Decoding the specificity of human T cell allorecognition - SUMMARY Direct CD8+ T cell recognition of allo-HLA plays a major role in acute cellular rejection (ACR) of transplanted tissues. However, much is unknown about the T cells responsible for direct ACR. For decades, alloreactive T cells have been viewed as generally nonspecific, responding either to a plethora of allopeptides in the context of allo-HLA, or unique determinants on allo-HLA independent of peptide. Recent data though has suggested that many, if not most, alloreactive T cells are allospecific, responding to individual peptide/HLA complexes. Despite these advances, and despite their critical role in transplant rejection, systematic studies of intragraft T cells, their specificities, and their fundamental biology are lacking. We now have an unprecedented opportunity to study the T cells driving direct ACR. Our initial work using scRNA sequencing on biopsies and urine from 10 patients undergoing kidney transplant rejection found a surprisingly limited number (~20/patient) of clonally expanded CD8+ T cells (CD8EXP) with unique CDR3 α/β sequences. Remarkably, some of these CD8EXP persist for months in rejecting allografts, despite histologically successful anti-rejection therapy. In some cases, these “rogue” cells can re-expand and contribute to additional rejection episodes. However, the specificities, transcriptomic programs, functionality, and environmental niches that favor persistence of these and other CD8EXP remain unclear. We are now poised to make substantial breakthroughs in these critical areas. Supported by a wealth of exciting preliminary data demonstrating both rigor and feasibility, the goals of this project are to capitalize on our discoveries to advance our understanding of the specificity and biology of CD8+ T cells driving ACR. In our 1st Aim, we will determine the specificities of intragraft CD8EXP from several patients undergoing kidney allograft rejection, allowing us to decode the underpinnings of allospecificity, including its structural and biochemical features, the contributions of TCR affinity and specificity to persistence of CD8EXP cells, and the potential for shared or immunodominant targets in patients with the same HLA mis-matches. In preliminary data for Aim 1, in what we believe is a first for the field, using yeast display coupled with state-of-the-art structural informatics, we identified a bona fide allopeptide that evokes a prominent response from one of these allospecific TCRs. In our 2nd Aim, we will define the spatially resolved environments and cell-cell communications that define the gene expression and functional states of persisting, intragraft, allospecific CD8EXP cells. Overall, we will greatly expand our knowledge of the T cells driving ACR. We will identify the ligands of T cells driving rejection, assess the potential for immunodominance, and deconstruct the biochemistry of TCR allospecificity. We will dissect the phenotypes and genetic programming of the allospecific T cells that are retained in various allograft niches. Beyond the advances in basic immunology and mechanistic biology of allorecognition, this work will significantly improve our understanding of the biology of ACR, allowing for more targeted prediction, monitoring, and control of rejection.
