Regulation of human B cell function by the inositol 5-phosphatases INPP5B and OCRL

Abstract

B cells are fundamental to adaptive immunity, driving antibody production, antigen presentation, and cytokine production. Their activation depends on the integration of B cell receptor (BCR) signalling with co-stimulatory inputs, with lipid phosphatases playing an important role in this process. While SHIP-1 and PTEN are well characterised in the immune system, the roles of the inositol 5-phosphatases INPP5B and OCRL in B cells remain unclear. Previous studies in DT40 and Ramos cells suggested effects on actin regulation and BCR signalling, but their function in primary human B cells has not been addressed. This thesis therefore investigated the expression and contribution of INPP5B and OCRL to B cell function, focusing on ex vivo primary B cells isolated from blood cones. Functional studies were performed using different approaches including a longitudinal ex vivo culture system maintained over 9 days. Pharmacological inhibition of INPP5B/OCRL was compared with shRNA-mediated knockdown of INPP5B, and outcomes were assessed sequentially for proliferation, activation, class switching, plasmablast differentiation, antibody secretion and signalling. Western blotting confirmed that INPP5B is broadly expressed across immune cells, predominantly as the short isoform, while OCRL is mainly detected in T cells as the long isoform. In Ramos cells, CRISPR-Cas9 knockout of INPP5B constrained proliferation, indicating a requirement for INPP5B to sustain responses in this model. In primary B cells, pharmacological inhibition of INPP5B/OCRL constrained proliferation, whereas shRNA knockdown of INPP5B enhanced proliferation at day 6. Despite these opposing effects on proliferation, both chemical inhibition and genetic knockdown increased activation, class switching, differentiation and antibody production. Signalling studies demonstrated that inhibition of INPP5B/OCRL amplified downstream Erk, Akt, NFκB-p65, and IKK activation, and these effects were found to be BCR-dependent. Knockdown further revealed a previously unrecognised co-dependence between INPP5B and OCRL as, surprisingly, depletion of one protein reduced the expression of the other. This study shows that INPP5B/OCRL acts as a brake on BCR dependent B cell functions in primary human B cells, but has distinct role in Ramos cells, suggesting potential different roles in cancer and healthy B cells. The newly uncovered co-dependency of INPP5B and OCRL provides a basis for future work on the mechanism and impact on immune regulation and disease.

Date of Award	15 Dec 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Lowe (Main Supervisor) & Madhvi Menon (Co Supervisor)