Development of a standardized protocol for reproducible generation of matured monocyte-derived dendritic cells suitable for clinical application

Development of a standardized protocol for reproducible generation of

matured monocyte-derived dendritic cells suitable for clinical application

H.R. Bohnenkamp and T. Noll*

Institute of Biotechnology 2, Research Center Juelich GmbH, 52428 Juelich, Germany; *Author for

correspondence (e-mail: [email protected]; phone: +49 2461 613955; fax: +49 2461 613870)

Received 21 March 2003; accepted in revised form 3 September 2003

Key words: Dendritic cells, Immunotherapy, Monocyte enrichment, Serum-free

Abstract

There is increasing interest in the generation of dendritic cells (DC) for cancer immunotherapy. In order to

utilize DC in clinical trials it is necessary to have standardized, reproducible and easy to use protocols. We

describe here the process development for the generation of DC as the result of investigation of culture

conditions as well as consumption rates of medium and cytokines. Our studies demonstrate that highly

viable DC (93 ± 2%) can be produced from CD14

enriched monocytes via immunomagnetic beads in a high

yield (31 ± 6%) with X-VIVO 15, 400 U ml

1

GM–CSF and 2000 U ml

1

IL-4 without serum and feeding.

For the maturation of DC different cocktails (TNF-, IL-1, IL-6, PGE

and TNF-, PGE

) were

compared. In both cases cells expressed typical surface molecules of mature DC and induced high prolif-

erative responses in mixed lymphocyte reactions which led to IFN- producing T-lymphocytes. The data

suggest that the use of this optimized, easy to use protocol results in highly mature DC.

Abbreviations: DC – dendritic cells; moDC – monocytes derived dendritic cells; FACS – fluorescence

activated cell sorter; FCS – fetal calf serum; GM–CSF – granulocyte macrophage–colony stimulating

factor; MLR – mixed lymphocyte reaction; PBMC – peripheral blood mononuclear cell; IL – Interleukin;

PGE

– Prostaglandin E

; TNF – tumor necrosis factor

Introduction

Dendritic cells (DC) are professional antigen

presenting cells, inducing immune responses while

stimulating na

€

ve T-lymphocytes and controlling

the activation of T-helper cells in the T

1–T

pathway (Banchereau and Steinman 1998;

O’Garra and Arai 2000).

The use of these potent stimulators as vaccines

for the immunotherapy of cancer is a very promis-

ing approach to overcome the tumor escape in

immune surveillance (Costello et al. 1999). To

date, several clinical trials have been performed

utilizing DC preparations, which demonstrated

anti-tumor responses (Nestle et al. 1998; Thurner

et al. 1999a; Kugler et al. 2000; Fong et al. 2001;

Kikuchi et al. 2001; Kobayashi et al. 2001; Schuler-

Thurner et al. 2002).

Different sources can be used for the generation

of DC: proliferating CD34

precursors in blood

(Caux et al. 1992) after G-CSF mobilization

(Mackensen et al. 2000), non-proliferating CD14

monocytes in peripheral blood after enrichment via

magnetic beads (Pickl et al. 1996; Dietz et al. 2000)

or adhesion (Bender et al. 1996; Romani et al. 1996;

Thurner et al. 1999b; Berger et al. 2002) and enrich-

ment of DC after cultivation of PBMC via elutria-

tion (Bernard et al. 1998; Goxe et al. 2000). Rare

circulating DC can also be isolated, but although

patients can be pretreated with Flt3 ligand the yield

121

Cytotechnology 121–131, 2003.

42:

2003 Kluwer Academic Publishers. Printed in the Netherlands.

of DC is comparably small (Fong et al. 2001). While

DC from CD34

cells require a prolonged culture

and special cytokine setup in order to increase

the small number of precursors, monocyte-derived

DC (moDC) are easy to obtain after enrichment

of monocytes by magnetic separation or adherence,

followed by differentiation using granulocyte

macrophage–colony stimulating factor (GM–

CSF) and interleukin-4 (IL-4). This method devel-

oped by Sallusto and Lanzavecchia (1994) and

Romani et al. (1994) is applied widely in experi-

mental protocols. Due to the ease of availability

of monocytes, rapidness of fully differentiation to

unmature (5–6 days) or mature (6–8 days) DC using

special maturation cocktails (e.g., TNF-,IL-1,

IL-6, PGE

(Jonuleit et al. 1997) or TNF-, PGE

(Kalinski et al. 1998)) and the possibility of avoid-

ing foreign antigens (fetal calf serum (FCS)), this

protocol was adapted for clinical applications

(Thurner et al. 1999b). For studies where DCs are

to be cultured ex vivo and then reintroduced to

the patient, it is advisable to avoid FCS or any

kind of foreign antigen due to possible infections

and immunogenicity. However, the use of autolo-

gous serum or plasma may cause non-standardized

cultivation conditions and so is best to avoid.

Here, we describe the development of a standard-

ized protocol, which circumvents the disadvantages

of non-uniform culture conditions caused by sup-

plemented serum, non-defined cell densities and

adherence steps. Furthermore, the protocol elimi-

nates the need for feeding the cells, which increases

the risk of contamination. In this study, we have

examined the influence of several important culti-

vation parameters to optimize and setup the serum-

free generation of DC in X-VIVO 15. Cell density,

GM–CSF and IL-4 concentration, medium com-

ponents like glucose, lactate and amino acids were

assessed. Moreover the influence of different

maturation cocktails (TNF-,IL-1, IL-6, PGE

and TNF-, PGE

) on the maturation status of DC

was investigated.

Material and methods

Medium, serum and cytokines

As standard medium X-VIVO 15 (Bio Whittaker,

Walkersville, MD) was used for the generation of

DC. RPMI 1640 (Gibco BRL, Eggenstein,

Germany) supplemented with 10% heat-

inactivated (56



C, 30 min) FCS (Gibco BRL,

Eggenstein, Germany) was used for the mixed

lymphocyte reaction (MLR). The differentiation of

monocytes into DC was performed using rhuGM–

CSF (Leucomax

, Norvartis, Nuernberg,

Germany) and rhuIL-4 (R&D, Wiesbaden,

Germany). Factors added for maturation of DC

included rhuTNF-, rhuIL-1, rhuIL-6 (all from

R&D) and PGE

(Sigma, Deisenhofen, Germany).

For the MLR rhuIL-2 (from BHK 21 cells, kindly

provided by Dr Wagner, GBF, Braunschweig,

Germany) was utilized.

Generation of human monocyte derived

dendritic cells

Peripheral blood mononuclear cells (PBMC) were

obtained from buffy coat preparations from

healthy donors (kindly provided by Dr T. Tonn,

Blutspendedienst Hessen, Germany) by standard

density gradient centrifugation on Biocoll (Ficoll

separating solution) (Biochrom KG, Berlin,

Germany). Monocytes CD14

were affinity-

purified utilizing the MACS

CD14 isolation kit

(Miltenyi Biotec, Bergisch Gladbach, Germany)

following the manufacturer’s instructions. Briefly,

PBMC were incubated in recommended buffer

with MACS

CD14 MicroBeads for 15 min at



C, centrifugated and resuspended in buffer.

Later cells were passed through a positive selection

column. This step was done twice to obtain highly

purified CD14 positive cells.

After resuspension of monocytes in X-VIVO 15

they were seeded at defined cell densities described

at the results’ section (Standard concentration:

1.3  10

1

) and placed in an incubator at



Cand5%CO

. Cytokines including rhuGM–CSF

and rhuIL-4 were added at day 0 (standard con-

centration: rhuGM–CSF (400 U ml

1

), rhuIL-4

(2000 U ml

1

)). All comparison experiments were

performed in 48 well plates (Greiner, Solingen,

Germany) and with optimized parameters up-

scaled in 75 cm

T-flasks (Greiner). After 6 days

of differentiation of monocytes into DC different

maturation cocktails were added: cocktail I (TNF-

(1000 U ml

1

), IL-1 (1000 U ml

1

), IL-6 (1000 U

1

), PGE

(1  gml

1

, 0.003 M)) and cocktail II

(TNF- (1000 U ml

1

), PGE

(18 gml

1

122

0.051 M)). After 8 days the resulting suspension-

cells were harvested and analyzed as described.

Cell counting and viability

After harvesting of DC on day 8, counting and via-

bility determination was performed using a hemo-

cytometer with standard trypan blue dye exclusion

and a CASY 1 particle counting system (model TT,

Schaerfe System, Reutlingen, Germany).

Phenotyping and fluorescence activated cell

sorter analysis

For analysing of PBMC and DC populations we

used the following mAbs (all mAbs from Becton

Dickinson, Heidelberg, Germany): CD1a

(CyChrome), CD3 (CyChrome), CD4 (PE), CD8

(FITC), CD14 (FITC), CD16 (PE), CD19 (PE),

CD25 (FITC), CD40 (FITC), CD54 (PE), CD56

(FITC), CD80 (FITC), CD83 (PE), CD86 (FITC),

anti-HLA-A,B,C (FITC), anti-HLA-DR

(CyChrome, PE). Cells (2  10

per specimen)

were suspended in 90 l of ice-cold PBS and incu-

bated with 5 l of corresponding mAb for 30 min at



C. After staining cells were washed once with

ice-cold PBS and fixed in 200 l of 1% paraformal-

dehyde in PBS. FACS analysis was performed

using a FACSCalibur (Becton Dickinson) and

CellQuest 3.1 software (Becton Dickinson).

Cytokine enzyme-linked immunosorbent assay

(ELISA) kits

Cytokine ELISA kits for rhuGM–CSF (detec-

tion limit: 4.7 pg ml

1

), rhuIL-4 (detection limit:

7.8 pg ml

1

) and rhuIL-12p70 (detection limit: 7.8

pg ml

1

) were purchased from Becton Dickinson

(OptEIA human ELISA Set, BD PharMingen,

Heidelberg, Germany) and were used following

the manufacturer’s instructions. The readout of

the ELISA-plates was performed using a photo-

meter (Wallac Victor

, PerkinElmer Life Science,

Bad Wildbad, Germany) reader at 450 nm with a

correction at 570 nm.

Metabolic analysis

Osmolality was measured using a freezing-point

osmometer Osmomat 030 (Gonotec, Berlin,

Germany). Glucose (Ebio compact, Eppendorf,

Hamburg, Germany), lactate (YSI 1500L, Yellow

Springs Instruments, Yellow Springs, USA), gluta-

mine and glutamate (YSI 2700 select, Yellow

Springs Instruments, Yellow Springs, USA) were

quantified enzymatically using the indicated auto-

matic analyzer according to the manufacturers

instructions. Amino acids analysis was realized

using HPLC (Amino Quant 1090 AX, Hewlett

Packard, Waldbronn, Germany).

The MLR

DC were added to 5  10

allogeneic PBMC at a

ratio of 1 : 10 in 6 well plates and co-incubated for

4 days in RPMI 1640 supplemented with 10% FCS.

After 4 and 7 days 50% of the culture medium were

replaced and 100 U ml

1

rhuIL-2 were added.

After 8 days the proliferating T-cells were counted,

phenotyped and an Interferon- secretion assay

was performed.

Interferon- secretion assay

For determination of the portion of IFN- produc-

ing T-cells an IFN- Cytokine Secretion Assay

(Miltenyi Biotec, Bergisch Gladbach, Germany)

was used according to the manufacturer’s instruc-

tions (Manz et al. 1995).

Results

Cell enrichment and starting population of

monocytes

There are different methods of monocyte enrich-

ment currently discussed in literature. Due to the

availability of GMP (Good Manufacturing

Practice) quality immunomagnetic beads from

Miltenyi (CliniMACS

) to isolate CD14

mono-

cytes (Dzionek et al. 2002), this method was used in

a laboratory setup with Mini- and Midi-MACS

magnets and appropriate columns. This magnetic

cell sorting approach was highly effective in isolat-

ing CD14

monocytes from PBMC with a yield of

12.5 ± 4.8% (n ¼ 6) and a purity of 98% (data not

shown).

123

Influence of different cell densities on yield and

maturation of moDC

To setup and optimize a protocol for the genera-

tion of moDC we first investigated the influence

of different cell densities on the consumption and

accumulation of medium components such as glu-

cose, lactate, glutamine, glutamate and supplemen-

ted cytokines. In different experiments 3.3  10

6.6  10

, 1.3  10

and 2.6  10

monocytes ml

1

were inoculated in X-VIVO15 supplemented with

800 U ml

1

GM–CSF and 500 U ml

1

IL-4 and

incubated for 6 days without feeding. A maturation

cocktail consisting of TNF- (1000 U ml

1

), IL-1

(1000 U ml

1

), IL-6 (1000 U ml

1

) and PGE

(1 gml

1

) was then added and the cells incubated

for further 2 days.

After 6 days of cultivation partly non-adherent

DC could be observed which did not express CD83

a marker of mature DC (Zhou and Tedder 1995).

The maturation cocktail added for two additional

days induced the expression of CD83 and an

increase in CD80 and CD86 as well as HLA-DR.

These non-adherent DC with many motile veils

showed the typical pattern of mature moDC. The

size of the cells increased from an average of 10 m

(monocytes) to 16 m (moDC), which was deter-

mined by a CASY 1 particle counter.

The yield (as defined by size of cells, morphology

and surface antigen expression) of matured moDC

was similar for the cultures initially inoculated with

3.3  10

, 6.6  10

, 1.3  10

monocytes ml

1

and

about 25% lower for the highest inoculated cell

density (Figure 1). All cell densities except for the

2.6  10

1

showed the typical mature DC

phenotype with high expression of HLA-A, B, C

(data not shown), HLA-DR, CD80, CD83

and CD86 (Figure 2). The DCs differentiated and

matured from 2.6  10

1

expressed reduced

HLA-DR, CD80, CD83 and CD86 antigens. The

HLA-DR/CD80 and HLA-DR/CD83 dot plots

for 6.6  10

and 1.3  10

1

showed two distinct

populations of DC: a population with higher HLA-

DR/CD80 and HLA-DR/CD83 expression and

a lower one, which may be caused by a different

maturation status. Especially for the HLA-DR/

CD80 dot plot for the cell density of 1.3  10

1

this observation was significant. Lower HLA-DR

expression seems to correlate with a decrease

in CD80 surface antigen expression. The mean

fluorescent intensity as an indicator for the level

of expression of CD40 decreased about 50% with

the highest cell density (data not shown). We could

not observe any differences in expression of CD1a

(data not shown).

We also analyzed medium components and

found an increase in lactate concentration at the

highest cell density at 25 mmol l

1

(Figure 3),

which caused a decreased culture pH and might

be responsible for the lower yield. Accumulation of

metabolic products like lactate produces an acidic

environment and therefore inhibits proliferation

(Bohnenkamp and Noll 2002; Patel et al. 2000).

The glucose concentration in all experiments

remains above limiting levels. Amino acid analysis

demonstrated no limitation for glutamine and

serine. The highest glutamate concentration was

1.4 mmol l

1

An ELISA for GM–CSF and IL-4 showed no

limitation for GM–CSF (GM–CSF residual con-

tent: 3.3  10

1

: 780 U ml

1

, 6.6  10

1

720 U ml

1

, 1.3  10

1

: 450 U ml

1

and 2.6 

1

: 500 U ml

1

, respectively) but IL-4 was

limiting (detection limit: 7.8 pg ml

1

) (data not

shown), which might explain the non-homogenous

DC population especially for the 6.6  10

and

1.3  10

1

inoculated monocytes.

Due to a cost-effective utilization of the cultiva-

tion system in terms of highest yield of matured

moDC per volume, all further experiments were

Figure 1. Influence of different cell densities on yield after

generation of moDC. Monocytes were enriched via

immunomagnetic beads, inoculated at specified cell densities

and differentiated with 800 U ml

1

GM–CSF and 500 U ml

1

IL-4. For maturation a cytokine cocktail consisting of TNF-,

IL-1, IL-6 and PGE

was used. The data represent the mean

(± SD) of triplicates from a single donor. Similar data were

obtained with two other donors.

124

performed at a cell density of 1.3  10

1

inoculated monocyte.

Optimization of GM–CSF and IL-4

concentrations

Based on the finding that no substrate feeding is

necessary for the generation of matured moDC at

a cell density of 1.3  10

1

inoculated mono-

cytes, we investigated the influence of different

concentrations of GM–CSF and IL-4 on yield

and phenotype of the cells in order to get a homo-

genous moDC population. Monocytes differen-

tiate in the presence of GM–CSF and IL-4 to

immature DC but both cytokines are also

necessary for maturation, therefore suggesting

that both cytokines are not exhausted after 8 days

of culture.

We did several experiments to test the stability,

half-life and consumption of the mentioned cyto-

kines. The stability and half-life test was estab-

lished utilizing triplicates of T-flasks we also used

for the generation of DC. We inoculated 800 U ml

1

GM–CSF and 500 U ml

1

IL-4 respectively in

X-VIVO 15 and incubated at 37



C and 5% CO

for 8 days. Samples were taken every [2

] day,

frozen and after collection thawed for quantifica-

tion of GM–CSF and IL-4. The GM–CSF used

(Leucomax

) showed no decrease of concentra-

tion after 8 days (data not shown). For the IL-4

Figure 2. Phenotype of moDC generated with in figure 1 specified cultivation parameters. HLA-DR/CD80, HLA-DR/CD86 and HLA-

DR/CD83 dot plots are shown for different cell densities (3.3  10

, 6.6  10

, 1.3  10

and 2.6  10

1

respectively). Decreased

CD80, CD83 and CD86 was expressed for the highest cell density, HLA-DR/CD80 and HLA-DR/CD83 dot plots for 6.6  10

and

1.3  10

1

featured a higher and a lower expressing population. The data shown are from one representative experiment out of three

performed.

125

(R&D) we could demonstrate that 80% of the cyto-

kine adhered to the surface of the flask after 2 h.

The remaining IL-4 concentration did not show

any further decay (data not shown). Therefore,

the monocytes were inoculated first and then the

cytokines were added to ensure that enough IL-4

remained in solution (data not shown).

The next step was to investigate the consumption

of GM–CSF and IL-4. Therefore we started with

200, 400 and 800 U ml

1

GM–CSF while IL-4 was

inoculated at 500 U ml

1

. The 200 U ml

1

were

exhausted completely while in the other two

experiments about 300 U ml

1

were consumed

(296 U ml

1

and 320 U ml

1

, respectively)

(Figure 4). The yield and phenotype was compar-

able to previous experiments, see Figure 2, 1.3 

1

inoculated monocytes. The 200 U ml

1

GM–CSF concentration resulted in the same

number of DCs but in a lower expression of

CD80, CD83 and CD86 (data not shown).

We then determined which concentration of IL-4

resulted in a homogenous populations of matured

moDC. We added different IL-4 concentrations

(500, 1000 and 2000 U ml

1

respectively) with a

constant concentration of 800 U ml

1

GM–CSF to

alter only one parameter. The IL-4 ELISA

indicated that only at an initial concentration of

2000 U ml

1

IL-4 was the not limiting, as we could

still measure 25 U ml

1

after 8 days of cultivation

(data not shown) and phenotypic analysis showed

Figure 4. GM–CSF consumption of cultivated cells after 8 days.

Data shown are the mean (± SD) of triplicate cultures from one

representative experiment of three performed.

Figure 3. Glucose and lactate analysis on day 0 (start concentration) and on day 8 (after generation of moDC). Shown is one

representative experiment out of three.

126

a homogenous population of matured moDC only

for the highest IL-4 concentration (Figure 5).

However, the number of DCs was similar for all

cytokine concentrations.

The data demonstrated that800 U ml

1

GM–CSF

and 2000 U ml

1

IL-4 resulted in homogenously

matured moDC. We also could show that

400 U ml

1

GM-CSF is sufficient for the genera-

tion of mature moDC without feeding.

Influence of different maturation cocktails

In previous experiments for maturation of moDC a

cocktail consisting of TNF-,IL-1, IL-6 and

Figure 5. Phenotype of moDC generated with different IL-4 concentrations (500, 1000 and 2000 U ml

1

). Only moDC generated with

2000 U ml

1

demonstrated a homogenous population of fully matured dendritic cells. The data shown are from one representative

experiment of three performed.

127

PGE

was used. In the following experiments, we

examined the feasibility to simplify this maturation

cocktail while obtaining the same yield, viability,

phenotype and distinct functional capacity of DC.

We used 400 U ml

1

GM–CSF, 2000 U ml

1

IL-4

and inoculated a cell density of 1.3  10

1

differentiate monocytes to dendritic cells.

Therefore, we compared two maturation cock-

tails: Cocktail I composed of 1000 U ml

1

TNF-,

1000 U ml

1

IL-1, 1000 U ml

1

IL-6 and 1-gml

1

PGE

and Cocktail II consisted of 1000 U ml

1

TNF- and 18 gml

1

PGE

. We found in 12

experiments similar results in yield, viability and

phenotype for both cocktails (data not shown).

A consequence of maturation is usually the

secretion of inflammatory cytokines by moDC

(Sallusto and Lanzavecchia 1999). However, it

has been demonstrated that PGE

induces the

final maturation of IL-12p70 deficient DC

(Kalinski et al. 1998). To investigate the amount

of bioreactive IL-12p70, we analyzed by ELISA the

cell culture supernatant of moDC after 2 days of

stimulation with the respective cytokine cocktail.

With both stimuli no detectable level of IL-12p70

was produced at any time point (data not shown).

Mixed lymphocyte reaction

The allostimulatory capacity of matured moDC

was tested using a DC T-cell ratio of 1 : 10. After

an induction phase cells were fed twice at 92 h and

163 h by replacement of 50% of the medium and

adding of 100 U ml

1

IL-2. Figure 6 illustrates the

potent stimulatory capacity produced by moDC

samples.

Testing of the supernatant of the MLR after

4 days for levels of IL-12p70 showed no detectable

amount of cytokine. Since PGE

suppressed

IL-12p70 production in matured moDC, we inves-

tigated whether a typical T

1orT

2 cytokine

pattern was being induced. After 190 h of cultiva-

tion and proliferation of T-lymphocytes the super-

natant was tested for secreted IFN-. Either T-cells

stimulated by moDC matured with cocktail II or I

were highly IFN- and CD25 positive (Figure 7).

ELISA for IL-4 showed no level of cytokine (data

not shown). These data suggest that T-lymphocytes

stimulated by moDC matured by either cytokine

cocktails were polarized towards T

1-type.

Figure 6. Allostimulatory capacity for PBMC from healthy donors. MoDC matured with different maturation cocktails (cocktail I:

TNF-, IL-1, IL-6 and PGE

; cocktail II: TNF-, PGE

) induced a similar stimulatory capacity in the allogeneic MLR. Shown are

mean values (± SD) from three experiments in triplicates.

Figure 7. Phenotype of PBMC after MLR. Either cocktail

I and II induced IFN- producing T-cells which were also

CD25 (IL-2 -chain) positive. Results are expressed as mean

(± SD) from three experiments in triplicates.

128

Generation of moDC with optimized parameters

Based on the optimized parameters, generation of

moDC was carried out in 75 cm

T-flasks.

Monocytes were inoculated at a cell density of

1.3  10

1

and differentiated with 400 U ml

1

GM–CSF and 2000 U ml

1

IL-4. On day 6

1000 U ml

1

TNF- and 18 gml

1

PGE

were

added to mature the cells and on day 8 the cells

were harvested.

No difference was observed regardless of using

48 well plates or 75 cm

T-flasks. The mean yield as

calculated from inoculated CD14+ monocytes was

31 ± 6% with a mean viability of 93 ± 2% (n ¼ 4).

Figure 8 illustrates the mean percentage of positive

DC for important surface antigens like HLA-DR,

HLA-A,B,C, costimulatory molecules like CD40,

CD80 and CD86, the intercellular adhesion mole-

cule (ICAM-1) CD54 and the standard maturation

marker CD83.

Discussion

The aim of this study was to determine optimal

conditions for the generation of moDC by devel-

oping a standardized, easy to use and reproducible

protocol. We accomplished within this study a non-

feeding protocol in serum-free conditions that is

suitable for clinical use. Culture of enriched

monocytes using serum-free conditions for 6 days

resulted in immature moDC with low levels of

CD83. Final maturation was induced for

additional 2 days by pro-inflammatory cytokines

TNF-,IL-1 and IL-6 supported by PGE

(Jonuleit et al. 1997) or TNF- and PGE

(Kalinski et al. 1998) alone, both of which resulted

in high levels of HLA-DR, CD80, CD83, CD86,

CD40 and CD54.

Using this protocol we could obtain moDC with

a yield of 31 ± 6% and a viability of 93 ± 2%. We

demonstrated that feeding the cells is not necessary

and that it is sufficient to only add 400 U ml

1

GM–CSF and 2000 U ml

1

IL-4 at the beginning

of the cultivation. Dietz et al. (2000), who gener-

ated moDC using MACS Micro-beads enriched

CD14

monocytes in X-VIVO 15, 1% human AB

serum, 800 U ml

1

GM–CSF and 1000 U ml

1

IL-4

fed the cells every 3rd day by replacing of one-third

with fresh medium containing 1600 U ml

1

GM–

CSF and 1000 U ml

1

IL-4 obtained a yield

between 11.4% and 31.2%. Berger et al. (2002)

reported a moDC yield of 19.9 ± 9.6% with a

viability of 94.0% ± 12.0%, using the adherent

fraction of PBMC: the cells were cultivated in

RPMI 1640 supplemented with 1% autologous,

heat-inactivated human plasma, 800 U ml

1

GM–CSF and 500 U ml

1

IL-4 and fed twice on

day 3 and 5 with additional medium, 800 U ml

1

GM–CSF and 500 U ml

1

IL-4.

Our data suggest that under serum-free con-

ditions the cytokine-cocktails consisting of either

TNF-,IL-1, IL-6 and PGE

or TNF- and

PGE

, a simplified cocktail with just two compo-

nents, are sufficient to induce the final maturation

of immature moDC into mature, homogenous

immunostimulatory DC. Stimulation of allogeneic

€

ve T-cells in MLR led to high proliferation with

either cytokine cocktail. Production of IFN- was

significantly induced, while no effect on the pro-

duction of IL-4 or IL-12p70 was seen. These find-

ings are in agreement with Jonuleit et al. (1997),

who demonstrated that addition of PGE

to a

cocktail of TNF-, IL-1 and IL-6 led to higher

IFN- production in allogeneic primary stimula-

tion. They also observed, that neither CD4

nor

CD8

T-cells produced IL-4 or IL-10, indicating

that these moDC could not support the develop-

ment of type-2 T-cells. Two studies showed

recently that PGE

regulates the migratory capa-

city of moDC (Luft et al. 2002; Scandella et al.

2002) and that these migratory-type DC produce

lower level of cytokines (including IL-12p70) and

induce IFN- production of T-cells in MLR. These

Figure 8. Phenotypic analysis of mature moDC generated with

the standardized protocol. Shown are results of surface antigen

expression as indicated as mean (± SD) from four independent

experiments.

129

conclusions were confirmed by several clinical

investigations using moDC matured with the

cytokine-cocktails utilized in this study (TNF-,

IL-1, IL-6, PGE

), which induced more potent

T-cell immune responses in undergoing immu-

notherapy patients (Schuler-Thurner et al. 2002;

Dhodapkar et al. 2001). These findings were in

contrast to Kalinski et al. (1998) who reported

that DC matured in the additional presence of

PGE

bias na

€

ve Th cell development toward the

Th2. However, this may be caused by different

cultivation parameters including FCS in the

cultivation setup.

In conclusion, we have shown that we could

further simplify the generation of fully mature

moDC while maintaining their high stimulatory

capacity. Although for the clinical application of

DCs the culture will need to take place in a fully

enclosed system, for instance cell bags (Guyre et al.

2002). Because of the simplicity of the protocol

described here the transfer to such a system should

be easily achieved.

Acknowledgements

The authors gratefully acknowledge Professor

Dr C. Wandrey for his support, B. Schwartzkopff

for performing medium analysis and cytokine

ELISA and C. Herfurth for amino acids analysis.

We also like to thank Dr Joy Burchell for critical

comments. This work was funded partly by the

European Commission (5th frame project, number:

QLK3-2002-01980).

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