Enhancing the TCR-induced Response of HLA-E-restricted Regulatory CD8 T Cells to Promote Allograft Tolerance

Abstract We pioneered a novel regulatory CD8 T subset (CD8 Treg) that promotes transplant tolerance by suppressing pathogenic CD4 T cells. CD8 Tregs use semi-variant TCRs that allow the selective killing of alloreactive CD4 T cells via recognizing the surface class-Ib MHC molecules (Qa-1 in mice and HLA-E in humans). These CD8 Tregs express distinctive co-signaling receptors, including inhibitory Killer Ig-like receptor (iKIR), a checkpoint that constrains the uncontrolled response of CD8 Tregs. The overall goal of this project is to leverage the expertise we gained through the study of mice CD8 Tregs to develop clinically relevant CD8 Treg immunotherapy. Our central hypothesis is that HLA-E-binding superagonists and iKIR inhibitors can selectively mobilize human CD8 Tregs and promote allograft tolerance. In Aim 1, I will exploit the semi- variant nature of HLA-E-restricted TCRs to develop a tolerogenic superagonist that specifically stimulates CD8 Tregs. This will be achieved by defining a dominant TCR used by human CD8 Tregs and reconstructing this TCR to screen for a potent inducer from a peptide-HLA-E presenting yeast library. Using a kidney transplant recipient's PBMCs, I will validate the efficacy of selected superagonists inducing CD8 Treg cytotoxicity against alloreactive CD4 T cells. In Aim 2, I will study the mechanism of iKIR controlling the CD8 Treg response. I will analyze the phosphorylation status of TCR signaling molecules and examine the change in CD8 Treg effector capacity upon blocking the iKIR, in vitro. To investigate the role of iKIR expressed by human CD8 Tregs in vivo, I will use a novel humanized kidney organoid transplant model we established; I will analyze the activation of CD8 Tregs, suppression of alloreactive CD4 T cells, and prolongation of kidney organoid allograft survival when humanized hosts are treated with an iKIR-inhibiting antibody. Successful completion of the project can transform therapeutic strategies to achieve tolerance by suggesting a human CD8 Treg vaccine, CD8 Treg-specific checkpoints, and a novel humanized mice model that allows studying CD8 Treg immunotherapy, in vivo. The K08 award is necessary and sufficient to advance my niche in catalyzing preclinical discoveries into phase-1 trials and to pursue my long-term goal of developing clinically relevant personalized tolerogenic therapies.