original papers - Advances in Clinical and Experimental Medicine

original papers - Advances in Clinical and Experimental Medicine

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ORIGINAL PAPERS
Adv Clin Exp Med 2009, 18, 3, 249–259
ISSN 1230−025X
© Copyright by Wroclaw Medical University
KAMILA WOJAS−KRAWCZYK1, 3, IWONA HUS2, PAWEŁ KRAWCZYK3, ANNA DMOSZYŃSKA2,
JANUSZ MILANOWSKI3, 4, JACEK ROLIŃSKI1
Comparison of Different Culture Microenvironments
for the Generation of Dendritic Cells from Peripheral
Blood Monocytes of Multiple Myeloma Patients
– Preliminary Report*
Porównanie różnych podłoży hodowlanych stosowanych do generowania
komórek dendrytycznych z monocytów krwi obwodowej
od chorych na szpiczaka plazmocytowego – doniesienie wstępne
1
Department of Clinical Immunology, Medical University of Lublin, Poland
Department of Hemato−oncology and Bone Marrow Transplantation, Medical University of Lublin, Poland
3
Department of Pneumonology, Oncology, and Allergology, Medical University of Lublin, Poland
4
Institute of Agricultural Medicine, Lublin, Poland
2
Abstract
Background. Many protocols regarding the ex vivo generation of dendritic cells (DCs) in culture medium with dif−
ferent protein supplementation have been described.
Objectives. This study attempted to generate autologous DCs from peripheral blood mononuclear cells (PBMCs)
of multiple myeloma (MM) patients and compare different culture conditions of DC generation.
Material and Methods. PBMCs from MM patients were cultured in medium supplemented with 2% human albu−
min, 10% autologous serum, or 10% allogenic serum from healthy donors in the presence of IL−4 and GM−CSF to
generate immature dendritic cells. Maturation of the generated cells was induced by addition of TNF−α. After−
wards, the course of apoptosis, phagocytic ability, and the immunophenotype of the generated autologous DCs we−
re analyzed. The concentrations of tumor−derived factors such as IL−6, TGF−β, VEGF, and IL−10 in serum from
the MM patients were also determined.
Results. There was a significantly higher percentage of CD1a+/CD14– cells in the culture with human albumin than
in the cultures with addition of autologous or allogenic serum. Moreover, the percentage of Annexin V−positive
(AV+) cells in the populations of CD1a+/CD14+ cells and CD1a+/CD14– cells was significantly higher in human al−
bumin−supplemented culture. Significantly lower percentages of AV+/propidium iodide− (AV+/PI–), AV+/PI+, and
AV–/PI+ cells were found in the culture supplemented with autologous serum than with human albumin. The pha−
gocytosis of FITC−dextran particles by DCs was higher in the cultures supplemented with autologous or allogenic
serum than with human albumin. The percentages of CD83+/CD1a+/HLA−DR+ and CD83+/CD1a–/HLA−DR+ cells
were lower in human albumin−supplemented than in allogenic serum−supplemented culture.
Conclusions. These data suggest the possibility of generating autologous DCs from PBMCs of MM patients in me−
dium supplemented with different kinds of serum supplementation. Of note, DC generation progressed in different
ways in the examined microenvironments, but supplementation of the cultures with human albumin resulted in the
highest percentage of apoptotic cells (Adv Clin Exp Med 2009, 18, 3, 249–259).
Key words: dendritic cells, multiple myeloma, phagocytic activity, immunophenotype, cell culture.
Streszczenie
Wprowadzenie. Metody pozyskiwania komórek dendrytycznych ex vivo są szeroko opisywane w światowej litera−
turze naukowej, wciąż nie ma jednak standardowego protokołu uzyskiwania tych komórek w warunkach in vitro.
* This study was supported by the State Committee for Scientific Research (KBN, Warsaw, Poland), grant no. 2 PO5B 120 27.
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Cel pracy. Zbadanie możliwości indukcji autologicznych komórek dendrytycznych od pacjentów ze szpiczakiem
plazmocytowym oraz porównanie skuteczności tej indukcji w różnych środowiskach hodowlanych.
Materiały i metody. Komórki mononuklearne izolowano z krwi obwodowej pacjentów ze szpiczakiem plazmo−
cytowym i hodowano w pożywce RPMI wzbogaconej interleukiną 4 (IL−4) i czynnikiem stymulującym tworzenie
kolonii granulocytów i makrofagów (GM−CSF) z dodatkiem następujących suplementów białka: 2% ludzkiej al−
buminy, 10% autologicznej surowicy lub 10% allogenicznej surowicy od zdrowych dawców. Dojrzałość komórek
dendrytycznych była wywołana czynnikiem martwicy nowotworów α (TNF−α). Oceniano następujące wskaźniki:
apoptozę komórek podczas hodowli DCs, zdolność do fagocytozy oraz immunofenotyp wygenerowanych autolo−
gicznych komórek dendrytycznych. Oceniono ponadto stężenie IL−6, TGF−β, VEGF i IL−10 w surowicy pacjen−
tów ze szpiczakiem plazmocytowym.
Wyniki. Odsetek komórek CD1a+/CD14– w hodowli z dodatkiem ludzkiej albuminy był istotnie wyższy w porów−
naniu z hodowlą z dodatkiem autologicznej lub allogenicznej surowicy. Odsetek komórek wykazujących ekspre−
sję fosfatydyloseryny (Annexyna V+, AV+) wśród populacji komórek CD1a+/CD14+ oraz komórek CD1a+/CD14–
był ponadto istotnie wyższy w hodowli z dodatkiem ludzkiej albuminy. Zaobserwowano również istotnie niższy
odsetek komórek AV+/PI–, AV+/PI+ i AV–/PI+ w hodowli wzbogaconej autologiczną surowicą w porównaniu do ho−
dowli stymulowanej ludzką albuminą. Komórki dendrytyczne generowane w hodowli wzbogaconej autologiczną
lub allogeniczną surowicą wykazywały większą zdolność fagocytozy cząsteczek dextranu−FITC w porównaniu
z komórkami dendrytycznymi generowanych w hodowlach z ludzką albuminą. Odsetek komórek
CD83+/CD1a+/HLA−DR+ i CD83+/CD1a–/HLA−DR+ był niższy w hodowlach wzbogaconych ludzką albuminą
w porównaniu z odsetkiem tych komórek uzyskanych w hodowlach stymulowanych allogeniczną surowicą.
Wnioski. Istnieje możliwość generowania autologicznych komórek dendrytycznych z PBMCs chorych na szpicza−
ka plazmocytowego w podłożach uzupełnionych auto−, allogeniczną surowicą lub ludzką albuminą. Należy jednak
zauważyć, że w każdym z tych podłoży proces generowania komórek dendrytycznych przebiega inaczej, a doda−
tek ludzkiej albuminy do podłoża hodowlanego jest związany z najwyższym odsetkiem komórek apoptotycznych
(Adv Clin Exp Med 2009, 18, 3, 249–259).
Słowa kluczowe: komórki dendrytyczne, szpiczak plazmocytowy, aktywność fagocytarna, immunofenotyp, ho−
dowle komórkowe.
Multiple myeloma (MM) is a B−cell malignan−
cy characterized by the expansion of plasma cells
and late B cells that are clonally committed to the
production and secretion of specific immunoglo−
bulin, which could be considered as tumor−asso−
ciated antigen [1, 2]. Many cytokines are involved
in the proliferation and differentiation of myeloma
cells. Interleukin 6 (IL−6) and transforming growth
factor β (TGF−β) are widely recognized as the ma−
jor cell−growth factors [1–3]. High concentrations
of these cytokines correlate with an aggressive co−
urse of disease and poor prognosis [3].
Dendritic cells (DCs) are a distinctive popula−
tion of leukocytes, but they are primarily known as
professional antigen−presenting cells (APCs)
[4–6]. Among other APCs, such as macrophages
and B cells, DCs are considered to have a key
function in generating primary and secondary im−
mune responses, maintaining immunological me−
mory, and having the exceptional ability to stimu−
late naïve T cells [4, 7]. They are believed to play
a pivotal role in antitumor immunity by taking
a tumor antigen and stimulating antigen-specific
T cells in a “cross-priming” fashion [5, 6]. This
suggests that adequate function of the DC system
is essential for the development of antitumor immunity [7]. On the other hand, a growing body of
evidence suggests that autologous DCs in a cancer-bearing host are not fully competent [8–12]. In
clinical cell-based cancer vaccine approaches,
DCs have also been used as a source of adjuvants
required to induce immunity against tumor-specific or tumor-selective antigens [13–17]. DC-based
cancer vaccines have been studied so far in patients with melanoma, myeloma, and prostate, renal, breast, ovarian, and gastrointestitial cancer
[15, 18]. One major focus has been on efficient ex
vivo generation of dendritic cells with the potential
to induce immune responses [18]. The in vitro DC
culture method is rather simple, but it commonly
needs the support of protein supplementation.
With regard to clinical application, the appropriate
choice of culture medium as well as potential serum supplements are important issues.
The aim of the present study was to verify the
ability to generate autologous dendritic cells from
peripheral blood mononuclear cells (PBMCs) of
multiple myeloma patients with different serum
supplementations. A culture was prepared with ad−
dition of 10% autologous serum to create a micro−
environment resembling the in vivo appearance.
DCs were also generated in medium supplemented
with 2% human albumin to avoid the possible ef−
fect of active tumor−derived factors. Additionally,
dendritic cells were generated with healthy human
serum to establish the influence of physiological
serum on DC cultures. The methodologies descri−
bed in here would be conducive to improve the
methods of generating autologous DCs from
PBMCs of multiple myeloma patients.
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DCs Generation from MM Patients
Material and Methods
Characteristics of the Multiple
Myeloma Patients
The study population comprised 15 newly dia−
gnosed MM patients (mean age: 65 ± 9.4 years).
The evaluation of the stage and the type of disease
manifestation was based on laboratory tests, bone
marrow biopsy, and bone X−ray examinations.
Preparation
of Autologous Serum
Autologous serum was obtained by centrifu−
gation (3000 rpm, 10 min) of peripheral blood
samples taken from each patient. After centrifuga−
tion, the serum was frozen in aliquots (10 aliquots
for 1 ml, 4 aliquots for 200 µl) at –80°C and successively thawed when examined. Allogenic serum obtained from 15 healthy blood donors (only
AB0) served as a control. The serum was used for
culture medium supplementation and for cytokine
measurement using ELISA.
Generation of Dendritic Cells
Peripheral blood was drawn from the MM pa−
tients into heparinized tubes according to the pro−
tocols accepted by the Local Ethics Committee.
PBMCs were isolated by density gradient centrifu−
gation (20 min 700 × g; Lymphoprep, Nycomed,
Norway). After isolation the PBMCs were resuspended in RPMI 1640 (BioWhittaker, Belgium)
medium supplemented with penicillin-streptomycin (1% v/v; Sigma, Germany), seeded in six-well
tissue culture plates, and left to adhere for 1.5 h at
37°C in a humidified 5% CO2 atmosphere. The
culture systems of the patients’ PBMCs carried out
were: 1) human albumin−supplemented culture: in
medium containing 2% human albumin, 2) autolo−
gous serum−supplemented culture: in medium con−
taining 10% autologous serum, and 3) allogenic
serum−supplemented culture: in medium contai−
ning 10% allogenic serum from the healthy do−
nors. After the adherence time, non−adherent cells
were removed and the culture plates were washed
by cold PBS (phosphate−buffered saline) without
Ca2+ and Mg2+ (Biochrom AG, Germany) to obta−
in a pure fraction of adherent CD14+ monocytes.
Adherent cells were then cultured in an adequate
medium supplemented with rhIL−4 (500 IU/ml;
Strathmann, Germany) and rhGM−CSF (1000 IU/ml;
Gentaur, Belgium).
The cells were fed every 3 days. On day 5, re−
combinant human tumor necrosis factor α (TNF−α,
50 ng/ml; Strathmann, Germany) was added to in−
duce terminal maturation of the generated DCs.
On day 7, at the end of the culture, the superna−
tants were collected and the adherent cells were
harvested by incubation with trypsin/EDTA solu−
tion (0.25%/0.02%; Biochrom AG, Germany),
washed in PBS without Ca2+ and Mg2+, and resu−
spended prior to immunophenotyping. The yield
of cultured DCs was 5–6% of the initial PBMCs
counts.
Phagocytosis Assay
To measure the phagocytic activity of the ge−
nerated DCs, 5−day cultures in the aforementioned
systems were also carried. Briefly, at the end of
culture the generated immature DCs were resu−
spended in RPMI 1640 and incubated with
FITC−dextran (ICN, Germany) at a concentration
of 1 mg/ml of medium at 37°C for 4 h and 24 h as
well as at 4°C for 4 h (negative controls). Incuba−
tion was stopped by adding cold RPMI 1640. The
cells were washed twice with cold RPMI 1640 and
immediately analyzed by a flow cytometer.
Apoptosis Analysis
During Cell Culture
To quantify the cells undergoing early pro−
grammed cell death, an Annexin V (AV) and pro−
pidium iodide (PI) apoptosis detection kit (R&D,
USA) was utilized according to the manufactu−
re’s protocols. The cells were removed from the
culture plates after 48 and 96 h of culture. Freshly
isolated PBMCs were also examined. The cells
were washed in the binding buffer provided. AV
(FITC−conjugated) and specific monoclonal anti−
bodies (anti−CD14 TC−conjugated and anti−
CD1a PE−conjugated; Caltag, USA) were added.
The cells were incubated for 15 min at room tem−
perature in the dark. PI was added in the last 5 min
of incubation to the tubes containing only AV; then
the cells were analyzed by flow cytometry.
Immunophenotyping
of the Generated Dendritic Cells
To characterize the phenotype of the generated
dendritic cells, a panel of FITC−, PE−, or Cy−chro−
me−conjugated monoclonal antibodies were used:
mouse anti−human CD45/CD14, mouse anti−hu−
man CD83/CD1a/HLA−DR, and mouse anti−hu−
man CD80/CD86/HLA−DR. Isotype controls were
also used. Non−specific staining was avoided by
adding 20 µl of FcR Blocking Reagent (Miltenyi−
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K. WOJAS−KRAWCZYK et al.
Statistical Analysis
Biotec, Germany). The cells were incubated for
20 minutes at 4°C in the dark and then washed in
PBS without Ca2+ and Mg2+.
The data are presented as the mean ± standard
deviation (SD). Statistical analysis was performed
using the Wilcoxon non−parametric test, the non−
parametric Kolmogorow−Smirnow test, the Mann−
whitney U test, and the Spearman correlation test.
A level of p < 0.05 was considered significant.
Flow Cytometric Analysis
Cells were analyzed using a FACSCalibur
flow cytometer (Becton Dickinson) equipped with
a 488−nm argon laser. An acquisition gate was
established based on FSC and SSC that included
only monocyte populations (mononuclear cells
and generated dendritic cells) and excluded lym−
phocytes, dead cells, and debris. After acquisition,
the cells were analyzed with CellQuest Software.
Results
The Influence of Culture
Supplementation on CD1a
and CD14 Antigen Expression
and Cell Apoptosis
β,
ELISA Analysis of IL−6, TGF−β
VEGF, and IL−10 Concentrations
in the Patients’ Serum
Early apoptotic cells were defined as AV+/PI–,
late apoptotic and necrotic cells as AV+/PI+, and
dead cells as AV–/PI+, as originally developed by
Vermes et al. [19]. After 48 h of culture there we−
re significantly (p < 0.05) higher percentages of
CD1a+/CD14+ and CD1a+/CD14– cells in the cul−
ture supplemented with human albumin than in
those with autologous or allogenic serum. There
were no significant differences between the cultu−
res supplemented with autologous and allogenic
serum after 96 h.
The concentrations of the cytokines were me−
asured in the patients’ serum using the commercial
detection kits High−Sensitivity Human IL−6 (Ben−
der, Austria), Human TGF−β (Bender, Austria),
Quantikine Human VEGF (R&D Systems, USA),
and High Sensitivity Human IL−10 ELISA Kit
(Bender, Austria) and an ELX 800 microplate rea−
der (Bio−Tek Instruments, USA).
60
freshly isolated cells
50
percentage of mononuclear cells
odsetek komórek
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autologous serum
p < 0.05
allogenic serum
human albumin
40
p < 0.05
30
20
10
0
AV+/CD1a+/CD14+
A
AV+/CD1a+/CD14+
AV+/CD1a+/CD14−
B
AV+/CD1a+/CD14−
C
+
+
D
+/
–
Fig. 1. Percentage of Annexin−V positive cells in CD1a /CD14 and CD1a CD14 populations in freshly isolated
cells and after 48 h (A, C) and 96 h (B, D) of culture in different microenvironments. Legend:
cells freshly isola−
ted from peripheral blood,
cells generated in culture supplemented with autologous serum,
cells generated in
culture supplemented with allogenic serum,
cells generated in culture supplemented with human albumin
Ryc. 1. Odsetek komórek Aneksyna−V−pozytywnych w grupie komórek CD1a+/CD14+ oraz w grupie CD1a+/CD14–
po 48−godzinnej (A, C) i 96−godzinnej (B, D) hodowli w różnych środowiskach hodowlanych oraz wśród świeżo izo−
lowanych PBMC. Legenda:
komórki świeżo izolowane z krwi obwodowej,
komórki hodowane w podłożu
z dodatkiem autologicznej surowicy,
komórki hodowane w podłożu z dodatkiem allogenicznej surowicy,
ko−
mórki hodowane w podłożu z dodatkiem ludzkiej albuminy
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DCs Generation from MM Patients
50
45
percentage of mononuclear cells
odsetek komórek
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freshly isolated cells
autologous serum
allogenic serum
human albumin
40
35
p < 0.05
30
25
20
15
p < 0.05
p < 0.05
10
5
0
AV+/PI−
A
AV+/PI+
B
AV−/PI+
C
AV+/PI−
D
AV+/PI+
AV−/PI+
E
F
Fig. 2. Percentages of apoptotic, late apoptotic, and necrotic cells in freshly isolated cells and after 48 h (A, B, C)
and 96 h (D, E, F) of culture in different microenvironments. Legend:
cells freshly isolated from peripheral blood,
cells generated in culture supplemented with autologous serum,
cells generated in culture supplemented with
allogenic serum,
cells generated in culture supplemented with human albumin
Ryc. 2. Odsetek komórek apoptotycznych, późnoapoptotycznych i martwych wśród świeżo izolowanych PBMC oraz
po 48−godzinnej (A, B, C) i 96−godzinnej (D, E, F) hodowli w różnych podłożach hodowlanych. Legenda:
komór−
ki świeżo izolowane z krwi obwodowej,
komórki hodowane w podłożu z dodatkiem autologicznej surowicy,
komórki hodowane w podłożu z dodatkiem allogenicznej surowicy,
komórki hodowane w podłożu z dodatkiem
ludzkiej albuminy
Table 1. Mean fluorescence intensity of freshly isolated CD1a−positive and CD14−positive cells and after 48 h and 96 h of
culture supplemented with autologous serum, allogenic serum, or human albumin
Tabela 1. Średnia intensywność fluorescencji świeżo izolowanych komórek CD1a−pozytywnych i CD14−pozytywnych
oraz komórek po 48− i 96−godzinnych hodowlach z dodatkiem: autologicznej surowicy, allogenicznej surowicy lub ludz−
kiej albuminy
Mean fluore−
scence intensity
(Średnia
intensywność
fluorescencji)
Freshly isolated cells
(Świeżo izolowane
PBMC)
Autologous serum
(Autologiczna
surowica)
48 h
96 h
48 h
96 h
48 h
96 h
MFI CD14
463.65 ± 129.71
211.27
± 146.91
631.2
± 406.92
133.29
± 69.5
337.46
± 186.57
171.42
± 125.79
460.26
± 326.63
MFI CD1a
74.47 ± 42.12
128.62
± 50.32
245.1
± 97.76
118.34
± 32.65
205.27
± 60.74
126.52
± 51.47
245.57
± 66.59
After 48 h of culture, the percentages of AV+
cells in the populations of CD1a+/CD14+ and
CD1a+/CD14– cells were significantly (p < 0.05)
higher in human albumin−supplemented culture
than in the allogenic and autologous serum supple−
mented cultures (Fig. 1). Significantly (p < 0.05)
lower percentages of AV+/PI–,, AV+/PI+, and
AV–/PI+ cells were also noted in the culture supple−
mented with autologous serum than in that supple−
mented with human albumin (Fig. 2).
The mean fluorescence intensity of freshly
isolated CD1a+ and CD14+ cells and after 48 h and
Allogenic serum
(Allogeniczna
surowica)
Human albumin
(Ludzka albumina)
96 h of culture supplemented with autologous se−
rum, allogenic serum, and human albumin are pre−
sented in Table 1.
Phagocytic Activity by
Generated Dendritic Cells
To test the phagocytic function of the immatu−
re DCs generated in the above culture systems,
FITC−dextran uptake by dendritic cells was evalu−
ated using flow cytometry. The results are presen−
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K. WOJAS−KRAWCZYK et al.
Table 2. Mean fluorescence intensity of FITC−dextran−positive cells generated in cultures supplemented with 10% autolo−
gous serum, 10% allogenic serum, or 2% human albumin
Tabela 2. Średnia intensywność fluorescencji FITC−dextran pozytywnych komórek dendrytycznych generowanych
w hodowlach z dodatkiem: 10% autologicznej surowicy, 10% allogenicznej surowicy lub 2% ludzkiej albuminy
Culture microenvironment
(Środowisko hodowlane)
Cells incubated for 4 h
at 4°C – control probes –
(Komórki inkubowane 4h
w 4°C – panel kontrolny)
Cells incubated for 4 h
at 37°C
(Komórki inkubowane
4 h w 37°C)
Cells incubated for 24 h
at 37°C
(Komórki inkubowane 24 h
w 37°C)
10% autologous serum
(10% autologiczna surowica)
14.37 ± 9.0 MFI
27.56 ± 11.6 MFI
36.08 ± 17.8 MFI
10% allogenic serum
(10% allogeniczna surowica)
14.99 ± 12.41 MFI
2% human albumin
(2% ludzka albumina)
11.72 ± 8.67 MFI
ted as the mean fluorescence intensity (MFI) of
FITC−dextran−positive cells and are shown in Ta−
ble 2 and an example of the phagocytosis in a re−
presentative patient is presented in Figure 3. In all
the cultures a significantly (p < 0.001) higher
expression of FITC−dextran was found in cultured
cells after 4 h of incubation at 37°C than in control
cells incubated at 4°C. Phagocytosis of FITC−de−
xtran after 4 h of incubation at 37°C was also si−
gnificantly (p < 0.05) higher in the cultures supple−
mented with autologous or allogenic serum than in
the cultures with human albumin. Of note, the
cells generated with autologous or allogenic serum
showed slightly lower FITC−dextran expression
after 24 h of incubation than the control cells. Only
in the 24−hour culture with human albumin was
the phagocytosis of FITC−dextran particles insi−
gnificantly higher in the cells incubated at
37°C than in the cells of the control.
Immunophenotype of Generated
Dendritic Cells
The generated dendritic cells were divided in−
to two groups according to leukocyte−monocyte−
specific marker expression, i.e. double−positive
cells (CD45+/CD14+), which did not lose their mo−
nocytic attribute (CD14 antigen) during culture,
and CD45+/CD14– cells, which appeared to lose
CD14 antigen and turn into dendritic cells. There
was a significantly (p < 0.05) higher percentage of
CD45+/CD14+ cells and a significantly (p < 0.01)
lower percentage of CD45+/CD14– cells in the cul−
ture with human albumin than that with allogenic
serum. Moreover, in the culture supplemented with
allogenic serum there was a significantly (p < 0.01)
higher percentage of CD45+/CD14– cells than in
the culture supplemented with autologous serum.
There was also significantly (p < 0.01) higher
CD14 expression in the culture supplemented with
24 ± 12.12 MFI
19.82 ± 9.04 MFI
36.03 ± 18.94 MFI
23.78 ± 11.33 MFI
autologous serum than in that with human albu−
min.
According to the expressions of CD1a and
CD83 antigen we recognized semi−mature dendri−
tic cells as CD83+/CD1a+/HLA−DR+ and mature
dendritic cells as CD83+/CD1a–/HLA−DR+ cells
[20, 21]. CD83 antigen was considered a marker
of mature and CD1a of immature DCs. The per−
centage of semi−mature DCs was significantly
(p < 0.05) lower in the culture supplemented with
2% human albumin than in the culture with alloge−
nic serum. Moreover, there was a significantly
(p < 0.05) higher percentage of CD83+/CD1a–/
/HLA−DR+ cells in the culture supplemented with al−
logenic serum than in that with autologous serum.
The generated dendritic cells were also divi−
ded according to the expression of co−stimulatory
molecules into CD80+/CD86+/HLA−DR+ and
CD80+/CD86–/HLA−DR+ cells [22]. There was
a significantly (p < 0.01) higher percentage of
CD80+/CD86+/HLA−DR+ cells in the culture sup−
plemented with human albumin than in those with
autologous serum or allogenic serum. The immu−
nophenotype analyses of the generated dendritic
cells are presented in Table 3.
β
IL−6, IL−10, VEGF, and TGF−β
Concentrations
in the Patients’ Serum
There was a significantly (p < 0.01) higher
concentration of IL−6 in patients’ serum than in the
allogenic serum from healthy donors. The IL−10,
VEGF, and TGF−β concentrations were similar in
the patients and healthy donors. The concentra−
tions of the cytokines are presented in Table 4.
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DCs Generation from MM Patients
R1
R1
R1
R3
R1
Fig. 3. Phagocytosis of FITC−dextran particles by cells generated in cultures supplemented with:
I – 10% autologous serum; II – 10% allogenic serum; III − 2% human albumin. — cells incubated
4 h at 4°C; — cells incubated 4 h at 37°C; — cells incubated 24 h at 37°C
Ryc. 3. Pochłanianie cząsteczek FITC−dekstranu przez komórki generowane w następujących środo−
wiskach hodowlanych: I – 10% autologicznej surowicy; II – 10% allogenicznej surowicy; III – 2%
ludzkiej albuminie. — komórki inkubowane 4 h w 4°C; — komórki inkubowane 4 h w 37°C;
— komórki inkubowane 24 h w 37°C
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K. WOJAS−KRAWCZYK et al.
Correlation Between Cytokine
Concentration in Patients’
Serum and the DC
Immunophenotype
There was a negative correlation (R = −0.64,
p < 0.05) between the concentration of TGF−β in
the patients’ serum and CD83 expression (Fig. 4).
The percentages of CD83–/CD1a+ cells generated
in the culture supplemented with autologous se−
rum negatively correlated with TGF−β concentra−
tion. Moreover, the percentages of CD83–/CD1a+
cells significantly negatively correlated (R = −0.88,
p < 0.01) with VEGF concentration. No significant
correlation between the concentrations of cytoki−
nes and the immunophenotype of the generated
dendritic cells was found.
Discussion
During the last decade, several protocols re−
garding in vitro dendritic cell generation have be−
ing described [23, 24]. Monocyte−derived dendri−
tic cells are becoming widely used as cell vaccines
for cancer treatment, but this procedure should be
optimized and standardized [23, 24]. In many cli−
nical trials, autologous plasma, fetal calf serum
(FCS), allogenic AB plasma, or human albumin
were used as serum or protein components to sup−
port dendritic cell generation [23, 25, 26]. The use
of FCS in cultures of dendritic cells precludes the−
ir practical application because FCS contains xe−
nogenic proteins which may affect DC function. It
has been proposed that human albumin is a kind of
non−antigenic culture substrate and provides the
clearest culture microenvironment, but one that is
destitute of natural growth factors [25]. These li−
mitations suggest that autologous plasma or serum
would be a less hazardous choice for DC genera−
tion. On the other hand, the active tumor−derived
factors in patients’ serum could impair monocytes,
which might result in their abrogated differentia−
tion into DCs in vitro [27, 28]. The present study
tried to verify the ability to generate DCs from
MM−bearing patients in different culture microe−
nvironments.
It is known that the induction of apoptosis
occurs not only by direct contact with tumor cells [29].
It has been demonstrated that murine DCs treated
with supernatants from tumor cell lines undergo
apoptosis [29, 30]. In the present study it was found
that the highest percentages of apoptotic, late apop−
totic, and dead cells were in the cultures supple−
mented with human albumin. Unexpectedly, the
percentages of apoptotic cells were lowest in the
autologous serum−supplemented cultures. This
suggests that autologous serum contains factors
which are crucial for the differentiation and gene−
ration of dendritic cells as well as for maintaining
their viability during culture. On the other hand, as
mentioned above, human albumin is the clearest
microenvironment for DC generation, but it seems
to be devoid of important cell viability factors [25].
Araki generated efficient DCs from CD14+ mono−
cytes of healthy volunteers in the presence of hu−
man albumin [25]. Moreover, the serum of cancer−
bearing patients could contain factors which po−
ssess a strong inhibitory capacity [12, 27].
However, Marten et al. showed strong stimulatory
activity of DCs generated with patient−derived
CA−19−9−antigen−containing serum [31]. The pre−
sent results rather argue against the use of human
albumin in efficient dendritic cell generation.
It is known that during their differentiation in−
to dendritic cells, monocytes lose their CD14 anti−
gen expression and turn into a CD1a+/CD14– cell
population [24, 25]. In the present experiment a si−
gnificantly decreasing expression of CD14 antigen
was found in all the cultures, but most of all in the
human albumin−supplemented ones. Therefore, the
expression of CD14 antigen was lower only in the
cells cultured for 96 h with allogenic serum than in
the freshly isolated cells. This suggests that supple−
mentation of DC cultures with allogenic serum cre−
ates the best condition for the differentiation of mo−
nocytes into DCs. Moreover, the highest percenta−
ge of CD45+/CD14+ double−positive cells
simultaneously with the lowest percentage of
CD45+/CD14– cells was found in cultures supple−
mented with autologous serum. It is widely known
that many cytokines or immunosuppressive cancer−
derived factors detected in the serum of cancer pa−
tients interfere in many ways with each stage of the
generation of dendritic cells [8, 9, 10, 27]. The pre−
sent authors suppose that serum from multiple my−
eloma patients could contain some factors which
disturb autologous dendritic cell differentiation and
which restrain the loss of CD14 expression.
Numerous clinical observations have demon−
strated that dendritic cells generated from tumor−
bearing host are defective [8–11, 32]. Ratta et al.
generated DCs from CD34+ cells from multiple
myeloma patients and showed reduced phagocytic
ability as well as impaired stimulatory function by
the generated autologous dendritic cells [32]. Mo−
reover, Brown et al. found a decreased percentage
of immature dendritic cells in the peripheral blood
of MM patients and also observed a lower expres−
sion of CD80 antigen on generated DCs from MM
patients compared with healthy donors [33].
Interestingly, the present study found the hi−
ghest percentages of CD83+/CD1a+/HLA−DR+ and
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DCs Generation from MM Patients
Table 3. Immunophenotype analysis of generated dendritic cells in cultures supplemented with 10% autologous serum, 10%
allogenic serum, or 2% human albumin after 7 days of culture
Tabela 3. Immunofenotyp komórek dendrytycznych uzyskanych po 7−dniowej hodowli z dodatkiem: 10% autologicznej
surowicy, 10% allogenicznej surowicy lub 2% ludzkiej albuminy
Immunophenotype of DCs – %
(Immunofenotyp DCs – %)
10% autologous serum
(10% autologiczna surowica)
10% allogenic serum
(10% allogeniczna surowica)
2% human albumin
(2% ludzka albumina)
CD45+/CD14−
74.9 ± 19.8
89.22 ± 7.14
CD45+/CD14+
20.3 ± 21.5
7.61 ± 5.6
CD83+/CD1a+
59.3 ± 30.44
58.45 ± 32.85
58.8 ± 27.9
98.3 ± 1.9
94.7 ± 6.24
38.35 ± 34.23
39.18 ± 27.15
HLA−DR+ cells amongst
CD83+/CD1a+ DCs
CD83+/CD1a−
HLA−DR+ cells amongst
CD83+/CD1a−
97.55 ± 2.7
38.2 ± 30.42
89.24 ± 8.5
CD80+/CD86+
73.9 ± 25.73
HLA−DR+ cells amongst
CD80+/CD86+
76.1 ± 26.8
76.5 ± 17.45
15.2 ± 10
95.82 ± 3.7
93.1 ± 9.53
81.19 ± 22.83
73.8 ± 20.4
79.1 ± 19.44
CD80+/CD86−
22.34 ± 24.4
16.94 ± 23.1
HLA−DR+ cells amongst
CD80+/CD86−
77.93 ± 16.4
79.46 ± 23.98
89.6 ± 11.65
23.2 ± 20.1
78.75 ± 23.6
Table 4. The concentrations of the examined cytokines in allogenic and autologous serum
Tabela 4. Stężenie badanych cytokin w allogenicznej i autologicznej surowicy
IL−6 (pg/ml)
IL−10 (pg/ml)
VEGF (pg/ml)
TGF−β1 (ng/ml)
Allogenic serum from
healthy donors
(Allogeniczna surowica
od zdrowych dawców)
0.15 ± 0.16*
2.96 ± 2.18
37.75 ± 12.68
5.32 ± 6.11
Autologous serum from
MM patients
(Autologiczna surowica
pacjentów ze szpiczakiem
plazmocytowym)
1.32 ± 1.32*
28.25 ± 40.03
42.89 ± 13.98
6.64 ± 6.62
*p < 0.01.
CD83+/CD1a–/HLA−DR+ cells in the culture sup−
plemented with allogenic serum compared with
those with human albumin and autologous serum.
Together with the results regarding the course of
apoptosis, this could support the thesis about the
deficiency of some essential factors in cultures sup−
plemented with human albumin. In addition, the
expression of FITC−dextran particles was also lo−
west in dendritic cells generated in human albu−
min−supplemented cultures. It is supposed that au−
tologous serum, even from cancer patients, could
not restrain the generation of dendritic cells. Ratta
et al. investigated the influence of specific factors
on dendritic cell generation; they generated fully
competent DCs from MM patients in medium sup−
plemented with antibodies against IL−6 [32].
Brown et al. also generated dendritic cells from
MM patients in medium supplemented with autolo−
gous serum and found that the autologous dendritic
cells possessed lower expression of CD80 and
CD86 despite the addition of antibodies against
TGF−β [33]. In the present study it was found that
only TGF−β in patients’ serum significantly negati−
vely correlated with CD83 expression. It seems
that TGF−β is a cytokine which inhibits dendritic
cell maturation. However, it is supposed that not
only TGF−β was responsible for the difference in
DC generation, but the interaction and connection
of all the cytokines and factors in both allogenic
and autologous serum, just the opposite to human
albumin. Especially since serum from healthy do−
nors consists of many effective immunological fac−
tors, a positive effect of mixed serum from healthy
donors on DC generation should be discussed [34].
The present study on culture microenviron−
ment rather argues against using human albumin
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K. WOJAS−KRAWCZYK et al.
Fig. 4. Negative correlation between
the concentration of TGF−β in patients’
serum and CD83 expression on mature
dendritic cells generated in cultures
supplemented with 10% autologous
serum
Ryc. 4. Ujemna korelacja między stę−
żeniem TGF−β w surowicy pacjentów
a ekspresją cząsteczki CD83 na ko−
mórkach dendrytycznych wygenerowa−
nych w hodowlach z dodatkiem 10%
autologicznej surowicy
as a protein supplementation, as it lacks specific
growth factors essential for dendritic cell diffe−
rentiation. This would suggest the superiority of
allogenic or autologous serum or commercially
available medium for protein supplementation of
culture medium. However, there are some disa−
dvantages of this method, including nonspecific
immune stimulation by proteins in allogenic se−
rum. Also, the efficacy of DC generation from
the PBMCs of MM patients should be tested
using serum−free media, such as CellGro DC.
Taken together, the presented study underlines
the necessity of determining the best culture
environment for the generation of functional
dendritic cells and further studies should be
undertaken.
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Address for correspondence:
Kamila Wojas−Krawczyk
Department of Pneumonology, Oncology, and Allergology
Medical University of Lublin
Jaczewskiego 8
20−954 Lublin
Poland
Tel.: +48 81 724 42 93
E−mail: [email protected]
Conflict of interest: None declared
Received: 26.01.2009
Revised: 4.05.2009
Accepted: 19.06.2009
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