Microbiota dictate T cell clonal selection to augment graft-vs-host disease after stem cell transplantation

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-MHC complexes, though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient TCR frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4 T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigen presented by gastrointestinal epithelium during an alloreactive response. The microbiota thus serves as a source of cognate antigen that contributes to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics. Male B6.Ptprca (CD45.1, Taconic) were divided into 3 groups representing exposure to different donor grafts (group 1, N=3: 0.5x106 CD4+ Marilyn (Allo) Tg (CD45.2+/CD90.1+); group 2, N=4: 0.5x106 Allo T with 1x106 CD4+ CBir1 T (CD45.2+/CD90.2+); group 3, N=3: 1x106 CD4+ CBir1 T). All recipients received 5x106 B6 (CD45.1+) bone marrow. Hashed and sorted splenocytes were harvested at day 6 to isolate Allo T (CD45.2+/CD45.1neg/CD90.1+) and CBir1 T (CD45.2+/CD45.1neg/CD90.1neg/Vβ14+) T cells for single cell RNA sequencing.