Using in vivo barcoding to capture T versus B cell fate commitment in the thymus

Abstract B cells are a rare subset in the thymus, an environment that supports T cell development and inhibits commitment to other immune cell lineages. Yet thymic B cells play an important role in the negative selection of autoreactive T cells. The origin of thymic B cells from either committed B cells or early thymocyte progenitors remains uncertain. We hypothesize that early thymic progenitor cells are a heterogenous population that can give rise to both T cells and B cells. In our study we combined the CRISPR/Cas9-dependent lineage recorder GESTALT with single cell RNAseq to capture cell lineage and type information to follow progressive cell fate decisions and to identify thymic B cell progenitors and transcriptional regulators permitting B cell differentiation in the thymus. Additionally, to evaluate the functional importance of thymic B cells in shaping the T cell repertoire, we performed TCR-seq on conventional CD4 T cells and Tregs. In the absence of B cells, amino acid usage within either TCR alpha or beta CDR3 regions remained unchanged. However, the dominant TCR alpha CDR3 length from the most abundant thymic Treg clones was reduced compared to wild type. Whether this repertoire shift impacts self-tolerance to B cell or AIRE-dependent antigens, both of which are expressed by thymic B cells, remains to be determined. Together, our data directly assesses lineage relationships leading to thymic T and B cell fate commitment and deepen our understanding of intra-thymic development and function of B cells that are required for central tolerance. Supported by AI089805 Burroughs-Wellcome Fellowship