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The Oncologist, Vol. 13, No. 1, 82-92, January 2008; doi:10.1634/theoncologist.2007-0127
© 2008 AlphaMed Press

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Myelomas

Peripheral Blood Dendritic Cell Subsets from Patients with Monoclonal Gammopathies Show an Abnormal Distribution and Are Functionally Impaired

Marta Martín-Ayusoa,b, Julia Almeidaa,b, Martín Pérez-Andrésa,b, Rebeca Cuelloc, Josefina Galended, Maria Isabel González-Frailee, Guillermo Martín-Nuñezf, Fernando Ortegag, Maria Jesús Rodríguezh, Jesús F. San Miguelb,i, Alberto Orfaoa,b

aServicio de Citometría & Departamento de Medicina, University of Salamanca, Salamanca, Spain; bInstituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer (CSIC-USAL), Salamanca, Spain; cService of Hematology, Hospital Clínico de Valladolid, Valladolid, Spain; dService of Hematology, Hospital del Bierzo, Ponferrada, Spain; eService of Hematology, Hospital Virgen Blanca, León, Spain; fService of Hematology, Hospital Virgen del Puerto, Plasencia, Spain; gService of Hematology, Hospital Rio Carrión, Palencia, Spain; hService of Hematology, Hospital Nstra Sra Sonsoles, Ávila, Spain; iServicio de Hematología, Hospital Universitario de Salamanca, Salamanca, Spain

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

Correspondence: Alberto Orfao, M.D., Ph.D., Servicio de Citometría, Centro de Investigación del Cáncer, Campus Miguel de Unamuno, 37007-Salamanca. Spain. Telephone: 34-923-29-4811; Fax: 34-923-29-4795; e-mail: orfao{at}usal.es

Received July 26, 2007; accepted for publication November 10, 2007.

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


   ABSTRACT
Top
 Footnotes
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgments
 References
 
Objectives. The information currently available about dendritic cells (DCs) in patients with different types of monoclonal gammopathy (MG) is limited and frequently controversial. In the present study, we analyzed the ex vivo distribution as well as the phenotypic and functional characteristics of peripheral blood (PB) DCs from different types of MG.

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

Results. Our results show an absolute overall higher number of all subsets of PB DCs in PCL, together with lower numbers of myeloid DCs in MM patients. From a phenotypic point of view, PB DC subsets from all types of MG expressed significantly higher levels of HLA molecules and altered patterns of expression of the CD2, CD11c, CD16, CD22, CD62L, and CD86 molecules, in association with altered patterns of secretion of inflammatory cytokines.

Conclusion. In summary, we show the existence of significant abnormalities in the distribution, phenotype, and pattern of secretion of inflammatory cytokines by different subsets of PB DCs from patients with MGs, which could reflect a potentially altered homing of DCs, together with a greater in vivo activation and lower responsiveness of PB DCs, which are already detectable in MGUS patients.


   INTRODUCTION
Top
 Footnotes
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgments
 References
 
Monoclonal gammopathies (MGs) are a heterogeneous group of diseases characterized by the expansion of clonal plasma cells (PCs) [1, 2]. Malignant MGs, for example, multiple myeloma (MM) and plasma cell leukemia (PCL), may develop from a previous MG of undetermined significance (MGUS)—the most common PC disorder—or emerge as a de novo disease without a previous diagnosis of MGUS [3–6]. In some MM cases, the number of circulating PCs, rarely represented in normal peripheral blood (PB), may increase in the advanced phases of the disease, leading to overt PCL [7].

In recent years, considerable progress has been made regarding the identification of critical events leading to malignant transformation of MGUS into MM [8–11]. Among other factors, those controlling the interaction of tumor cells with the bone marrow (BM) microenvironment, including signaling through cellular and soluble components of 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 interaction [12–14]. In turn, an increasingly higher number of reports have focused on the study of T cells and their relationship with clonal PCs; the occurrence of idiotype-specific CD4 and CD8 T-cell responses is being repeatedly reported [15–19] in MM patients. Despite this, the information currently available about cells from the immune system other than T lymphocytes in patients with MGs and their role in the behavior of the disease is very limited [20, 21, 22].

Dendritic cells (DCs) are a key component of the immune system, because they play a critical role in priming naive T cells and inducing tumor-specific protective immune responses [23–26]. DCs can process both endogenous and exogenous protein-derived antigens and present the corresponding antigenic peptides through the HLA class I and class II pathways, respectively, leading to the activation of specific T cells; the immunomodulatory capabilities of DCs further contribute to polarization of T cells into either a type 1 helper T cell (Th1) or Th2 cytokine secretion pattern. In turn, growing evidence supports the notion that, under specific conditions, DCs also promote tolerogenic responses [27].

At present, the knowledge about DCs from MM patients remains limited, and contradictory results have been reported regarding their phenotypic and functional characteristics [21, 22, 28, 29]. Accordingly, while some studies found 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-presenting capacity and failure to upregulate CD80 expression [21, 22]. Some of these discrepancies could be a result, at least to a certain extent, of either the different subpopulations of DCs analyzed after in vitro culture or the distinct technical approaches employed [21, 22, 28, 29].

In the present study, we comparatively analyzed the ex vivo distribution as well as the phenotypic and functional characteristics of circulating PB DCs from patients with MGUS, MM, and PCL in comparison with a group of healthy individuals. Our results show that the overall absolute number of all subsets of PB DCs is significantly greater in PCL whereas it is normal in MGUS; in turn, MM patients showed lower numbers of myeloid DCs. From a phenotypic point of view, PB DC subsets from patients with MGs expressed significantly higher levels of antigen-presenting HLA-II molecules and altered expression of several adhesion/costimulatory molecules, in associating with an abnormal pattern of secretion of inflammatory cytokines.


   MATERIALS AND METHODS
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgments
 References
 
Patients, Controls, and Samples
In total, 61 patients with untreated MGs (29 MGUS, 28 MM, and 4 PCL) and 10 age- and sex-matched healthy subjects were included in this study. The diagnostic criteria for MGUS were as follows [1, 2, 30]: (a) the presence of serum M-component <3 g/dl or a small amount of urine Ig light chain protein excretion and (b) ≤10% BM PCs, in the absence of clinical symptoms, lytic bone lesions, anemia, hypercalcemia, and renal function impairment. Patients with MM were diagnosed according to previously described criteria [30]. A diagnosis of PCL required the presence of >2 x 109/l circulating PCs in PB [7]. The mean ages of patients with MGUS (24 male and 5 female), MM (18 male and 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], MM patients were distributed as follows: stage I, 22%; stage II, 37%; and stage III, 41%.

From each individual both EDTA- and heparin-anticoagulated PB samples were collected and immediately processed for the immunophenotypical analysis of the different subsets of PB DCs and the evaluation of cytokine production, respectively. All samples were obtained according to the recommendations of the ethical committee of the University Hospital of Salamanca (Salamanca, Spain), after informed consent had been given by each individual.

Enumeration of PB DCs
For the enumeration of PB DCs, a single platform flow cytometry method [32] with counting beads, combined with a direct immunofluorescence lyse-nonwash technique, was used. Briefly, 100 µl of a premixed PB sample was stained for 15 minutes in the dark at room temperature (RT) with the following four-color staining—fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/peridinin chlorophyll protein (PerCP)/allophycocyanin (APC)—Dendritic Cell Exclusion 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-conjugated monoclonal antibodies (mAbs) (Dendritic Cell Exclusion Kit) were purchased from Cytognos (Salamanca, Spain), and all other reagents were obtained from Becton-Dickinson Biosciences (BDB; San Jose, CA). Afterward, 2 ml of the fixative-free QuicklysisTM solution (Cytognos) was added and another 10-minute incubation was performed under the same conditions as described above. Immediately prior to data acquisition, 100 µl of Perfect-Count MicrospheresTM (Cytognos) was added and samples were homogeneously mixed in a roller mixer.

Data acquisition was performed using a FACSCalibur flow cytometer (BDB) in two sequential steps. Initially, information was collected on 105 events per test corresponding to the whole sample cellularity and the counting beads; in a second step, information was stored exclusively for HLA-DR+ events through an electronic "gate." In this latter step, a minimum of 103 HLA-DR+/lineage– cells was analyzed per sample. For data acquisition and analysis, the CellQuestTM and Paint-A-Gate ProTMsoftware programs (BDB) were used, respectively. Enumeration of DCs and their subsets was performed for each PB sample according to well-established methods, as previously described in detail [32]. The following subsets of HLA-DR+/lineage– DCs were identified: myeloid DCs (mDCs), plasmacytoid DCs (pDCs), and monocyte-associated DCs (CD16+ DCs).

Immunophenotypic Analysis of PB DC Subsets
For the immunophenotypic characterization of the different DC subsets, whole PB samples (approximately 1 x 106 cells in 100 µl per test) were stained in replicates with saturating concentrations of the Dendritic Cell Exclusion Kit anti-HLA-DR PerCP and CD33-APC, as previously described [33, 34]. Each aliquot replicate was also stained with one of the PE-conjugated mAbs directed against the molecules under study, whose specificities and sources are shown in Table 1. Briefly, PB samples were incubated for 15 minutes in the dark (at RT), in the presence of 5–20 µl of the above-mentioned mAbs, according to the manufacturer's specifications. Afterward, 2 ml per tube of FACS lysing solution (BDB) was added and the samples were incubated for another 10 minutes under the above-mentioned conditions. Then, cells were centrifuged (5 minutes at 540 x g), washed twice with 4 ml of phosphate-buffered saline (PBS, pH = 7.6) and resuspended in 0.5 ml of PBS until analyzed in the flow cytometer. Data acquisition was performed in a FACSCalibur flow cytometer as described above. For each antigen analyzed, the intensity of expression was evaluated as reflected by its mean fluorescence intensity (MFI) expressed in arbitrary relative linear units scaled from 0 to 10,000. Isotype-matched negative controls were used to establish cutoff values for positivity.


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

 
Analysis of Cytokine Production by PB DCs
For the analysis of cytokine production by PB DCs, 1 ml of each heparin-anticoagulated PB sample was diluted 1/1 (vol/vol) in RPMI 1640 culture medium (BioWhittaker, Walkersville, MD) supplemented with 2 mM L-glutamine and placed in a tube to which 100 ng/ml of lipopolysaccharide (LPS, from Escherichia coli, serotype 055:B5; Sigma, St Louis, MO) and 10 ng/ml of human recombinant interferon (IFN)-{gamma} (Promega, Madison, WI) were added as DC stimulants. In addition, 10 µg/ml of brefeldin A (BFA; Sigma) was added to block cytokine secretion in cytokine-producing cells. An unstimulated sample aliquot, containing BFA in the absence of both LPS and IFN-{gamma}, was processed in parallel, in an identical way. Afterward, PB samples were incubated for 6 hours at 37°C in a 5% CO2 and 95% humidity sterile environment. Once this incubation period was completed, the sample was aliquoted in different tubes (approximately 200 µl per tube) and stained with the Dendritic Cell Exclusion Kit anti-HLA-DR-PerCP and anti-CD33-APC. After gently mixing, cells were incubated for 15 minutes in the dark (at RT). Following this incubation period, cells were fixed, permeabilized, and stained with mAbs directed against different human cytokines (Table 1), using the FIX & PERM® reagent kit (Invitrogen, Carlsbad, CA), according to the manufacturer's recommendations. Data acquisition and analysis were performed as described above. Evaluation of cytokine production was based on both the percentage of positive cells and their MFI. A DC population was considered to secrete a given cytokine when ≥3% of the DCs were positive for that cytokine.

Statistical Analyses
Mean values and their standard deviations (SDs) and ranges, as well as the 25th and 75th percentiles and 95% confidence intervals, were calculated for all variables under study using the SPSS 10.0 software program (SPSS Inc., Chicago, IL). The Mann-Whitney U and Kruskal-Wallis nonparametric tests were used for the evaluation of the statistical significance of the differences observed between two or more groups, respectively. p-values ≤.05 were considered to be statistically significant.


   RESULTS
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgments
 References
 
Distribution of PB DCs in Patients with MGs
The overall numerical distributions of PB DCs and their subsets in MGUS and MM patients were similar to that of the control group, except for a lower number of myeloid DCs detected in MM cases (p ≤ .007) (Fig. 1). In turn, the absolute number of PB DCs, but not its percentage, was significantly (p = .005) greater in the PCL patient group (237 ± 91 versus 60 ± 15 cells/µl); this higher absolute number of PB DCs in PCL cases was associated with higher numbers of all three PB DC subsets (p = .005 for CD16+ DCs, p = .02 for mDCs, and p = .005 for pDCs), despite the significantly lower percentage of mDCs (p = .04) (Fig. 1).


Figure 1
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  		Figure 1. Absolute (A) and relative (B) numbers of circulating DCs and their subsets in PB samples from MGUS, MM, and PCL patients 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.

 
Immunophenotypic Characteristics of PB DCs and Their Subsets in Patients with MGs
The immunophenotypic features of PB DCs from both MM and MGUS patients in comparison with control subjects are summarized in Figures 2–4. As shown there, CD11b, CD58, CD64, CD80, and CD154 (CD40L) were consistently negative in all PB DC subsets from both the patient and control groups. In contrast, CD38, CD86, CD123, and HLA-DR were positive in all PB DC subsets from all individuals. Upon comparing the different groups of subjects studied, a statistically significant higher expression level of HLA-DR was observed for the three different subsets of PB DCs in both MGUS (p < .001) and MM (p < .001) patients, as compared with the control group (Fig. 3). In turn, expression of the CD86 costimulatory molecule was also significantly greater in mDCs from MGUS patients (p = .009) and in both mDCs (p = .03) and pDCs (p = .002) from MM patients, whereas it was normal among CD16+ DCs (Fig. 2). Regarding CD38 and CD123, similar patterns of expression were observed in the different groups of individuals studied, except for a greater reactivity for CD38 detected among CD16+ DCs from MGUS patients with respect to the control group (p = .003) (Fig. 3).


Figure 2
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  		Figure 2. Expression of the CD5, CD2, CD22, CD62L, and CD86 costimulatory and adhesion molecules on different subsets of PB DCs from patients with MGUS and MM in comparison with healthy controls. Results are expressed as MFI (arbitrary relative linear units). CD11b, CD58, CD64, CD80, and CD154 expression was consistently negative and is not displayed. Dotted lines correspond to the cutoff value for positivity for the different markers studied. *p ≤ .05 versus all other 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, peripheral blood.

 


Figure 3
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  		Figure 3. Immunophenotypic expression of CD11c, CD16, CD38, CD123, and HLA-DR on the different subsets of PB DCs from patients with MGUS and MM in comparison with healthy controls. Results are expressed as MFI (arbitrary relative linear units). Dotted lines correspond to the cutoff value for positivity for the different markers studied. #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, peripheral blood.

 


Figure 4
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  		Figure 4. Expression of the CXCR1, CXCR4, and CCR5 chemokine receptors on different subsets of PB DCs from patients with MGUS, 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 chemokine receptor) are shown. No statistically significant differences were found among the different groups of individuals studied for any chemokine receptor.

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

 
The other markers studied (CD5, CD2, CD62L, CD22, CD11c, CD16) showed a variable pattern of expression on the different subsets of PB DCs. Accordingly, expression of CD5 (Fig. 2) and CD16 (Fig. 3) was restricted to myeloid DCs and CD16+ DCs, respectively; in turn, expression of CD2, CD62L, and CD22 was almost always absent in CD16+ DCs, whereas it was present in the other two subsets of DCs from all groups of patients and controls analyzed (Fig. 2). Finally, expression of CD11c was restricted to CD16+ and myeloid DCs (Fig. 3). Upon comparing the expression levels of these molecules in PB DCs from the different groups of individuals studied, pDCs from MGUS, but not MM, patients showed a significantly lower reactivity for the CD2 (p < .001), CD62L (p = .003), and CD22 (p = .005) adhesion molecules. In contrast, mDCs from MGUS patients showed a normal pattern of expression of CD2, CD62L, and CD22, with the expression of this latter marker being significantly greater (p = .008) in mDCs from MM patients. Reactivity for CD11b and CD11c was significantly greater among CD16+ DCs and both CD16+ DCs and mDCs, respectively, from both MGUS (p ≤ .01) and MM (p ≤ .01) patients (Fig. 3). No statistically significant differences were observed among the different groups of individuals studied regarding the expression of CD5 (Fig. 2).

Except for a few PCL cases, PB DCs from all cases studied were CXCR1– and CXCR4+. In turn, CCR5 was expressed by the majority of mDCs and pDCs in all groups of individuals analyzed; a minor proportion (10% ± 15%) of CD16+ DCs from control individuals, but not MG patients, was CCR5+. No statistically significant differences were detected in the expression levels of CXCR1, CXCR4, and CCR5 among the different groups of individuals studied (Fig. 4).

Cytokine Secretion by PB DCs in Patients with MGs
As shown in Table 2, no statistically significant differences were found among the different groups of individuals analyzed regarding the percentage of cases showing spontaneous ex vivo secretion of cytokines. Despite this, all subsets of PB DCs analyzed showed spontaneous ex vivo production of inflammatory cytokines in a variable proportion of MGUS and MM patients, but not among either the PCL or the control group. Of note, CD16+ DCs were found to spontaneously produce all inflammatory cytokines explored (IL-6, IL-8, IL-12, tumor necrosis factor [TNF]-{alpha}, IL-15, transforming growth factor [TGF]-β), with the exception of IL-1β, in a variable proportion of MGUS patients; in turn, pDCs and mDCs from this patient group only produced IL-6 together with TNF-{alpha} or IL-12 in a smaller proportion of cases, respectively. In MM patients, a lower proportion of cases showed spontaneous production of IL-6 and IL-8 by mDCs and of TNF-{alpha} by pDCs. As in MGUS patients, CD16+ DCs from around one fourth of all MM patients (23%) showed secretion of IL-6 and TNF-{alpha}; in turn, secretion of IL-8 and IL-15 by CD16+ DCs was only detected in one MM patient.


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  		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)

 
After in vitro stimulation with LPS plus IFN-{gamma}, an overall increased production of inflammatory cytokines over baseline levels was observed in all groups of individuals for both PB CD16+ DCs and mDCs (Fig. 5), but not pDCs. Interestingly, secretion of all cytokines except IL-12, by both CD16+ DCs and mDCs was significantly lower in the three patient groups with respect to controls. Accordingly, a lower proportion of IL-1β, IL-6, and IL-8–secreting PB CD16+ DCs was observed in both MGUS (p ≤ .002) and MM (p ≤ .001) patients together with a lower percentage of IL-8+ PB CD16+ DCs (p = .04) in PCL cases. In addition, CD16+ DCs showed lower amounts of IL-1β (MFI, 40 ± 16 versus 79 ± 28; p = .003) and TNF-{alpha} (MFI, 1,156 ± 810 versus 2,862 ± 1,281; p = .001) produced per cell in MGUS patients and a lower amount of TNF-{alpha} produced per cell (MFI, 1,718 ± 1,232 versus 2,863 ± 1,281; p = .003) in MM patients. In turn, mDCs showed a lower percentage of IL-1β, IL6, IL8, and TNF-{alpha}–secreting cells in both MGUS (p ≤ .009) and MM (p ≤ .01) patients and of IL-1β in PCL cases (p = .04). Finally, a lower percentage of IL-1β+ mDCs was also detected among MM versus MGUS patients (p = .02) (Fig. 5).


Figure 5
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  		Figure 5. LPS- plus IFN-{gamma}–induced production of IL-1β, IL-6, IL-8, IL-12, and TNF-{alpha} by PB CD16+ DCs and mDCs from patients with MGUS, MM, and PCL in comparison with healthy controls. Results are expressed as percentage of cytokine-producing cells. #p ≤ .05 versus healthy controls; {oplus} p ≤ .05 for comparison between MGUS and MM patients.

Abbreviations: CTRL, control; DC, dendritic cell; IFN interferon; IL, interleukin; LPS, lipopolysaccharide; mDC, myeloid dendritic cell; MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma; PB, peripheral blood; PCL, plasma cell leukemia; TNF-{alpha}, tumor necrosis factor {alpha}.

 

   DISCUSSION
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgments
 References
 
Limited information has been reported so far about the features of DCs from MG patients. Here we show the presence of numerical and phenotypic abnormalities, together with an altered pattern of cytokine secretion by PB DCs from patients with MGs. Interestingly, DC abnormalities were detected in all three subtypes of MG analyzed, with slight variations.

Regarding the numerical distribution of PB DCs in MG patients, discrepant results have been reported in the literature [21, 22, 28, 29, 35]. Accordingly, while some studies have shown the presence of lower numbers of circulating mDCs [35] or both mDCs and pDCs in MM patients [21], others did not find significant differences with regard to healthy individuals [22, 28, 29]. In the present study, we found lower absolute and relative numbers of mDCs in MM patients but not in MGUS patients, together with greater numbers of all subsets of PB DCs in PCL patients. The lower numbers of PB mDCs observed in MM patients might be explained by either decreased production or increased homing into the BM. In line with the latter possibility, it has been shown that both mDCs and pDCs can be recruited to lymphoid/inflamed tissues [36–39] and the tumor microenvironment [40, 41], and higher percentages of BM T cells together with lower numbers of circulating T lymphocytes have been reported in patients with MGs [18]. In line with this, our results could also suggest the existence of greater homing of DCs into tissues containing high numbers of clonal PCs in PCL (e.g., PB). Certainly, the number of PCL cases included in this study is rather small, as a result of the rarity of this entity [42], and conclusions from this study on PCL must be drawn with appropriate caution.

Data on the phenotypic characteristics of PB DCs from patients with MGs are almost all restricted to MM patients and usually controversial. Accordingly, while some studies did not show differences in the expression levels of costimulatory molecules between MM patients and healthy donors [28, 29], others found lower expression levels of HLA-DR, CD40, and CD80, together with a lower antigen-presenting capacity [21] and failure to upregulate expression of CD80 [22] by MM PB-derived cultured DCs. In the present study, using freshly obtained PB DCs, we found significant phenotypic differences between MG patients and controls, including, among others: (a) higher expression levels of HLA-DR by all subsets of PB DCs from both MM and MGUS patients, (b) higher expression levels of CD11c on both CD16+ DCs and mDCs from both patient groups, and (c) upregulation of CD86 expression on mDCs alone in MGUS patients and of both mDCs and pDCs in MM patients. In contrast, we found no expression of the CD80 costimulatory molecule on PB DCs from MG patients. Interestingly, it has been reported, in certain inflammatory processes, that CD86 and CD40, but not CD80, are upregulated under stimulation of DCs, which led those authors to suggest that DCs would acquire a state of aberrant responsiveness to stimuli [43, 44]. Presumably, this behavior might contribute to the impaired functionality of DCs in MG. Expression levels of the CD2, CD62L, and CD22 adhesion/costimulatory molecules were significantly lower on pDCs from MGUS patients, whereas reactivity for CD2 and CD22 was greater in CD16+ DCs and mDCs from MM patients, respectively. Altogether, these observations support the notion that development of MG could be associated with specific changes in the interaction of PCs and their immunological microenvironment, which would vary between the benign and malignant disease states. Supporting our findings in MG patients, it has been reported that circulating DCs from other B-cell disorders are also numerically lower [45] and display an altered phenotype, showing lower expression levels of some costimulatory molecules, with a potential functional impact on the regulation of T-cell responses [46].

Discrepant results have been reported so far regarding the functional status of DCs from MG patients. In the present study, we analyzed, for the first time, the ex vivo ability of PB DCs from MGUS, MM, and PCL patients to produce inflammatory cytokines after a short-term culture. Interestingly, all PB DC subsets showed spontaneous production of inflammatory cytokines in MGUS and MM patients, but not in PCL patients or healthy controls; this was particularly evident for CD16+ DCs, which is consistent with the involvement of this cell subset in different inflammatory disorders [47]. The higher rate of spontaneous production of inflammatory cytokines observed in the present study could reflect in vivo activation of PB DCs from MG patients, resulting from the chronic immune stimulation status that is characteristic of the disease [13, 18, 27, 48, 49]. In contrast, markedly lower secretion of inflammatory cytokines after in vitro stimulation was observed in the three patient groups with respect to controls. In this sense, chronic in vivo stimulation could lead to immunologic exhaustion of DCs upon short-term in vitro stimulation; alternatively, the lower secretion of inflammatory cytokines by PB DCs after in vitro stimulation could be explained by the existence 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 functional impairment of DCs correlates with the production of tumor-derived soluble factors such as IL-6, which clearly affect DC maturation [21].

Despite the altered secretion of most inflammatory cytokines (e.g., IL-1β, IL-6, IL-8, and TNF-{alpha}), production of IL-12 after short-term in vitro culture by both CD16+ DCs and mDCs was similar in MG patients and control individuals. At present, it is well known that DC-derived IL-12 plays an essential role in polarizing T cells toward a Th1 pattern of cytokine secretion, which has been associated with protective antitumor responses [50–52]. Therefore, preservation of IL-12 secretion at normal levels could be associated with an attempt by the immune system to control the disease through stimulation of tumor-associated antigen-specific T cells. In line with this, recent results have shown a predominant Th1/cytotoxic phenotype with a squed T-cell receptor vβ repertoire among T lymphocytes from the tumor microenvironment in patients with MG, which already occurs by the early stages of the disease [18]. Further studies are necessary to establish the exact relationship between functional abnormalities of DCs and T-cell responses in these patients.

In summary, our results show the existence of significant abnormalities in the distribution, phenotype, and pattern of secretion of inflammatory cytokines by different subsets of PB DCs from patients with MGs. Overall, our findings support the existence of an altered homing of DCs in MM and PCL patients, together with greater in vivo activation and lower responsiveness of circulating DCs, which are already detectable in MGUS patients.


   ACKNOWLEDGMENTS
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgments
References
 
This work was partially supported by the Spanish Red de mieloma multiple y otras gammapatías (Contract Grant # RTIC G03/136), the Spanish Red de Centros de Cáncer (Contract Grant # RTIC C03/10) from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Madrid, Spain), and RETICC RD061002010035 from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Madrid, Spain).


   FOOTNOTES
 
Conception/design: Alberto Orfao

Provision of study materials or patients: Rebeca Cuello, Josefina Galende, Maria Isabel González-Fraile, Guillermo Martín-Nuñez, Fernando Ortega, Maria Jesús Rodriguez, Jesús F. San Miguel

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

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

Manuscript writing: Marta Martín-Ayuso

Final approval of manuscript: Julia Almeida, Jesús F. San Miguel, Alberto Orfao


   REFERENCES
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgments
 References
 

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