Multi-omic profiling of human thymic B cells reveals intrathymic Ig-class switching and differentiation into multiple memory B cell subsets

Primarily recognized as the site for T cell development, the thymus supports a complex interplay between thymic stromal cells and developing thymocytes, which is essential for T cell maturation and the establishment of central tolerance. Emerging evidence indicates that thymic B cells contribute to tolerance induction by functioning as antigen-presenting cells. However, their developmental pathways and functional roles remain poorly understood. Using tissue mass cytometry, we localized B cells in the thymus and their orientation towards other cells in the medulla. We characterized the heterogeneity of thymic B cells using single-cell RNA sequencing of cells isolated from human thymi, identifying naive, germinal center-like, plasma cell, and multiple memory B cell populations. We identified a distinct pre-B cell subset with local thymic B cell development potential, that can develop due to the inhibition of Notch signaling by Deltex1, a Notch antagonist. Using BCR repertoire analysis, we explored clonal diversity, somatic hypermutation patterns and class switch recombination of thymic B cells. Spectral flow cytometry further validated the surface phenotype of thymic B cell populations and confirmed the presence of distinct CD21-CD27- memory compartments with heterogeneous surface immunoglobulin isotype usage. In doing so we provide novel insights into the unique biology of human thymic B cells, their development, and their differentiation in the human thymus. We conclude that the human thymus supports local B cell development and differentiation into medullary memory-like populations that undergo class switching with limited somatic hypermutation, suggesting secondary lymphoid-like B cell programs adapted to central tolerance induction. One Sentence SummaryThe human thymus is not only a primary lymphoid organ for T cell development, but also a secondary site for the development and maturation of unique populations of B cells.