mTECs and B cells form a thymic microniche associated with B-cell licensing

Thymic B cells rely on T cells and type-III IFN (IFN-λ) signaling for class switch recombination and effective licensing as APCs, a process essential for their role in establishing central T-cell tolerance. IFN-λ is produced exclusively by a subset of medullary thymic epithelial cells (mTECs); however, the spatial layout between B cells and IFN-λ–producing mTECs remain unclear. Here, we studied the distribution of thymic B cells and IFN-λ⁺ mTECs, alongside other mTEC populations, to better understand the niche required for thymic B-cell licensing. Using 26 target multiplex immunofluorescence imaging of the thymus, we identified populations of naïve B cells, licensed B cells, plasmacytoid dendritic cells, plasma cells, mTECs, and mimetic mTECs within the thymus. Spatial analysis revealed licensed B cells closely interacted with both IFN-λ⁺ mTEC and GP2⁺ microfold mTECs, with transcriptional analysis predicting CCL20-CCR6–mediated B-cell interactions with microfold and IFN-λ⁺ mTECs. However, studies in CCR6-deficient mice showed CCR6-CCL20 interaction was not required for B-cell licensing. To further investigate the role of GP2⁺ microfold mTECs in B-cell licensing, we studied the NOD/ShiLtJ mouse model, which we confirmed to have a natural reduction in microfold mTECs. Interestingly, the thymic B cells in NOD mice demonstrated reduced licensing, which was attributed to reductions in IFN-λ receptor expression on thymic B cells. These data indicate that microfold mTECs modulate the capacity of thymic B cells to undergo IFN-λ–induced licensing.