMCs.27(D–F) Serum samples were incubated with donor PBMCs, and the degree of IgG binding to either MHC I or MHC II was analyzed by flow cytometry to detect alloantibody response (n = 2 technical replicates). The MFI readings when the animal is naive is defined as 1 for each animal. MHC I (blue bar) and MHC II (red bar) at different time points demonstrated no donor-specific activation for animals 1 (D) and 2 (E) during the study except for animal 2, which had an increase of MHC II at 1 month posttransplant, possibly due to the residual anti-CD20 administered. For animal 3, anti-donor-specific antibodies were detected at high titer at 1 month posttransplant, especially IgG-MHC II was elevated at about 50 times relative to naive (F) when the animal was ill.(G–J) Mixed lymphocyte reaction to CD4+ (G and I) and CD8+ (H and J) to the donor (red bar) and third-party (blue bar) stimulators demonstrated marked decreases in both CD4+ and CD8+ compartments compared with pretransplant levels when exposed to the irradiated donor or third-party PBMCs (n = 2 technical replicates).