CD38 in the age of COVID-19: a medical perspective.

This medical review addresses the hypothesis that CD38/NADase is at the center of a functional axis (i.e., intracellular Ca2+ mobilization/IFNγ response/reactive oxygen species burst) driven by severe acute respiratory syndrome coronavirus 2 infection, as already verified in respiratory syncytial virus pathology and CD38 activity in other cellular settings. Key features of the hypothesis are that 1) the substrates of CD38 (e.g., NAD+ and NADP+) are depleted by viral-induced metabolic changes; 2) the products of the enzymatic activity of CD38 [e.g., cyclic adenosine diphosphate-ribose (ADPR)/ADPR/nicotinic acid adenine dinucleotide phosphate] and related enzymes [e.g., poly(ADP-ribose)polymerase, Sirtuins, and ADP-ribosyl hydrolase] are involved in the anti-viral and proinflammatory response that favors the onset of lung immunopathology (e.g., cytokine storm and organ fibrosis); and 3) the pathological changes induced by this kinetic mechanism may be reduced by distinct modulators of the CD38/NAD+ axis (e.g., CD38 blockers, NAD+ suppliers, among others). This view is supported by arrays of associative basic and applied research data that are herein discussed and integrated with conclusions reported by others in the field of inflammatory, immune, tumor, and viral diseases.

Molecular dynamics of targeting CD38 in multiple myeloma.

Multiple functions of CD38 need exploring to expand clinical application of anti-CD38 antibodies in multiple myeloma (MM). We investigated membrane dynamics of MM cells and subsequent events when CD38 is targeted by therapeutic antibodies. Human MM cells (BF01) were co-cultured in vitro with therapeutic antibody (or control immunoglobulin G) and analysed using gene expression profiling. Microvesicles from antibody-exposed cells were analysed for differential gene and microRNA (miRNA) expression, and for phenotypic characterisation. Exposure of BF01 cells to anti-CD38 antibody resulted in CD38 membrane redistribution, upregulation of metabolism-related genes and downregulation of genes involved in cell cycle processes. Microvesicles derived from antibody-exposed cells showed increased CD73 and CD39 expression, presence of programmed death-ligand 1 and significant up-/down-modulation of miRNAs. Microvesicles accumulated around immunoglobulin Fc receptor-positive (FcR+ ) cells. Upon internalisation, natural killer cells displayed significantly increased expression of genes related to activation and immune response, and downregulation of genes involved in the cell cycle. Cells may use microvesicles to transmit signals distally as part of a survival strategy. Microvesicles are equipped on their surface with enzymatic machinery leading to production of tolerogenic adenosine. Further, they are internalised in FcR+ cells with significant functional modifications. These observations have relevance for improving anti-CD38 therapeutic antibodies through targeting this mechanism and its sequelae.

The Circular Life of Human CD38: From Basic Science to Clinics and Back.

Monoclonal antibodies (mAbs) were initially considered as a possible "magic bullet" for in vivo elimination of tumor cells. mAbs represented the first step: however, as they were murine in nature (the earliest experience on the field), they were considered unfit for human applications. This prompted the development of techniques for cloning the variable regions of conventional murine antibodies, genetically mounted on human IgG. The last step in this years-long process was the design for the preparation of fully human reagents. The choice of the target molecule was also problematic, since cancer-specific targets are quite limited in number. To overcome this obstacle in the planning phases of antibody-mediated therapy, attention was focused on a set of normal molecules, whose quantitative distribution may balance a tissue-dependent generalized expression. The results and clinical success obtained with anti-CD20 mAbs revived interest in this type of strategy. Using multiple myeloma (MM) as a tumor model was challenging first of all because the plasma cells and their neoplastic counterpart eluded the efforts of the Workshop on Differentiation Antigens to find a target molecule exclusively expressed by these cells. For this reason, attention was turned to surface molecules which fulfill the requisites of being reasonably good targets, even if not specifically restricted to tumor cells. In 2009, we proposed CD38 as a MM target in virtue of its expression: it is absent on early hematological progenitors, has variable but generalized limited expression by normal cells, but is extremely high in plasma cells and in myeloma. Further, regulation of its expression appeared to be dependent on a variety of factors, including exposure to all-trans retinoic acid (ATRA), a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells that are now approved for in vivo use. This review discusses the history of human CD38, from its initial characterization to its targeting in antibody-mediated therapy of human myeloma.

CD38 in Adenosinergic Pathways and Metabolic Re-programming in Human Multiple Myeloma Cells: In-tandem Insights From Basic Science to Therapy.

Tumor microenvironments are rich in extracellular nucleotides that can be metabolized by ectoenzymes to produce adenosine, a nucleoside involved in controlling immune responses. Multiple myeloma, a plasma cell malignancy developed within a bone marrow niche, exploits adenosinergic pathways to customize the immune homeostasis of the tumor. CD38, a multifunctional protein that acts as both receptor and ectoenzyme, is overexpressed at all stages of myeloma. At neutral and acidic pH, CD38 catalyzes the extracellular conversion of NAD+ to regulators of calcium signaling. The initial disassembly of NAD+ is also followed by adenosinergic activity, if CD38 is operating in the presence of CD203a and CD73 nucleotidases. cAMP extruded from tumor cells provides another substrate for metabolizing nucleotidases to signaling adenosine. These pathways flank or bypass the canonical adenosinergic pathway subjected to the conversion of ATP by CD39. All of the adenosinergic networks can be hijacked by the tumor, thus controlling the homeostatic reprogramming of the myeloma in the bone marrow. In this context, adenosine assumes the role of a local hormone: cell metabolism is adjusted via low- or high-affinity purinergic receptors expressed by immune and bone cells as well as by tumor cells. The result is immunosuppression, which contributes to the failure of immune surveillance in cancer. A similar metabolic strategy silences immune effectors during the progression of indolent gammopathies to symptomatic overt multiple myeloma disease. Plasma from myeloma aspirates contains elevated levels of adenosine resulting from interactions between myeloma and other cells lining the niche and adenosine concentrations are known to increase as the disease progresses. This is statistically reflected in the International Staging System for multiple myeloma. Along with the ability to deplete CD38+ malignant plasma cell populations which has led to their widespread therapeutic use, anti-CD38 antibodies are involved in the polarization and release of microvesicles characterized by the expression of multiple adenosine-producing molecules. These adenosinergic pathways provide new immune checkpoints for improving immunotherapy protocols by helping to restore the depressed immune response.

Microvesicles expressing adenosinergic ectoenzymes and their potential role in modulating bone marrow infiltration by neuroblastoma cells.

Metastatic diffusion of Neuroblastoma (NB) cells in the bone marrow (BM) represents the most negative prognostic factors for NB patients. Multiple immune escape mechanisms are postulated as responsible. Our working hypothesis is that adenosine (ADO), an immunosuppressive molecule along with the ectoenzymatic pathways (CD39-CD73 and CD38-CD203a/PC-1-CD73) controlling its production, are involved in the dynamics of NB cells in the BM. The results indicate that ectonucleotidases are expressed by i) NB cell lines, ii) metastatic NB cells isolated from NB patients' BM, iii) microvesicles (MV) derived from both NB cell types and iv) resident BM cell populations. BM infiltration by NB cells increased CD203a/PC-1 and CD73 expression on lymphoid and myeloid cells, respectively. Expressions of ectoenzymes and GD2 (NB-associated marker) were higher on MV from NB patients' BM than in controls. Moreover, CD203a/PC-1 expression on BM-derived MV provide a basis for distinguishing NB patients with high or low BM infiltration. ADO production and consumption of related by-products were significantly higher when assessed on NB patients' MV than those from controls. MV isolated from NB patients' BM significantly downregulated in vitro T cell proliferation. Lastly, NB patients with worse prognosis are identified by a high percentage of CD38+ or CD73+ MV in the BM. In conclusion, ectonucleotidases are present and functional on NB cells, as well as in NB-infiltrated BM and in MV derived from BM. It is reasonable that MV are involved in BM infiltration by NB cells. Therefore, targeting these molecules may widen the therapeutic armamentarium for metastatic NB patients.

Ectonucleotidase Expression on Human Amnion Epithelial Cells: Adenosinergic Pathways and Dichotomic Effects on Immune Effector Cell Populations.

This study investigates the mechanism(s) underlying the immunoregulatory activities of placenta-derived human amnion epithelial cells (hAEC). The working hypothesis is that NAD+ and ATP, along with ectoenzymes involved in their metabolism, play a significant role in hAEC-mediated immune regulation. Proof of principle of the hypothesis was obtained by analyzing the interactions between hAEC and the main human leukocyte populations. The results obtained indicate that hAEC constitutively express a unique combination of functional ectoenzymes, driving the production of adenosine (ADO) via canonical (CD39, CD73) and alternative (CD38, CD203a/PC-1, CD73) pathways. Further, the picture is completed by the observation that hAEC express A1, A2a, and A2b ADO receptors as well as ADO deaminase, the enzyme involved in ADO catabolism. The contribution of the purinergic mediator to immunomodulation was confirmed by exposing in vitro different immune effector cells to the action of primary hAECs. B cells showed an enhanced proliferation and diminished spontaneous apoptosis when in contact with hAEC. T cell proliferation was partially inhibited by hAEC through ADO production, as confirmed by using specific ectoenzyme inhibitors. Further, hAEC induced an expansion of both T and B regulatory cells. Last, hAEC inhibited NK cell proliferation. However, the involvement of ADO-producing ectoenzymes is less apparent in this context. In conclusion, hAEC exert different in vitro immunoregulatory effects, per se, as a result of interactions with different populations of immune effector cells. These results support the view that hAEC are instrumental for regenerative medicine as well as in therapeutic applications for immune-related diseases.

CD38: A Target for Immunotherapeutic Approaches in Multiple Myeloma.

Multiple Myeloma (MM) is a hematological cancer characterized by proliferation of malignant plasma cells in the bone marrow (BM). MM represents the second most frequent hematological malignancy, accounting 1% of all cancer and 13% of hematological tumors, with ~9,000 new cases per year. Patients with monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic smoldering MM (SMM) usually evolve to active MM in the presence of increased tumor burden, symptoms and organ damage. Despite the role of high dose chemotherapy in combination with autologous stem cell transplantation and the introduction of new treatments, the prognosis of MM patients is still poor, and novel therapeutic approaches have been tested in the last years, including new immunomodulatory drugs, proteasome inhibitors and monoclonal antibodies (mAbs). CD38 is a glycoprotein with ectoenzymatic functions, which is expressed on plasma cells and other lymphoid and myeloid cell populations. Since its expression is very high and uniform on myeloma cells, CD38 is a good target for novel therapeutic strategies. Among them, immunotherapy represents a promising approach. Here, we summarized recent findings regarding CD38-targeted immunotherapy of MM in pre-clinical models and clinical trials, including (i) mAbs (daratumumab and isatuximab), (ii) radioimmunotherapy, and (iii) adoptive cell therapy, using chimeric antigen receptor (CAR)-transfected T cells specific for CD38. Finally, we discussed the efficacy and possible limitations of these therapeutic approaches for MM patients.

Cytokine-Induced Killer Cells Express CD39, CD38, CD203a, CD73 Ectoenzymes and P1 Adenosinergic Receptors.

Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by in vitro differentiation of peripheral blood mononuclear cells (PBMC), represent a promising immunological approach in cancer. Numerous studies have explored the role of CD38, CD39, CD203a/PC-1, and CD73 in generating extracellular adenosine (ADO) and thus in shaping the tumor niche in favor of proliferation. The findings shown here reveal that CIK cells are able to produce extracellular ADO via traditional (CD39/CD73) and/or alternative (CD38/CD203a/CD73 or CD203a/CD73) pathways. Transcriptome analysis showed the mRNA expression of these molecules and their modulation during PBMC to CIK differentiation. When PBMC from normal subjects or cancer bearing patients were differentiated into CIK cells under normoxic conditions, CD38 and CD39 were greatly up-regulated while the number of CD203a, and CD73 positive cells underwent minor changes. Since hypoxic conditions are often found in tumors, we asked whether CD39, CD38, CD203a, and CD73 expressed by CIK cells were modulated by hypoxia. PBMC isolated from cancer patients and differentiated into CIK cells in hypoxic conditions did not show relevant changes in CD38, CD39, CD73, CD203a, and CD26. CIK cells also expressed A1, A2A, and A2B ADO receptors and they only underwent minor changes as a consequence of hypoxia. The present study sheds light on a previously unknown functional aspect of CIK cells, opening the possibility of pharmacologically modulated ADO-generating ectoezymes to improve CIK cells performance.

CD38 modulates respiratory syncytial virus-driven proinflammatory processes in human monocyte-derived dendritic cells.

Respiratory syncytial virus (RSV) is the most common cause of hospitalization due to bronchiolitis in infants. Although the mechanisms behind this association are not completely elucidated, they appear to involve an excessive immune response causing lung pathology. Understanding the host response to RSV infection may help in the identification of targets for therapeutic intervention. We infected in-vitro human monocyte-derived dendritic cells (DCs) with RSV and analysed various aspects of the cellular response. We found that RSV induces in DCs the expression of CD38, an ectoenzyme that catalyses the synthesis of cyclic ADPR (cADPR). Remarkably, CD38 was under the transcriptional control of RSV-induced type I interferon (IFN). CD38 and a set of IFN-stimulated genes (ISGs) were inhibited by the anti-oxidant N-acetyl cysteine. When CD38-generated cADPR was restrained by 8-Br-cADPR or kuromanin, a flavonoid known to inhibit CD38 enzymatic activity, RSV-induced type I/III IFNs and ISGs were markedly reduced. Taken together, these results suggest a key role of CD38 in the regulation of anti-viral responses. Inhibition of CD38 enzymatic activity may represent an encouraging approach to reduce RSV-induced hyperinflammation and a novel therapeutic option to treat bronchiolitis.

Expression of CD38 in myeloma bone niche: A rational basis for the use of anti-CD38 immunotherapy to inhibit osteoclast formation.

It is known that multiple myeloma (MM) cells express CD38 and that a recently developed human anti-CD38 monoclonal antibody Daratumumab mediates myeloma killing. However, the expression of CD38 and other functionally related ectoenzymes within the MM bone niche and the potential effects of Daratumumab on bone cells are still unknown. This study firstly defines by flow cytometry and immunohistochemistry the expression of CD38 by bone marrow cells in a cohort of patients with MM and indolent monoclonal gammopathies. Results indicate that only plasma cells expressed CD38 at high level within the bone niche. In addition, the flow cytometry analysis shows that CD38 was also expressed by monocytes and early osteoclast progenitors but not by osteoblasts and mature osteoclasts. Indeed, CD38 was lost during in vitro osteoclastogenesis. Consistently, we found that Daratumumab reacted with CD38 expressed on monocytes and its binding inhibited in vitro osteoclastogenesis and bone resorption activity from bone marrow total mononuclear cells of MM patients, targeting early osteoclast progenitors. The inhibitory effect was not observed from purified CD14+ cells, suggesting an indirect inhibitory effect of Daratumumab. Interestingly, all-trans retinoic acid treatment increased the inhibitory effect of Daratumumab on osteoclast formation. These observations provide a rationale for the use of an anti-CD38 antibody-based approach as treatment for multiple myeloma-induced osteoclastogenesis.

Roles and Modalities of Ectonucleotidases in Remodeling the Multiple Myeloma Niche.

Ectoenzymes are cell surface molecules, which represent functional bridges between the environment and the cytoplasm. One set of ectoenzymes-CD39, CD38, CD203a, and CD73-leads to the generation of adenosine (ADO) by metabolizing ATP and NAD+. While ADO is known to control inflammation and suppress immune responses, other aspects of ADO function are still obscure, mainly due to its short half-life in biological fluids. Human multiple myeloma (MM) grows in the closed system of the bone marrow (BM) niche representing an ideal setting for studying ectoenzymes and their products. Another source of information on ectoenzyme function may derive from in vivo results of anti-CD38 antibody therapy in MM. Current results, obtained from in vitro models and from preliminary in vivo findings, indicate that ectoenzymes produce ADO locally in the BM niche. Furthermore, MM cells release microvesicles (MV), which thanks to their molecular cargo and surface ectoenzymes may function as particulate communicators outside of the niche. During anti-CD38 antibody therapy, the MV carry therapeutic IgG, determining that the prevalent orientation of MV will be toward cells and tissues expressing receptors for the IgG Fc domain. The resulting picture is one where MM adopts an immune escape strategy based on reshaping the environmental niche. This adaptation is followed by actions of MV that are exerted in biological fluids and circulating immune cells. By coating FcRs+ cells, MV modify pericellular spaces, reproducing the metabolic halo generated by ectoenzymes within closed systems.

Antibody mimicry, receptors and clinical applications.

This review focuses on the concept of antibodies acting as receptor agonists and antagonists, and on the potential relevance of this notion in applied medicine. Antibodies are composed of three functional units: two antigen-binding fragments (Fabs) that confer antigen specificity and one constant fragment (Fc) linking antibodies to immune effector functions. The proof-of-concept that large amounts of highly specific and homogeneous antibodies could be produced was provided in 1975 by César Milstein and Georges Köhler. These monoclonal antibody (mAb) reagents started a revolution in medical research, diagnostics, and clinical applications. Alongside diagnostic applications, mAbs were successfully used in vivo: (i) to bind (neutralize/antagonize) antigens expressed on the surface of tumor cells; (ii) to activate immune effector mechanisms; (iii) to crosslink plasma membrane receptors and hence activate therapeutic signaling pathways; and lastly, (iv) the technique was expanded to produce bispecific mAbs, which can bind two different antigens while retaining the ability to activate immune effector functions. The abilities of mAbs to bind, transduce signals, and exert immunostimulatory agonistic capacities are the central issues of this review. The starting point is that some mAbs operate as molecular agonists, substituting for the natural ligand of the receptor. Our analysis is restricted to mAbs that act as receptor agonist/antagonists by either mimicking ligand binding, or through allosteric modulation mediated by binding sites that are topographically distinct from the orthosteric binding site. Functional considerations based on the agonistic stimulation of human CD38 by specific mAbs as surrogate ligands are described as examples of the features of such molecules.

Adenosine Generated in the Bone Marrow Niche Through a CD38-Mediated Pathway Correlates with Progression of Human Myeloma.

Human myeloma cells express CD38 at high levels and grow in hypoxic niches inside the bone marrow. Myeloma cells respond to hypoxia with metabolic changes leading to aerobic glycolysis, thus reducing ATP and increasing NAD+. Our hypothesis is that these conditions favor the enzymatic pathways involved in the production of adenosine in the niche. Within the niche, NAD+ is able to activate a discontinuous adenosinergic pathway that relies upon CD38, CD203a, and CD73 or TRACP, according to the environmental pH. The observed variability in adenosine concentrations in bone marrow aspirates is a result of the interactions taking place among myeloma and other cells in the bone marrow niche. A pilot study showed that adenosine profiles differ during disease progression. Adenosine levels were significantly higher in the bone marrow plasma of patients with symptomatic myeloma and correlated with ISS staging, suggesting that adenosine is produced in the myeloma niche at micromolar levels by an ectoenzymatic network centered on CD38. Adenosine levels increase with disease aggressiveness, a finding that supports adenosine as a potential marker of myeloma progression.

A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing ß-Glucan and MP65 Determinants in Candida Species.

There is a real medical need of new diagnostic tools for the early recognition of invasive Candida infections. We exploited a rather simple and rapid redox methodology to construct a bispecific monoclonal antibody (bsmAb) that combines a monoclonal antibody (mAb) directed against 1,3-ß-D-glucan, a well-known, pan-fungal diagnostic biomarker, with a mAb recognizing MP65, a major immunogenic mannoprotein secreted by C.albicans and other Candida species. The bsmAb (MP65/bglu mAb) was successfully produced and purified at high yields and proved to bind and reveal simultaneously, with high sensitivity, the ß-glucan and MP65 antigens in both purified and native forms. The MP65/bglu mAb is the first bispecific antibody generated against a fungal microorganism and may prove useful for the concurrent detection of different and clinically significant Candida biomarkers in patient sera.

A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation.

Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD(+ )T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4(+) T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8(+) T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role.

NAD⁺-Metabolizing Ectoenzymes in Remodeling Tumor-Host Interactions: The Human Myeloma Model.

Nicotinamide adenine dinucleotide (NAD⁺) is an essential co-enzyme reported to operate both intra- and extracellularly. In the extracellular space, NAD⁺ can elicit signals by binding purinergic P2 receptors or it can serve as the substrate for a chain of ectoenzymes. As a substrate, it is converted to adenosine (ADO) and then taken up by the cells, where it is transformed and reincorporated into the intracellular nucleotide pool. Nucleotide-nucleoside conversion is regulated by membrane-bound ectoenzymes. CD38, the main mammalian enzyme that hydrolyzes NAD⁺, belongs to the ectoenzymatic network generating intracellular Ca(2+)-active metabolites. Within this general framework, the extracellular conversion of NAD⁺ can vary significantly according to the tissue environment or pathological conditions. Accumulating evidence suggests that tumor cells exploit such a network for migrating and homing to protected areas and, even more importantly, for evading the immune response. We report on the experience of this lab to exploit human multiple myeloma (MM), a neoplastic expansion of plasma cells, as a model to investigate these issues. MM cells express high levels of surface CD38 and grow in an environment prevalently represented by closed niches hosted in the bone marrow (BM). An original approach of this study derives from the recent use of the clinical availability of therapeutic anti-CD38 monoclonal antibodies (mAbs) in perturbing tumor viability and enzymatic functions in conditions mimicking what happens in vivo.

Unconventional, adenosine-producing suppressor T cells induced by dendritic cells exposed to BPZE1 pertussis vaccine.

BPZE1 is a live attenuated pertussis vaccine that successfully completed a phase 1 safety trial. This article describes the induction of unconventional suppressor T cells-producing ADO by MDDCs exposed to BPZE1 (BPZE1-DC) through distinct ectoenzymatic pathways that limit the damaging effect of inflammation. BPZE1-DC induces CD4(+) and CD8(+) T lymphocytes to express 2 sets of ectoenzymes generating ADO: 1 set is part of the conventional CD39/CD73 pathway, which uses ATP as substrate, whereas the other is part of the CD38/CD203a/CD73 pathway and metabolizes NAD(+). The contribution of the ADO-generating ectoenzymes in the regulatory response was shown by: 1) selective inhibition of the enzymatic activities of CD39, CD73, and CD38; 2) the ability of suppressor T cells to convert exogenously added ATP and NAD(+) to ADO; and 3) a positive correlation between ectoenzyme expression, ADO levels, and suppression abilities. Thus, T lymphocytes activated by BPZE1-DC shift to a suppressor stage, through the expression of ectoenzyme networks, and are able to convert extracellular nucleotides into ADO, which may explain the potent anti-inflammatory properties of BPZE1 observed in several murine models.

CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases.

Unraveling the contribution of ectoenzymes to myeloma life and survival in the bone marrow niche.

The bone marrow provides a protected environment for generating a vast array of cell types. Bones are thus a dynamic source of structural components and soluble factors used either locally or at a distance from their site of production. We discuss the role of ectoenzymes in the bone niche where human myeloma grows. Selected ectoenzymes have been tested for their ability to promote production of substrates involved in signaling, synthesis of growth factors and hormones, and modulation of the immune response. Because of the difficulty of simultaneously tracking all these activities, we narrow our focus to events potentially influencing synthesis of adenosine (ADO), an important regulator of multiple biological functions, including local immunological tolerance. Our working hypothesis, to be discussed and partially tested herein, is that CD38, and likely BST1/CD157--both NAD(+) -consuming enzymes, are active in the myeloma niche and lead a discontinuous chain of ectoenzymes whose final products are exploited by the neoplastic plasma cell as part of its local survival strategy. Coadjuvant ectoenzymes include PC-1/CD203a, CD39, and CD73, which control the production of ADO. Results discussed here and from ongoing experiments indicate that the myeloma niche hosts the canonical, as well as alternative, pathways of ADO generation. Other possibilities are presented and discussed.

The ADP-ribosyl cyclases--the current evolutionary state of the ARCs.

The major ADP-ribosylating enzyme families are the focus of this special issue of Frontiers in Bioscience . However, there is room for another family of enzymes with the capacity to utilize nicotinamide adenine dinucleotide (NAD): the ADP-ribosyl cyclases (ARCs). These unique enzymes catalyse the cyclization of NAD to cyclic ADP ribose (cADPR), a widely distributed second messenger. However, the ARCs are versatile enzymes that can manipulate NAD, NAD phosphate (NADP) and other substrates to generate various bioactive molecules including nicotinic acid adenine dinucleotide diphosphate (NAADP) and ADP ribose (ADPR). This review will focus on the group of well-characterized invertebrate and vertebrate ARCs whose common gene structure allows us to trace their origin to the ancestor of bilaterian animals. Behind a facade of gene and protein homology lies a family with a disparate functional repertoire dictated by the animal model and the physical trait under investigation. Here we present a phylogenetic view of the ARCs to better understand the evolution of function in this family.