Peripheral Blood Dendritic Cell Subsets from Patients withMonoclonal Gammopathies Show an Abnormal Distribution and Are

Functionally Impaired

MARTA MARTIN-AYUSO,a,b JULIA ALMEIDA,a,b MARTIN PEREZ-ANDRES,a,b REBECA CUELLO,c

JOSEFINA GALENDE,d MARIA ISABEL GONZALEZ-FRAILE,e GUILLERMO MARTIN-NUNEZ,f

FERNANDO ORTEGA,g MARIA JESUS RODRIGUEZ,h JESUS F. SAN MIGUEL,b,i ALBERTO ORFAOa,b

aServicio de Citometrıa & Departamento de Medicina, University of Salamanca, Salamanca, Spain; bInstitutode Biologıa Molecular y Celular del Cancer, Centro de Investigacion del Cancer (CSIC-USAL), Salamanca,Spain; cService of Hematology, Hospital Clınico de Valladolid, Valladolid, Spain; dService of Hematology,

Hospital del Bierzo, Ponferrada, Spain; eService of Hematology, Hospital Virgen Blanca, Leon, Spain;fService of Hematology, Hospital Virgen del Puerto, Plasencia, Spain; gService of Hematology, Hospital Rio

Carrion, Palencia, Spain; hService of Hematology, Hospital Nstra Sra Sonsoles, Avila, Spain; iServicio deHematologıa, Hospital Universitario de Salamanca, Salamanca, Spain

Key Words. Circulating dendritic cell subsets • Monoclonal gammopathies • Multiple myeloma • MGUSPlasma cell leukemia

Disclosure: No potential conflicts of interest were reported by the authors, planners, reviewers, or staff managers of this article.

ABSTRACT

Objectives. The information currently available aboutdendritic cells (DCs) in patients with different types ofmonoclonal gammopathy (MG) is limited and fre-quently controversial. In the present study, we analyzedthe ex vivo distribution as well as the phenotypic andfunctional characteristics of peripheral blood (PB) DCsfrom different types of MG.

Methods. For this purpose, 61 untreated patients in to-tal with MG were analyzed—MG of undetermined sig-nificance (MGUS), 29 cases; multiple myeloma (MM),28 cases; and plasma cell leukemia (PCL), 4 cases—incomparison with a group of 10 healthy controls.

Results. Our results show an absolute overall highernumber of all subsets of PB DCs in PCL, together withlower numbers of myeloid DCs in MM patients. From a

phenotypic point of view, PB DC subsets from all typesof MG expressed significantly higher levels of HLA mol-ecules and altered patterns of expression of the CD2,CD11c, CD16, CD22, CD62L, and CD86 molecules, inassociation with altered patterns of secretion of inflam-matory cytokines.

Conclusion. In summary, we show the existence ofsignificant abnormalities in the distribution, pheno-type, and pattern of secretion of inflammatory cyto-kines by different subsets of PB DCs from patientswith MGs, which could reflect a potentially alteredhoming of DCs, together with a greater in vivo acti-vation and lower responsiveness of PB DCs, which arealready detectable in MGUS patients. The Oncologist2008;13:82–92

Correspondence: Alberto Orfao, M.D., Ph.D., Servicio de Citometrıa, Centro de Investigacion del Cancer, Campus Miguel de Unamuno,37007-Salamanca. Spain. Telephone: 34-923-29-4811; Fax: 34-923-29-4795; e-mail: orfao@usal.es Received July 26, 2007; acceptedfor publication November 10, 2007. ©AlphaMed Press 1083-7159/2008/$30.00/0 doi: 10.1634/theoncologist.2007-0127

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INTRODUCTION

Monoclonal gammopathies (MGs) are a heterogeneousgroup of diseases characterized by the expansion of clonalplasma cells (PCs) [1, 2]. Malignant MGs, for example,multiple myeloma (MM) and plasma cell leukemia (PCL),may develop from a previous MG of undetermined signif-icance (MGUS)—the most common PC disorder— oremerge as a de novo disease without a previous diagnosis ofMGUS [3–6]. In some MM cases, the number of circulatingPCs, rarely represented in normal peripheral blood (PB),may increase in the advanced phases of the disease, leadingto overt PCL [7].

In recent years, considerable progress has been made re-garding the identification of critical events leading to ma-lignant transformation of MGUS into MM [8–11]. Amongother factors, those controlling the interaction of tumorcells with the bone marrow (BM) microenvironment, in-cluding signaling through cellular and soluble componentsof the immune system, appear to be particularly relevant.Different soluble and cellular proteins such as cytokines(e.g., interleukin [IL]-6), chemokines, adhesion molecules,and cell surface receptors are typically involved in this in-teraction [12–14]. In turn, an increasingly higher number ofreports have focused on the study of T cells and their rela-tionship with clonal PCs; the occurrence of idiotype-spe-cific CD4 and CD8 T-cell responses is being repeatedlyreported [15–19] in MM patients. Despite this, the informa-tion currently available about cells from the immune systemother than T lymphocytes in patients with MGs and theirrole in the behavior of the disease is very limited [20,21, 22].

Dendritic cells (DCs) are a key component of the im-mune system, because they play a critical role in primingnaive T cells and inducing tumor-specific protective im-mune responses [23–26]. DCs can process both endoge-nous and exogenous protein-derived antigens and presentthe corresponding antigenic peptides through the HLAclass I and class II pathways, respectively, leading to the ac-tivation of specific T cells; the immunomodulatory capabil-ities of DCs further contribute to polarization of T cells intoeither a type 1 helper T cell (Th1) or Th2 cytokine secretionpattern. In turn, growing evidence supports the notion that,under specific conditions, DCs also promote tolerogenic re-sponses [27].

At present, the knowledge about DCs from MM patientsremains limited, and contradictory results have been re-ported regarding their phenotypic and functional character-istics [21, 22, 28, 29]. Accordingly, while some studiesfound no alterations in MM DCs from these patients [28,29], others have reported lower expression levels of HLA-DR, CD40, and CD80, together with a lower antigen-pre-

senting capacity and failure to upregulate CD80 expression[21, 22]. Some of these discrepancies could be a result, atleast to a certain extent, of either the different subpopula-tions of DCs analyzed after in vitro culture or the distincttechnical approaches employed [21, 22, 28, 29].

In the present study, we comparatively analyzed the exvivo distribution as well as the phenotypic and functionalcharacteristics of circulating PB DCs from patients withMGUS, MM, and PCL in comparison with a group ofhealthy individuals. Our results show that the overall abso-lute number of all subsets of PB DCs is significantly greaterin PCL whereas it is normal in MGUS; in turn, MM patientsshowed lower numbers of myeloid DCs. From a phenotypicpoint of view, PB DC subsets from patients with MGs ex-pressed significantly higher levels of antigen-presentingHLA-II molecules and altered expression of several adhe-sion/costimulatory molecules, in associating with an abnor-mal pattern of secretion of inflammatory cytokines.

MATERIALS AND METHODS

Patients, Controls, and SamplesIn total, 61 patients with untreated MGs (29 MGUS, 28MM, and 4 PCL) and 10 age- and sex-matched healthy sub-jects were included in this study. The diagnostic criteria forMGUS were as follows [1, 2, 30]: (a) the presence of serumM-component �3 g/dl or a small amount of urine Ig lightchain protein excretion and (b) �10% BM PCs, in the ab-sence of clinical symptoms, lytic bone lesions, anemia, hy-percalcemia, and renal function impairment. Patients withMM were diagnosed according to previously described cri-teria [30]. A diagnosis of PCL required the presence of�2 � 109/l circulating PCs in PB [7]. The mean ages of pa-tients with MGUS (24 male and 5 female), MM (18 maleand 10 female), and PCL (1 male and 3 female) were 70 �11 years, 70 � 10 years, and 69 � 6 years, respectively.According to the International Staging System [31], MMpatients were distributed as follows: stage I, 22%; stage II,37%; and stage III, 41%.

From each individual both EDTA- and heparin-antico-agulated PB samples were collected and immediately pro-cessed for the immunophenotypical analysis of the differentsubsets of PB DCs and the evaluation of cytokine produc-tion, respectively. All samples were obtained according tothe recommendations of the ethical committee of the Uni-versity Hospital of Salamanca (Salamanca, Spain), after in-formed consent had been given by each individual.

Enumeration of PB DCsFor the enumeration of PB DCs, a single platform flow cy-tometry method [32] with counting beads, combined with a

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direct immunofluorescence lyse-nonwash technique, wasused. Briefly, 100 �l of a premixed PB sample was stainedfor 15 minutes in the dark at room temperature (RT) withthe following four-color staining—fluorescein isothiocya-nate (FITC)/phycoerythrin (PE)/peridinin chlorophyll pro-tein (PerCP)/allophycocyanin (APC)—Dendritic CellExclusion Kit, which includes the CD3 (clone 33–2A3),CD14 (clone FWKW-1), CD19 (clone A3/B1), and CD56(clone C5.9) reagents, CD16 (clone Leu-11), anti-HLA-DR(clone L243), and CD33 (clone Leu-M9). FITC-conjugatedmonoclonal antibodies (mAbs) (Dendritic Cell ExclusionKit) were purchased from Cytognos (Salamanca, Spain),and all other reagents were obtained from Becton-Dickin-son Biosciences (BDB; San Jose, CA). Afterward, 2 ml ofthe fixative-free Quicklysis™ solution (Cytognos) wasadded and another 10-minute incubation was performed un-der the same conditions as described above. Immediatelyprior to data acquisition, 100 �l of Perfect-Count Micro-spheres™ (Cytognos) was added and samples were homo-geneously mixed in a roller mixer.

Data acquisition was performed using a FACSCaliburflow cytometer (BDB) in two sequential steps. Initially, in-formation was collected on 105 events per test correspond-ing to the whole sample cellularity and the counting beads;in a second step, information was stored exclusively forHLA-DR� events through an electronic “gate.” In this lat-ter step, a minimum of 103 HLA-DR�/lineage� cells wasanalyzed per sample. For data acquisition and analysis, theCellQuest™ and Paint-A-Gate Pro™software programs(BDB) were used, respectively. Enumeration of DCs andtheir subsets was performed for each PB sample accordingto well-established methods, as previously described in de-tail [32]. The following subsets of HLA-DR�/lineage�

DCs were identified: myeloid DCs (mDCs), plasmacytoidDCs (pDCs), and monocyte-associated DCs (CD16� DCs).

Immunophenotypic Analysis of PB DC SubsetsFor the immunophenotypic characterization of the differentDC subsets, whole PB samples (approximately 1 � 106

cells in 100 �l per test) were stained in replicates with sat-urating concentrations of the Dendritic Cell Exclusion Kitanti-HLA-DR PerCP and CD33-APC, as previously de-scribed [33, 34]. Each aliquot replicate was also stainedwith one of the PE-conjugated mAbs directed against themolecules under study, whose specificities and sources areshown in Table 1. Briefly, PB samples were incubated for15 minutes in the dark (at RT), in the presence of 5–20 �l ofthe above-mentioned mAbs, according to the manufactur-er’s specifications. Afterward, 2 ml per tube of FACS lys-ing solution (BDB) was added and the samples wereincubated for another 10 minutes under the above-men-

tioned conditions. Then, cells were centrifuged (5 minutesat 540 � g), washed twice with 4 ml of phosphate-bufferedsaline (PBS, pH � 7.6) and resuspended in 0.5 ml of PBSuntil analyzed in the flow cytometer. Data acquisition wasperformed in a FACSCalibur flow cytometer as describedabove. For each antigen analyzed, the intensity of expres-sion was evaluated as reflected by its mean fluorescence in-tensity (MFI) expressed in arbitrary relative linear unitsscaled from 0 to 10,000. Isotype-matched negative controlswere used to establish cutoff values for positivity.

Analysis of Cytokine Production by PB DCsFor the analysis of cytokine production by PB DCs, 1 ml ofeach heparin-anticoagulated PB sample was diluted 1/1(vol/vol) in RPMI 1640 culture medium (BioWhittaker,Walkersville, MD) supplemented with 2 mM L-glutamineand placed in a tube to which 100 ng/ml of lipopolysaccha-ride (LPS, from Escherichia coli, serotype 055:B5; Sigma,St Louis, MO) and 10 ng/ml of human recombinant inter-feron (IFN)-� (Promega, Madison, WI) were added as DCstimulants. In addition, 10 �g/ml of brefeldin A (BFA;Sigma) was added to block cytokine secretion in cytokine-producing cells. An unstimulated sample aliquot, contain-ing BFA in the absence of both LPS and IFN-�, wasprocessed in parallel, in an identical way. Afterward, PBsamples were incubated for 6 hours at 37°C in a 5% CO2

and 95% humidity sterile environment. Once this incuba-tion period was completed, the sample was aliquoted in dif-ferent tubes (approximately 200 �l per tube) and stainedwith the Dendritic Cell Exclusion Kit anti-HLA-DR-PerCPand anti-CD33-APC. After gently mixing, cells were incu-bated for 15 minutes in the dark (at RT). Following this in-cubation period, cells were fixed, permeabilized, andstained with mAbs directed against different human cyto-kines (Table 1), using the FIX & PERM� reagent kit (In-vitrogen, Carlsbad, CA), according to the manufacturer’srecommendations. Data acquisition and analysis were per-formed as described above. Evaluation of cytokine produc-tion was based on both the percentage of positive cells andtheir MFI. A DC population was considered to secrete agiven cytokine when �3% of the DCs were positive for thatcytokine.

Statistical AnalysesMean values and their standard deviations (SDs) andranges, as well as the 25th and 75th percentiles and 95%confidence intervals, were calculated for all variables understudy using the SPSS 10.0 software program (SPSS Inc.,Chicago, IL). The Mann-Whitney U and Kruskal-Wallisnonparametric tests were used for the evaluation of the sta-tistical significance of the differences observed between

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two or more groups, respectively. p-values �.05 were con-sidered to be statistically significant.

RESULTS

Distribution of PB DCs in Patients with MGsThe overall numerical distributions of PB DCs and theirsubsets in MGUS and MM patients were similar to that ofthe control group, except for a lower number of myeloidDCs detected in MM cases (p � .007) (Fig. 1). In turn, theabsolute number of PB DCs, but not its percentage, was sig-nificantly (p � .005) greater in the PCL patient group(237 � 91 versus 60 � 15 cells/�l); this higher absolutenumber of PB DCs in PCL cases was associated with highernumbers of all three PB DC subsets (p � .005 for CD16�

DCs, p � .02 for mDCs, and p � .005 for pDCs), despite thesignificantly lower percentage of mDCs (p � .04) (Fig. 1).

Immunophenotypic Characteristics of PB DCsand Their Subsets in Patients with MGsThe immunophenotypic features of PB DCs from both MMand MGUS patients in comparison with control subjects aresummarized in Figures 2– 4. As shown there, CD11b,CD58, CD64, CD80, and CD154 (CD40L) were consis-tently negative in all PB DC subsets from both the patientand control groups. In contrast, CD38, CD86, CD123, andHLA-DR were positive in all PB DC subsets from all indi-viduals. Upon comparing the different groups of subjectsstudied, a statistically significant higher expression level ofHLA-DR was observed for the three different subsets of PB

Table 1. mAbs used for the analysis of surface antigen expression and intracellular cytokines

Specificity mAb conjugates Clone Source

Costimulatory molecules CD2-PE T11-RD1 Beckmann Coultera

CD5-PE Leu-1 BDBb

CD80-PE Anti-B7 BDBb

CD86-PE 1T2.2 Pharmingenc

CD154 (CD40L)-PE TRAP1 Dakod

Adhesion molecules CD22-PE Leu-14 BDBb

CD58-PE Anti-LFA-3 BDBb

CD62L-PE Leu-8 BDBb

CD11b-PE Leu-15 BDBb

CD11c-PE Leu-M5 BDBb

FcIgG receptors CD16-PE Leu-11c BDBb

CD64-PE 10.1 Caltage

MHC Anti HLA DR-PerCP L243 BDBb

Cytokine receptors CD123-PE 9F5 Pharmingenc

Chemokine receptors CD181 (CXCR1)-PE 5A12 Pharmingenc

CD184 (CXCR4)-PE 12G5 BDBb

CD195 (CCR5)-PE 2D7/CCR5 BDBb

Cytokines Anti-IL-1�-PE AS10 BDBb

Anti-IL-6-PE MQ2–6A3 Pharmingenc

Anti-IL-8(anti-CXCL8)-PE AS14 BDBb

Anti-IL-12-PE C11.5 Pharmingenc

Anti-TNF-�-PE Mab11 Pharmingenc

Anti-IL-15-PE G243–935 Pharmingenc

Anti-TGF-�-PE A75–3 Pharmingenc

Other CD38-PE Leu-17 BDBb

CD45RO-PE Leu-45RO BDBb

CD87-PE VIM5 Pharmingenc

aBeckmann Coulter, Miami, FL; bBecton-Dickinson Biosciences, San Jose, CA; cPharmingen, San Diego, CA; dDako,Glostrup, Denmark; eCaltag Laboratories, San Francisco, CA.Abbreviations: IL, interleukin; mAb, monoclonal antibody; MHC, major histocompatibility complex; PE, phycoerythrin;PerCP, peridin chlorophyll protein; TNF-�, tumor necrosis factor �; TGF-�: transforming growth factor �.

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DCs in both MGUS (p � .001) and MM (p � .001) patients,as compared with the control group (Fig. 3). In turn, expres-sion of the CD86 costimulatory molecule was also signifi-cantly greater in mDCs from MGUS patients (p � .009) andin both mDCs (p � .03) and pDCs (p � .002) from MMpatients, whereas it was normal among CD16� DCs (Fig.2). Regarding CD38 and CD123, similar patterns of expres-sion were observed in the different groups of individualsstudied, except for a greater reactivity for CD38 detectedamong CD16� DCs from MGUS patients with respect tothe control group (p � .003) (Fig. 3).

The other markers studied (CD5, CD2, CD62L, CD22,CD11c, CD16) showed a variable pattern of expression onthe different subsets of PB DCs. Accordingly, expression ofCD5 (Fig. 2) and CD16 (Fig. 3) was restricted to myeloidDCs and CD16� DCs, respectively; in turn, expression ofCD2, CD62L, and CD22 was almost always absent inCD16� DCs, whereas it was present in the other two sub-sets of DCs from all groups of patients and controls ana-lyzed (Fig. 2). Finally, expression of CD11c was restrictedto CD16� and myeloid DCs (Fig. 3). Upon comparing theexpression levels of these molecules in PB DCs from thedifferent groups of individuals studied, pDCs from MGUS,but not MM, patients showed a significantly lower reactiv-ity for the CD2 (p � .001), CD62L (p � .003), and CD22(p � .005) adhesion molecules. In contrast, mDCs fromMGUS patients showed a normal pattern of expression ofCD2, CD62L, and CD22, with the expression of this latter

marker being significantly greater (p � .008) in mDCs fromMM patients. Reactivity for CD11b and CD11c was signif-icantly greater among CD16� DCs and both CD16� DCsand mDCs, respectively, from both MGUS (p � .01) andMM (p � .01) patients (Fig. 3). No statistically significantdifferences were observed among the different groups of in-dividuals studied regarding the expression of CD5 (Fig. 2).

Except for a few PCL cases, PB DCs from all cases stud-ied were CXCR1� and CXCR4�. In turn, CCR5 was ex-pressed by the majority of mDCs and pDCs in all groups ofindividuals analyzed; a minor proportion (10% � 15%) ofCD16� DCs from control individuals, but not MG patients,was CCR5�. No statistically significant differences weredetected in the expression levels of CXCR1, CXCR4, andCCR5 among the different groups of individuals studied(Fig. 4).

Cytokine Secretion by PB DCs in Patientswith MGsAs shown in Table 2, no statistically significant differenceswere found among the different groups of individuals ana-lyzed regarding the percentage of cases showing spontane-ous ex vivo secretion of cytokines. Despite this, all subsetsof PB DCs analyzed showed spontaneous ex vivo produc-tion of inflammatory cytokines in a variable proportion ofMGUS and MM patients, but not among either the PCL orthe control group. Of note, CD16� DCs were found to spon-taneously produce all inflammatory cytokines explored

Figure 1. Absolute (A) and relative (B) numbers of circulating DCs and their subsets in PB samples from MGUS, MM, and PCLpatients in comparison with healthy controls. #p � .05 versus the control group; *p � .05 versus all other groups.

Abbreviations: DC, dendritic cell; MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma;PB, peripheral blood; PCL, plasma cell leukemia.

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(IL-6, IL-8, IL-12, tumor necrosis factor [TNF]-�, IL-15,transforming growth factor [TGF]-�), with the exception ofIL-1�, in a variable proportion of MGUS patients; in turn,pDCs and mDCs from this patient group only producedIL-6 together with TNF-� or IL-12 in a smaller proportionof cases, respectively. In MM patients, a lower proportionof cases showed spontaneous production of IL-6 and IL-8

by mDCs and of TNF-� by pDCs. As in MGUS patients,CD16� DCs from around one fourth of all MM patients(23%) showed secretion of IL-6 and TNF-�; in turn, secre-tion of IL-8 and IL-15 by CD16� DCs was only detected inone MM patient.

After in vitro stimulation with LPS plus IFN-�, an over-all increased production of inflammatory cytokines overbaseline levels was observed in all groups of individuals forboth PB CD16� DCs and mDCs (Fig. 5), but not pDCs. In-terestingly, secretion of all cytokines except IL-12, by bothCD16� DCs and mDCs was significantly lower in the threepatient groups with respect to controls. Accordingly, a

Figure 2. Expression of the CD5, CD2, CD22, CD62L, andCD86 costimulatory and adhesion molecules on different sub-sets of PB DCs from patients with MGUS and MM in compar-ison with healthy controls. Results are expressed as MFI(arbitrary relative linear units). CD11b, CD58, CD64, CD80,and CD154 expression was consistently negative and is notdisplayed. Dotted lines correspond to the cutoff value for pos-itivity for the different markers studied. *p � .05 versus allother groups; #p � .05 versus healthy controls.

Abbreviations: CTRL, control; DC, dendritic cell; MGUS,monoclonal gammopathy of undetermined significance; MFI,mean fluorescence intensity; MM, multiple myeloma; PB, pe-ripheral blood.

Figure 3. Immunophenotypic expression of CD11c, CD16,CD38, CD123, and HLA-DR on the different subsets of PBDCs from patients with MGUS and MM in comparison withhealthy controls. Results are expressed as MFI (arbitrary rela-tive linear units). Dotted lines correspond to the cutoff valuefor positivity for the different markers studied. #p � .05 versushealthy controls.

Abbreviations: CTRL, control; DC, dendritic cell; MGUS,monoclonal gammopathy of undetermined significance; MFI,mean fluorescence intensity; MM, multiple myeloma; PB, pe-ripheral blood.

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lower proportion of IL-1�, IL-6, and IL-8 –secreting PBCD16� DCs was observed in both MGUS (p � .002) andMM (p � .001) patients together with a lower percentage ofIL-8� PB CD16� DCs (p � .04) in PCL cases. In addition,

CD16� DCs showed lower amounts of IL-1� (MFI, 40 �16 versus 79 � 28; p � .003) and TNF-� (MFI, 1,156 �810 versus 2,862 � 1,281; p � .001) produced per cell inMGUS patients and a lower amount of TNF-� produced per

Figure 4. Expression of the CXCR1, CXCR4, and CCR5 chemokine receptors on different subsets of PB DCs from patients withMGUS, MM, and PCL in comparison with healthy controls. For each chemokine receptor, both the percentage of positive cells(upper panels for each chemokine receptor) and their MFI (arbitrary relative linear units) (bottom panels for each chemokinereceptor) are shown. No statistically significant differences were found among the different groups of individuals studied for anychemokine receptor.

Abbreviations: CTRL, control; DC, dendritic cell; MGUS, monoclonal gammopathy of undetermined significance; MFI, meanfluorescence intensity; MM, multiple myeloma; PB, peripheral blood; PCL, plasma cell leukemia.

Table 2. Spontaneous ex vivo cytokine secretion by different subsets of PB DCs in patients with MGUS (n � 27) and MM(n � 26) in comparison with control individuals (n � 8)

Myeloid DCs Plasmacytoid DCs CD16� DCs

Controls MGUS MM Controls MGUS MM Controls MGUS MM

IL-1� 0% 0% 0% 0% 0% 0% 0% 0% 0%

IL-6 0% 15% 4% 0% 7% 0% 0% 15% 23%

IL-8 0% 0% 4% 0% 0% 0% 0% 4% 4%

IL-12 0% 4% 0% 0% 0% 0% 0% 4% 0%

TNF-� 0% 0% 0% 0% 11% 4% 0% 22% 23%

IL-15 0% 0% 0% 0% 0% 0% 0% 7% 4%

Results are expressed as a percentage of cases. No statistically significant differences were found among the differentgroups of individuals for any of the cytokines analyzed.None of the plasma cell leukemia cases showed spontaneous secretion of inflammatory cytokines by PB DCs.Abbreviations: DC, dendritic cell; IL, interleukin; MGUS, monoclonal gammopathy of undetermined significance; MM,multiple myeloma; PB, peripheral blood; TNF-�, tumor necrosis factor �.

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cell (MFI, 1,718 � 1,232 versus 2,863 � 1,281; p � .003)in MM patients. In turn, mDCs showed a lower percentageof IL-1�, IL6, IL8, and TNF-�–secreting cells in bothMGUS (p � .009) and MM (p � .01) patients and of IL-1�

in PCL cases (p � .04). Finally, a lower percentage of IL-1�� mDCs was also detected among MM versus MGUSpatients (p � .02) (Fig. 5).

DISCUSSION

Limited information has been reported so far about the fea-tures of DCs from MG patients. Here we show the presenceof numerical and phenotypic abnormalities, together withan altered pattern of cytokine secretion by PB DCs from pa-tients with MGs. Interestingly, DC abnormalities were de-tected in all three subtypes of MG analyzed, with slightvariations.

Regarding the numerical distribution of PB DCs in MGpatients, discrepant results have been reported in the litera-ture [21, 22, 28, 29, 35]. Accordingly, while some studieshave shown the presence of lower numbers of circulatingmDCs [35] or both mDCs and pDCs in MM patients [21],others did not find significant differences with regard tohealthy individuals [22, 28, 29]. In the present study, wefound lower absolute and relative numbers of mDCs in MMpatients but not in MGUS patients, together with greaternumbers of all subsets of PB DCs in PCL patients. Thelower numbers of PB mDCs observed in MM patientsmight be explained by either decreased production or in-creased homing into the BM. In line with the latter possi-bility, it has been shown that both mDCs and pDCs can berecruited to lymphoid/inflamed tissues [36–39] and the tu-mor microenvironment [40, 41], and higher percentages ofBM T cells together with lower numbers of circulating Tlymphocytes have been reported in patients with MGs [18].In line with this, our results could also suggest the existenceof greater homing of DCs into tissues containing high num-bers of clonal PCs in PCL (e.g., PB). Certainly, the numberof PCL cases included in this study is rather small, as a re-sult of the rarity of this entity [42], and conclusions fromthis study on PCL must be drawn with appropriate caution.

Data on the phenotypic characteristics of PB DCs frompatients with MGs are almost all restricted to MM patientsand usually controversial. Accordingly, while some studiesdid not show differences in the expression levels of co-stimulatory molecules between MM patients and healthydonors [28, 29], others found lower expression levels ofHLA-DR, CD40, and CD80, together with a lower antigen-presenting capacity [21] and failure to upregulate expres-sion of CD80 [22] by MM PB-derived cultured DCs. In thepresent study, using freshly obtained PB DCs, we foundsignificant phenotypic differences between MG patients

Figure 5. LPS- plus IFN-�–induced production of IL-1�, IL-6,IL-8, IL-12,andTNF-�byPBCD16� DCsandmDCsfrompatientswith MGUS, MM, and PCL in comparison with healthy controls.Results are expressed as percentage of cytokine-producing cells.#p � .05 versus healthy controls; Q p � .05 for comparison betweenMGUS and MM patients.

Abbreviations: CTRL, control; DC, dendritic cell; IFN inter-feron; IL, interleukin; LPS, lipopolysaccharide; mDC, myeloiddendritic cell; MGUS, monoclonal gammopathy of undeterminedsignificance; MM, multiple myeloma; PB, peripheral blood;PCL, plasma cell leukemia; TNF-�, tumor necrosis factor �.

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and controls, including, among others: (a) higher expres-sion levels of HLA-DR by all subsets of PB DCs from bothMM and MGUS patients, (b) higher expression levels ofCD11c on both CD16� DCs and mDCs from both patientgroups, and (c) upregulation of CD86 expression on mDCsalone in MGUS patients and of both mDCs and pDCs inMM patients. In contrast, we found no expression of theCD80 costimulatory molecule on PB DCs from MG pa-tients. Interestingly, it has been reported, in certain inflam-matory processes, that CD86 and CD40, but not CD80, areupregulated under stimulation of DCs, which led those au-thors to suggest that DCs would acquire a state of aberrantresponsiveness to stimuli [43, 44]. Presumably, this behav-ior might contribute to the impaired functionality of DCs inMG. Expression levels of the CD2, CD62L, and CD22 ad-hesion/costimulatory molecules were significantly lower onpDCs from MGUS patients, whereas reactivity for CD2 andCD22 was greater in CD16� DCs and mDCs from MM pa-tients, respectively. Altogether, these observations support thenotion that development of MG could be associated with spe-cific changes in the interaction of PCs and their immunologi-cal microenvironment, which would vary between the benignand malignant disease states. Supporting our findings in MGpatients, it has been reported that circulating DCs from otherB-cell disorders are also numerically lower [45] and display analtered phenotype, showing lower expression levels of somecostimulatory molecules, with a potential functional impact onthe regulation of T-cell responses [46].

Discrepant results have been reported so far regarding thefunctional status of DCs from MG patients. In the presentstudy, we analyzed, for the first time, the ex vivo ability of PBDCs from MGUS, MM, and PCL patients to produce inflam-matory cytokines after a short-term culture. Interestingly, allPB DC subsets showed spontaneous production of inflamma-tory cytokines in MGUS and MM patients, but not in PCL pa-tients or healthy controls; this was particularly evident forCD16� DCs, which is consistent with the involvement of thiscell subset in different inflammatory disorders [47]. Thehigher rate of spontaneous production of inflammatory cyto-kines observed in the present study could reflect in vivo acti-vation of PB DCs from MG patients, resulting from thechronic immune stimulation status that is characteristic of thedisease [13, 18, 27, 48, 49]. In contrast, markedly lower secre-tion of inflammatory cytokines after in vitro stimulation wasobserved in the three patient groups with respect to controls. Inthis sense, chronic in vivo stimulation could lead to immuno-logic exhaustion of DCs upon short-term in vitro stimulation;alternatively, the lower secretion of inflammatory cytokinesby PB DCs after in vitro stimulation could be explained by theexistence of maturation-associated functional abnormalities,such as upregulation of CD86 but not CD80, as discussed

above. In line with this, previous studies have shown that func-tional impairment of DCs correlates with the production of tu-mor-derived soluble factors such as IL-6, which clearly affectDC maturation [21].

Despite the altered secretion of most inflammatory cyto-kines (e.g., IL-1�, IL-6, IL-8, and TNF-�), production ofIL-12 after short-term in vitro culture by both CD16� DCs andmDCs was similar in MG patients and control individuals. Atpresent, it is well known that DC-derived IL-12 plays an es-sential role in polarizing T cells toward a Th1 pattern of cyto-kine secretion, which has been associated with protectiveantitumor responses [50–52]. Therefore, preservation ofIL-12 secretion at normal levels could be associated with anattempt by the immune system to control the disease throughstimulation of tumor-associated antigen-specific T cells. Inline with this, recent results have shown a predominant Th1/cytotoxic phenotype with a squed T-cell receptor v� repertoireamong T lymphocytes from the tumor microenvironment inpatients with MG, which already occurs by the early stages ofthe disease [18]. Further studies are necessary to establish theexact relationship between functional abnormalities ofDCs and T-cell responses in these patients.

In summary, our results show the existence of signifi-cant abnormalities in the distribution, phenotype, and pat-tern of secretion of inflammatory cytokines by differentsubsets of PB DCs from patients with MGs. Overall, ourfindings support the existence of an altered homing of DCsin MM and PCL patients, together with greater in vivo ac-tivation and lower responsiveness of circulating DCs,which are already detectable in MGUS patients.

ACKNOWLEDGMENTS

This work was partially supported by the Spanish Red demieloma multiple y otras gammapatıas (Contract Grant #RTIC G03/136), the Spanish Red de Centros de Cancer (Con-tract Grant # RTIC C03/10) from the Instituto de Salud CarlosIII (Ministerio de Sanidad y Consumo, Madrid, Spain), andRETICC RD061002010035 from the Instituto de Salud Car-los III (Ministerio de Sanidad y Consumo, Madrid, Spain).

AUTHOR CONTRIBUTIONSConception/design: Alberto OrfaoProvision of study materials or patients: Rebeca Cuello, Josefina Galende,

Maria Isabel González-Fraile, Guillermo Martın-Nuñez, Fernando Ortega,Maria Jesús Rodriguez, Jesús F. San Miguel

Collection/assembly of data: Marta Martın-Ayuso, Martın Pérez-Andrés,Maria Isabel González-Fraile

Data analysis and interpretation: Marta Martın-Ayuso, Julia Almeida, Mar-tın Pérez-Andrés, Alberto Orfao

Manuscript writing: Marta Martın-AyusoFinal approval of manuscript: Julia Almeida, Jesús F. San Miguel, Alberto

Orfao

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