CD38 CD49d Are Physically and Functionally Associated in B-Cell Chronic Lymphocytic Leukemia Cells.

The simultaneous over-expression of CD38 and CD49d (integrin α4) was recently shown to be part of the molecular signature characterizing a chronic lymphocytic leukemia (CLL) subgroup with bad prognosis. The high correlation between CD38 and CD49d expression levels in CLL, and the known propensity of both CD38 and integrins to form supramolecular complexes, prompted us to investigate whether specific CD49d and CD38 interactions occur on the CLL membranes, and whether the resulted molecular complexes have a pathogenetic role in part explaining the aggressiveness of CD38/CD49d expressing CLL cells.

Confocal microscopy and biochemical approaches were used to study the membrane organization of CD49d and CD38 in primary CLL cells and other B cell lines. Detection of apoptosis was performed by flow cytometry.

Co-capping experiments in CLL cells and in the cell line models Raji and RPMI-8226 demonstrated a membrane relationship between CD38 and CD49d. Anti-CD49d monoclonal antibodies (mAbs) induced capping in about 75% of CLL cells, with a 80% redistribution of CD38 in the context of the capping area. The same capping area stained for CD29 (integrin β1) indicating that CD49d is part of the α4β1 (CD49d/CD29) complex. It also contained CD81, confirming the known association between this tetraspanin and α4 integrins. Similar results were obtained with the cell lines Raji and RPMI-8226, both constitutively expressing CD38 and CD49d at high levels. The lateral association between CD38 and CD49d was next confirmed at the biochemical level by co-immunoprecipitation experiments with anti-CD49d mAbs and subsequent staining of immunoprecipitates by anti-CD38 mAbs. To further check whether the integrity of lipid rafts is necessary for the physical association between CD38 and CD49d, CLL cells were treated with methyl-β-cyclodestrin (MβCD) to deprive cell membranes of cholesterol, and cell lysates with octyl-D-glucopyranoside (ODG) to solubilize raft-associated proteins. Treatment with ODG revealed that a discrete amount of CD38/CD49d complexes could also be constitutively found outside raft domains. Consistently, co-capping experiments showed that CD49d-CD38 association was unaffected by lipid rafts disruption by MβCD. CD38-CD49d association was also maintained after polarization of CD49d to its natural ligand, as witnessed by a striking co-localization of CD49d and CD38 in cell uropods formed by CLL and Raji cells left to adhere and spread onto CS-1 fibronectin (FN) fragments, which specifically bind CD49d integrins. Finally, we tested whether the simultaneous engagement of CD38 and CD49d could enhance the protection against spontaneous apoptosis of cultured CLL. Purified CLL cells from six patients were cultured in the presence of anti-CD38 mAb or CS-1 FN fragment, either alone or in combination, in order to engage CD38 and CD49d alone or simultaneously. Analysis of cell apoptosis after a 72-hour culture, showed 74%±1.7 viable cells in the absence of any stimuli, and a substantial improvement of cell viability (94%±0.5) after exposure to both anti-CD38 mAb and CS-1 fragment, compared to a slight protection from apoptosis obtained by CD38 (80±1.1) or CD49d (79±1.1) independent engagements (p<0.01). Conclusions: CD49d and CD38 are physically associated both inside and outside the membrane cholesterol-rich raft domains. Such a physical association suggests a cooperative role for CD38 and CD49d which may explain at least in part the bad clinical outcome of the CLL subset coexpressing these molecules.

No relevant conflicts of interest to declare.