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CD38 is highly and uniformly expressed on multiple myeloma (MM) cells, and at relatively low levels on normal lymphoid and myeloid cells, and in some tissues of nonhematopoietic origin. CD38 is a transmembrane glycoprotein with ectoenzymatic activity, and also functions as a receptor and adhesion molecule. Altogether, this has triggered the development of several CD38 antibodies including daratumumab (fully human), isatuximab (chimeric), and MOR202 (fully human). CD38 antibodies have pleiotropic mechanisms of action including Fc-dependent immune-effector mechanisms, direct apoptotic activity, and immunomodulatory effects by the elimination of CD38 + immune-suppressor cells. CD38-targeting antibodies are generally well tolerated and induce partial response or better in ∼30% of heavily pretreated MM patients as monotherapy. Based on their distinct mechanisms of action, favorable toxicity profile, and single-agent activity, CD38 antibodies are attractive partners in combination regimens. Indeed, deep responses and prolonged progression-free survival can be achieved in relapsed/refractory MM patients when CD38 antibodies are combined with immunomodulatory agents or proteasome inhibitors. Infusion-related reactions, which typically occur during the first infusion, are the most frequent adverse events. Attention should also be paid to the interference of CD38 antibodies with certain laboratory assays, which may complicate response evaluation and blood compatibility testing. Several studies are currently examining the role of CD38-based therapies in newly diagnosed and high-risk smoldering MM. Furthermore, CD38 antibodies are currently also under investigation in other hematologic malignancies, including acute lymphoblastic leukemia, natural killer/T-cell lymphoma, and acute myeloid leukemia, as well as in solid tumors., (© 2018 by The American Society of Hematology.)

Immunotherapeutic strategies are emerging as promising therapeutic approaches in multiple myeloma (MM), with several monoclonal antibodies in advanced stages of clinical development. Of these agents, CD38-targeting antibodies have marked single agent activity in extensively pretreated MM, and preliminary results from studies with relapsed/refractory patients have shown enhanced therapeutic efficacy when daratumumab and isatuximab are combined with other agents. Furthermore, although elotuzumab (anti-SLAMF7) has no single agent activity in advanced MM, randomized trials in relapsed/refractory MM have demonstrated significantly improved progression-free survival when elotuzumab is added to lenalidomide-dexamethasone or bortezomib-dexamethasone. Importantly, there has been no significant additive toxicity when these monoclonal antibodies are combined with other anti-MM agents, other than infusion-related reactions specific to the therapeutic antibody. Prevention and management of infusion reactions is important to avoid drug discontinuation, which may in turn lead to reduced efficacy of anti-MM therapy. Therapeutic antibodies interfere with several laboratory tests. First, interference of therapeutic antibodies with immunofixation and serum protein electrophoresis assays may lead to underestimation of complete response. Strategies to mitigate interference, based on shifting the therapeutic antibody band, are in development. Furthermore, daratumumab, and probably also other CD38-targeting antibodies, interfere with blood compatibility testing and thereby complicate the safe release of blood products. Neutralization of the therapeutic CD38 antibody or CD38 denaturation on reagent red blood cells mitigates daratumumab interference with transfusion laboratory serologic tests. Finally, therapeutic antibodies may complicate flow cytometric evaluation of normal and neoplastic plasma cells, since the therapeutic antibody can affect the availability of the epitope for binding of commercially available diagnostic antibodies., (© 2016 by The American Society of Hematology.)

Extracellular adenosine (ADO), generated from ATP or ADP through the concerted action of the ectoenzymes CD39 and CD73, elicits autocrine and paracrine effects mediated by type 1 purinergic receptors. We have tested whether the expression of CD39 and CD73 by chronic lymphocytic leukemia (CLL) cells activates an adenosinergic axis affecting growth and survival. By immunohistochemistry, CD39 is widely expressed in CLL lymph nodes, whereas CD73 is restricted to proliferation centers. CD73 expression is highest on Ki-67(+) CLL cells, adjacent to T lymphocytes, and is further localized to perivascular areas. CD39(+)/CD73(+) CLL cells generate ADO from ADP in a time- and concentration-dependent manner. In peripheral blood, CD73 expression occurs in 97/299 (32%) CLL patients and pairs with CD38 and ZAP-70 expression. CD73-generated extracellular ADO activates type 1 purinergic A2A receptors that are constitutively expressed by CLL cells and that are further elevated in proliferating neoplastic cells. Activation of the ADO receptors increases cytoplasmic cAMP levels, inhibiting chemotaxis and limiting spontaneous drug-induced apoptosis of CLL cells. These data are consistent with the existence of an autocrine adenosinergic loop, and support engraftment of leukemic cells in growth-favorable niches, while simultaneously protecting from the action of chemotherapeutic agents.

This review highlights a decade of investigations into the role of CD38 in CLL. CD38 is accepted as a dependable marker of unfavorable prognosis and as an indicator of activation and proliferation of cells when tested. Leukemic clones with higher numbers of CD38(+) cells are more responsive to BCR signaling and are characterized by enhanced migration. In vitro activation through CD38 drives CLL proliferation and chemotaxis via a signaling pathway that includes ZAP-70 and ERK1/2. Finally, CD38 is under a polymorphic transcriptional control after external signals. Consequently, CD38 appears to be a global molecular bridge to the environment, promoting survival/proliferation over apoptosis. Together, this evidence contributes to the current view of CLL as a chronic disease in which the host's microenvironment promotes leukemic cell growth and also controls the sequential acquisition and accumulation of genetic alterations. This view relies on the existence of a set of surface molecules, including CD38, which support proliferation and survival of B cells on their way to and after neoplastic transformation. The second decade of studies on CD38 in CLL will tell if the molecule is an effective target for antibody-mediated therapy in this currently incurable leukemia.

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-zeta 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-gamma production, PKC phosphorylation at Thr538, and PKC 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.

Our interest in chronic lymphocytic leukemia (CLL) derives primarily from the exploitation of human diseases as strategic models for defining the in vivo biological roles of CD38. Using this model, we showed that CD38 triggers robust proliferation/survival signals modulated through the interactions with the CD31 ligand expressed by nurse-like cells and by the stromal/endothelial components. By analyzing a cohort of 56 patients with clinically and molecularly characterized CLL, we show that (1) patients with CD38(+)/ZAP-70(+) are characterized by enhanced migration toward Stromal derived factor-1alpha (SDF-1alpha)/CXCL12; (2) CD38 ligation leads to tyrosine phosphorylation of ZAP-70, showing that these markers are functionally linked; (3) ZAP-70 represents a limiting factor for the CD38 pathway in the CLL context, as shown by studying CD38-mediated signal transduction in 26 molecularly characterized patients; and (4) the CLL subgroup of patients defined on the basis of migratory potential is marked by a specific genetic signature, with a significant number of differentially expressed genes being involved in 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 as determined in several clinical trials results in more dependable identification of patients with CLL who have aggressive disease.

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.

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.

The absence of mutations in the IgV genes, together with the presence of ZAP-70 and CD38, are the most reliable negative prognostic markers for chronic lymphocytic leukemia (CLL) patients. Several lines of evidence indicate that CD38 may be not only a diagnostic marker but also a key element in the pathogenetic network in CLL. First, CD38 is a receptor that induces proliferation and increases survival of CLL cells. Second, CD38 signals start upon interaction with the CD31 ligand expressed by stromal and nurse-like cells. Third, CD38/CD31 contacts up-regulate CD100, a semaphorin involved in sustaining CLL growth. Fourth, evidence that nurselike cells express high levels of CD31 and plexin-B1, the high-affinity ligand for CD100, offers indirect confirmation for this model of receptor cross-talk. Elements of variation in the clinical course of CD38(+) CLL patients include (1) potential intersection with ZAP-70, a kinase involved in the CD38 signaling pathway in T and natural killer (NK) cells, and (2) the effects of genetic polymorphisms of the receptors involved, at least of CD38 and CD31. Consequently, CD38 together with ZAP-70 appear to be the key elements of a coreceptor pathway that may sustain the signals mediated by the B-cell receptor and potentially by chemokines and their receptors. This would result in acquisition of increased survival potential, providing clues to the poorer prognosis of CD38(+) patients.

CD38, an ectoenzyme and a signaling receptor, is a novel marker of human mature monocyte-derived dendritic cells (MDDCs). The working hypothesis is that CD38 is not only a marker but also contributes to functions specifically gained by MDDCs with maturation. This was tested by assessing the role(s) of CD38 after signaling with agonistic anti-CD38 monoclonal antibodies or by blocking the interactions taking place between CD38 and CD31, its counterreceptor. The results indicate the following: (1) CD38 engagement in MDDCs ensures efficient chemotaxis and transendothelial migration driven by CC chemokine ligand 21 (CCL21); (2) CD38 is laterally associated with the CCL21-specific CC chemokine receptor 7 and with CD83 and CD11b; (3) CD38 localizes in membrane lipid domains; (4) CD38 signaling contributes to support longevity of lipopolysaccharide (LPS)-matured MDDCs after growth factor withdrawal; and (5) IFN-gamma is produced by cocultured T lymphocytes, thus affecting T-helper 1 (Th1) polarization. These data suggest that the localization of CD38 in lipid rafts and its multiple interactions with signaling receptors rule innate and adaptive immune responses by tuning DC migration, survival, and Th1-polarization ability. These findings may lay out the basis to assess the functional role(s) of human CD38 in infections, autoimmune diseases, and neoplastic disorders.

The immune system works through leukocytes interacting with each other, with other cells, with tissue matrices, with infectious agents, and with other antigens. These interactions are mediated by cell-surface glycoproteins and glycolipids. Antibodies against these leukocyte molecules have provided powerful tools for analysis of their structure, function, and distribution. Antibodies have been used widely in hematology, immunology, and pathology, and in research, diagnosis, and therapy. The associated CD nomenclature is commonly used when referring to leukocyte surface molecules and antibodies against them. It provides an essential classification for diagnostic and therapeutic purposes. The most recent (8th) Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), held in Adelaide, Australia, in December 2004, allocated 95 new CD designations and made radical changes to its aims and future operational strategy in order to maintain its relevance to modern human biology and clinical practice.

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 Ca(2+) 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 beta(2) 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.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical course. Recent studies have shown that CD38 surface expression on the malignant cell clone may serve as a prognostic marker in that CD38(+) patients with B-CLL are characterized by advanced disease stage, lesser responsiveness to chemotherapy, and shorter survival than CD38(-) patients. To further investigate the molecular phenotype of these 2 clinical subgroups, we compared the gene expression profiles of CD38(+) (n = 25) with CD38(-) (n = 45) B-CLL patients using oligonucleotide-based DNA chip microarrays representative of approximately 5600 genes. The results showed that B-CLLs display a common gene expression profile that is largely independent of CD38 expression. Nonetheless, the expression of 14 genes differed significantly between the 2 groups, including genes that are involved in the regulation of cell survival. Furthermore, unsupervised hierarchical cluster analysis of 76 B-CLL samples led to the separation of 2 major subgroups, comprising 20 and 56 patients. Clustering to the smaller group was due in part to the coordinate high expression of a large number of ribosomal and other translation-associated genes, including elongation factors. Importantly, we found that patients with high expression of translation factors were characterized by a more favorable clinical course with significantly longer progression-free survival and reduced chemotherapy requirements than the remaining patients (P <.05). Our data show that gene expression profiling can help identify B-CLL subtypes with different clinical characteristics. Furthermore, our results suggest a role of translation-associated genes in the pathogenesis of B-CLL.

Human transferrin receptor-2 (TFR-2) is a protein highly homologous to TFR-1/CD71 and is endowed with the ability to bind transferrin (TF) with low affinity. High levels of TFR-2 mRNA were found in the liver and in erythroid precursors. Mutations affecting the TFR-2 gene led to hemochromatosis type 3, a form of inherited iron overload. Several issues on distribution and function of the receptor were answered by raising a panel of 9 monoclonal antibodies specific for TFR-2 by immunizing mice with murine fibroblasts transfected with the human TFR-2 cDNA. A polyclonal antiserum was also produced in mice immunized with 3 peptides derived from the TFR-2 sequence, exploiting an innovative technique. The specificity of all the reagents produced was confirmed by reactivity with TFR-2(+) target cells and simultaneous negativity with TFR-1(+) cells. Western blot analyses showed a dominant chain of approximately 90 kDa in TFR-2 transfectants and HepG2 cell line. Analysis of distribution in normal tissues and in representative cell lines revealed that TFR-2 displays a restricted expression pattern--it is present at high levels in hepatocytes and in the epithelial cells of the small intestine, including the duodenal crypts. Exposure of human TFR-2(+) cells to TF-bound iron is followed by a significant up-regulation and relocalization of membrane TFR-2. The tissue distribution pattern, the behavior following exposure to iron-loaded TF, and the features of the disease resulting from TFR-2 inactivation support the hypothesis that TFR-2 contributes to body iron sensing.

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.

Previously, we demonstrated that B-chronic lymphocytic leukemia (B-CLL) cells could be divided into 2 groups depending on the expression of CD38 by the malignant cells. The 2 groups differed in their signal-transducing capacities initiated by cross-linking of surface IgM; only in CD38-positive cells was an efficient signal delivered, invariably resulting in cell apoptosis. In this study, we investigated the effect of surface IgD cross-linking in 10 patients with CD38-positive B-CLL. Exposure of the malignant cells to goat antihuman delta-chain antibodies (Gadelta-ab) caused [Ca(++)]i mobilization and tyrosine kinase phosphorylation in a manner not different from that observed after goat antihuman mu-chain antibody (Gamu-ab) treatment in vitro. However, Gadelta-ab-treated cells failed to undergo apoptosis and instead displayed prolonged survival in culture and differentiated into plasma cells when rIL2 was concomitantly present. Cross-linking of surface IgD failed to induce proliferation of the malignant cells in vitro. Moreover, treatment with Gadelta-ab did not prevent apoptosis of B-CLL cells induced by Gamu-ab. Collectively, these experiments demonstrated that IgM and IgD expressed by the same cell may deliver opposite signals under particular circumstances and provide some clues for the understanding of the pathophysiology of B-CLL. (Blood. 2000;95:1199-1206)

Receptors used by natural killer (NK) cells to mediate natural cytotoxicity are poorly defined, although it is now clear that a number of adhesion molecules can serve this function. CD38 transduces signals on T- and B-cell lines, and we asked whether it could trigger lytic and secretory responses in human NK cells. By using an anti-CD38 monoclonal antibody in reverse antibody-dependent cellular cytotoxicity experiments, it is shown that CD38 engagement triggers cytotoxic responses by activated NK cells, but not by cytotoxic T lymphocytes or fresh NK cells. Cross-linking with anti-CD38 F(ab')(2) caused activated NK cells to release granzymes and cytokines, but did not trigger an increase in intracellular Ca(2+). Fresh NK cells acquired CD38-dependent lytic function during activation with interleukin-2 (IL-2), and inhibitor studies suggested that IL-2 stimulated the de novo expression of proteins that act between CD38 and the lytic machinery in NK cells. The induction of proteins that link commonly expressed adhesion molecules to effector mechanisms could provide a paradigm for pathogen recognition by the innate immune system.

Interleukin-15 (IL-15) is a potent regulator of T-, B-, and natural killer cell proliferation and displays unusually tight controls of secretion. Even though IL-15 mRNA is constitutively expressed in monocytes/macrophages and is upregulated by a variety of stimuli, evidence for IL-15 cytokine secretion is only found exceptionally, eg, conditions of pathological, chronic inflammation. This raises the possibility that monocytes express membrane-bound IL-15 rather than secrete it. The current study explores this hypothesis. We demonstrate here that biologically active IL-15 is indeed detectable in a constitutively expressed, membrane-bound form on normal human monocytes, as well as on monocytic cell lines (MONO-MAC-6, THP-1, and U937), but not on human T or B cells (MT4, M9, C5966, JURKAT, DAUDI, RAJI, and Epstein-Barr virus-immortalized B-cell clones). Furthermore, cell surface-bound IL-15 is upregulated upon interferon-gamma stimulation. Interestingly, monocyte/macrophage inhibitory cytokines such as IL-4 and IL-13 fail to downregulate both constitutive and induced cell-surface expression of IL-15. Membrane-bound IL-15 does not elute with acetate buffer or trypsin treatment, suggesting that it is an integral membrane protein and that it is not associated with the IL-15 receptor complex. Finally, membrane-bound IL-15 stimulates T lymphocytes to proliferate in vitro, indicating that it is biologically active. These findings enlist IL-15 in the fairly small family of cytokines for which the presence of a biologically active membrane-bound form has been demonstrated (eg, IL-1, tumor necrosis factor-alpha, and IL-10) and invites the speculation that most of the biological effects of IL-15 under physiological conditions are exerted by the cell surface-bound form.

Recent studies performed in mice knocked out for the tumor necrosis factor (TNF ), the lymphotoxin-alpha, or the type I TNF receptor (R), genes have shown that these animals display gross defects in germinal center (GC) formation, suggesting that members of the TNF and TNFR superfamilies are involved in the control of B-cell migration. Based on these premises, we have here investigated the effects of human recombinant (r) TNF on the polarization and locomotion of tonsillar B cells. rTNF increased the spontaneous polarization and locomotion of unfractionated tonsillar B lymphocytes in a dose-dependent manner by inducing a true chemotactic response. Memory (IgD-, CD38(-)) and naive (IgD+, CD38(-)), but not GC (IgD-, CD38(+)) B cells purified from total tonsillar B lymphocytes, showed a significantly higher locomotion in the presence than in the absence of rTNF. Accordingly, type I and II TNF receptors (TNFRs) were detected by flow cytometry on the surface of memory and naive, but not GC, B lymphocytes. Blocking experiments with monoclonal antibodies to type I or II TNFR showed that rTNF enhanced the spontaneous chemotaxis of memory and naive B cells through the selective engagement of type II TNFR. Finally, the TNF gene was found to be expressed in memory, naive and GC B lymphocytes; the cytokine was released in culture supernatants from the three B-cell subsets after stimulation. These data may support the hypothesis that human TNF is involved in the paracrine and perhaps autocrine control of B-cell migration in secondary lymphoid tissues.