Generation of antigen-loaded dendritic cells in a serum-free medium using different cytokine combinations

Vaccine 21 (2003) 877–882

Generation of antigen-loaded dendritic cells in a serum-free medium using different cytokine combinations Tomas Büchler a,b,∗ , Roman Hajek a,b,c , Lida Bourkova a , Lucie Kovarova a,b , Romana Musilova a , Alena Bulikova a , Michal Doubek b , Adam Svobodnik c , Iveta Mareschova a,c , Pavlina Vanova a , Eva Tuzova a , Petra Vidlakova a , Jiri Vorlicek b , Miroslav Penka a a

b

Laboratory of Experimental Hematology and Cell Immunotherapy, Department of Clinical Hematology, Masaryk University Hospital, Brno, Czech Republic Department of Internal Medicine—Hematooncology, Masaryk University Hospital, Jihlavska 20, 639 00 Brno, Czech Republic c University Oncology Center, Masaryk University, Brno, Czech Republic Accepted 19 August 2002

Abstract Dendritic cells (DCs) are antigen-presenting cells that play a critical role in the induction of cytotoxic T-lymphocytes. An optimal method for the generation of DC for clinical use remains to be established. The aim of our study was to find an optimal cytokine combination for DC generation from peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) in serum-free conditions. Serial immunophenotyping enabled us to observe changes in DC content during the culture as well as the development of maturation and activation markers. As a source for DC culture, we used PBSC from patients with multiple myeloma after stem cell mobilization using cyclophosphamide and G-CSF, or PBMC from healthy donors without mobilization. The cells were cultured in a serum-free medium with different cytokine combinations including GM-CSF, TNF-␣, Flt-3, CD40L, IFN-␥, IL-1␣, IL-6, PGE1, and IL-4. The cell cultures were evaluated by immunophenotyping. For PBMC, interleukin-12 assay was performed. For PBSC, the yield of DC as determined by CD83+ cell count ranged from 0.6 × 105 to 30.1 × 104 (mean: 9.4 × 104 ) of DC generated per 1 × 106 of initially plated nucleated cells from apheresis. This yield corresponded to (0.3–19.1) × 105 (mean: 4.3 × 105 ) per 1 × 106 of CD34+ cells in the apheresis products. For PBMC, the yield was (0.4–24.8) × 104 (mean: 2.4 × 104 ) of DC generated per 1 × 106 of initially plated mononuclear cells from venous blood. The cultured cells expressed the mature immunophenotype. No significant differences in cell yield or immunophenotype were detected when comparing different cytokine combinations. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Dendritic cells; Immunotherapy; Cytokines; In vitro cell culture

1. Introduction Dendritic cells (DCs) are extremely effective in capturing and presentation of antigens to T cells and play a key role in the induction of cytotoxic T lymphocytes (CTLs) [1]. Immunotherapy using antigen-loaded DCs represents an attractive anticancer strategy, particularly in the setting of minimal residual disease. Large quantities of DCs presenting a tumor-associated antigen are needed to induce

∗ Corresponding author. Tel.: +420-54719-2633; fax: +420-54719-3603. E-mail addresses: [email protected], [email protected] (T. Büchler).

clinically significant immune response directed against malignant cells [2]. There is no simple, reliable method for generation of sufficient quantities of DCs for vaccination purposes. Based on previous studies, a number of protocols for DC culture and ex-vivo expansion have been described, using various sources of DC precursors such as bone marrow [3,4], cord blood [5], peripheral blood mononuclear cells (PBMCs) [6,7], peripheral blood stem cells (PBSCs) [8,9], and CD14+ monocytes [10]. In human bone marrow, a minor subset of hematopoietic progenitor cells has been identified. It is phenotypically distinct (CD34+, Lin-, CD45RA+, CD38+, Thy-1-, c-kit-) and gives rise to T, B, NK cells and DCs, but does not produce myeloid and erythroid cells [11]. Although DCs are

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 5 3 5 - 2

878

T. Büchler et al. / Vaccine 21 (2003) 877–882

extremely diverse depending on their stage of differentiation and activation, the following properties are attributable to DCs: (a) the ability to stimulate primary T cell responses; (b) spontaneous and rapid clustering with T cells; (c) marked cell motility and the ability to migrate/home to T cell areas within lymphoid tissue [12]; (d) specialized phagocytic activity and antigen capture by receptor-mediated uptake and macropinocytosis [13]; (e) a phenotype that is distinct from other cell types. Several immunophenotypic markers are expressed on cultured DCs, however, none has been universally accepted for DC enumeration. The markers used to identify DCs include the presumably specific molecules including CMRF-44, CMRF-56, CD83; co-stimulatory molecules such as CD40, CD80, CD86; adhesion molecules such as CD11a, CD11c, CD44, CD50, CD54, CD58, CD102; common leukocyte antigens such as CD45RA, CD45RO; class I and II major histocompatibility complex (MHC) antigens; and activation markers such as CD25 [2]. DCs differ functionally and phenotypically depending on their stage of activation [12]. Immature DCs are extremely efficient at capturing and processing antigens [14]. Maturation of DCs is induced by inflammatory mediators and results in reduced capacity to endocytose antigens and increased levels of adhesion and co-stimulatory molecules for optimal presentation of peptide–MHC complexes to T cells [15]. Roles of several cytokines in the process of DC generation have been established. Their impact on DC culture is reviewed by Syme and Gluck [16]. The aim of our study was to find an optimal cytokine combination for DC generation from PBSC and PBMC in serum-free conditions suitable for clinical anti-tumor vaccination studies. Serial immunophenotyping enabled us to observe changes in DC content during the culture as well as the development of maturation and activation markers.

2. Materials and methods 2.1. Cell culture As a source of PBSCs for DC generation, leftover apheresis products from patients with multiple myeloma (Table 1) after mobilization with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) were cryopreserved in a solution containing 10% dimethylsulphoxide (DMSO). Prior to use, PBSC preparations were thawed, washed twice in Hanks’ balanced salt solution (Sigma–Aldrich, St. Louis, MO, USA) with 4% human albumin (USOL, Prague, Czech Republic), 10 U/ml heparin (Lé´civa, Prague, Czech Republic) and 20 U/ml DNAse (Boehringer Mannheim, Germany). The cell suspension was centrifuged at 800 g for 10 min and the supernatant was discarded. PBMCs from venous blood of three healthy volunteers, 30, 36, and 40 years old, without previous mobilization were prepared by standard gradient centrifugation using Histopaque (Sigma–Aldrich) and immediately plated for DC culture. The cells were cultured in X-VIVO 10 medium (Bio Whittaker, Walkersville, MD, USA) with 80 U/ml DNAse (Boehringer Mannheim), and 1 mM glutamin (Sigma– Aldrich), in six 3-ml wells at 37 ◦ C in the atmosphere of 5% CO2 and 4.5% O2 . The initial cell concentration was 3.3 × 06 cells per ml of X-VIVO 10. Taking advantage of adherence of DC precursors after 2-h culture, non-adherent cells were discarded with the supernatant as described [7]. The cells were fed every 3 days, adding glutamin and cytokines. Various combinations of cytokines were tested (Tables 2 and 3), including granulocyte and macrophage colony-stimulating factor (GM-CSF; Schering Plough), tumor necrosis factor-␣ (TNF-␣; Bender Medsystems Diagnostics, Vienna, Austria), Flt-3 (Genzyme, Cambridge, MA, USA), CD40L (Bender Medsystems Diagnostics, Vienna,

Table 1 Characteristics of the patients whose apheresis products were used for dendritic cell generation Patient no.

Sex

Age at apheresis

Date of diagnosis (month/year)

CD34+ cells in the apheresis product (%)

1 2 3 4

F M F F

59 53 49 53

9/96 5/96 4/96 5/96

3.3a , 3.8a 5.0 1.6 1.3

a

Two different apheresis products from patient no. 1 were used.

Table 2 Combinations of cytokines that were used for a two-step dendritic cell culture from peripheral blood stem cells (PBSCs) after mobilization with cyclophosphamide and G-CSF Cytokine combination for PBSCs Days 1–5 Days 6–9

GM-CSF, IL-4 GM-CSF, TNF-␣

GM-CSF, IL-4, Flt-3 GM-CSF, TNF-␣

GM-CSF, IL-4, Flt-3 GM-CSF, TNF-␣, CD40L

GM-CSF, TNF-␣ GM-CSF, TNF-␣

Doses of cytokines: GM-CSF, 800 U/ml; TNF-␣, 10 ng/ml; IL-4, 1000 IU/ml; Flt-3, 25 ng/ml; CD40L, 50 ng/ml.

GM-CSF, TNF-␣, Flt-3 GM-CSF, TNF-␣

T. Büchler et al. / Vaccine 21 (2003) 877–882

879

Table 3 Combinations of cytokines that were used for a one-step dendritic cell culture from venous blood mononuclear cells (PBMCs) Cytokine combinations for PBMCs Days 1–9

TNF-␣, IL-1 ␣, IL-6, PGE1

GM-CSF, IL-4

GM-CSF, PGE1

GM-CSF, PGE1, IFN-␥

Doses of cytokines: GM-CSF, 800 U/ml; TNF-␣, 10 ng/ml; IL-4, 1000 IU/ml; PGE1, 1 ␮g/ml; IFN-␥, 1000 U/ml; IL-1 ␣, 10 ng/ml; IL-6, 1000 IU/ml.

Austria), interferon-␥ (IFN-␥; Boehringer Ingelheim, Germany), interleukin-1␣ (IL-1␣; PeproTech, London, UK), interleukin-6 (IL-6; PeproTech), prostaglandin E1 (PGE1 ; Schwartz Pharma, Monheim, Germany), interleukin-4 (IL-4; Sigma–Aldrich). The antigen prepared as described later was added on day 5. The culture time was 10 days in all the cases. 2.2. Antigen preparation

Besançon, France) according to the manufacturer’s instructions. Briefly, supernatant from DC cultures (100 ␮l) was added into anti-IL-12 antibody-coated plates followed by 50 ␮l of biotinylated anti-IL-12 antibody. The mixture was incubated for 3 h and washed. The plates were then incubated with streptavidin–horseradish peroxidase (100 ␮l) for further 30 min. The reaction was evaluated following the addition of a chromogen at an absorbance of 450 nm.

The antigen for DC pulsing was prepared as previously described [17]. Briefly, cells from a leukemia cell line (K562) and a myeloma cell line (8226) were lysed by four freeze–thaw cycles. The samples were centrifuged at 500 × g for 15 min, the supernatant from each sample collected, filtered through a 0.22 ␮m membrane, and added to the DC culture. The number of tumor cells used to prepare the lysate was calculated to be the same as the number of CD83+ cells generated on day 6, i.e. about 10×106 cells for PBSC-derived DC and 1×106 cells for PBMC-derived DCs.

2.5. Statistical analysis

2.3. Immunophenotyping

3.1. DC culture

Samples for immunophenotyping were taken at the start of the culture, and after 7 and 10 days. Immunophenotyping was performed using the following fluorescence-marked mononuclear antibodies to characterize the cultured cells: CD1a, CD11c, CD54, CD86, CD83, HLA-DR (Caltag, Burlingame, CA, USA). The antigens CD83, HLA-DR, and CD86 were determined as a combination. Cell yield after 7 and 10 or 11 days of culture was calculated after correction for volumes taken for sampling.

We have generated mature, antigen-loaded DCs from PBSCs or PBMCs under different culture conditions to evaluate the yield and maturation. The yields of DCs as determined by the number of CD83+ cells generated from PBSCs and PBMCs are summarized in Tables 4 and 5, respectively. For PBSCs, the yield of DCs as determined by CD83+ cell count ranged from 0.6×104 to 30.1×104 (mean: 9.4 ×104 ) of DCs generated per 1 × 106 of initially plated nucleated cells from apheresis. This yield corresponded to (0.3–19.1) × 105 (mean: 4.3 × 105 ) per 1 × 106 of CD34+ cells in the apheresis products. For PBMC, the yield was (0.4–24.8) × 104 (mean: 2.4 × 104 ) of DCs generated per 1 × 106 of initially plated mononuclear cells from venous blood. The growth of CD83+ cells was higher in the media containing IL-4 during the first 5 days of PBSC culture, whereas the growth was somewhat lower in the media that did not

2.4. Interleukin-12 assay Enzyme-linked immunosorbent assay (ELISA) was used for the assessment of cytokine production in PBMC-derived cells. Interleukin-12 (IL-12) content was measured on days 0, 7, and 10 using the IL-12 p70 ELISA Kit (Diaclone,

Descriptive statistics (arithmetic mean, median, range, standard deviation) was used do describe various groups of data. Statistical significance was assessed by the non-parametric Mann–Whitney test or the non-parametric Wilcoxon test for pairs.

3. Results

Table 4 Comparison of different cytokine combinations used for dendritic cell culture from peripheral blood stem cells Cytokine combination

No. of experiments

CD83 + cells generated from 1 × 106 MCs (× 103 )

CD83 + cells generated from 1 × 106 CD34+ cells (× 105 )

GM-CSF, GM-CSF, GM-CSF, GM-CSF, GM-CSF,

3 6 3 3 3

19.1 5.7 14.1 3.5 4.3

7.9 2.3 7.3 1.0 3.2

IL-4, TNF-␣ IL-4, Flt-3, TNF-␣ IL-4, Flt-3, TNF-␣, CD40L TNF-␣ TNF-␣, Flt-3

MCs, mononuclear cells in the apheresis product.

880

T. Büchler et al. / Vaccine 21 (2003) 877–882

PBSC- and PBMC-derived DCs, respectively. The expression of CD83 increased significantly between days 7 and 10 for PBSC-derived DCs (P < 0.05). For PBMC-derived DCs, the expression of DC markers on day 7 was not significantly different from that on day 10. The observed increase in DC-specific maturation markers and in the adhesion molecules in cells cultured from PBSCs between days 7 and 10 provides evidence that cytokines used in the second phase of two-step culture protocols effectively stimulate maturation in DCs generated from mobilized blood cells.

Table 5 Comparison of different cytokine combinations used for dendritic cell generation from peripheral blood mononuclear cells (PBMCs) Cytokine combination

No. of experiments

CD83 + cells generated from 1 × 106 PBMCs (× 103 )

GM-CSF, IL-4 TNF-␣, IL-1 ␣, IL-6, PGE1 GM-CSF, PGE1 GM-CSF, PGE1 , IFN-␥

3 3 2 3

2.0 1.6 2.6 3.5

3.3. IL-12 production assay

contain IL-4. The yield of DCs was highest in the medium with GM-CSF and IL-4 with TNF-␣ added on day 5. We did not detect any significant differences between the tested cytokine combinations regarding the yield or the phenotype of cultured DCs. However, we were able to detect some trends. Our results suggest that the medium containing GM-CSF, IL-4 in the first 6 days with the addition of TNF-␣ as a maturation stimulus on day 6 may be superior to other cytokine combinations if PBSCs are used as a source for DC generation. Wide inter-individual differences were identified both within the group of patients with multiple myeloma and that of healthy donors.

ELISA to determine IL-12 production by cultured cells was performed on the PBMC cultures with samples taken on days 0, 7, and 10. IL-12 levels produced by cells cultured with different cytokines did not vary significantly. On day 0, the production of IL-12 was below the range of the used ELISA set. The IL-12 production ranged from 5.6 to 93.8 pg/ml (mean: 74.1 pg/ml) on day 7, and 11.1 to 98.3 pg/ml (mean: 79.9 pg/ml) on day 10.

4. Discussion 3.2. Immunophenotyping Serial immunophenotyping enabled us to observe the changes in DC content during the culture as well as the development of maturation and activation markers. DC yields achieved in our experiments were similar to those reported earlier by other investigators [6,7,18]. Significant inter-individual variability was seen in patients with hematological malignancies as well as in healthy donors. The

The CD83+ antigen was considered as the principal marker of DC expansion. It was determined together with the expression of HLA-DR, co-stimulatory molecules (CD80 and CD86) and adhesion molecules (CD54 and CD11c). The results of immunophenotyping at various stages of the culture are as shown in Tables 6 and 7, for

Table 6 Immunophenotypic analysis of cells in cultures on days 0, 7, and 10 using PBSCs as a source for dendritic cell generation Cytokine combination

Mean percentage of positive cells CD83 0

GM-CSF, GM-CSF, GM-CSF, GM-CSF, GM-CSF,

7

HLA DR 10

0

7

CD11c 10

0

7

CD86 10

0

IL-4, TNF-␣ 1.8 33.3 59.7 56.5 67.9 73.3 6.6 36.6 58.5 7.9 IL-4, Flt-3, TNF-␣ 2.5 18.3 39.1 52.2 48.0 59.9 21.4 24.6 49.9 12.8 IL-4, Flt-3, TNF-␣, CD40L 1.8 27.9 63.1 56.5 48.3 78.1 6.6 39.6 49.4 7.9 TNF-␣ 3.1 8.4 27.0 47.8 53.5 56.7 36.2 25.0 49.1 17.6 TNF-␣, Flt-3 3.1 8.0 13.6 47.8 40.1 35.0 36.2 6.6 55.3 17.6

CD1a 0

CD54

7

10

7

10

0

28.4 34.2 38.0 27.8 15.1

45.5 1.1 21.4 45.5 29.2 51.0 12.4 15.2 51.0 26.9 58.2 1.1 16.4 58.2 29.2 38.5 23.7 30.0 38.5 24.6 16.8 23.7 9.4 16.8 24.6

7

10

26.8 33.1 20.3 56.9 61.6

34.4 48.0 42.9 65.8 78.8

Table 7 Immunophenotypic analysis of cells in cultures on days 0, 7, and 10 using venous blood PBMCs as a source for dendritic cell generation Cytokine combination

Mean percentage of positive cells CD83

GM-CSF, IL-4 TNF-␣, IL-1␣, IL-6, PGE1 GM-CSF, PGE1 GM-CSF, PGE1 , IFN-␥

HLA DR

CD11c

CD86

CD1a

CD54

0

7

10

0

7

10

0

7

10

0

7

10

0

7

10

0

7

10

0.5 0.5 0.4 0.5

2.2 1.4 2.8 2.1

2.2 1.3 1.9 1.3

15.7 17.2 16.7 13.4

43.0 34.7 37.6 22.5

50.0 23.3 30.4 22.6

7.1 7.7 8.9 4.7

7.2 6.2 9.0 6.3

8.1 9.6 7.2 6.2

2.1 1.7 2.7 2.0

5.6 7.5 8.7 7.1

6.4 6.4 8.4 7.6

0.7 0.8 0.7 0.7

5.9 5.5 7.1 6.2

7.2 5.0 5.0 3.9

5.2 6.0 5.9 3.9

6.2 7.6 9.3 6.2

5.7 6.0 7.4 6.2

T. Büchler et al. / Vaccine 21 (2003) 877–882

basis for this heterogeneity or its impact on immune system functions are not known. The optimal number of DCs for adequate, specific T-cell activation has not been established. However, in first clinical studies in patients with hematological malignancies, DCs were applied subcutaneously or intravenously in doses ranging from 1 × 106 to 180 × 106 [19–22]. Our results suggest that reasonably high numbers of DCs can be obtained from apheresis products after hematopoietic progenitor cell mobilization. It is much more difficult to generate mature DCs from non-mobilized peripheral blood. The yield of dendritic cells is often disappointing when using PBMCs as a source for their generation and the development of maturation markers is less evident than with PBSC-derived DCs. DC quantification in a cell culture remains controversial as no cell antigen or antigen combination has been universally agreed to represent DCs. The approaches used by various investigators include methods based on a lin-HLA-DR+ phenotype [23], the CD83 antigen [24] and the CMRF44 antigen [25]. In our experiments, we used the CD83+ cell counts to determine the yield of mature, activated DCs. We believe that the evaluation of DC cultures by cell morphology cannot be used for DC enumeration. The “dendritic” shape is not specific for DCs and other cells that accompany DCs in culture, such as activated macrophages or fibroblasts may be mistaken for DCs. A consensus method for DC enumeration would greatly facilitate the comparison of results achieved by different investigators. The observed increases in maturation and adhesion markers in the PBSC cultures contrasting with little change in the surface expression of these molecules critically important for the activation of CTLs may be due to two factors—firstly, we used one-step culture protocols in the latter which probably do not enhance the maturation of DCs as much as the two-step ones and, secondly, the response of PBMC-derived DCs to maturation-inducing cytokines may be different from that seen in PBSC-derived DCs. However, the IL-12 production was high in PBMC-derived DCs, an evidence of their activation by cytokines as suggested Mosca et al. [26]. In conclusion, mature DCs can be generated from various cell sources under serum-free conditions in quantities sufficient for clinical application in cancer vaccination protocols. However, the culture of DCs is a costly process where some as yet unknown variables may play a part. No particular cytokine combination described to-date appears to provide a significant advantage over others. Based on our results, the cytokine combination including GM-CSF and IL-4 with TNF-␣ added on day 5 was chosen for our vaccination protocol in patients with multiple myeloma. This combination provides optimal cell yield and immunophenotype for DCs cultured under serum-free conditions. Importantly, such culture method does not require the addition of the more expensive cytokines, such as Flt-3 or CD40L. Next, in our experiments we will concentrate on the identification of factors that determine the yield of DCs in an

881

in vitro culture, a question of great importance for the design of clinical trials that include multiple vaccinations with antigen-loaded DCs. Future studies should also clarify the molecular mechanisms behind the activation, maturation, and expansion of DCs, using methods such as comparative gene expression and protein cell content profiling.

Acknowledgements ˇ This project was supported by the Grants IGA MZ CR 6152-3 and 6763-3, MŠMT VZ J07/98-141100003 and ˇ 301/00/0405. J07/98-6700008, and GACR References [1] Steinman RM. Dendritic cells and immune-based therapies. Exp Hematol 1996;24(8):859–62. [2] Hajek R, Butch AW. Dendritic cell biology and the application of dendritic cells to immunotherapy of multiple myeloma. Med Oncol 2000;17(1):2–15. [3] Lardon F, Snoeck HW, Berneman ZN, et al. Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: antagonistic effects of IL-4 and IFN-gamma and selective involvement of TNF-alpha receptor. Immunology 1997;91(4):553–9. [4] Szabolcs P, Avigan D, Gezelter S, et al. Dendritic cells and macrophages can mature independently from a human bone marrowderived, post-colony-forming unit intermediate. Blood 1996;87(11): 4520–30. [5] Caux C, Vanbervliet B, Massacrier C, et al. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF-alpha. J Exp Med 1996;184(2):695–706. [6] Bender A, Sapp M, Schuler G, Steinman RM, Bhardwaj N. Improved methods for the generation of dendritic cells from non-proliferating progenitors in human blood. J Immunol Methods 1996;196(2):121– 35. [7] Romani N, Reider D, Heuer M, et al. Generation of mature dendritic cells from human blood: an improved method with special regard to clinical applicability. J Immunol Methods 1996;196(2):137–51. [8] Bernhard H, Disis ML, Heimfeld S, Hand S, Gralow JR, Cheever MA. Generation of immuno-stimulatory dendritic cells from human CD34+ hematopoietic progenitor cells of the bone marrow and peripheral blood. Cancer Res 1995;55(5):1099–104. [9] Herbst B, Kohler G, Mackensen A, Veelken H, Mertelsmann R, Lindemann A. CD34+ peripheral blood progenitor cell and monocyte derived dendritic cells: a comparative analysis. Br J Haematol 1997;99(3):490–9. [10] Chapuis F, Rosenzwajg M, Yagello M, Ekman M, Biberfeld P, Gluckman JC. Differentiation of human dendritic cells from monocytes in vitro. Eur J Immunol 1997;27(2):431–41. [11] Galy A, Morel F, Hill B, Chen BP. Hematopoietic progenitor cells of lymphocytes and dendritic cells. J Immunother 1998;21(2):132–41. [12] Hart DNJ. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997;90(9):3245–87. [13] Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: down-regulation by cytokines and bacterial products. J Exp Med 1995;182(2):389–400. [14] O’Doherty U, Peng M, Gezelter S, et al. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology 1994;82(3):487–93.

882

T. Büchler et al. / Vaccine 21 (2003) 877–882

[15] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6673):245–52. [16] Syme R, Glück S. Effect of cytokines on the culture and differentiation of dendritic cells in vitro. J Hematother Stem Cell Res 2001;10(1):43–51. [17] Yang S, Darrow TL, Vervaert CE, Seigler HF. Immunotherapeutic potential of tumor antigen-pulsed and unpulsed dendritic cells generated from murine bone marrow. Cell Immunol 1997;179(1):84– 95. [18] Tarte K, Fiol G, Rossi JF, Klein B. Extensive characterization of dendritic cells generated in serum-free conditions: regulation of soluble antigen uptake, apoptotic tumor cell phagocytosis, chemotaxis and T cell activation during maturation in vitro. Leukemia 2000;14(12):2182–92. [19] Reichardt VL, Okada CY, Liso A, et al. Proliferating dendritic cell progenitors in human blood. J Exp Med 1994;180(7):83–93. [20] Bohlen H, Thielemanns K, Tesch H, et al. Idiotype vaccination strategies against a murine B-cell lymphoma: dendritic cells loaded with idiotype and bi-specific idiotype × anti-class II antibodies can protect against tumor growth. Cytokines Mol Ther 1996;2(4):231.

[21] Lim SH, Bailey-Wood R. Idiotypic protein-pulsed dendritic cell vaccination in multiple myeloma. Int J Cancer 1999;83(2):215–22. [22] Wen YJ, Ling M, Bailey-Wood R, Lim SH. Idiotypic protein-pulsed adherent peripheral blood mononuclear cell-derived dendritic cells prime immune system in multiple myeloma. Clin Cancer Res 1998;4(4):957–62. [23] McCarthy DA, Macey MG, Bedford PA, Knight SC, Dumonde DC, Brown KA. Adhesion molecules are up-regulated on dendritic cells isolated from human blood. Immunology 1997;92(2):244–51. [24] Weissman D, Li Y, Orenstein JM, Fauci AS. Both a precursor, and a mature population of dendritics cells can bind HIV: however, only the mature population that expresses CD80 can pass infection to unstimulated CD4+ T cells. J Immunol 1995;155(8):4111–7. [25] Fearnley DB, Whyte LF, Carnoutsos SA, Cook AH, Hart DN. Monitoring human blood dendritic cell numbers in normal individuals and in stem cell transplantation. Blood 1999;93(2):728–36. [26] Mosca PJ, Hobeika AC, Clay TM, et al. A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment. Blood 2000;96(10):3499–504.