INPP5K limits PtdIns (4,5)P2 accumulation in CD19 microclusters and restrains CD19 co-receptor membrane proximity and BCR-proximal signaling in B cells

Introduction CD19 is a key B-cell co-receptor that amplifies B-cell receptor (BCR) signaling and tunes B-cell activation thresholds. How the conformation of the CD19 cytoplasmic domain is regulated to restrain basal and BCR-induced signaling remains incompletely understood. We investigated whether the phosphoinositide 5-phosphatase INPP5K controls CD19 conformation and CD19-amplified BCR-proximal signaling through local regulation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]. Methods A B-cell-specific Inpp5k knock-down (KD) mouse model was generated using CD21-Cre–mediated recombination. CD19–phospholipid interactions were assessed by tryptophan fluorescence with synthetic CD19 peptides and lipid bicelles. CD19 cytoplasmic domain proximity to the plasma membrane was measured by indirect FRET. PtdIns(4,5)P₂ distribution within CD19 microclusters, BCR-proximal signaling, and microcluster dynamics were analyzed by Airyscan microscopy and phospho-flow cytometry. Peripheral B-cell subsets and serum immunoglobulins were quantified by flow cytometry and ELISA. Results The CD19 juxtamembrane cytoplasmic domain contains a conserved polybasic region that interacts with acidic phospholipids, including PtdIns(4,5)P 2 . INPP5K KD B cells displayed increased PtdIns(4,5)P 2 density within CD19 microclusters and a constitutively membrane-proximal CD19 cytoplasmic domain in resting transitional T1/T2 B cells. This was associated with elevated basal phosphorylation of CD19^Y531^, BTK^Y223^ and PLCγ2^Y759^, with preserved inducibility upon BCR engagement, whereas ITAM-proximal components (CD79A, LYN, SYK) were unchanged or reduced. INPP5K deficiency also increased basal CD19 and BCR microcluster formation, impaired CD19–BCR colocalization after stimulation, and altered ezrin phosphorylation and actin organization. These changes coincided with accumulation of T1 B cells, reduced T2, follicular and marginal-zone B cells, and decreased serum IgM and IgG. Discussion INPP5K limits local PtdIns(4,5)P 2 accumulation in CD19 microclusters, thereby restraining membrane association of the CD19 cytoplasmic domain and basal CD19-amplified BCR-proximal signaling. Loss of INPP5K skews the spatial organization and balance of BCR-proximal signaling, with downstream consequences for B-cell microcluster dynamics, peripheral differentiation and humoral output.