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The prognosis for patients with B-cell chronic lymphocytic leukemia (B-CLL) is generally less favorable for those expressing CD38. Our working hypothesis is that CD38 is not merely a marker in B-CLL, but that it plays a receptor role with pathogenetic potential ruling the proliferation of the malignant clone. CD38 levels were generally low in the patients examined and monoclonal antibody (mAb) ligation was inefficient in signaling. Other cellular models indicated that molecular density and surface organization are critical for CD38 functionality. Interleukin 2 (IL-2) induced a marked up-modulation and surface rearrangement of CD38 in all the patients studied. On reaching a specific expression threshold, CD38 becomes an efficient receptor in purified B-CLL cells. Indeed, mAb ligation is followed by

IntroductionHuman CD38 is a pleiotropic membrane glycoprotein with ectoenzymatic properties 1,2 mediating cell-cell interactions by binding the nonsubstrate CD31 ligand. 3,4 CD38 is also a powerful negative prognostic marker for patients with chronic lymphocytic leukemia (CLL): CD38 ϩ CLL cells are characterized by a greater proliferative potential and by diminished sensitivity to chemotherapies (reviewed in Matrai 5 ). CD38 expression by leukemic cells is higher in peripheral lymphoid organs and in bone marrow (BM) than in circulating CLL cells of the same patient. 6 Recent data indicate that peripheral lymphoid organs host the proliferative core of the disease: results from in vivo measurements of B-cell kinetics showed that patients with CLL have definable and often substantial birth rates, varying from 0.1% to more than 1% of the entire clone per day. 7 Along with histologic findings that have demonstrated the presence of proliferation centers in lymph nodes and in BM, 8 the aforementioned study also substantiates the notion that the bulk of the leukemic clone resides in solid lymphoid organs.The lesson inferred from these observations is that CLL cells recirculate from blood to peripheral lymphoid organs, where a favorable microenvironment provides growth and survival signals mediated-at least in part-by CD38. 9 Chemokines and their receptors regulate CLL cell trafficking between the 2 compartments. [10][11][12] Stromal derived factor-1␣ (SDF-1␣)/CXCL12, 13 which is abundantly produced by stromal and nurselike cells (NLCs), 14 is effective in recruiting circulating CLL lymphocytes toward secondary lymphoid organs via the specific CXCR4 receptor. 15 Together with CD38, ZAP-70, a member of the syk tyrosine kinase family, has recently been recognized as a reliable negative prognostic marker for patients with CLL. 16,17 Here, we show that CD38 ϩ /ZAP-70 ϩ patients are characterized by enhanced migration toward SDF-1␣. Further, CD38 ligation leads to phosphorylation of the activatory tyrosines in ZAP-70 in both a panel of 12 patients with CLL and in an ad hoc modified B-cell line model. These results show that the 2 markers are functionally linked and confirm previous data in T and natural killer (NK) cell models. 18,19 ZAP-70 represents a limiting factor for the CD38 pathway in the CLL context, likely serving as a cross-point where migratory signals mediated via CXCR4 receptor intersect with growth signals mediated via CD38. Last, a specific genetic signature marking migratory potential was identified with microarray studies. Many of the differentially expressed genes modulate cell-cell interactions and movement.Altogether, the results of this work provide biological evidence for why the combined analysis of CD38 and ZAP-70 expression experienced in clinical trials 20 results in more dependable identification of patients with CLL who have aggressive disease. Patients, materials, and methods Patients and cellsAll samples used in this study were acquired after informed consent was obtained in accordance with the ...

Homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth, a critical step in disease progression, is principally coordinated by the CXCL12/CXCR4 axis. A cohort of 62 CLL patients was divided into migrating and nonmigrating subsets according to chemotaxis toward CXCL12. Migrating patients phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) proteins more than nonmigrating patients (Po0.0002). CD38 expression was the parameter most strongly associated with heightened CXCL12 signaling (Po0.0001), confirmed by independent statistical approaches. Consistent with this observation, CD38À CLL cells in samples with bimodal CD38 expression responded less to CXCL12 than the intact clone (P ¼ 0.003). Furthermore, lentivirus-induced de novo expression of CD38 was paralleled by increased responses to CXCL12, as compared with cells infected with a control virus. CD38 ligation with agonistic monoclonal antibodies (mAbs) enhanced CXCL12 signaling, whereas blocking anti-CD38 mAbs inhibited chemokine effects in vitro. This is attributed to physical proximity on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by (i) coimmunoprecipitation and (ii) confocal microscopy experiments. Blocking anti-CD38 mAbs significantly compromised homing of CLL cells from blood to lymphoid organs in a mouse model. These results indicate that CD38 synergizes with the CXCR4 pathway and support the working hypothesis that migration is a central step in disease progression.

The present work deals with the mechanisms of signal transduction mediated via CD38 in normal and neoplastic human B lymphocytes. The results indicate that CD38 is a receptor and that CD38-mediated signals are tightly regulated at 3 distinct levels. The first concerns the structural organization of CD38, which is clearly divided into monomeric and dimeric forms. The second level of regulation is based on the dynamic localization of CD38 molecules in lipid microdomains within the plasma membrane. Lateral associations with other proteins, namely with the CD19/CD81 complex, determine the third level of control. Raft localization and association with the CD19 complex are prerequisites for CD38-mediated signals in tonsillar B cells and in continuous lines. Lastly, the results indicate that lipid microdomain disruption and silencing of CD19 directly impacts on CD38's ability to mediate Ca(2+) fluxes, while leaving its surface expression unchanged. CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites including cyclic adenosine diphosphate ribose (cADPR). Our inability to identify a correlation between the production of cADPR and the receptorial functions support the hypothesis that CD38 is a pleiotropic molecule whose behavior as a receptor is independent from its enzymatic activity.