Human Leukocyte Antigen-E-restricted Killer Immunoglobulin-like Receptor-expressing CD8+ Regulatory T cells in Allograft Rejection

Abstract

T lymphocytes from CD8 lineage that express natural killer cell receptors have been identified to have regulatory functions. In the context of transplantation, previous studies from our laboratory have shown that murine alloreactive CD4+ T cells upregulate Qa-1 (HLA-E in humans) stress peptide complex on their surface, becoming targets for Qa-1-restricted Ly49+ CD8+ regulatory T cells. Immunization of the host with stress peptides (FL9 and Hsp60) mobilized Ly49+ CD8+ regulatory T cell-mediated killing of alloreactive CD4+ T cells in vivo and improved graft survival in a murine model by inhibiting donor-specific antibody responses. Moreover, inducing a single mutation in Qa1 that prevents TCR binding led to allograft rejection in mice. Preservation of self-tolerance by CD8+ regulatory T cell-mediated pathogenic target cell suppression via self-peptide-MHC1b complex interaction inhibits alloimmune rejection without causing generalized immune suppression. Translation of murine findings to humans identified inhibitory KIR receptors in humans to be homologous to murine Ly49 in CD8+ regulatory T cells. Our characterization of these KIR+ CD8+ regulatory T cell subsets (KIR+ CD8+ Tregs) in transplantation revealed significantly higher frequency of KIR+ CD8+ Tregs in peripheral blood following antibody-mediated allograft rejection compared to healthy controls. KIR2DL2/3 and KIR3DL1 markers were highly enriched in CD8+ T cells during allograft rejection (median >1%) relative to other inhibitory KIR markers. Inhibitory KIR+ (KIR2DL2/3 and KIR3DL1) subsets in CD8+ T cells were significantly higher following transplantation when compared to healthy controls. We also demonstrated that KIR+ CD8+ Tregs isolated from human peripheral blood mononuclear cells (PBMCs) can be expanded in vitro using IL-15 and Hsp60 peptide in an antigen-dependent manner. The expanded KIR+ CD8+ Tregs suppressed activated HLA-E-upregulated CD4+ T cells. Additionally, to study human alloimmunity, we developed a humanized mouse model transplanted with human kidney organoid. Using this pre-clinical model, we demonstrated that ex vivo–expanded KIR+ CD8+ Tregs killed alloreactive CD4+ T cells.