CD49d governs immune synapse formation through actin rearrangements and synchronizes BCR signaling in CLL

B cell receptor (BCR) signaling is a key determinant of chronic lymphocytic leukemia (CLL) pathophysiology. CD49d, the alpha4 subunit of the very late antigen-4 (VLA-4) integrin, can be activated by BCR signals; however, its role in modulating BCR functionality remains unknown. We used mouse models and primary human CLL to address this aspect. We found CD49d being not only required for bone marrow infiltration in TCL1 transgenic mice but also shaping human bone marrow infiltration patterns, along with patient outcome. In mice transplanted with TCL1 transgenic cells, CD49d loss altered leukemic cell localization in splenic niches. CD49d-deficient murine cells and human CD49d-low CLL cells could not form immune synapses to antigen-presenting membranes. CD49d-loss-related F-actin polymerization and transcriptomic defects in cytoskeleton-associated pathways were found in mouse and human. The requirement of CD49d for engraftment and actin organization could be recapitulated in a second murine model with highly aggressive transformed CLL. RNAseq analyses and kinome profiling revealed impaired antigen responsiveness due to kinase transcription and activity shifts in CD49d-deficient murine leukemic cells, recapitulated in human while applying several human BCR signatures. Finally, in human CLL expressing low CD49d, actin organization was deregulated, along with a desynchronization of Syk and PLCgamma activation. In summary, our findings establish CD49d as a key regulator of BCR functionality, linking integrin signaling to cytoskeletal dynamics and antigen responsiveness in CLL.