One year old reconstituted mice were vaccinated with a plasmid encoding for the full length OVA. (A) The percentage of circulating T lymphocytes was evaluated in each indicated condition (upper panel) to monitor the long-term persistence of T cells in reconstituted mice. The function of CD8+ T cells was assessed by an antigen presentation assay of OT-I peptide and the percentage of produced IFN-γ was determined by intracellular FACS staining (lower panel). (B) The CD4+ T cell response following immunization was indirectly evaluated by measuring the level of anti-OVA IgG produced in reconstituted vs. WT mice (right panel). (C-I) Reconstituted C57BL/6 ZAP-/- mice rapidly rejected fully MHC-mismatched BALB\c skin grafts. (C) Survival curves for allogeneic BALB/c skin grafts on C57BL/6 seven and sixteen months following reconstitution ZAP-/- recipients (•) show rejection between day 10 and day 14, as fast as the allogeneic control (○). However, survival curves for syngeneic C57BL/6 skin grafts are similar in both the C57BL/6 WT (⧫) and the untreated ZAP-/- recipients (▵) to allogeneic BALB/c skin grafts on untreated ZAP-70-/- mice (▾), which don't show any graft rejection. Histological sections were performed of syngeneic (C57BL/6) skin that was grafted on WT C57BL/6 recipients (D, G) and allogeneic (BALB/c) skins which were grafted on a seven months electroporated ZAP-/- recipient (E, H) and on an untreated ZAP-/- recipient (F, I). These sections have been analyzed by hematoxylin-eosin (H/E, 200X view), to observed the presence of infiltrating cells (G, H, I), and by immunofluorescence (100X view), using antibodies against the epithelial marker Keratin 5 (K5, green) and the T lymphocyte marker CD3, to characterize these infiltrating cells (D, E, F). Contrary to the non-rejected skins (D, F, G, I) harvested 100 days after transplantation, the dermis of BALB/c allogeneic skin graft rejected at 50% by the reconstituted C57BL/6 ZAP70-/- recipient contains numerous infiltrating cells (dark purple staining; H), characterized as CD3+ cells (E). TCR Vαn specific primer with a specific TCR constant alpha primer = ADVn