RECOGNITION OF INTACT DONOR CLASS I BY RECIPIENT CD8+ T CELLS IS REQUIRED FOR TOLERANCE INDUCTION FOLLOWING LIVER-DIRECTED TRANSGENE EXPRESSION

Mario Leong ⁰; Alexandra Sharland ⁰; Moumita Paul-Heng ⁰; Eithne Cunningham ⁰; Daniel Bunker ⁰; Kate Bremner ⁰; Zane Wang ⁰; Chuanmin Wang ⁰; Szun Szun Tay ⁰; Claire McGuffog ⁰; Grant Logan ⁰; Ian Alexander ⁰; Min Hu ⁰; Stephen Alexander ⁰; Tim Sparwasser ⁰; Patrick Bertolino ⁰; David Bowen ^0,0; Alex Bishop ⁰

²Sydney Medical School, The University of Sydney, Sydney, Germany; ³Gene Therapy Group, Children's Medical Research Institute, Sydney, Germany; ⁴Liver Immunobiology Group, Centenary Institute, Sydney, Germany; ⁵Twincore Instititute for Infection Immunology, Hannover Medical School, Hannover, Germany

Introduction: AAV-mediated expression of donor MHC I (H-2K? or H-2K?) 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-K?-D227K and K?-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-K?-injected mice survived indefinitely (p<0.001). rAAV-K?-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 K?-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.
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