Recognition of Intact Donor Class I by Recipient CD8+ T Cells is Required for Tolerance Induction Following Liver-Directed Transgene Expression

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Introduction: AAV-mediated expression of donor MHC I (H-2Kb or H-2Kd) in recipient liver induces donor-specific tolerance in a mouse skin transplant model where Kb or Kd are mismatched between donor and host [1]. Recognition of both intact and processed allogeneic class I could contribute to tolerance induction. Aims and Methods: To determine the relative importance of intact and processed class I in tolerance induction, we generated rAAV-Kb-D227K and Kd-D227K vectors where a point mutation in the class I alpha 3 domain abrogates CD8 binding [2] and direct allorecognition but does not interfere with recognition of processed peptides. Expression of WT or mutant class I on hepatocytes was assessed by FACS and IHC. TCR-transgenic T cells recognizing intact (Des-RAG) or processed (TCR75) allogeneic class I were used as reporter cells in adoptive transfer experiments. Skin bearing the mismatched MHC I was grafted onto uninjected recipients or mice inoculated with rAAV encoding WT or D227K mutant Kb or Kd. In some recipients, Tregs were depleted using anti-FR4 or Diphtheria toxin (DT). Interferon gamma production by responding T cells was determined using ELISpot. Results: WT and D227K class I were expressed on hepatocytes at comparable levels. Adoptive transfer assays confirmed that the D227K mutation abrogated recognition of intact Kb by CD8+ Des-RAG T cells, whilst recognition of processed Kd peptides by TCR75-RAG cells was unimpaired. Uninjected B10.BR mice rejected Kb-mismatched 178.3 skin (MST = 16 d), while grafts onto rAAV-Kb-injected mice survived indefinitely (p < 0.001). rAAV-Kb-D227K only slightly prolonged graft survival (MST = 27d, p < 0.05). These results were recapitulated in the B6.Kd to C57BL/6 model. The modest survival prolongation of skin grafts in mice inoculated with Kb-D227K was abrogated when the recipients were treated with anti-FR4 (clone TH6) to deplete Tregs. Similarly, the survival prolongation of Kd-mismatched B6.Kd grafts onto DEREG recipients treated with rAAV-Kd-D227K (MST = 28d compared with 20d in mice receiving DT alone) was blocked when mice receiving Kd-D227K were also treated with DT (MST 18 days, p = 0.0046). Treg depletion did not shorten survival of skin grafts in mice inoculated with WT MHC class I vectors. Interferon-gamma production by unfractionated or CD8-enriched splenocytes from primed C57BL/6 mice in response to B6.Kd stimulators was over fourfold lower in mice inoculated with WT Kd than in those receiving Kd-D227K (p = 0.038). Conclusions: Recognition of intact MHC class I by alloreactive CD8+ T cells is required for tolerance induction via liver-directed expression of donor MHC class I. Indirect recognition of class I allopeptides and generation of Tregs can produce a modest prolongation of class I-mismatched skin graft survival but does not induce tolerance.FigureFigure