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.

CD73/Adenosine Pathway Involvement in the Interaction of Non-Small Cell Lung Cancer Stem Cells and Bone Cells in the Pre-Metastatic Niche.

Adenosinergic signaling is an important regulator of tissue homeostasis and extracellular accumulation of adenosine (Ado) and is associated with different pathologies, such as cancer. In non-small-cell lung cancer (NSCLC), a subset of CD133/CXCR4+ cancer stem cell (CSCs) has been demonstrated to initiate bone metastases. Here we investigated how NSCLC CSCs interact with osteoclasts (OCs) and osteoblasts (OBs) by modulating Ado production and OC activity. We proved that CSC-spheres, generated in vitro from NSCLC cell lines, express CD38, PC-1, and CD73, enzymes of the non-canonical adenosinergic pathway, produce high level of Ado, and down-regulate A1R and A3R inhibitory receptors, while expressing A2AR and A2BR. To address the Ado role and modulation of the in-bone pre-metastatic niche, we performed co-cultures of CSC-spheres with OCs and OBs cells. Firstly, we verified that active OCs do not activate non-canonical the adenosinergic pathway, conversely to OBs. OCs co-cultured with CSC-spheres increase Ado production that is related to the OC resorption activity and contributes to T-cell suppression. Finally, we proved the efficacy of anti-CD73 agents in blocking NSCLC cell migration. Overall, we assessed the importance of adenosinergic signaling in the interaction between CSCs and OCs at the pre-metastatic niche, with therapeutic implications related to Ado production.

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.

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.

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 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.

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.

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.

Expression of P2X7 ATP receptor mediating the IL8 and CCL20 release in human periodontal ligament stem cells.

ATP is released by human periodontal ligament cells (hPDLCs) and has been shown to regulate PDL regeneration and responses to mechanical stress through activation of P2Y receptors. This nucleotide, however, has also been reported to trigger the pro-inflammatory cascade by inducing the maturation and/or release of chemokines/cytokines from various cell types mainly via P2X7 receptors. Much less is known on the possible role of ATP in stem cells deriving from PDL (hPDLSCs) which are considered to be a promising tool for cell-based therapy to restore lesions. Given the role played by P2X7 in pathophysiological conditions, in this study we investigated the expression of P2X7 ATP receptors in hPDLSCs. The results obtained showed that hPDLSCs express P2X7 receptors evaluated by means of cytofluorimetric, immunohistochemistry, reverse transcriptase-PCR, and Western blot analyses. P2X7 ligation by 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), a specific receptor agonist, was followed by an increase in intracellular Ca2+ and in the uptake of ethidium bromide. These effects were dramatically reduced by oxidized ATP (oATP), the P2X7 irreversible inhibitor, suggesting that the P2X7 is the functional receptor involved. At 24 h treatment of hPDLSCs with BzATP it enhanced the release of the pro-inflammatory agents IL8 and CCL20, without influencing cell viability. These effects were counteracted by pre-treating the cells with oATP or with A-740003, a selective and potent P2X7 competitive antagonist. Collectively, these results indicated that extracellular ATP mediate a pro-inflammatory response via P2X7 receptors in hPDLSCs opening a further approach to control hPDLSCs behavior in their possible application as therapeutic tool.

Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule.

In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule's regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

CD73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death.

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.

Stable coordination of the inhibitory Ca2+ ion at the metal ion-dependent adhesion site in integrin CD11b/CD18 by an antibody-derived ligand aspartate: implications for integrin regulation and structure-based drug design.

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg(2+) ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking mAb 107 binds MIDAS of integrin CD11b/CD18 A domain (CD11bA), but in contrast, it favors the inhibitory Ca(2+) ion over the Mg(2+) ion at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of the Ca(2+) ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca(2+) ion. Binding of the Fab fragment of mAb 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that the denticity of the ligand Asp/Glu can modify the divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca(2+) ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists.

CD38 ligation in peripheral blood mononuclear cells of myeloma patients induces release of protumorigenic IL-6 and impaired secretion of IFNγ cytokines and proliferation.

CD38, a surface receptor that controls signals in immunocompetent cells, is densely expressed by cells of multiple myeloma (MM). The immune system of MM patients appears as functionally impaired, with qualitative and quantitative defects in T cell immune responses. This work answers the issue whether CD38 plays a role in the impairment of T lymphocyte response. To this aim, we analyzed the signals implemented by monoclonal antibodies (mAb) ligation in peripheral blood mononuclear cells (PBMC) obtained from MM patients and compared to benign monoclonal gammopathy of undetermined significance (MGUS). PBMC from MM both failed to proliferate and secrete IFNγ induced by CD38 ligation while it retained the ability to respond to TCR/CD3. The impaired CD38-dependent proliferative response likely reflects an arrest in the progression of cell cycle, as indicated by the reduced expression of PCNA. CD38 signaling showed an enhanced ability to induce IL-6 secretion. PBMC from MM patients displays a deregulated response possibly due to defects of CD38 activation pathways and CD38 may be functionally involved in the progression of this pathology via the secretion of high levels of IL-6 that protects neoplastic cells from apoptosis.

The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of ß(1) and ß(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

The Key Role of NAD+ in Anti-Tumor Immune Response: An Update.

Nicotinamide adenine dinucleotide (NAD+) is an important molecule that functions as a co-enzyme in numerous metabolic processes. Generated both through de novo synthesis and via salvage pathways, NAD+ is the substrate for a variety of NAD+-consuming enzymes. Among them is CD38, a cell surface ecto-enzyme widely expressed on different types of cells and endowed with the function of cADP-ribose synthases/NAD+ glycohydrolase. Surface CD38 expression is increased in different hematological and solid tumors, where it cooperates with other ecto-enzymes to produce the immunosuppressive molecule adenosine (ADO). Few studies have explored the correlation of NAD+ levels with T-cell mediated anti-tumor response in preclinical models. We therefore discuss these novel findings, examining the possible contribution of NAD+ depletion, along with ADO production, in the immunosuppressive activities of CD38 in the context of human tumors. Lastly, we discuss the use of pharmacological inhibitors of CD38 and supplementation of different NAD+ precursors to increase NAD+ levels and to boost T cell responses. Such molecules may be employed as adjuvant therapies, in combination with standard treatments, for cancer patients.

CXCR4 Inhibition Counteracts Immunosuppressive Properties of Metastatic NSCLC Stem Cells.

Cancer stem cells (CSCs) are functionally defined as the cell subset with greater potential to initiate and propagate tumors. Within the heterogeneous population of lung CSCs, we previously identified highly disseminating CD133+CXCR4+ cells able to initiate distant metastasis (metastasis initiating cells-MICs) and to resist conventional chemotherapy. The establishment of an immunosuppressive microenvironment by tumor cells is crucial to sustain and foster metastasis formation, and CSCs deeply interfere with immune responses against tumors. How lung MICs can elude and educate immune cells surveillance to efficiently complete the metastasis cascade is, however, currently unknown. We show here in primary tumors from non-small cell lung cancer (NSCLC) patients that MICs express higher levels of immunoregulatory molecules compared to tumor bulk, namely PD-L1 and CD73, an ectoenzyme that catalyzes the production of immunosuppressive adenosine, suggesting an enhanced ability of MICs to escape immune responses. To investigate in vitro the immunosuppressive ability of MICs, we derived lung spheroids from cultures of adherent lung cancer cell lines, showing enrichment in CD133+CXCR4+MICs, and increased expression of CD73 and CD38, an enzyme that also concurs in adenosine production. MICs-enriched spheroids release high levels of adenosine and express the immunosuppressive cytokine IL-10, undetectable in an adherent cell counterpart. To prevent dissemination of MICs, we tested peptide R, a novel CXCR4 inhibitor that effectively controls in vitro lung tumor cell migration/invasion. Notably, we observed a decreased expression of CD73, CD38, and IL-10 following CXCR4 inhibition. We also functionally proved that conditioned medium from MICs-enriched spheroids compared to adherent cells has an enhanced ability to suppress CD8+ T cell activity, increase Treg population, and induce the polarization of tumor-associated macrophages (TAMs), which participate in suppression of T cells. Treatment of spheroids with anti-CXCR4 rescued T cell cytotoxic activity and prevented TAM polarization, likely by causing the decrease of adenosine and IL-10 production. Overall, we provide evidence that the subset of lung MICs shows high potential to escape immune control and that inhibition of CXCR4 can impair both MICs dissemination and their immunosuppressive activity, therefore potentially providing a novel therapeutic target in combination therapies to improve efficacy of NSCLC treatment.

CD38 and CD157 ectoenzymes mark cell subsets in the human corneal limbus.

Nicotinamide adenine dinucleotide (NAD(+)), a precursor of molecules involved in cell regulatory processes, is released in extra-cellular compartments after stress or inflammation.This study investigates the expression in the human cornea of CD38 and CD157, two NAD(+)-consuming ectoenzymes and surface receptors. The analysis in corneal epithelial and stromal cells was performed by means of multiple approaches, which included immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and confocal microscopy. The presence of enzymatically active NAD(+)-consumers in intact corneal cells was analyzed by high performance liquid chromatography (HPLC)-based assays. The results obtained show that CD38 and CD157 are expressed constitutively by corneal cells: CD38 appears as a 45-kDa monomer, while CD157 is a 42- to 45-kDa doublet. The molecules are enzymatically active, with features reminiscent of those observed in human leukocytes. CD38 is expressed by cells of the suprabasal limbal epithelium, whereas it is not detectable in central corneal epithelium and stroma. CD157 is expressed by basal limbal clusters, a p63(+)/cytokeratin 19(+) cell subset reported to contain corneal stem cells, and by stromal cells. The results of the work indicates that the human cornea is equipped with molecular tools capable of consuming extracellular NAD(+), and that CD157 is a potential marker of corneal limbal cells in the stem cell niche. The presence and characteristics of these ectoenzymes may be exploited to design drugs for wound repair or for applications in tissue transplantation.

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.

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.