Search

Recently, the introduction of anti-CD38 monoclonal antibody, Daratumumab (DARA), in multiple myeloma (MM) therapy has improved the response rate of relapsed MM patients. However only a fraction of the DARA-treated patients respond, thus further studies on DARA mechanisms of action are needed. Because the antibody dependent cellular phagocytosis (ADCP) mediated by monocyte, is one of the mechanisms through DARA exerts its anti-MM activity, an ex-vivo approach was established in order to investigate which mechanisms or patient's immunological characteristics could influence DARA-mediated killing of MM cells. Bone marrow mononuclear cells (BM-MNCs) obtained from 25 MM patients (12 newly diagnosed and 13 relapsed MM) were analyzed at time 0 (T0) by flow cytometry. We checked the % of plasma cells (PCs) (CD138+ cells), % of total monocytes (CD14+ cells) and their two subsets (CD14+CD16- or CD14+CD16+ cells), the ratio between % of CD14+ and % of CD138+ cells (CD14+:CD138+ ratio) and the median fluorescence intensity (MFI) of CD38 and of the immuno-check points CD47 on PCs and CD172a (SIRPα) on monocytes. Subsequently, BM-MNCs were treated with control IgG (10µg/ml), DARA (10µg/ml) and the F(Ab)2 portion of DARA (DARA F(Ab)2) (10µg/ml) for 48 hours and then the % variation of surviving 7AAD- CD138+ cell, the modification of the % of monocyte or subsets analyzed and the % of PCs that are attached to monocyte (identified as CD138+ cells also positive to CD14) were evaluated by flow cytometry. Firstly, we found that DARA significantly exploited its anti-MM activity (median % variation of surviving CD138+ cells: 69.05%, p=0.0007) compared to IgG control while DARA F(Ab)2 did not have any killing effect (median % variation of surviving CD138+ cells: 97.33%) compared to the control. Secondly, we detected that the % of total of monocytes and their subsets (CD14+CD16+ or CD14+CD16-) composition were not modulated by DARA or DARA F(Ab)2 treatment compared to IgG. Indeed, only in presence of DARA, we observed that a double positive population of CD138+CD14+ cell significantly increased compared to IgG control (mean %: 15.76 vs 3.14, p=69.05% of surviving PCs). Interestingly, in the HR group compared to LR group were significantly increased the % of double positive CD138+CD14+ (median % HR:21.59 vs LR: 7.41, p=0.035), the % of CD138+ cells bonded to CD14+CD16+ (median % HR:13.28 vs LR: 3.93, p=0.048) and the CD14+:CD138+ ratio measured at T0 (median HR:0.63 vs LR: 0.32, p=0.0158). Moreover, in 5 relapsed MM patient, we have correlated the ex-vivo response to DARA with the type of in vivo response after DARA single-agent treatment. In addition in our cohort of patients, we observed that the % of surviving CD138+ cells after DARA treatment negatively correlate with the CD14+:CD138+ ratio at T0 (r:-0.628, p=0.0023), with the % of CD138+CD14+ population (r:-0.602, p=0.0039) and with the % of CD138+CD14+CD16+ population (r:- 0.657, p=0.0238) but did not correlate with the MFI of CD38 expression on PCs and the % of CD14+CD16+ at T0. Finally, to go further inside the mechanism involved in DARA response, we have explored the role of the inhibitory axis CD47-CD172a in the ADCP DARA-induced in 5 ex-vivo samples from our cohort of MM patients. We found that the MFI of CD47 strongly positively correlated with the % of surviving CD138+ cells (r: 0.9897; p=0.010) after DARA treatment; on the other hand, we have not reported any correlation with the MFI of CD172a on monocytes. The results of these analyses are continuously updating increasing the number of samples tested. In conclusion, these data highlight that monocyte binding to PCs, in particular those CD14+CD16+, plays a central role in DARA killing effect independently of % subset composition in the pre-treatment samples, suggesting that there are mechanisms that regulate the effectiveness of the monocyte-based killing effect on malignant PCs, as the immune-suppressive axis CD47-CD172a, giving the rationale design to identify new strategies to increase the efficiency of DARA-based therapeutic regimen. Disclosures Malavasi: Takeda Pharmaceutical Co: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceutica: Membership on an entity's Board of Directors or advisory committees, Research Funding. Aversa:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP-ribose from NAD+, and hydrolysis of cyclic ADP- ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP- ribose hydrolase activities. This finding indicates that the calcium- mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation.

Human CD38 is a target of different therapeutic antibodies and the results from in vivo applications are favorable. However, the use of CD38 targeting may be improved due to insights still being gleaned from basic research, also covering its paralogue CD157. Myeloma was adopted to confirm in vivo our in vitro models of CD38, which is a receptor and an ectoenzyme. Our hypothesis is that ectoenzymes involved in NAD+ metabolism are part of the myeloma escape strategy. Extracellular nucleotides (ATP, NAD+), high in tumor environments, also serve as intracellular signal transducers, with their degradation products modulating the communications between myeloma and normal cells. Hypoxia helps reprogram the local metabolism. In the extracellular milieu, nucleotides bind purinergic type P2 receptors or are metabolized by ectoenzymes and converted to adenosine (ADO), an immunosuppressor that may also bind different P1 receptors. The resulting anergic status helps the tumor evade the host immune response by promoting Tregs and MDSC, and depressing NK and T effectors. Another question is whether therapeutic antibodies interfere with the survival mechanisms of myeloma. The unprecedented fact that anti-CD38 antibody therapy targets not only a molecule expressed by the tumor but also by effectors and inhibitory cells is a further source of complication. The direct action of the antibodies on the tumor is being investigated in depth, but little is known about their presentation to the target cells. The steps of Fc domain presentation of the antibodies to the target molecule dissected in vitro using CHO cells expressing 4 distinct human FcRs. Exposure of CD38 to Daratumumab (DARA) bound to CHO/FcRs causes at 37 °C a selective aggregation to one pole of the myeloma cells. Such aggregations (rich in CD38 and bound DARA) are released as microvesicles (MVs) into the culture medium or in vivo into biological fluids. These MVs differ from those spontaneously released simply in the presence of the antibody on their surface. In conclusion, MVs are equipped with an enzymatic network potentially capable of metabolizing both ATP and NAD+ to produce ADO. Secondly, they may fuse with neighboring cells and leave the myeloma niche, eventually reaching the blood. The therapeutic IgG on their surface acts as a link which favors the capture by FcRs+ cells. This model was confirmed by tracking the migrated MVs which accumulate around NK cells (>30%) and monocytes (>90%). The same MVs then enter the cytoplasm of NK, monocytes and MDSCs, even if the functional effects induced by their internalization remain to be defined. Preliminary results conducted on purified NK cells exposed in vitro to MV from myeloma membranes treated with DARA indicate that they contain gene products of potential relevance. Indeed, a set of genes involved in the immune response and in the regulation of cell death is up-modulated. Instead, another set of genes related to mitosis and cell cycle is down-modulated. Under evaluation is what happens when MVs are taken up by FcR+-dendritic cells, which may reveal possible vaccinal effects. The transfer of the results of basic research on CD38 to therapy is just beginning: exploration of its multiple functions are expected to provide new, valuable insights. Disclosures Malavasi: Tusk Therapeutics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

The surface glycoprotein CD36 (GPIV) is known to mediate the adhesion of Plasmodium falciparum malaria-infected red blood cells and to be a receptor for extracellular matrix proteins such as collagen and thrombospondin. The murine monoclonal IgM antibody NL07, which is specific for CD36, has now been shown to also be a potent inhibitor of the adhesion of P falciparum malaria-infected red blood cells to C32 melanoma cells. Treatment of platelets with NL07 monoclonal antibody resulted in rapid degranulation, release of ATP and serotonin, increase in [Ca2+]i, and tyrosine phosphorylation of a substrate protein of 130 kD. In about one-half of the experiments, activation with NL07 resulted in the formation of small aggregates of 10 to 30 platelets, whereas in the other half of the experiments, large aggregates were seen similar to those induced by adenosine diphosphate (ADP) and these large aggregates could be converted to the small aggregates by ATP alpha S or by AP-2 or other antibodies against GPIIb and/or IIIa. Microaggregates of 2 to 5 platelets were seen with Glanzmann's platelets that constitutively lack GPIIb/IIIa. Aggregate formation was not seen with heat-treated serum, in the presence of anti C1q antibodies, or when using C5-, C8-, or C9-deficient human sera. Although activation of platelets with purified complement components results in a slow morphologic change without aggregation, involvement of CD36 results in rapid complement-mediated activation leading to formation of small aggregates that is largely independent of GPIIb/IIIa and that, under certain circumstances, proceeds to the formation of large ADP-dependent aggregates.

CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n = 232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter syndrome (RS). Five-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.

During immunologic synapse (IS) formation, human CD38 redistributes to the contact area of T cell–antigen-presenting cell (APC) conjugates in an antigen-dependent manner. Confocal microscopy showed that CD38 preferentially accumulated along the contact zone, whereas CD3-ζ redistributed toward the central zone of the IS. APC conjugates with human T cells or B cells transiently expressing CD38–green fluorescent protein revealed the presence of 2 distinct pools of CD38, one localized at the cell membrane and the other in recycling endosomes. Both pools were recruited to the T/APC contact sites and required antigen-pulsed APCs. The process appeared more efficient in T cells than in APCs. CD38 was actively recruited at the IS of T cells by means of Lck-mediated signals. Overexpression of CD38 in T cells increased the levels of antigen-induced intracellular calcium release. Opposite results were obtained by down-regulating surface CD38 expression by means of CD38 siRNA. CD38 blockade in influenza HA-specific T cells inhibited IL-2 and IFN-γ production, PKCθ phosphorylation at Thr538, and PKCθ recruitment to the IS induced by antigen-pulsed APCs. These results reveal a new role for CD38 in modulating antigen-mediated T-cell responses during IS formation.

The relationship between multiple myeloma (MM) cells and osteoblast (OB)s and osteoclast (OCL)s into the bone marrow (BM) niche has a critical role in the pathophysiology of MM and in the development of bone disease in MM patients. Recently, Daratumumab (DARA), a human anti-CD38 monoclonal antibody has been developed with broad-spectrum killing activity including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and, at least in part, modulation of CD38 enzymatic activity. In pre-clinical studies, DARA has been shown to effectively kill MM cells and clinical trials are ongoing. However, the effects of DARA in the context of the MM bone niche and on MM-induced bone remodeling alterations are unknown. In this study, we have firstly evaluated the expression profile of CD38 and its related ectoenzymes by bone niche cells and, thereafter, we investigated the effect of DARA on OCL and OB formation and bone remodeling. The study design included immunohistochemistry for CD38, CD73, CD39, CD203a (PC-1), CD157 and CD31 on bone biopsies in a cohort of 38 patients with newly diagnosed MM and 14 patients with monoclonal gammopathy of uncertain significance (MGUS). Moreover, the expression profile of these antigens was performed by flow cytometry on primary BM CD138+ cells (sample number=16), mesenchymal stromal cells, OBs, monocytes and OCLs and on the related human cell lines. As expected, we found that CD38 was highly expressed by MM cells that were also positive for CD203a and for CD39 and CD31 at variable level but not for CD157 and CD73. CD38 was also expressed by BM monocytes but not by OBs, OCLs and BM stromal cells. Interestingly, we found that OBs expressed CD73 and CD203a. Any significant difference was not observed in the expression of CD38 and related ectoenzymes between MM and MGUS patients. In line with CD38 expression profile by MM cells and the niche, we further demonstrated that DARA binds both MM cells and monocytes, but not OBs and OCLs. Consistently, we lack to find any significant effect of DARA on OB formation from BM stromal cells or OBs proliferation and survival. Thus, we investigated the effect of DARA (1-25 ug/ml) as compared to human IgG isotype control on OCL formation and activity in the presence of RANKL and M-CSF, using either CD138- cell fraction or purified CD14+ cells from MM BM samples. OCLs were evaluated by both TRAP staining and a fluorimetric osteolysis assay. We found that DARA, between 10 and 25 ug/ml, with a dose dependent effect, significantly inhibited OCL formation and activity from BM mononuclear cells and from the CD138- cell fraction, but not from purified CD14+ cells. The inhibitory effect on OCL formation by DARA was observed when the antibody was present for all the culture period (21-28 days). On the other hand DARA did not show any effect on late OCL progenitors and mature OCLs. Accordingly CD38 expression by monocytes cultured in a pro-osteoclastogenic medium disappeared after 7 days. In conclusion, our data indicate that DARA inhibit osteoclastogenesis, likely mediated by ADCC, targeting monocytes and early OCL progenitors. This evidence supports the use of an anti-CD38 based approach as a treatment for MM bone disease. Disclosures Malavasi: Janssen: Honoraria, Research Funding. Giuliani:Celgene Italy: Other: Research Grant; Janssen Pharamceutical: Other: Research Grant.

B-cell chronic lymphocytic leukemia (CLL) is one of the most prevalent B cell malignancies in adults and characterized by expansion of monoclonal mature B cells. Survival and proliferation of CLL cells depends on microenvironmental contact in lymphoid organs. The transmembrane glycoprotein CD38 acts as an important mediator of survival, proliferation and migration signals for CLL cells, and its expression is associated with poor prognosis. Spleen tyrosine kinase (SYK) is a central element of the B-cell receptor signal transduction pathway and has additionally been shown to be involved in cytokine and integrine signaling. In this study we demonstrate direct involvement of SYK in the CD38 signaling pathway in primary CLL samples. CD38-stimulation of primary CLL cells by its ligand CD31 induced a consistent phosphorylation of the tyrosine residue Y352, the first activation site that releases SYK from its autoinhibitory conformation (p In conclusion, our data show that SYK acts as a central downstream effector of CD38 signaling regulating survival-, proliferation-, and migration pathways in CLL and also functions as a strong regulator of CD38 expression. The interaction of CD38 and SYK involves the BCR pathways, where CD38 enhances the response to BCR-stimulation and, moreover, may act as an enhancer of autonomous BCR-signaling in CLL. Additionally, the CD38-SYK interaction involves BCR-independent microenvironmental pathways as shown for CD40 and CXCL12. CD38 expression not only serves as a negative prognostic marker but has also been shown to possess biological significance in CLL. We therefore propose that disruption of the CD38-SYK axis may represent a promising therapeutic option in CLL. Disclosures No relevant conflicts of interest to declare.

Daratumumab (DARA) is an anti-CD38 human mAb in phase III clinical trials in myeloma patients. DARA binding induces killing of tumor cells via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis. This work reports on the results obtained while dissecting the events following in vivo CD38 ligation by DARA. Treatment of myeloma cells with DARA + anti-human IgG at 37 °C influenced myeloma cytoskeleton, with redistribution of the CD38 molecules and a formation of distinct polar aggregates visualized by confocal microscopy. DARA effects are different from those observed using 8 different murine anti-human CD38 mAbs, which tended to internalize. The findings observed after DARA ligation were confirmed by exposing myeloma cells to DARA immobilized on CHO cells modified to express 4 distinct human FcRs. CHO cells adhere to plastic and mimics the events that take place in the myeloma niche. First, the interaction between Dara and the different FcRs was determined according to a biosensor-based approach on an IAsys Plus equipment (Affinity Sensors). Results show that DARA possesses the highest avidity for CD64, due to the higher kinetic stability of the complex. Conversely, the differences in the FcR-DARA recognition phase (kass) had only minor effects on final stability of the complex. The results also indicated that NK cells and monocytes are the blood populations with higher kdiss for DARA. The effects observed at 37 °C on myeloma cells in the presence of immobilized DARA were amplified when compared to those with soluble mAb. Results indicate that DARA induces CD38 target to aggregate, polarize and to release microvesicles (MV) from extrusions of the myeloma membrane. MV in culture supernatants and in bone marrow plasma of myeloma patients were characterized for concentration (particles/ml) and size by means of Malvern NanoSight NS300 equipment. DARA treatment was followed by high amounts of MV of different sizes released from myeloma cells. Same experiments repeated using DARA + anti-human IgG labeled with Alexa 488 analyzed by Malvern equipment highlighted the presence of DARA on the surface of MV. The induction of MV may be relevant for in vivo therapy: MV are outward buds of the membrane, which host molecules clustered in microdomains. The presence of CD38 has been confirmed. MV phenotype was analyzed looking for the ectoenzymes that join CD38 in the regulation of adenosine in the myeloma niche. MV phenotype included not only the presence of the expected CD38, but also of CD203a/PC-1, CD39 and CD73, the ectoenzymatic pathway leading to ADO production. A first conclusion is that DARA treatment is followed in vivo by a marked release of MV at the tumor site. The fate of the MV bearing DARA on their surface is multiple: on one side, MV may interact locally with different cells and populations of the niche. Another possibility is that MV are released into the blood stream and interact with cell populations therein. The lipid bilayer of MV consents passive movements even through tissues. MV appear as minicellular signals delivering instructions at a distance from their place of origin. Further, the ectoenzymes analyzed are also involved in cell migration or in interaction with countereceptors (e.g., CD31) expressed by endothelial cells. This issue, was investigated by testing FITC-conjugated DARA on a Laboratory-established human myeloma line. MV-DARA-FITC were then exposed to PBMC preparations obtained from normal donors. The results from a cytofluorimetric analysis highlighted the tendency of the labeled MV to cluster around CD16+ (NK cells) and CD14+ subsets (monocytes). At the moment, it is only possible to conclude that MV are associated with cell membranes, a binding likely mediated by FcRs. Not known yet whether MV interact with other cell types (e.g., macrophages, dendritic cells or lymphocytes). DARA shows a high affinity to FcRs of immune cell types (NK cells, monocytes, B cells). Given clinical data that indicate a robust increase in T cell counts, activation and clonality following DARA treatment should be expected. MV containing DARA and portions of myeloma cell membranes could help drive antigen presentation and T cell response in some patients. This is being investigated further. Disclosures Mark: Bioinvent International: Consultancy, Research Funding. Giuliani:Janssen Pharmaceutica: Research Funding; Celgene Italy: Research Funding. Sasser:Janssen Pharmaceuticals: Employment. Malavasi:Janssen: Honoraria, Research Funding.

Paracellular diapedesis, a key step in leukocyte recruitment to the site of inflammation, occurs at endothelial junctions and is regulated by highly coordinated interactions between leukocytes and endothelium. We found that CD157, a glycosylphosphatidylinositol-anchored ectoenzyme belonging to the NADase/ADP-ribosyl cyclase family, plays a crucial role for neutrophil diapedesis, because its ligation with specific monoclonal antibodies (both on neutrophils or endothelial cells) results in altered neutrophil movement on the apical surface of endothelium and, ultimately, in loss of diapedesis. Real-time microscopy revealed that CD157 behaves as a sort of compass during the interaction between neutrophils and endothelial cells; indeed, following CD157 ligation, neutrophils appear disoriented, meandering toward junctions where they eventually stop without transmigrating. These findings are relevant in vivo because CD157-deficient neutrophils obtained from patients with paroxysmal nocturnal hemoglobinuria are characterized by a severely impaired diapedesis.

This work addresses the question whether CD38, a negative prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL), plays a role in neoplastic B-cell growth and survival. We show that CD38+ B-CLL cells bind to murine fibroblasts transfected with the CD31 ligand. The interaction triggers an extensive remodeling of the B-CLL membrane, with relocalization of BCR/CD19 to the CD38/CD31 contact areas, and it also increases cell survival and proliferation. A second event is the up-modulation of the survival receptor CD100, restricted to proliferating cells, and a concomitant decrease of CD72 (low-affinity CD100 ligand and negative regulator of immune responses). The most efficient signals are delivered through sequential interactions between CD38/CD31 and CD100/plexin-B1 (high-affinity CD100 ligand), as inferred by coculture experiments using specific transfectants and blocking monoclonal antibodies (mAbs). The finding that nurselike cells from B-CLL patients express CD31 and plexin-B1, which deliver growth and survival signals to CD38+/CD100+ B-CLL cells, further confirms the model proposed. These findings show that a set of normal receptors and ligands ruling physiologic signaling pathways in B lymphocytes becomes detrimental when expressed in the context of B-CLL cells, ultimately leading to the generation of a tumor reservoir.

CD157, a glycosylphosphatidylinositol (GPI)–anchored protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, is expressed on the surface of most human circulating neutrophils. This work demonstrates that CD157 is a receptor that induces reorganization of the cytoskeleton and significant changes in cell shape, and that signals mediated by CD157 act through modulation of cytosolic Ca2+ concentration. These signals are independent of the products of CD157's enzymatic activities (ie, cyclic adenosine diphosphate [ADP]–ribose and ADP-ribose). Indeed, the enzymatic activities of CD157 in circulating neutrophils as well as in dimethyl sulfoxide (DMSO)–differentiated (CD157+/CD38-) HL-60 cells, are hardly detectable. This work also shows that the receptorial activity relies on cross-talk between CD157 and β2 integrin. CD157 localizes in GM1-enriched lipid rafts and, upon activation, it migrates to the uropod, a structure specialized in motility and adhesive functions. Indeed, CD157 is involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP). These findings were consistent with the results obtained in neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH), in which CD157 is deficient. These neutrophils showed constant defects in adhesion and migration. Our data attribute specific and crucial roles to CD157 in the regulation of innate immunity during inflammation.

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 Ca2+ fluxes and by a markedly increased proliferation. The unsuitability of CD38 to perform as a receptor is obviated through close interaction with the B-cell–receptor (BCR) complex and CD19. On mAb binding, CD38 translocates to the membrane lipid microdomains, as shown by a colocalization with the GM1 ganglioside and with CD81, a raft-resident protein. Finally, CD38 signaling in IL-2–treated B-CLL cells prolonged survival and induced the appearance of plasmablasts, providing a pathogenetic hypothesis for the occurrence of Richter syndrome.

CD38, a surface glycoprotein of unrestricted lineage, is an ectoenzyme (adenosine diphosphate [ADP] ribosyl cyclase/cyclic ADP-ribose hydrolase) that regulates cytoplasmic calcium. The molecule also performs as a receptor, modulating cell-cell interactions and delivering transmembrane signals, despite showing a structural ineptitude to the scope. CD38 ligation by agonistic monoclonal antibodies induced signals leading to activation of the lytic machinery of natural killer (NK) cells from adults; similar signals could not be reproduced in YT and NKL, 2 CD16(-) human NK-like lines. It was hypothesized that CD38 establishes a functional cooperation with professional signaling molecules of the NK cell surface. The present work answers the question about the molecule exploited by CD38 for signaling in NK cells, using as a model CD16(-) NK lines genetically corrected for CD16 expression. Our results indicate that a functional CD16 molecule is a necessary and sufficient requisite for CD38 to control an activation pathway, which includes calcium fluxes, tyrosine phosphorylation of ZAP70 and mitogen-activated protein kinase, secretion of interferon-gamma, and cytotoxic responses. Fluorescence resonance energy transfer and cocapping experiments also showed a surface proximity between CD38 and CD16. These results were confirmed by using the NKL cell line, in which CD16(+) and CD16(-) variants were obtained without genetic manipulation. Together, our findings show CD38 to be a unique receptor molecule that cannot signal by itself but whose receptor function is rescued by functional and physical associations with a professional signaling structure that varies according to lineage and environment. This molecule is CD16 in NK cells.

Development of human multiple myeloma (MM) in the bone niche derives from multiple factors. Besides cells and soluble factors, hypoxia triggers anaerobic glycolysis, reduces ATP and simultaneously increases NAD+. This condition likely influences the ectoenzymatic pathways working in the niche. ATP is the substrate for the CD39/CD73 pathway leading to adenosine (Ado) production. NAD+ activates an alternative pathway, where Ado is obtained by the articulated actions of the CD38/CD203a/CD73 axis. The working hypothesis is that Ado plays a significant role in tumor immune escape and growth in closed systems, such as the myeloma niche. The two distinct pathways were confirmed by immunohistochemistry and immunofluorescence on cells derived from the bone marrow (BM) myeloma niche, where they operate in a discontinuous way. The first step of the study was to design a simple and reliable assay for detecting Ado at nanomolar levels in the fluids of the niche, taking into account i) the very short half-life of Ado, ii) the degradation of Ado to inosine, and iii) the fast cellular uptake of Ado. The technique adopted relied on HPLC with UV detection, which simultaneously monitored nucleotide and nucleoside concentrations. Ado was identified by retention time and by enzymatic peak shift assay using adenosine deaminase. The HPLC assay showed a good linearity for Ado (R2=0.9972) as well as a low value of detection (5 ng/ml) and quantitation (10 ng/ml). BM plasma derived from osteomedullary biopsies from monoclonal gammopathy of unknown significance (MGUS) (3), from smoldering MM (3) and from symptomatic MM (27) were immediately treated with acetonitrile to extract Ado and avoid its degradation or use by the cells present in the biopsy. The results demonstrate that Ado is detectable in BM plasma, taken as representative of the niche, where all the products converge. Results are summarized in the Table. Table Adenosine ( µ M) MGUS(n=3) SMM(n=3) MM(n=27) Mean value 19 34 59 Limit values 9.58 – 28.33 17.05 – 46.50 24.88 - 193.07 Range value 18.75 29.45 168.19 The variability in the levels of Ado detected suggest that they are the result of multiple heterotypic interactions taking place between myeloma cells on the one side, and osteoblasts (OB), osteoclasts (OC) and stromal cells (SC) on the other. The individual contributions of the most important components of the niche were assessed by using cell and cell line models, which consented the design of selected co-cultures. Human myeloma cells (JJN3 and KSM12BM lines) were challenged by: i) osteoblasts (HOBIT and HOB01 lines); ii) fully differentiated human osteoclasts (PBMCs isolated from BM samples of MM patients exposed to MCSF and RANKL) and iii) stromal cells (HS5 line). Ado levels were assayed as described. Co-cultures of MM with OCs displayed a significant increase in Ado levels (88%±10). On the contrary, co-cultures of MM with OBs and SCs lines were characterized by decreased Ado levels (41%±12 and 29%±14, respectively). Schematically, this study indicates that significant levels of Ado are present in BM, which robustly increase in MM conditions. This conclusion was made possible by the use of an ad hoc designed HPLC assay, which defines adenine nucleotides, nucleosides and their metabolites. Extracellular Ado concentrations ranged from 10-200 µM in the biopsies from different monoclonal gammopathies disorders. This observation supports the view that bone and MM are equipped with the ability to maintain Ado at micromolar levels, with a profile that may appear characteristic of different pathological conditions. An appealing hypothesis is that myeloma cells usurp the normal organization of bone, replacing it with a niche that maximizes local growth and protection from immune defenses. Relevant for translational medicine is that Ado levels in the myeloma niche might be an early indicator of an aggressive form of the disease. Consequently, Ado levels could become a valuable marker of disease progression in plasma cell dyscrasias. Disclosures Giuliani: Celgene Italy: Research Funding. Malavasi:Jensen Research & Development: Consultancy, Research Funding.