MHC Molecules on B Cell Microvilli Are Spatially Associated with IL-15Rα

Abstract

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor α (IL-15Rα) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B-cell–T-cell contact, the distribution of IL-15Rα and MHC molecules within these structures may contribute to the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15Rα with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy–based Förster resonance energy transfer (FLIM-FRET). Both MHC class I and MHC class II molecules showed a significant spatial association with IL-15Rα; however, the extent of colocalisation decreased as the spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15Rα and MHC class I, whereas STORM did not detect this association. In contrast, IL-15Rα and MHC class II remained significantly colocalised at both resolutions. FLIM–FRET further demonstrated molecular proximity between IL-15Rα and both MHC class I and MHC class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15Rα is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B cell–T cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B cell–T cell interactions.