CD38 and CD157: A long journey from activation markers to multifunctional molecules

Cytometry Part B (Clinical Cytometry) 84B:207–217 (2013)

CD38 and CD157: A Long Journey fromActivation Markers to Multifunctional Molecules

Valeria Quarona,1 Gianluca Zaccarello,1 Antonella Chillemi,1 Enrico Brunetti,1

Vijay Kumar Singh,2 Enza Ferrero,1,3 Ada Funaro,1,3 Alberto L. Horenstein,1,3 andFabio Malavasi1,3,4*

1Department of Medical Sciences, Laboratory of Immunogenetics, University of Torino Medical School,TORINO, Italy

2Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia, BIELLA, Italy3Centro di Ricerca in Medicina Sperimentale (CeRMS), University of Torino Medical School, Torino, Italy

4Transplantation Immunology Service, Citta della Salute Hospital, TORINO, Italy

CD38 (also known as T10) was identified in the late 1970s in the course of pioneering work carriedout at the Dana-Farber Cancer Center (Boston, MA) that focused on the identification of surface mole-cules involved in antigen recognition. CD38 was initially found on thymocytes and T lymphocytes, buttoday we know that the molecule is found throughout the immune system, although its expression levelsvary. Because of this, CD38 was considered an ‘‘activation marker,’’ a term still popular in routine flowcytometry. This review summarizes the findings obtained from different approaches, which led to CD38being re-defined as a multifunctional molecule. CD38 and its homologue CD157 (BST-1), contiguousgene duplicates on human chromosome 4 (4p15), are part of a gene family encoding products that mod-ulate the social life of cells by means of bidirectional signals. Both CD38 and CD157 play dual roles asreceptors and ectoenzymes, endowed with complex activities related to signaling and cell homeostasis.The structure-function analysis presented here is intended to give clinical scientists and flow cytomet-rists a background knowledge of these molecules. The link between CD38/CD157 and human diseaseswill be explored here in the context of chronic lymphocytic leukemia, myeloma and ovarian carcinoma,although other disease associations are also known. Thus CD38 and CD157 have evolved from simpleleukocyte activation markers to multifunctional molecules involved in health and disease. Future taskswill be to explore their potential as targets for in vivo therapeutic interventions and as regulators of theimmune response. VC 2013 International Clinical Cytometry Society

Key terms: leukocyte markers; tissue distribution; cell receptor; ectoenzymes; calcium homeostasis

How to cite this article: Quarona V, Zaccarello G, Chillemi A, Brunetti E, Singh VK, Ferrero E, Funaro A, Horen-stein AL, Malavasi F. CD38 and CD157: A Long Journey from Activation Markers to Multifunctional Molecules.Cytometry Part B 2013; 84B: 207–217.

CD38, also known as T10, epitomizes a story sharedby several other molecules, identified within a projectaimed at probing the cell surface of human leukocytesusing murine monoclonal antibodies (mAbs), reagentswhich became available in the late 1970s. Both probesand targets were unknown, prompting scientists todevise original strategies in order to identify the struc-ture and functions of the target molecules. The Leuko-cyte Workshop adopted a strategy that provedsuccessful in the early days of HLA studies: when two ormore mAbs reacted with the same target, they were said

Grant sponsor: Associazione Italiana Ricerca Cancro (AIRC); Grantnumbers: 11602, AIRC 5x1000 (partial); Grant sponsor: PRIN(Ministry of Education, University, and Innovation, Rome, Italy), fromFIRB (Fondo per gli Investimenti della Ricerca di Base, Rome, Italy),from ex-60% Project (University of Torino).

*Correspondence to: Fabio Malavasi, Lab of Immunogenetics, Uni-versity of Torino Medical School, Via Santena 19, 10126, Torino,Italy.

E-mail: [email protected] 17 January 2013; Revision 6 March 2013; Accepted 21

March 2013Published online 10 April 2013 in Wiley Online Library

(wileyonlinelibrary.com).DOI: 10.1002/cyto.b.21092

Review Article

VC 2013 International Clinical Cytometry Society

to form a cluster centered on an unknown molecule.This approach (Cluster Designation, CD) made it possi-ble to identify an amazing number of surface moleculesin a limited number of years. However, it took manymore years to identify the functions exerted in vivo bythe majority of these molecules, an effort still far frombeing concluded.

CD38 was identified during the pioneering work ofE.L. Reinherz (Dana-Farber Cancer Center, Boston, MA)(1), focused on the identification of membrane mole-cules involved in antigen recognition. The fact thatthose studies were performed in a medical institutioninfluenced the first part of CD380s life history. The mole-cule was found in thymocytes and in activated normallymphocytes, and also in selected leukemias and myelo-mas (2). Its initial deployment in clinical diagnosis gaveCD38 the label of ‘‘activation marker,’’ a term still quitepopular in routine flow cytometry.

This review will attempt to summarize and update thefindings obtained from different areas of research, whichled to the redefinition of CD38 as a multifunctional mol-ecule. We now know that CD38 is joined by CD157(also referred to as bone marrow stromal cell antigen 1,BST-1) in being part of a gene family coding for productsthat modulate the social life of cells by means of bidirec-

tional signals (from outside to inside and vice versa).Besides being receptors, the same molecules act also asectoenzymes, endowed with complex activities.

A complete analysis of the structure and functions ofCD38 and CD157 in humans is intended to give clinicalscientists and flow cytometrists access to backgroundknowledge which is usually found only within the realmof basic science (3). The hope is that this set of informa-tion may improve the reading and comprehension ofresults obtained daily in clinics.

THE CD38 GENE FAMILY

CD38 and CD157 genes are located on human chro-mosome 4 (4p15) (4,5) (Fig. 1). Over 98% of the 70 kbCD38 gene is represented by intronic sequences. Thegene encompasses 8 exons: the largest, exon 1, deter-mines the intracytoplasmic and transmembrane regionsand the 33 membrane-proximal amino acids of theextracellular region (6,7). CD38 expression appears tobe under quite complex multilayered transcriptional reg-ulation. The first layer of control lies in the promoterregion characterized by the lack of a TATA box and thepresence of a CpG island (6), a methylation-controlledregion more frequently associated with housekeepingthan tissue-specific genes. A second layer of control is

FIG. 1. Schematic representation of human chromosome 4, with attention to CD38 (and its regulatory elements) and CD157. The right side showsthe schematic features of membrane CD38 and CD157 proteins.

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likely to be upstream of the CpG island, where potentialbinding site for transcription factors (e.g., T cell tran-scription factor-1, nuclear factor for interleukin-6) lie(6), while a third level of gene control lies within thefirst intron of the CD38 gene. The 50-end of the intron 1contains responsive elements for retinoic acid (8) andperoxisome proliferator-activated receptor c (PPARc)(9). Intron 1 is also the location of a single nucleotidepolymorphism (SNP) (10) that binds the transcriptionfactor E2A (11) and whose variants are differentiallyexpressed in pathology (vide infra).

The CD157 gene extends for over 35 kb, very closeto its paralogue CD38 and consists of nine exons (4,12).CD38 and CD157 are highly conserved, as demonstratedby the fact that exons 2-8 are similar in length and main-tain the same phase of intron insertion (13). The50-flanking region of the human CD157 gene shows con-sensus sequences for interferon responsive elements(c-IREs and ISRE-like), for nuclear transcription factors(E2A, AP2, AP3, PEA3, C/EBP, CREB, Sp1), for cytokine-responsive factors (NF-IL-6, NF-jB), and for p53 (4). Thisgene also lacks a TATA box, suggesting multiple tran-scription start sites (4,14). All these elements suggestthat the CD157 gene may be upregulated by eventssuch as DNA damage, inflammation and infection,whereas NF-IL-6 and NF-jB binding sites may explainthe increased expression of the CD157 molecule inpatients affected by rheumatoid arthritis (12).

TISSUE DISTRIBUTION OF CD38 AND CD157

CD38 expression was first observed on thymocytesand T lymphocytes (15). Today, CD38 is considered vir-tually ubiquitous, at least in the immune system, butwith variable expression levels. Table 1 lists the main tis-sues and cells where CD38 and CD157 are detectable.Because CD157 joined the family relatively later, data onits distribution is still limited.

Flow cytometric analyses indicate that human CD157is constitutively expressed by myeloid cells in peripheralblood mononuclear cells (PBMCs). The molecule is alsoexpressed by synovial, vascular endothelial and folliculardendritic cells (16). Moreover, CD157 is also present onother cell types and tissues, such as dermal fibroblasts,human mast cells from lung, uterus, foreskin, and perito-neal mesothelial cells, among others (17–21). CD157was recently reported to be expressed in Paneth cells,where it mediates the effects of calorie restriction andrapamycin on murine intestinal stem cell function (22).

CD38 AS A RECEPTOR

An initial function attributed to CD38 was the regula-tion of activation and proliferation of human T lympho-cytes. Early functional studies of unidentified moleculeswere pursued by monitoring the effects induced by theengagement of different domains of the target by a panelof specific mAbs. The rationale was that effects inducedby ligation with a surrogate mAb would mimick thoseinduced by a ligand still unknown at the time. CD38engagement was followed by the activation of selected

PBMC populations (23). The identification of a first puta-tive ligand was obtained by exploiting the observationthat human T lymphocytes tended to adhere to endothe-lial cells (24). Experiments blocking this adhesion con-cluded that CD31 [also known as PECAM-1, a memberof the immunoglobulin (Ig) superfamily crucial to leuko-cyte adhesion and transmigration (25)] was a non-sub-strate ligand for CD38. It was later demonstrated thatCD38/CD31 interactions trigger the same signaling cas-cade and recapitulate the biological events observedusing agonistic mAbs (26,27). The interplay betweenCD38 and CD31 is crucial for leukocyte migration

Table 1Distribution of CD38 and CD157 in normal human tissues

CD38 CD157

LymphoidBlood T cells (precursors,

activated)Neutrophils

B cells (precursors,activated)

Eosinophils

Myeloid cells (mono-cytes, macrophages,dendritic cells)

Basophils

NK cells MonocytesErythrocytes MacrophagesPlatelets Plasmocytoid

dendritic cellsCord Blood T and B lymphocytes,

monocytesBone Marrow Precursors B cells precursors

Plasma cells Myeloid precursorsStromal cells

Thymus Cortical thymocytesLymph nodes Germinal center B

cellsFollicular dendritic

cellsSpleen Reticular cells

(white pulp)Non-lymphoidLiver Fetal B cell

progenitorsVessels Mast cellsBrain Purkinje cells

Cultured astrocytesCerebellum

Eye CorneaRetinal gangliar cells

Prostate Epithelial cellsGut Intraepithelial

lymphocytesBrush border, epi-

thelial cells ofvilli

Lamina proprialymphocytes

Peyer’s patches

Stromal cells incryptopatches

Isolated lymphoidfollicles

Peritoneum Mesothelial cellsMacrophages/

peritonealexudates

Pancreas b-cells a and b-cellsMuscle Sarcolemma (smooth

and striatedmuscle)

Bone OsteoclastsKidney Glomeruli Collecting tubuli

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through the endothelium (28). The CD38/CD31 cross-talk has been extensively analyzed in a number of differ-ent environments ranging from T to B, NK, and myeloidcells, in normal and pathological conditions (29). CD38-mediated signals are regulated at distinct levels: the firstlevel concerns the ultrastructural organization of themolecule, which exists both in monomeric (2) and di-meric (30) (or multimeric) type I forms (31). A flip-flopmechanism of membrane positioning has been recentlyproposed, with a type III form of CD38 displaying itscatalytic site in the cytoplasm (32). The second level isbased on the dynamic localization of CD38 in lipidmicrodomains within the plasma membrane. Lateralassociations with other proteins, which vary accordingto the cell lineage, determine a third level of control.Lipid raft localization and association with professionalsignaling complexes are pre-requisites for signals medi-ated through CD38 (33).

CD157 AS A RECEPTOR

The receptor and signaling features of CD157 havealso been investigated by using agonistic mAbs to mimicputative ligand(s). By doing so, it has been demonstratedthat CD157 ligation induces tyrosine phosphorylation ofa 130 kDa protein, identified as focal adhesion kinase(FAK), in myeloid cell lines (34,35), and that CD157engagement regulates Ca2þ homeostasis and mediatessuperoxide (O2�) production in the human myelomono-cytic U937 line (36). Accumulating evidence indicatesthat CD157 is a key player in the control of leukocyteadhesion, migration and diapedesis (37,38). In this con-text, CD157 behaves as a receptor by establishing lateralinteractions with other transmembrane molecules, thusovercoming its structural limitation (i.e., of being a GPI-anchored molecule) and acquiring the ability to trans-duce signals (13). In more detail, CD157 interacts withb1 and b2 integrins, and Ab-induced cross-linking ofCD157 promotes relocation of these complexes into de-tergent-resistant membrane domains (21). Moreover,CD157 effectively contributes to the integrin-driven sig-naling network, which is critical during leukocyte trans-migration, leading to optimal phosphorylation oftyrosine kinase receptors and activation of PI3K andMAPK signaling cascades (39).

CD38 AND CD157 ENZYMATIC FUNCTIONS

The enzymatic functions of the two proteins wereinvestigated after the enzyme ADP-ribosyl cyclase(ADPRC, purified from the mollusk Aplysia californica)was observed to display a striking similarity in proteinsequence with human CD38 (40). CD38 is a multifunc-tional enzyme that catalyzes the synthesis of cyclic ADP-ribose (cADPR) from nicotinamide adenine dinucleotide(NADþ ) and also mediates the hydrolysis of cADPR toADPR (41–44). In acidic conditions, CD38 catalyzes thegeneration of nicotinic acid adenine dinucleotide phos-phate (NAADP) from nicotinamide adenine dinucleotidephosphate (NADPþ ) (45). cADPR, ADPR, and NAADPbind different receptors and channels involved in the

regulation of cytoplasmic Ca2þ fluxes, activating signal-ing pathways critical for several biological processes[e.g., lymphocyte proliferation (46,47), cardiac (48) andintestinal longitudinal muscle contraction (49), glucose-induced insulin release in pancreas (50)]. The role ofCD38 and of its products in regulating a wide range ofphysiological functions is indicated by the multipledefects revealed in CD38 knock-out (KO) mice. Theseinclude impairment of neutrophil chemotaxis, defectiveoxytocin (OT) release, and aberrant social behavior (51).

The recombinant soluble extracellular domain ofCD38 mediates ADP-ribosylation of cysteine residues ofseveral proteins, including CD38 itself (52). CD38 is alsomodified by ecto-ADP-ribosyltransferases (ARTs). Argi-nine ADP-ribosylation results in inactivation of both cy-clase and hydrolase activities, whereas cysteine ADP-ribosylation leads only to inhibition of the hydrolase ac-tivity. Arginine ADP-ribosylation causes a decrease in in-tracellular cADPR and a subsequent decrease in Ca2þ

influx, with consequent death of activated T lympho-cytes (53). Moreover, CD38 is the major NAD glycohy-drolase (NADase) in mammalian cells, regulatingintracellular NADþ levels. CD38 thus modulates the ac-tivity of sirtuins, intracellular NADþ -dependent deacety-lases implicated in ageing, cell protection and energymetabolism (54,55).

CD157 also cleaves extracellular NADþ , generatingcADPR and ADPR (13,36). However, the catalytic effi-ciency of CD157 in generating cADPR is even lowerthan that of CD38 (13).

The products derived from NADþ cleavage operatedby CD38 or CD157 can also act as extracellular immuno-modifiers (56). Emerging data indicate that these prod-ucts may operate outside the cell as paracrine factors(57). Moreover, the catalytic reactions generate sub-strates for ARTs and poly-ADP-ribose polymerases(PARPs) involved in cell signaling, DNA repair and apo-ptosis (56).

CD38 is also found in soluble form in normal andpathological fluids (58) and in exosomes, which aremembrane vesicles secreted by B cells and likely a com-ponent of an intercellular communication network (59).

CD38 AND DISEASE

Chronic Lymphocytic Leukemia (CLL). CLL is acommon adult leukemia which results from the accumu-lation of small B (CD19þ /CD5þ /CD23þ ) lymphocytesin blood, bone marrow (BM), lymph nodes (LN), andother lymphoid tissues (60). The latter districts repre-sent permissive niches, where lymphocytes can prolifer-ate in response to microenvironmental signals (61).Elevated expression of CD38 in CLL cells is generallyassociated with advanced disease stage, higher incidenceof lymphadenopathy, high-risk cytogenetics, shorter lym-phocyte doubling time, shorter time to first treatmentand poorer response to therapy. Besides being a prog-nostic marker, CD38 is a component of a molecular

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network which delivers growth and survival signals toCLL cells (62). CD38 acts as a receptor in leukemic cellsand its signals are mediated by ZAP70, another negativeprognostic factor for the disease and a limiting factor forCD38-mediated activation (33,63). CD38 can work inassociation with chemokines and their receptors, mainlyCXCL12/CXCR4, influencing the migratory responsesand contributing to the recirculation of neoplastic cellsfrom blood to lymphoid organs (64) and with specificadhesion molecules belonging to the integrin family(65,66). CD38 engagement by means of the ligand CD31(or surrogate agonistic mAb) triggers the activation of anintracellular signaling pathway and these signals increasechemotaxis as well as proliferation of neoplastic B cells.Together, this evidence contributes to the current viewof CLL as a chronic disease in which the host’s microen-vironment promotes leukemic cell growth and also con-trols the sequential acquisition and accumulation ofgenetic alterations (62).

Multiple Myeloma (MM). MM is a malignancy char-acterized by accumulation of monoclonal plasma cells inBM, a high concentration of monoclonal Igs in serumand urine, and lytic bone lesions. The proliferation ofneoplastic plasma cells in MM interferes with the normalproduction of blood cells, while the monoclonal Igimpairs humoral immunity.

There are several issues suggesting that CD38 playssignificant role(s) in MM. First, CD38 is expressed byplasma cells (normal and tumoral) at top levels of cellsurface density. Secondly, experiments in murine modelsshowed that CD38 is a key regulator of OT levels in bio-logical fluids while OT is also released by human osteo-blasts (67). A receptor for OT (OTR) is detectable onmyeloma cells and derived lines (Malavasi F., unpub-lished results, 2012). CD38 is expressed by osteoblastsand osteoclasts, where it implements signals leading toIL-6 release and inhibition of bone resorption (68,69).

In light of these considerations, plasma cells (andtheir malignant counterpart) and bone niches are goodtesting grounds for assessing the presence of a connec-tion between ectoenzymes and neuropeptides (70). Thesystem is closed, and nucleosides represent additionalsignals to those led by cytokines/chemokines and otherconventional regulators: ATP and NADþ operating inloco may complement the physiological regulatory sys-tems of plasma cells.

Acute Promyelocytic Leukemia (APL). APL is aunique subtype of acute leukemia, which causes anarrest of leukocyte differentiation at the promyelocytestage. Retinoic acid (RA) is included in therapeuticalprotocols for its ability to induce the differentiation ofleukemic cells into mature granulocytes. This therapymay be associated with retinoic acid syndrome (RAS), aclinical manifestation characterized by fever, dyspnea,pulmonary edema and infiltrates (71). Normal granulo-cytes are CD38-, while RA-treated APL cells express highamounts of the molecule (72,73). The aberrant expres-sion of CD38 on leukemic cells enhances their propen-sity to interact with CD31, expressed by lung

endothelial cells, resulting in a local production ofinflammatory cytokines, apoptosis of endothelial cellsand eventually contributing to the development of RAS(71).

CD157 AND DISEASE

Ovarian Cancer. Leukocyte extravasation is a pro-cess which shares similarities with metastatic infiltrationto secondary organs. CD157 is expressed by mesothelialcells, which share biological properties and embryologi-cal origin with ovarian surface epithelial cells (18). Aworking hypothesis was that CD157 might be expressedby epithelial ovarian cancer cells, guiding interactionsbetween tumor cells and mesothelium. If confirmed,CD157 could be involved in the control of ovarian can-cer dissemination. This hypothesis was independentlysupported by a report that BST-1/CD157 was amonggenes that were differentially expressed in primary cul-tures of epithelial ovarian cancer cells when comparedto their normal counterparts (74). The results obtainedhave confirmed the hypothesis: indeed, CD157 isexpressed by >90% of epithelial ovarian cancers and isinvolved in the interactions between epithelial ovariancancer cells, extracellular matrix proteins and mesothe-lial cells. All these steps ultimately control migration oftumor cells and invasion of surrounding tissues. Highexpression of CD157 in human ovarian cancers is associ-ated with clinical aggressiveness, confirming the role ofCD157 as an independent prognostic factor of tumorrelapse shortly after surgery (75). The functional contri-bution of CD157 to the progression of epithelial ovariancancer relies on its ability to switch on a differentiationprogram, which allows neoplastic cells to overcome therules of epithelial tissue architecture, turning it towardsa more mesenchymal state. The outcome of clinical rele-vance in clinics is that these events boost the malignantprogression of the disease (76).

Parkinson’s Disease. The BST-1/CD157 gene hasrecently been associated with Parkinson’s disease.Indeed, BST-1 SNPs rs11931532, rs12645693,rs4698412, and rs4538475 were identified as risk factorsin sporadic late-onset Parkinson’s disease in a Japanesegenome-wide association (GWA) study. rs4538475showed the strongest association (77). The associationbetween BST-1 (rs4698412 SNP) and Parkinson’s diseasewas confirmed in the European population (78), even ifnot present in a Northern Han Chinese population (79).A meta-analysis of GWA studies performed on a North-ern American and European population indicates thatBST-1 did surpass the threshold for genome-wide signifi-cance (80). A conclusion is that ethnicity significantlyinfluences the association between BST-1/CD157 locusand Parkinson’s disease.

ROLE OF THE CD38 FAMILY IN OTHER HUMAN DISEASES

Other human diseases showing correlation with CD38or CD157 are reviewed in (13). The polymorphismsassociated with genetic susceptibility to CLL have also

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been studied in other diseases, including systemic lupuserythematosus (SLE), where the CC genotype increasessusceptibility to and the GC genotype confers protectionfrom discoid rash development (81).

High ratios of CD8þ /CD38þ T lymphocytes predictdisease progression in HIV-infected adults (82). HIVinfection is clinically characterized by increased expres-sion of CD38, apparently not directly caused by theinfection (83). Highly active anti-retroviral therapy(HAART), the standard treatment in both pediatric andadult HIV infection, leads to viral suppression withdecreased CD38 expression, increased CD4þ T lympho-cyte counts, recovery of immune functions againstopportunistic infections and a good clinical outcome inmost patients.

Recent studies have combined traditional diagnostictests (viral load, CD4þ T lymphocyte counts, and immu-nophenotyping) with an evaluation of CD38 activation.A study involving small sample of patients has shownthat (i) CD38 expression levels in CD8þ T lymphocytesand (ii) lymphoproliferative assays in response tomycotic antigens provide additional parameters for mon-itoring patients with discordant immune-virologicalresponses to HAART (84).

CD38 expression has been also matter of technologi-cal debate, in terms of mAb used [see the discrepancyreported (3)] or protocols adopted. To this end, con-certed efforts are being made to standardize CD38 pro-tocols in order to obtain comparable evaluation of themarker molecule in HIV-infected patients. A recent pa-per provided a platform based on flow cytometry withfluorosphere MFI values, color compensation along withbiological controls which assure technologically stableanalysis. This standardized protocols have been adoptedby the laboratories of South African National HealthService for the evaluation of the CD8 þ /CD38þ ratiosfor monitoring HIV patients (85).

CD38 is also reported as target of autoantibodies intype 1 and type 2 diabetes mellitus (86,87), as well as inSLE (Pavon et al., 2013 in press).

Results from CD38 KO mice have highlighted a rolefor CD38 in the release of OT from the neurohypophysis(51). The clinical diagnosis of a deficit in short-termsocial memory in these mice has drawn the attention tohuman conditions sharing this feature, such as autismspectrum disorder (ASD). Indeed, some polymorphismsof the CD38 gene (rs6449197, rs3796863, andrs1800561) are associated with ASD (88,89). Beside this,it has recently been reported that a quantitative trait ofCD38 expression correlates with social functions in ASD(90). The study was performed by analyzing CD38expression in lymphoblastoid cell lines derived fromPBMCs of ASD patients, which has been showed to belower than CD38 expression in similar lines obtainedfrom the parents. A possible explanation is that reducedcell surface expression negatively influences the enzy-matic performances of CD38 in ASD, leading to a mal-function of the CD38/OT axis in this disorder (51,91). Afurther step of the study investigated the role of reti-

noids in upregulating cell surface CD38, potentially sug-gesting a new therapeutic approach (92,93).

EMERGING WORKING HYPOTHESIS

From a phylogenetic perspective, the human CD38

and BST-1 genes share gene structure (6). A similarintron/exon organization is also shared by their respec-tive orthologs in mouse, chicken and frog, and by theADPRCs from Aplysia, Schistosoma mansoni and thepurple sea urchin (Strongylocentrotus purpuratus)(NCBI and Ensembl genome databases). Thus theADPRCs are believed to derive from a common ancestralgene. As the common taxonomic denominator of theADPRC-bearing species is that they all belong to thebilateria (i.e., animals with front/back and left/right sym-metry), this suggests that the origin of the ADPRCs datesback to the last common bilaterian ancestor, about 555million years ago (mya).

Read from an applied perspective, the evidencederived from a phylogenetic analysis of the CD38 familyprompts the hypothesis that early CD38/CD157 precur-sors were components of innate immunity, and thattheir passage to the surface of immune cells evolved inparallel with the transition to adaptive immunity (Fig.2). We hypothesized that CD38 (CD157 having not beenconsidered in this context yet) might be part of a circuitgenerating activation or suppression of immuneresponses according to the environment. CD38 wouldalso be a component of one of the multiple strategiesadopted by tumoral cells to fool the immune system.The enzymes CD39 (ectonucleoside triphosphatediphosphohydrolase 1) and CD73 (ecto-50 nucleotidase)govern a metabolic pathway leading to the generation ofadenosine (ADO) and likely to immunosuppressionwhen ADO is taken up by specific receptors expressedby lymphocytes. This pathway is flanked by differentmechanisms guided by NADþ , which is consumed bythe CD38/CD157 ectoenzymes and—in some systems—by ART2. Endogenous signals released during physiologi-cal or pathological conditions may contribute to alarmthe innate immune system, accompanied by the produc-tion of pro-inflammatory cytokines. In addition, extracel-lular NADþ may influence the immune system byaltering the balance between activation and suppressionled by specific lymphocyte subsets in different districtsand organs.

CD38 has recently been associated with functionsexerted by regulatory T cells (Tregs) in murine models.High CD38 espression in Foxp3þ /CD4þ T cell popula-tions correlates with extremely powerful modulatoryproperties of CD4þ regulatory T lymphocytes (94). Fur-thermore, CD38 is part of the Treg transcriptional signa-ture (95,96). The role of CD38 has been confirmed inCD38 KO mice, where NADþ influences the survival,phenotype and function of Treg cells and providesproof-of-principle that acting on the ART2/P2X7 systemmay be a new strategy for manipulating these cells invivo (97). A recent report states that CD8þ /CD38high

T lymphocytes have strong immunosuppressive

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capabilities in vitro and in vivo. This subset possesses aregulatory potential that could work together with theinnate immune response and control immune homeosta-sis during inflammation (98).

PC-1 (also known as CD203a) is a cell surface enzymewith nucleotidase pyrophosphatase phosphodiesterase(ENPP1) activity (99). We have recently verified the exis-tence of a new pathway led by the CD38/PC-1 network,which provides substrates to CD73 and consequentlyfeeds the production of ADO in different organs (Fig. 3).

Detailed knowledge about this pathway has been hin-dered by the fact that PC-1/CD203a had only been studiedin human cells to answer questions mainly related to dia-betes, and by technical difficulties in analyzing ADO anddifferent substrates in vitro and in biological fluids (Hor-enstein et al., 2013 submitted). However, a link betweenCD38 and CD73 was highlighted some years ago (100).

It is not known to what extent this unconventionalectoenzyme network will be able to contribute to thegeneration of local tolerance in different disease models,

FIG. 3. Distribution of CD38, PC-1/CD203a, CD39, and CD73 in human thymus, lymph node and spleen assessed by immunohistochemistry.

FIG. 2. Phylogeny of the CD38 family.

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such as the BM microenvironment in MM (Fig. 4) andthe mesenchymal stem cell (MSC) niche in recurrentpregnancy losses (Cecati et al., 2013 submitted). Prelimi-nary results demonstrate the existence of the CD38/PC-1/CD73 axis in different cells, where tolerance plays apart in maintaining homeostasis. Studies are currentlybeing conducted in MM and melanoma patient samplesto determine a possible strategy of immune escape oper-ated by CD38 and CD157. In these samples, a chain ofectoenzymes capable of generating ADO independentlyfrom CD39 has already been confirmed (Morandi et al.,in preparation).

There are still several unanswered questions concern-ing CD38 and CD157. One deals with CD38 tissue distri-bution, which ranges from discrete expression duringlymphocyte differentiation to a limited presence during

the physiological resting state of both T and B cells. Themolecule is strongly re-expressed by cells undergoingactivation and in selected leukemias. In contrast to thenotion of CD38 as an activation marker, terminally differ-entiated plasma cells (and derived tumors) express thehighest surface density among human cells. This meansthat ontogeny has still a lot to teach and to unveil.

Furthermore, the fact that CD38 and CD157 areectoenzymes should no longer be considered oddities inleukocyte biology; on the contrary, more than 4% of themolecules expressed on the surface of human cellsshow enzymatic features (101).

Nucleotide-metabolizing ectoenzymes constitute a sub-group of a larger family of ectoenzymes, involved in thecatabolism and scavenging of extracellular nucleotides.This process results in the synthesis of compounds that

FIG. 4. Schematic model of human bone marrow highlighting crosstalk taking place among plasma cells, osteoblasts, osteoclasts, endothelium,and stroma.

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play critical roles in cell homeostasis and metabolism,and not simply nucleotide recycling. Initially it wasthought that nucleotide-metabolizing ectoenzymeswould operate in environments containing only traceamounts of the substrate and that the final productwould be used prevalently inside the cell. This view haslater been revised, and it is now known that substratesand final products are not topologically confined to oneside or other of the plasma membrane.

The author’s laboratory tackled these issues by (i) fo-cusing exclusively on human model, (ii) using the ontog-eny and phylogeny of these molecules as a source ofphysiological clues, and (iii) trying to infer informationfrom disease, the best experiments performed by natureitself.

The aim of this paper was to review old and newfacts enriching the field related to the CD38 family prod-ucts and on the role of ectoenzymes in general. Eventhough interpretations are kept to a minimum, the per-spective of this Laboratory has inevitably pervaded thevision of the gene family.

In conclusion, CD38 and CD157 have shifted frombeing considered simple activation markers of leukocytepopulations to being recognized as molecules exertingmultiple functions in health and disease. A last dauntingtask is now to demonstrate that these surface structuresmay become appropriate targets for in vivo therapeuticinterventions (Chillemi et al., 2013 submitted) and regu-lators of the immune response (Horenstein et al., 2013submitted).

ACKNOWLEDGMENTS

Valeria Quarona and Antonella Chillemi are studentsof the PhD Program ‘‘Biomedical Sciences and Oncol-ogy’’, University of Torino. Andrea Zito provided much-appreciated technical assistance. The FondazioneRicerca Medicina Sperimentale (FIRMS) provided valua-ble assistance and support to this research project.

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