Disruption of CD8-Coreceptor Binding Abrogates Tolerance Induction Via Liver-Directed Expression of Donor MHC Class I - A Role for PD-L1?

Abstract

Introduction. We previously used an adeno-associated viral (rAAV) vector to express donor MHC (H-2Kb) in recipient livers and induce donor-specific tolerance in a mouse skin transplant model. Kb expression in B10.BR mice led to impaired production of IFN-γ in response to Kb, but no substantial deletion of CD8 T cells, suggesting that tolerance was associated with functional silencing. Methods. To determine the role of direct recognition of class I by CD8-dependent T cells in tolerance induction, we generated rAAV encoding a mutant Kb (rAAV-D227K), where Asp at position 227 of the α3 domain is replaced with Lys, abrogating CD8 binding. Expression of Kb, D227K-Kb and PD-L1 on hepatocytes was assessed by FACS and IHC. Kb-bearing 178.3 skin was grafted onto uninjected recipients and mice injected with rAAV-D227K or rAAV-Kb ± PD-L1 blockade. Results. Uninjected B10.BR mice rejected 178.3 skin (MST=16 d, n=6), while grafts onto rAAV-Kb-injected mice survived long term (MST>250, n=5). Survival of 178.3 grafts onto mice injected with rAAV-D227K was only slightly prolonged (MST=27, n=6), suggesting that CD8 coreceptor engagement is needed for tolerance induction. Kb expression in B10.BR livers resulted in increased PD-L1, but this was not seen following Kb-D227K expression, or with Kb expression in C57BL/6 livers, suggesting that alloreactive CD8 T cell activation results in PD-L1 upregulation. Antibody blockade of PD-L1 plus rAAV-Kb treatment resulted in increased ALT levels (721±73) compared to treatment with rAAV-Kb (33±4) or anti-PD-L1 (35±2) alone. Experiments to determine the effect of PD-L1 blockade on skin graft survival are ongoing. Conclusion. Disruption of CD8 coreceptor engagement abolishes tolerance induction via liver-directed expression of donor MHC class I, indicating that direct recognition by CD8-dependent T cells is essential for the process. PD-L1 upregulation accompanied alloreactive CD8 T cell activation and blockade of PD-L1 resulted in hepatitis, suggesting that this molecule might play a role in the functional silencing of CD8 T cells in this model.