A library-based examination of T cell allorecognition 2259104

Abstract Introduction Alloreactive T cells drive acute transplant rejection and Graft-versus-Host-Disease (GvHD) by reacting to peptides displayed on non-self MHC (allo-MHC). Despite their critical role, the molecular mechanisms of T cell allorecognition of peptide-allo-MHC complexes are not well understood. It is unclear how peptide-specific or cross-reactive alloreactive T cells are, and how they compare to conventional T cells. Additionally, little is known about the antigens recognized in an alloresponse–whether they are tissue-specific or ubiquitous peptides, and whether there are immunodominant antigens. Here, we use high-throughput T cell receptor (TCR) sequencing, cloning, and antigen profiling tools to conduct a repertoire-scale examination of T cell allorecognition. Methods We investigate the molecular mechanisms of allorecognition in a single-MHC mismatch murine skin graft model. We conduct single-cell TCR sequencing, use a pooled TCR cloning method to recapitulate the TCR repertoire for functional testing, and conduct high-throughput antigen screens to identify the targets of alloreactive TCRs. Results Single-cell TCR sequencing of the alloreactive repertoire in this model reveals dominant usage of a specific TRBV gene segment. Initial testing of select expanded TCRs indicates that they exhibit some degree of specificity for the peptide, and that they may recognize ubiquitously expressed antigens that are not skin-specific. We are further characterizing the specificity of these TCRs and the rest of our dataset by constructing a TCR library of the entire TCR dataset for functional testing. Future work will include high-throughput library-on-library screens to pair alloreactive TCRs with the antigens they recognize. Conclusion This work will provide a mechanistic examination of allorecognition at an unprecedented scale, providing insight into the molecular rules of allorecognition and the structure of an alloresponse, which may inform the development of improved therapies for transplant rejection and GvHD. Funding Source Cancer Grand Challenges partnership financed by CRUK (CGCATF-2023/100001), the National Cancer Institute (OT2CA297463), and The Mark Foundation for Cancer Research; Ludwig Center at MIT’s Koch Institute for Integrative Cancer Research Topic Categories Transplantation Immunology (TRAN)