Transient T-cell depletion induces donor-specific central tolerance with preserved T-cell responses to third-party MHC and viral pathogens. (A) T-cell receptor (TCR) deletion analysis. To assess clonal deletion consistent with thymic deletion (central tolerance induction), expression of TCR clones was measured at 24 weeks after injection among donor-derived cells in the B6^GFP→Balb/c strain combination and among host-derived cells in the Balb/c→B6^GFP strain combination. In both cases, a partial deletion significantly below the baseline frequency in uninjected B6^GFP controls was observed in mice receiving TCD BM + anti-CD3mAb. The degree of deletion was comparable with that observed among mice injected at 14 DPC with TCD BM only. (B) Kaplan-Meier curve of skin graft survival. Mice injected with TCD BM + anti-CD3 mAb at 20 DPC demonstrated uniform, long-term survival of donor-derived skin grafts at 28 days postoperatively, comparable with mice injected with TCD BM only at 14 DPC. Mice injected at 20 DPC with TCD BM only uniformly rejected the skin grafts by 12 days postoperatively, comparable with uninjected controls. (C) Skin graft stereomicroscopy and histology. Skin grafts were harvested for stereomicroscopy and H&E histology 28 days postoperatively or on the day of rejection. Donor-strain (B6^GFP) skin grafted onto Balb/c mice injected with TCD BM + anti-CD3 mAb at 20 DPC and with TCD BM only at 14 DPC showed de novo hair growth with bright green skin under ultraviolet light. Histologic examination demonstrated normal skin architecture, including hair follicles (green arrows), in the absence of lymphocytic infiltration (red arrows) at the interface between the donor and recipient skin. In contrast, skin grafted onto Balb/c uninjected controls and Balb/c mice injected with TCD BM only at 20 DPC demonstrated erythema, desiccation, and dehiscence of skin grafts with loss of green fluorescence at 11 to 13 days postoperatively, consistent with allograft rejection. Histologic examination demonstrated significant lymphocytic infiltration (red arrows) at the interface between the donor and recipient skin, with neoepithelialization by the recipient underneath the graft (blue arrows). (D) In vivo MLR. T cells harvested from mice injected with TCD BM + anti-CD3 mAb at 20 DPC demonstrated decreased proliferation in the presence of donor strain MHC class I comparable to T cells harvested from mice injected with TCD BM only at 14 DPC. However, they proliferated normally in the presence of third-party MHC class I. This demonstrated that our transient T cell deletion strategy resulted in donor-specific tolerance rather than pervasive immunocompromise. (E) Pathogen humoral immune response. Anti-adeno–associated virus serotype 9 (AAV9) antibodies were measured using enzyme-linked immunosorbent assay 28 days after primary inoculation. Comparable amounts of both IgM and IgG were measured in uninjected controls and mice injected at 21 DPC with TCD BM + anti-CD3 mAb, demonstrating normal humoral immune responses to viral pathogens, including unimpaired B cell class switching from IgM to IgG, which is T-cell-dependent. (F) Pathogen T-cell immune response. Anti-AAV9 T-cell responses were measured using interferon gamma (IFN-γ) enzyme-linked immunospot assay 28 days after primary inoculation. Spleen-derived T cells harvested from mice injected at 21 DPC with TCD BM + anti-CD3 mAb secreted IFN-γ in the presence of the AAV9 peptide library in equal amounts as uninjected controls, demonstrating no long-term detriment in T-cell response to viral pathogens. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, not significant (ns). SFU, spot-forming units.