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. 2004 Nov 20;54(5):506–512. doi: 10.1007/s00262-004-0633-6

In vitro dendritic cell generation and lymphocyte subsets in myeloma patients: influence of thalidomide and high-dose chemotherapy treatment

Philipp Schütt 1,✉, Ulrike Buttkereit 1, Dieter Brandhorst 1, Monika Lindemann 2, Sven Schmiedl 1, Hans Grosse-Wilde 2, Siegfried Seeber 1, Mohammad Resa Nowrousian 1, Bertram Opalka 1, Thomas Moritz 1
PMCID: PMC11032805  PMID: 15750834

Abstract

While vaccination with antigen-pulsed dendritic cells (DCs) represents a promising therapeutic strategy in multiple myeloma (MM), clinical benefit, so far, has been limited to individual patients. To identify potential problems with this approach, we have analyzed the influence of treatment parameters, in particular high-dose chemotherapy (HD-CTX) and thalidomide, on in vitro DC generation and peripheral blood lymphocyte subsets in MM patients. From a total of 25 MM patients, including 14 patients on thalidomide treatment and 11 after HD-CTX, in vitro DC generation from peripheral blood monocytes under serum-free condition was investigated. In addition, peripheral blood lymphocyte subsets were assessed in 17 patients including 10 patients on thalidomide treatment and 9 patients after HD-CTX. Efficient in vitro generation of DCs (median 7.1×106/100 ml peripheral blood; range 0.1–42.5×106/100 ml peripheral blood) expressing DC-typical surface markers was observed in 23 MM patients (92%), although reduced expression of CD1a, CD40, CD83, and HLA-DR was observed in patients treated with thalidomide. With respect to lymphocyte subsets, MM patients showed significantly (p<0.05) reduced B and CD4+ lymphocytes in the peripheral blood. This effect was most prominent within 6 months of HD-CTX and in patients receiving thalidomide (usually in combination with CTX). CD8+ lymphocytes were significantly increased in MM patients. Thus, despite the well-known deficiencies in their immune system, adequate numbers of DCs can be generated in most myeloma patients. In patients treated with thalidomide, however, it remains to be seen whether the reduced expression of co-stimulatory molecules has functional relevance.

Keywords: Dendritic cells, Multiple myeloma, Thalidomide, High-dose chemotherapy

Introduction

Multiple myeloma (MM) is a malignant lymphoproliferative disease characterized by the accumulation of plasma cells in bone marrow, osteolytic bone lesions, production of a monoclonal immunoglobulin, renal insufficiency, and progressive bone marrow failure. The treatment of choice for symptomatic disease is chemotherapy, and in patients younger than 65 years high-dose chemotherapy (HD-CTX) with peripheral blood stem cell support has been shown to prolong disease-free survival and overall survival [1]. However, in most, if not all, patients even intensified chemotherapy is not able to offer cure or long-term disease-free survival, so complementary therapeutic strategies are needed. The curative potential of allogeneic stem-cell transplantation in selected patients suggests that, in principle, myeloma cells are sensitive to immunological interventions [2]. Several antigens on myeloma cells have been identified as potential targets for immunotherapeutic strategies [5, 10], and especially the idiotype protein, i.e. the immunoglobulin produced by the myeloma cells, has been investigated in this respect [15, 23, 34]. Thus, it has been demonstrated that mice could be protected against the outgrowth of transplanted myeloma cells by vaccination with the idiotype protein [23]. Idiotype-responsive T lymphocytes have been described in early-stage MM [3, 12, 47], and more recently, successful generation of idiotype-specific cytotoxic T lymphocytes with the capacity to lyse autologous idiotype-pulsed dendritic cells (DCs) and autologous myeloma cells has been reported [44].

Effective immunotherapeutic strategies in myeloma patients will most likely require the induction of a robust cytotoxic T-lymphocyte response against antigens presented by the malignant clone. However, MM is frequently associated with generalized immunological deficits [43], including functional deficiencies of endogenous antigen-presenting cells [6, 32]. Therefore, vaccination with ex vivo-generated DCs pulsed with tumor antigens such as the idiotype protein appears as an attractive therapeutic approach and, several phase I/II clinical studies have been performed along this line. Most of these studies, however, have shown limited success, and the induction of idiotype-responsive T lymphocytes has been restricted to a minority of patients. This failure has been attributed to the generalized immunodeficiency of myeloma patients or to the relatively weak antigenicity of the idiotype protein [19, 43, 45]. Definite answers, however, are difficult to come by, as clinical studies performed so far substantially differed in their design. This applied to the vaccination schedule, the methods used to generate protein-pulsed DCs, and to the disease and treatment status of the patients recruited for the studies. Thus, a detailed analysis of the functional status of the immune system of myeloma patients at various stages of disease and treatment seems warranted to improve the results of further immunotherapeutic interventions. We addressed this question in a total of 25 myeloma patients, focusing in particular on the effects of thalidomide application and/or previous conventional or HD-CTX. Using a clinically applicable protocol for in vitro DC generation, we measured the yield and phenotype of DCs generated from the peripheral blood of myeloma patients. In addition, lymphocyte subsets in the peripheral blood were assessed.

Materials and methods

Patients and samples

In vitro generation of DCs was investigated in 25 patients. Of these 25 patients, 6 were previously untreated, 11 were after HD-CTX, 11 were after conventional chemotherapy, and 14 were on current treatment with thalidomide. Quantitative analysis of lymphocyte subsets in the peripheral blood was performed in a total of 17 patients including 3 untreated patients, 9 patients with previous HD-CTX, 7 patients after conventional chemotherapy and 10 patients receiving current treatment with thalidomide. From some patients, samples were collected at different stages of disease or therapy; thus, the sum of patients allocated to individual stage/treatment subgroups may exceed the total number of patients. In 3 patients more than one sample was available for one specific subgroup. In these cases, the median value was used for data analysis. All samples were taken at least 3 weeks after the last application of chemotherapy. Healthy volunteers (5 for DC generation, 21 for lymphocyte subsets) served as controls.

Generation of dendritic cells

An amount of 20–100 ml of peripheral blood was obtained from MM patients after informed consent, and peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Lymphoprep (Axis Shield, Oslo, Norway). Monocytes were isolated by culturing PBMCs (2×106 /ml) in X-Vivo 15 medium (BioWhittaker, Walkerwill, MD, USA) at 37°C, 5% CO2 for 2 h in 6-well plates (Falcon, Becton Dickinson, Heidelberg, Germany) and then nonadherent cells were removed by gentle washing with phosphate-buffered saline (PBS). For DC generation, the adherent cells were used and cultured in X-Vivo 15 medium supplemented with 1,000 U/ml IL-4 (Cell Genix, Freiburg, Germany) and 1,000 U/ml GM-CSF (Leukomax, Novartis, Basel, Switzerland) for 7 days. On day 7, the culture medium was exchanged and maturation of DCs was induced by culture in X-Vivo 15 medium supplemented with 1,000 U/ml IL-4, 1,000 U/ml GM-CSF, 10 ng/ml TNF-α (Cell Genix), and 10 ng/ml IL-1ß (Cell Genix) for another 7 days. In some experiments, DCs were loaded with antigens such as the autologous idiotype protein and in these experiments 40 μg/ml of antigen was added at days 1, 4 and 7 of culture.

Phenotypic characterization of dendritic cells

Phenotypic characteristics of monocytes as well as immature (day 7) and mature (day 14) DCs were assessed by flow cytometric analysis of cell populations gated for typical forward and side-scatter characteristics (Coulter EPICS XL, Beckman Coulter, Krefeld, Germany) using fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or PE/cyanin 5.1 (Cy5)-labeled monoclonal antibodies (mAbs) specific for CD1a, CD14, CD40, CD83, CD86, and HLA-DR (Becton Dickinson).

Lymphocyte subset analysis in the peripheral blood

Lymphocyte subsets in the peripheral blood were assessed by flow cytometry using four-color immunofluorescence analysis on a Coulter EPICS XL. In brief, 4.5 ml of peripheral blood were collected in EDTA-treated tubes. Samples of 100 μl were incubated with combinations of FITC-, PE-, energy-coupled dye (ECD; phycoerythrin-Texas Red conjugate)- or PE/Cy5-labeled mAbs specific for CD45/CD4/CD8/CD3, CD45/CD56/CD19/CD3, CD45RA/CD4/CD45RO/CD8, TCRγ/δ/TCRα/β/CD45 or CD23/CD19/CD3/HLA-DR (Beckman Coulter). After 15 min, red cells were lysed and the samples were fixed using the Multi-Q-Prep device and the Immunoprep reagent kit (both Beckman Coulter). At least 1×104 cells were analyzed for each marker combination.

Statistical analysis

All statistical analyses were performed using the SPSS 11.0 for Windows software (SPSS Inc., Chicago, IL, USA). To estimate the significance of differences, the Mann–Whitney U-test or the Wilcoxon test were used throughout the study as indicated. To test for the relationship between variables, Spearman’s rank correlation coefficients were calculated. Linear multivariate regression analysis was performed to define the influence of individual parameters on DC yield using “previous treatment with HD-CTX”, “current treatment with thalidomide”, and “previous treatment with chemotherapy” as dichotomous parameters while “paraprotein levels” and “peripheral blood monocyte counts” were introduced as continuous parameters. P values <0.05 were considered significant.

Results

Generation of dendritic cells

Using a 7-day culture of monocytes with IL-4 and GM-CSF followed by a 7-day culture in the presence of IL-4, GM-CSF, TNF-α, and IL-1ß, 17–99% (median 88%) of cells displayed DC-typical morphology and high expression of CD40, CD83, CD86, and HLA-DR, and downregulation of CD14. While the total yield of mature DCs obtained from 100 ml of peripheral blood varied considerably for individual myeloma patients, median yield did not differ significantly from healthy controls (Table 1). No significant effect of conventional (within 6 weeks) or HD-CTX was observed, and DC yield was independent of disease stage. However, in untreated myeloma patients and in patients without current thalidomide treatment, DC yields were significantly (p< 0.05) reduced as compared to healthy controls. Most likely, the latter observation reflects a technical problem during the monocyte isolation process, as these subgroups were characterized by high paraprotein levels in the serum, which can interfere with the isolation of mononuclear cells. Peripheral blood monocyte counts were normal in these subgroups of patients, and, when we reanalyzed the recovery of mononuclear cells after density centrifugation in an exemplary patient with a high paraprotein level (IgG 55 g/l), loss of approximately 80% of mononuclear cells was observed. This could be prevented by washing the blood cells twice with PBS before density centrifugation.

Table 1.

In vitro DC generation from myeloma patients

  Number of patients DC Yield (106/100 ml) median (range) Samples with DC yield ≥2×106/100 ml Monocytes in PB (106/100 ml) median (range) DC/monocytes ratio median (range)
Healthy controls 5 9.5 (7.5–12.8) 5/5 50 (40–60) 0.20 (0.13–0.26)
Myeloma patients 25 7.1 (0.1–42.5) 23/25 60 (20–230) 0.13 (0.00–0.39)*
+ Thalidomide 14 8.2 (2.8–42.5) 14/14 60 (20–230) 0.16 (0.04–0.39)
− Thalidomide 14 5.5 (0.1–10.6)** 12/14 65 (25–131) 0.09 (0.00–0.21)**
HD-CTX < 6 months 6 14.4 (5.2–34.7) 6/6 70 (35–230) 0.11 (0.08–0.30)
HD-CTX >12 months 5 8.1 (2.8–12.0) 5/5 50 (30–70) 0.20 (0.09–0.22)
Conv. CTX < 6 weeks 8 8.6 (0.1–42.5) 7/8 90 (20–220) 0.10 (0.00–0.39)
Conv. CTX > 6 weeks 3 5.0 (1.9–8.1) 2/3 60 (50–100) 0.05 (0.03–0.16)
Untreated patients 6 4.4 (2.3–7.5)* 6/6 65 (25–131) 0.08 (0.02–0.18)*
Stage 2 4 5.8 (2.3–42.5) 4/4 85 (25–110) 0.09 (0.05–0.39)
Stage 3 21 7.5 (0.1–34.7) 19/21 60 (20–230) 0.13 (0.00–0.30)*
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+ Thalidomide current treatment with thalidomide of at least 100 mg daily, - Thalidomide no treatment with thalidomide within the last two months, HD< 6 months and HD> 12 months treatment with high-dose chemotherapy (HD-CTX) within the last 6 months or longer than 12 months prior to DC generation, Conv. CTX< 6 weeks and Conv. CTX> 6 weeks treatment with conventional dose chemotherapy within the last 6 weeks or longer than 6 weeks prior to DC generation (patients after HD-CTX excluded), Stages 2 and 3 relate to the classification of Durie and Salmon, PB peripheral blood

*Significant differences (p< 0.05) to healthy controls; **Significant differences (p< 0.05) to myeloma patients treated with thalidomide

As expected, DC yield was strongly correlated with peripheral blood monocyte counts (r=0.387; p=0.026). DC yield was negatively correlated with plasma protein (r=−0.404; p=0.033) and myeloma paraprotein levels (r=−0.424; p=0.025). In multivariate analysis, however, only the peripheral blood monocyte count was significantly (p=0.048) correlated with DC yield, while other parameters such as paraprotein level, previous treatment with HD-CTX (yes/no), current treatment with thalidomide (yes/no), or previous treatment with chemotherapy (yes/no) failed to reach statistical significance.

During the maturation process, the expression of CD1a, CD14, CD40, CD83, CD86, and HLA-DR was monitored weekly, and the typical pattern of differentiation and maturation from monocytes to mature DCs was observed with upregulation of CD1a, CD40, CD83, CD86, and HLA-DR, and downregulation of CD14 (Table 2). In all groups investigated, more than 89% of mature DCs exhibited high expression of CD40, CD83, and CD86, and no significant differences in the percentage of positive cells were observed between different study groups. Interestingly, expression of CD1a, CD40, CD83, CD86, and HLA-DR on mature DCs was not significantly different between myeloma patients and healthy controls. Monocytes from myeloma patients had significantly increased expression of CD1a and HLA-DR, and immature DCs from myeloma patients had significantly higher expression levels of CD1a and CD83. Interestingly, thalidomide treatment had substantial effects on DC phenotype. Surface expression of CD1a, CD40, CD83, CD86, and HLA-DR was reduced in myeloma patients treated with thalidomide when compared to patients without thalidomide treatment or to healthy controls, and these differences achieved statistical significance for CD1a, CD40, CD83, and HLA-DR (Fig. 1).

Table 2.

Surface marker expression of monocytes, immature, and mature DCs

  n CD1a CD14 CD40 CD83 CD86 HLA-DR
Controls
Mo 5 0.0 (0.0–4.5) 9.1 (4.7–10.4) 1.5 (1.3–9.8) 1.9 (1.2–2.8) 1.2 (1.1–5.7) 2.4 (1.9–2.5)
DC (d7) 5 5.2 (4.3–6.2) 1.6 (1.3–4.3)* 6.3 (5.3–7.6) 1.5 (1.4–1.6) 12.9 (5.8–19)* 65 (46–90)*
DC (d14) 5 13 (5.4–78)* 1.6 (1.5–1.9)* 27 (26–28)* ** 9.1 (8.3–9.7)* ** 103 (85–123)* ** 228 (136–435)* **
MM
Mo 9 4.9 (0.0–7.8)*** 6.7 (5.1–17) 1.6 (1.2–13) 1.9 (1.3–18) 1.4 (1.3–1.8) 3.9 (2.7–5.6)***
DC (d7) 25 11 (3.4–57)* *** 1.6 (0.8–7.2)* 9.1 (3.3–34) 2.2 (1.3–8.5)*** 7.4 (0.7–30)* 32 (7.9–215)*
DC (d14) 25 9.0 (2.6–78) 1.5 (0–12)* 26 (10–105)* ** 9.3 (4.8–66)** 87 (20–234)* ** 149 (61–895)* **
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Median values and ranges of mean fluorescence intensity (MFI) of monocytes (Mo), immature (d7), and mature (d14) dendritic cells (DCs) are given

*Denotes significant differences (p<0.05) to naïve monocytes; **Denotes significant differences (p<0.05) to immature (d7) DCs; ***Denotes significant differences (p<0.05) between healthy volunteers (controls) and myeloma patients (MM)

Fig. 1.

Fig. 1

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Surface phenotype of in vitro-generated DCs. Median values (bold lines) and interquartile ranges (boxes) of mean fluorescence intensity (MFI) for surface expression of CD1a, CD14, CD40, CD83, CD86, and HLA-DR of mature DCs (day 14) are depicted. Whiskers shown above and below the boxes represent the largest and smallest observed scores that are less than 1.5 box length from the end of the box, i.e. in practice, the lowest and highest values expected. Data are given for mature DCs from myeloma patients with (+Th) and without (−Th) thalidomide treatment in comparison with DCs from healthy volunteers (Co). * Denotes significant differences (p<0.05) to healthy volunteers, # denotes significant differences (p< 0.05) to myeloma patients without current treatment with thalidomide.

Analysis of the lymphoid system

To screen for deficiencies within the lymphoid system, total lymphocytes and lymphocyte subsets were assessed in the peripheral blood of myeloma patients and were compared to healthy volunteers. Median values for total lymphocytes (1.4/nl, range 0.4–2.3/nl vs. 1.7/nl, range 1.3–3.6/nl), CD19+ B cells (0.05/nl, range 0.00–0.44/nl vs. 0.2/nl, range 0.05–0.37/nl), and CD4+ T-cells (0.37/nl, range 0.05–0.89/nl vs. 0.79/nl, range 0.41–1.4/nl) were significantly (p<0.05) reduced in myeloma patients when compared to healthy controls. This reduction was most prominent for B lymphocytes within 6 weeks of conventional (0.002/nl, range 0.00–0.05/nl) or 6 months of HD-CTX (0.07/nl, range 0.02–0.17/nl), and for CD4+ lymphocytes within 6 months of HD-CTX (0.21/nl, range 0.05–0.69/nl). With respect to thalidomide treatment, patients on thalidomide had significantly (p<0.05) reduced counts of CD19+ B cells (0.01/nl, range 0.00–0.17/nl vs. 0.17/nl, range 0.05–0.44/nl) and CD4+ T-cells (0.31/nl, range 0.05–0.97/nl vs. 0.49/nl, range 0.20–0.89/nl) in comparison to patients without current thalidomide treatment. In contrast, CD8+ lymphocytes were significantly (p<0.05) increased in all myeloma patients (0.68/nl, range 0.03–1.7/nl) and in myeloma patients within 6 months after HD-CTX (1.08/nl, range 0.22–1.7/nl) when compared to healthy controls (0.45/nl, range 0.11–1.2/nl).

Discussion

Using a clinically applicable protocol, we describe here the efficient generation of mature DCs from peripheral blood monocytes in the majority of myeloma patients, as other groups have before [7, 29, 31]. In our protocol, GM-CSF and IL-4 were present during the first week of culture, while maturation was induced by adding IL-1ß and TNF-α for the second week. Despite potentially positive effects of prostaglandin E2 (PGE2), such as stimulation of DC migration and IFN-γ production [16], IL-6 and PGE2 were omitted from our maturation cocktail as these cytokines may increase the production of IL-10 and decrease the production of IL-12 by DCs [11, 17, 18, 42], and thus reduce the desired TH1 and favor a TH2 response of CD4+ lymphocytes [20, 22, 26, 28]. On the whole, DC yield for myeloma patients was similar to healthy controls and adequate numbers of DCs could be generated from most myeloma patients irrespective of disease or treatment status. Only on more detailed analysis some subgroups with reduced DC yield emerged, such as untreated myeloma patients and patients without current thalidomide therapy. These reduced DC yields could be attributed to technical problems in the process of monocyte purification due to high paraprotein levels.

In our hands, DCs generated from myeloma patients in vitro displayed a mature phenotype characterized by high expression of HLA-DR and co-stimulatory molecules CD40, CD83, and CD86, similar to DCs generated from healthy controls. While these data are in accordance with work published by other groups, in previous studies, however, expression data have often been confined to a few patients or to the frequency of marker-positive DCs, rather than actual expression levels of surface markers [29, 30]. The high expression of HLA-DR, CD40, CD83, and CD86 on DCs observed in our study is in contrast to the surface phenotype of DCs obtained directly from the peripheral blood of myeloma patients. For these cells, low expression of co-stimulatory molecules and HLA-DR as well as impaired functional activity have been reported [32, 46]. This observation has been attributed to increased IL-6 serum concentrations [6, 32], but increased beta-2 microglobulin (ß2M) levels may be another explanation, as Xie et al. [46] have demonstrated a negative impact of ß2M on DC maturation.

A functionally impaired lymphoid system has repeatedly been described in MM patients with reduced numbers of lymphocytes, in particular CD4+ cells, and decreased levels of polyclonal immunoglobulins in the peripheral blood [4, 19, 43]. This has been attributed to progressive disease with high tumor burden, but also has been interpreted as a side effect of previous conventional or HD-CTX [19, 24, 25, 41]. These findings were affirmed in our study. We observed significantly decreased numbers of CD19+ lymphocytes following high-dose or conventional chemotherapy, and CD4+ lymphocytes were significantly decreased within 6 months of HD-CTX. CD8+ lymphocyte counts, however, were increased in myeloma patients, and this effect was most prominent in patients after HD-CTX. Along this line, substantial skewing of the CD4+/CD8+ ratio during lymphoid resconstitution post-transplantion has been described before in myeloma patients and also for other malignancies and may indicate a preferential toxicity of chemotherapy for B and CD4+ cells in comparison with CD8+ lymphocytes [19, 21, 33, 41].

In our study, patients treated with thalidomide had significantly reduced peripheral blood B cells and CD4+ lymphocytes when compared to healthy volunteers or myeloma patients without thalidomide treatment. These data tie in with other work documenting multiple immunomodulatory properties of thalidomide therapy, which include activation of CD8+ T lymphocytes [13, 14], stimulation of IL-2 production by mononuclear cells [36, 38, 39], or enhancement of natural killer cell activity [8]. For CD4+ cells, co-stimulatory activity in the context of anti-CD3 stimulation and also suppression of CD4+ cells has been described [13, 37, 40]. In addition, in our study DCs from patients treated with thalidomide (mostly in combination with conventional chemotherapy) showed markedly reduced expression of co-stimulatory molecules and HLA-DR in comparison with healthy controls and MM patients receiving no thalidomide. While the functional relevance of these findings presently remains unknown, our preliminary studies indicate adequate T-lymphocyte stimulation by these cells (data not shown). So far, effects of thalidomide on DCs or DC-related cells have been reported mainly from in vitro experiments clearly using supratherapeutic doses [9, 27, 35]. These effects include suppression of TNF-α production by stimulated monocytes and Langerhans cells, inhibition of antigen presentation by Langerhans cells to responsive TH1 clones, or upregulation of allostimulatory and T-helper cell type 1 (Th1) responses. Thus, to the best of our knowledge the study presented here is the first report on the yield and phenotype of DCs generated in a larger cohort of actually thalidomide treated patients.

Acknowledgements

The authors thank C. Wartchow and I. Demirer for their help in preparing the manuscript and D. Dilloo for valuable comments and discussion.

Footnotes

The study was supported by “Internes Forschungsförderungsprogramm Essen” (IFORES) and “Förderverein Essener Tumorklinik e. V.”

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