RNA-seq-based transcriptome analysis identifies TCR-expressing B1 cells as a multipotent lymphoid intermediate

Classically, B and T lymphocytes are regarded as distinct lineages that can be readily distinguished by the receptors expressed on their surface. However, our recent study established that peritoneal B1 cells serve as precursors for extrathymic T cells through a TCR⁺CD19⁺ double-positive (DP) intermediate. To investigate the biological characteristics of these TCR-expressing B1 cells, we isolated them from the murine peritoneal cavity and compared their transcriptomes with those of B1 cells. DP cells displayed transcriptional signatures more closely associated with innate T cell immunity than with canonical B1 identity, while maintaining expression of B cell lineage regulators such as Pax5 and Ebf1. Guided by transcriptomic profiling, we identified surface receptors that were more highly expressed in DP cells and assessed their differentiation potential using receptor-specific ligands. Differentiation assays demonstrated that stimulation with dTAT-WKYMVm, butyrate, ApoE, CypA, and Ephrin-A1 promoted the development of αβ or γδ T cells, with ephrin-A1 inducing a dose-dependent enhancement of T cell differentiation in co-culture with OP9 stromal cells. Notably, stimulation with UTP and MST1 induced acquisition of an NK cell phenotype, highlighting that TCR-expressing B1 cells possess broader lymphoid plasticity beyond T-lineage differentiation. Collectively, these findings suggest that TCR-expressing B1 cells may represent an intermediate lymphoid population with multipotent differentiation capacity, providing new insights into lymphocyte diversification and developmental plasticity.