Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy

Monitoring and isolation of blood dendritic cells from

apheresis products in healthy individuals: a platform

for cancer immunotherapy

J. Alejandro Lopez a, Georgina Crosbie a, Cathryn Kelly b, Ann Marie McGee a,Katrina Williams b, Slavica Vuckovic a, Robert Schuyler c, Robyn Rodwell b,

Sue J. Wright b, Kerry Taylor b, Derek N.J. Hart a,*

aMater Medical Research Institute, Aubigny Place, South Brisbane 4101, AustraliabMater Adult Hospital, Raymond Terrace, South Brisbane 4101, Australia

cGambro BCT, Lakewood, CO 80215, USA

Received 25 January 2002; received in revised form 19 April 2002; accepted 21 May 2002

Abstract

The fundamental role of dendritic cells (DC) in initiating and directing the primary immune response is well established.

Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and

infectious diseases. As blood DC (BDC) physiology differs from that of the DC homologues generated in vitro from monocyte

precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation

of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this

study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential

procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package

(Version 6.0 AutoPBPC) provides an operator-independent reliable source of mononuclear cells (MNC) for BDC preparation.

Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating

BDC predicted by blood volume. An average of 66 million (range, 17–179) BDC per 10-l procedure were obtained, largely

satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 15 l. BDC were not

activated by the isolation procedure and, more importantly, both BDC subsets (CD11c+CD123low and CD11c�CD123high) were

equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection

and demonstrated that fully functional BDC can be obtained by this procedure.

D 2002 Published by Elsevier Science B.V.

Keywords: Immunotherapy; Dendritic cells; Apheresis; Isolation

0022-1759/02/$ - see front matter D 2002 Published by Elsevier Science B.V.

PII: S0022 -1759 (02 )00185 -0

Abbreviations: APC, antigen-presenting cell; BDC, Blood DC; DC, dendritic cells; MNC, mononuclear cells; Mo-DC, monocyte-derived

DC; PBMC, peripheral blood mononuclear cells; SCF, stem cell factor; TBV, total blood volume.* Corresponding author. Tel.: +61-7-3840-2557; fax: +61-7-3840-2550.

E-mail address: [email protected] (D.N.J. Hart).

www.elsevier.com/locate/jim

Journal of Immunological Methods 267 (2002) 199–212

1. Introduction

Dendritic cells (DC) due to their unique properties

as initiators and modulators of the immune system

have become the focus of studies for the development

of vaccination strategies for malignant disease (Hart

and Hill, 1999; Fong and Engleman, 2000). Emerging

data from Phase I and Phase II studies show very

promising results (Hsu et al., 1996; Nestle et al., 1997;

Murphy et al., 1999; Geiger et al., 2000; Kugler et al.,

2000). However, there is a lack of consensus with

respect to the methods of DC isolation, the source cell

population for derivation of DC, the need for and

influence of exogenous cytokine stimulation and the

effects of the isolation strategy on the DC product

(Nestle et al., 2001). It is also now important to

employ Good Manufacturing Practice principles to

ensure consistency of the DC product and compliance

with an increasingly stringent regulatory environment.

DC plays a fundamental role both in innate and

cognate immune responses (Hart, 1997; Clark et al.,

2000). They circulate in blood as precursors with an

intermediate differentiation/activation phenotype and

represent a small (less than 1%) proportion of blood

mononuclear cells (MNC). DC moves from the cir-

culation into the tissues, where they acquire a fully

mature antigen-presenting cell (APC) phenotype after

interaction with pathogens or other stimuli. This is

characterized by the expression of key surface mole-

cules such as CD40, CD80 and CD86, which are

involved in costimulation as well as other markers of

DC differentiation/activation (Banchereau et al., 2000;

Hart et al., 2000). Various subsets of DC have been

described to date and they appear to have different

physiological functions (Rissoan et al., 1999); they are

defined by the expression of the CD123 (IL-3 recep-

tor) and the CD11c antigens (Olweus et al., 1997;

Robinson et al., 1999).

Various approaches have been used to achieve DC

isolation resulting in differences in the properties of the

generated DC preparations (Ferlazzo et al., 1999). DC-

like cells can be produced via transformation of blood

monocytes in vitro in the presence of various cytokines

and are referred to as monocyte-derived DC (Mo-DC)

(Romani et al., 1994; Sallusto and Lanzavecchia,

1994); additionally, DC can be expanded from

CD34+ progenitors obtained from bone marrow (Reid

et al., 1992; Egner et al., 1993; Szabolcs et al., 1995;

Young et al., 1995), peripheral blood (Mackensen et

al., 1995; Siena et al., 1995; Strunk et al., 1996; Monji

et al., in press) or neonatal cord blood (Caux et al.,

1992, 1996). Alternatively, DC and their precursors

present in the blood (blood dendritic cells, BDC) can

be enriched using density gradients (Mehta-Damani et

al., 1994; McLellan et al., 1995) and selected further

using monoclonal antibodies (Fearnley et al., 1997;

Schakel et al., 1998; Dzionek et al., 2000). This latter

approach encompasses potential theoretical advan-

tages over cytokine-mediated DC in as much as it

yields homogenous DC in a defined state of differ-

entiation, capable of responding to physiological stim-

uli and free from the influence of exogenous cytokines

(Hart and Hill, 1999). We have observed differences

between BDC and Mo-DC in their expression of

surface markers such as DC-SIGN, DEC-205 and the

mannose receptor (Hart et al., 2000; Kato et al., 2000).

More importantly, BDC appeared to be more efficient

in the induction of in vitro primary immune responses

(Osugi et al., in press. Blood, 2002), as well as in vitro

antigen uptake and processing (Ho et al., 2002).

Apheresis products have been used previously as a

source of BDC for cancer immunotherapy (Fong et

al., 2001), but no detailed studies on BDC phenotype

and yields have been published to date. Furthermore,

as the phenotype and function (e.g. adhesion mole-

cules and chemokine receptors) of BDC can be easily

modulated, it is important to investigate the effect of

apheresis on BDC phenotype and function. We have

demonstrated previously that BDC numbers can be

rapidly increased as a result of external modulators

such as surgical and physical stress (Ho et al., 2001)

and it is possible that BDC might ‘‘mobilize’’ during

the procedure.

Given the potential advantages of BDC, we have

developed a method for the generation of DC from the

mononuclear cell apheresis product via a single mag-

netic bead separation procedure using the monoclonal

antibodies CMRF-44 (Hock et al., 1994; Fearnley et

al., 1997) and 56 (Hock et al., 1999). Further descrip-

tion of the kinetics of CMRF-44/56 expression on

BDL and their purification will be provided elsewhere

(Lopez et al., submitted JIM, 2002). In order to

improve BDC yields and maximize their use in cancer

vaccination, it is essential to determine optimal aphe-

resis conditions. We describe a clinically applicable

technique using apheresis of mononuclear cells from

J. Alejandro Lopez et al. / Journal of Immunological Methods 267 (2002) 199–212200

unstimulated healthy donors for the purification of

BDC from the apheresis product via a single magnetic

bead separation procedure.

2. Materials and methods

2.1. Study population

The Mater Misericordiae Health Services Ethics

Committee approved the study. Healthy volunteers

(n=14; 10 males, 4 females), average age 41 (range

21–56), participated in this study. Medical evaluation

and complete blood cell counts were performed prior

to apheresis. No prior cytokine stimulation/mobiliza-

tion was attempted.

2.2. Apheresis isolation software

Two different programs on the Cobe Spectra

(Gambro BCT, Lakewood CO, USA) were compared

for BDC collection. The semi-automated (Version 4.7

software MNC Program) ‘‘manual’’ version, with

variable inlet flow rate from 20 to 150 ml/min allows

a continuous collection of the mononuclear cell

(MNC) layer forming at the interface between the

red cells and the plasma. The product is collected

when the operator judges the interface position to be

optimal based on collect line appearance (judged by

comparison with the Spectra WBC colorgram). The

hematocrit range of the collected products was 3–5%.

This protocol takes into account hematocrit, sex,

height and weight to determine the flow rates

required. The second protocol, the automated (Version

6.1 software, AutoPBPC ) version, accumulates MNC

at the interface position and periodically harvests them

according to the rate of movement of MNC into the

centrifuge channel. Input of subject MNC count,

hematocrit, height, weight and sex enables the Spectra

software to automatically control the collection over

three phases: accumulation, harvest and chase. We

configured the system to harvest 4-ml volumes of

MNC and a platelet-rich plasma chase of 7- or 9-ml

volumes.

Fig. 1. FACS profile of MNC labeled and analyzed for DC. Forward and side scatter profile selected viable cells (left panel) and live cells were

labeled with lineage markers and HLA-DR+. Lin�HLA-DR+ cells (region R2 in middle panel) were analyzed for BDC subsets in dot plots

(right panel).

J. Alejandro Lopez et al. / Journal of Immunological Methods 267 (2002) 199–212 201

2.3. Apheresis sample collection

Peripheral venous blood samples (20 ml) were taken

from a peripheral vein before apheresis (time 0), 30

min, 24 and 48 h after completion of the procedure.

Apheresis was performed in the Mater Hospital Clin-

ical Apheresis Unit. Additionally, at various time

points during the procedure, the products correspond-

ing to total processed blood volumes of 2.5, 5, 7.5, 10,

12.5 and 15 l were collected. Unless specifically stated,

samples were harvested at every planned volume,

except when no product had been collected by the

Fig. 2. Selection of harvesting software protocol. Data corresponding to seven experiments run with the automatic protocol Spectra Version 6.1,

AutoPBPC (shown in open bars, 1, 4, 10, 16, 19, 21 and 22) and four experiments run with the manual protocol Spectra Version 4.7, MNC (shown

in full bars, 2, 3, 9 and 20). (A) Bars show total MNC, volumes, platelets and DC recovered in 10-l apheresis products for each experiment. (B)

BDC efficiency of recovery in repeated experiments performed in three different donors, using the two software options. Placed horizontally,

experiments performed on the same individual with a minimum of 2 weeks interval. Calculated BDC (106) present in TBV before the procedure

(as described in Materials and methods) were compared with the total BDC recovered after apheresis and expressed as total DC recovered.


machine. In some experiments, samples were taken

from the access vein immediately before each product

harvest. ACD-Awas used as anticoagulant at a ratio of

1:12 to the inlet flow. Normal healthy volunteers

participated in the study and they received standard

clinical apheresis care. The Mater Adults Hospital’s

Ethical Committee approved their participation.

2.4. Isolation of peripheral blood mononuclear cells

(PBMC) and labeling of BDC

MNC were isolated by Ficoll–Hypaque (Pharma-

cia, Sweden) density gradient and the interphase cells

washed three times. Cells were labeled in a 50-Almixture of hybridoma supernatants containing anti-

bodies specific for: CD14 (CMRF-31, IgG2a; pro-

duced in this laboratory), CD3 (OKT3, IgG2a,

American Type Tissue Collection, Rockville, MD),

CD16 (HuNK2, IgG2a) and CD19 (FMC63, IgG1),

gifts of Prof H. Zola, Adelaide, Australia. After one

washing step, fluorescein-conjugated sheep–mouse

immunoglobulin-specific (FITC-SAM, Amrad Bio-

tech, Victoria, Australia) was added, followed by

saturating concentrations of mouse immunoglobulin

(Sigma, St. Louis). Finally, PE.Cyanin5-conjugated

HLA-DR specific antibody (Immu375, IgG1, Immu-

notech, Marseille, France) was added together with

either PE-conjugated CD11c (S-HCL-3, IgG2a, BD

Bioscience, BDIS, San Jose, CA) or PE-conjugated

CD123 (a chain specific, 9F5, IgG2b, PharMingen

International, San Diego, CA). IgG1 (MOPC-21),

IgG2a (G155-178) and IgG2b (27-35) isotype con-

trols were purchased from PharMingen. For certain

experiments, the following antibodies were used: PE-

conjugated CD14 (leuM3, IgG2b), CD19 (leuM12,

IgG1) and CD80 (L307.4, IgG1) from BDIS and PE-

conjugated CD40 (5C3, IgG1), CD86 (24F, IgG1,

PharMingen). Labeled samples were analyzed by

FACSCalibur flow cytometer (BDIS).

2.5. DC counts

BDC counts (expressed as 106/l) were calculated

from the number of MNC/L blood (determined by the

automated cell counter Advia 120, Hematology Sys-

tem, Bayer, Tarrytown, NY) multiplied by the percent-

age of BDC (Ho et al., 2001). Alternatively, values

were also expressed as the total content of BDC

calculated on total blood volume (TBV) of every

volunteer.

2.6. DC isolation

The BDC purification procedure will be described

elsewhere (Lopez et al., submitted. JIM, 2002).

Briefly, magnetic bead separation of BDC was under-

taken using biotinylated CMRF-44 (Hock et al., 1994)

antibody and biotin-specific beads (Miltenyi Biotech,

Bergisch Gladbach, Germany). Positive and negative

fractions were collected and evaluated by flow cytom-

etry (level of BDC enrichment) and MLR (function).

For the experiment with frozen cells, apheresis mate-

rial was diluted in ice-cold DMEM medium to 50% of

the total final volume and supplemented with 10%

DMSO and 40% AB pooled serum, stored at �70 jCin an insulated styrofoam container and tested after 2

weeks of storage.

2.7. MLR with isolated BDC

A titration of irradiated MNC (cultured and fresh),

CMRF-44+ magnetically selected BDC or the nega-

tive fraction of the CMRF-44 isolation (5�103–

3�105 cells) was incubated with allogeneic T-lym-

Table 1

Efficiency of recovery of BDC: blood dendritic cells (BDC)

recovered after apheresis expressed as a percentage of total blood

volume (TBV)a

Experiment Software used

Automatic Manual

1 179

2 126

3 50

4 120

9 44

10 23

16 28

19 65

20 65

21 40

22 101

Mean 79.4 71.3

SE 21.6 18.8

a Percentage was established on the basis of the TBV (before

starting the procedure) and the calculated total DC recovered in the

apheresis product.


phocytes (5�104 cells) for 5 days in 96-well U-

bottom plates. Sixteen hours prior to harvesting the

cells, 0.5 ACi of 3H-thymidine was added to each

well. 3H-thymidine uptake was counted in a liquid h-scintillation counter (Wallac, MicroBeta Trilux Scin-

tillation Counter, Turku, Finland).

2.8. Statistical analysis

Paired statistical analysis was performed using the

Student’s two-tailed t-test.

3. Results

3.1. Definition of BDC for counting

Quantification of BDC was performed defining

class II expressing cells, lacking markers for specific

hemopoietic lineages. DC subsets were identified as

CD11c+ or CD123+ cells within the BDC gated

population (Fig. 1). For the purpose of calculations,

BDC were defined as the percentage of lineage

negative HLA-DR high expressing cells.

Fig. 3. Total BDC recovery. (A) Detailed evaluation of BDC present in 10-l (open bars) and 15-l apheresis (hatched) products; the numbers of

BDC (106) present at various harvesting points are shown as independent bars (right axis) and the percentages of BDC as a line (left axis). (B)

Percentage of BDC present in blood before, 0.5, 24 and 48 h after the procedure.


3.2. Compliance with the apheresis procedure

As reported in the literature (Furuta et al., 1999;

Sato et al., 2001), apheresis was safe and well

tolerated. Of the 24 procedures undertaken, 1 was

aborted due to difficult venous access and 1 due to

mild hypocalcaemia symptoms (spontaneously revert-

ing); the rest were well tolerated with only local

discomfort that ceased at the end of the procedure.

Only a small (average 13%) reduction in the platelet

counts was detected at the end of the apheresis (mean

235�109/ml before and mean 201�109/ml after).

3.3. Optimal software for BDC isolation

Ten-liter collections were performed using the

semi-automated (Version 4.7 software MNC Program)

‘‘manual’’ or fully ‘‘automatic’’ (AutoPBSC) software

versions to control the COBE Spectra. A comparison

of the volume, total of MNC recovered, number of

platelets and total BDC recovered in the apheresis

product is shown in Fig. 2A. The automatic software

(n=7) resulted in consistently lower product volumes

(meanFS.E.=automatic: 45F5 ml versus manual:

116F8 ml; p<0.005) and the number of contaminating

platelets was also significantly lower than in the

manual (n=4) collections (automatic: 57F10�109

versus manual: 459F104�109; p<0.05). Although

the total number of MNC recovered from the manual

procedures was, as expected, higher than that obtained

with the automatic software (11.088F1.415�109 and

5.982F1.148�109, respectively; p<0.05), there was

no statistically significant difference in the number of

BDC recovered (automatic: 69F16�106 versus man-

ual: 102F35�106; p=0.34). Furthermore, the effi-

ciency to recover the available circulating BDC

(predicted blood volume�BDC count) was compara-

ble using the two software protocols (automatic:

80F22% versus manual: 71F19%; p=0.77). As

shown in Table 1, the efficiency of recovery differed

Fig. 3 (continued).


considerably between individual experiments possibly

as a reflection of the variations in BDC counts

documented previously (Ho et al., 2001). Three sets

of experiments were performed on the same individ-

uals utilizing automatic and manual software with a

minimum 2-week interval (experiments 1 and 2; 10,19

and 9; 21,22 and 20). In this group of experiments

(Fig. 2B), the automatic software (experiments 1, 10,

19, 21 and 22) produced an average 82% BDC

recovery as opposed to 78% with the manual proce-

dure (experiments 2, 9 and 20). The automatic soft-

ware was considered advantageous because of the

smaller product volume, lower platelet contamination

and reduced operator dependency, appropriate for

multicenter trials.

3.4. Optimal harvesting volume

We next evaluated the recovery of BDC obtained

using the automatic software at different total inlet

volumes. Firstly, we studied four 10-l and five 15-l

Fig. 4. Efficiency of BDC recovery. (A) The total BDC (106) present in the apheresis product from 10-l (open bars) and 15-l (shaded bars)

apheresis. (B) The percentage of the theoretical total BDC available obtained based on TBV is plotted.

Table 2

Activation status of blood dendritic (BDC) before, during and after

apheresis procedure: percentage of BDC

Sample % CD40+ % CD80+ %CD86+

Experiment

2 3 4 2 3 4 2 3 4

Before 7 47.4 25.4 0 4.5 1.1 0.97 38.8 57.5

Product 14 23.5 37.6 0.12 5.0 2.9 1.33 41.5 39.0

After 30 min 6 57.3 22.8 0 3.6 1.2 0.33 34.2 56.7

24 h 11.9 0.5 44.5

48 h 21.2 8.4 0.5 0.3 43.9 62.5

Fig. 5. BDC subset distribution in apheresis product. MeanFS.E. of

the percentage of CD11c+ and CD123+ in blood samples before and

after apheresis procedure (n=6, experiments 1, 2, 3, 4, 9 and 10) is

shown.


procedures in detail. As shown in Fig. 3A, more than

50% of all the BDC recovery took place during the

later part of the procedure; this observation was made

both in the 10-l and the 15-l procedures. The higher

yields towards the end of the procedure resulted from

the combined effect of a slight increase in the

percentage of BDC (Fig. 3A, lines) and an increase

in the MNC in the product (not shown). There were

only minor changes in the percentage of BDC in

blood after the procedure. Although there was not a

defined pattern in all of the experiments, small

decreases in BDC% were detected 30 min after the

end of the procedure with a fast rebound at 24 h in

experiments 6, 8, 13 and 17 (Fig. 3B), i.e. four of

nine procedures.

3.5. Efficiency of BDC recovery

The efficiency of BDC recovery by the automatic

procedure was evaluated further by extending the

number of volunteers. As shown in Fig. 4A, the

efficiency of recovery was variable (n=17; mean

efficiency 73%; range 17–179%) and it reflected

volunteer variations. Recoveries that were greater than

Fig. 6. Isolation of CMRF-44 BDC from apheresis product. Dot plots show FACS analysis of cells before magnetic bead separation (upper

panels), in negative (middle panels) and positive (lower panels) fractions. Percentage of cells present in every quadrant of the dot plot is shown.

Left panels: experiments with freshly obtained apheresis product. Right panels: results obtained with the same cells frozen and thawed after 2

weeks.


100% of the predicted number of BDC in TBV

indicated possible BDC mobilization during the

procedures. Furthermore, the finding reinforced this

observation that higher recoveries were observed at

the latter harvesting points (data not shown). The

yield from the 5 15-l apheresis procedure was

112F21�106 (meanFS.E.), while that of the 12

10-l procedure was 78F17�106 ( p=0.2999). The

efficiency of the 15-l procedures was 97F10% and

that of the 10-l procedure, 66F14% ( p=0.202).

Evaluation of the BDC percentage and BDC counts

in peripheral blood obtained from the access line was

performed at times coinciding with the different

apheresis volume harvests. These remained constant

throughout the procedure (data not shown). This

finding was also observed in the 15-l experiments.

These data further reinforce the point that active

BDC mobilization or recruitment occurred during the

apheresis.

3.6. BDC costimulatory phenotype is not altered by

the apheresis procedure

As shown in Table 2, the apheresis procedure does

not generate significant changes in the expression of

CD40, CD80 and CD86 on lineage�HLA-DR+ DC

(n=3).

3.7. DC subset distribution remains unchanged after

apheresis

The BDC subset proportions remained unchanged

after the procedure. Fig. 5, summarizes the percent-

age CD11c+ DC and CD123+ subset distribution

observed in six experiments. In these experiments,

the CD11c+/CD123+ DC ratio before apheresis was

0.52F0.17 versus 0.45F0.18 in the product ( p=

0.502).

3.8. BDC can be successfully isolated from apheresis

product

We have optimized an isolation protocol for BDC,

based on the use of the CMRF-44 monoclonal anti-

body and magnetic bead technology to purify BDC

after overnight culture (Lopez et al., submitted for

publication). Following apheresis, CMRF-44 antigen

expression in vitro was not altered and purification

of the BDC was readily achieved (Fig. 6). Further-

more, the separation of BDC from both fresh and

frozen apheresis products yielded comparable BDC

purity (Fig. 6). Furthermore, repeated BDC isolations

(n=4) performed on the same frozen apheresis

material yielded very consistent results (data not

shown). The BDC purified in this way from aphe-

resis material were fully functional in an allogeneic

MLR (Fig. 7).

4. Discussion

Clinical use of DC requires a highly reproducible

and GMP applicable method that provides sufficient

yields of nonactivated cells that are fully functional

upon cryopreservation. Here, we evaluated the use of

two software protocols for the collection of BDC

using the Cobe Spectra apheresis machine and estab-

lished that the automated version (Auto 6.1) provides

a lesser volume product and reduced platelet contam-

inant. We also demonstrated that the apheresis product

Fig. 7. Allogeneic MLR evaluation of enriched BDC prepared from

apheresis MNC. Freshly harvested apheresis cells (fresh) were

cultured overnight (cultured), labeled with CMRF-44 biotinylated

antibody and enriched with a positive immunoselection column.

Both positive and negative fraction cells were collected and tested.

A titration of APC from each origin was cultured with isolated

allogeneic T-cells, as described in Materials and methods.


contains sufficient numbers of BDC in a nonactivated

state suitable for immunotherapy. No skewing of

subset composition was observed allowing further

subset fractionation, if appropriate. The data suggests

that BDC are mobilized during the procedure and

although a 10-l apheresis procedure is sufficient, if

necessary, a 15-l apheresis procedure will generate an

improved BDC yield. Finally, we showed that BDC

can be isolated from the apheresis product and that

they are also fully functional after cryopreservation. A

detailed functional evaluation of BDC purified after a

magnetic bead separation following apheresis is pre-

sented elsewhere (Lopez et al., in preparation).

The selection of an automated protocol for the

harvest of BDC has various advantages. The repro-

ducibility and the operator independence permits the

standardization of the method across laboratories, a

particularly relevant issue when multi-center proto-

cols are envisaged, as will be important in evaluating

DC cancer immunotherapy protocols. Apheresis

using automated Cobe AutoPBSC software has also

been used to obtain MNC for the preparation of Mo-

DC and these were likewise fully functional; how-

ever, details on the apheresis procedure are not

available for comparison (Lewalle et al., 2000).

Apheresis MNC products have been used success-

fully for the production of Mo-DC from monocytes

(Thurner et al., 1999; Goxe et al., 2000; Lewalle et

al., 2000) and Langerhans cells from CD34+ progen-

itors but again, data on yields were not provided

(Gatti et al., 2000). Apheresed MNC preparations

have also been used for density gradient separation

of BDC but no information has been published

regarding the yields (Fong et al., 2001).

The reduced product volume facilitates further

BDC isolation procedures including the labeling with

monoclonal antibodies. The Cobe AutoPBPC pro-

gram provided lesser platelet contamination of the

sample. This feature is particularly relevant, since

platelets are known for their capacity to activate

BDC; for example, high platelet contamination has

been shown to hinder Mo-DC generation from aphe-

resis material both in melanoma patients and healthy

volunteers (Glaser et al., 1999).

Critical to the clinical use of the pheresed material

is the ability to handle the products in a closed clinical

grade environment. Clinical grade Mo-DC has been

prepared from pheresed MNC after processing with

the Cobe Spectra cell separator (Rouard et al., 2000).

This methodology can now be applied to BDC. The

product can then be enriched significantly for BDC,

whilst maintaining sterile Good Manufacturing Prac-

tice (GMP) procedures, using a BDC-specific mAb for

positive cell selection.

Cryopreservation of DC preparations will also be

highly advantageous for clinical scheduling. Protocols

examining the generation of Mo-DC have shown that

they may be stored safely frozen either as PBMC or as

matured Mo-DC (Thurner et al., 1999; Lewalle et al.,

2000). It has also been demonstrated that antigen-

loaded Mo-DC obtained from apheresis protocols can

be stored frozen, retaining their stimulatory function

(Feuerstein et al., 2000; Lewalle et al., 2000). Our

data shows that BDC can also be obtained from frozen

PBMC collected by apheresis. Thawing cells prior to

an overnight incubation before the antibody-mediated

separation of DC makes this a very convenient pro-

tocol.

Mo-DC has been generated from the apheresis

products obtained from patients with multiple mye-

loma (Tarte et al., 1997) and chronic myeloid leuke-

mia (Zheng et al., 2000). Indeed, we now have data

that establishes the optimal BDC harvesting time in

G-CSF and cyclophosphamide-treated patients with

non-Hodgkin lymphoma and multiple myeloma, who

are undergoing mobilization for blood stem cell col-

lection (Vuckovic et al., in preparation). The data also

predict that sufficient numbers of BDC will be

obtained from apheresis in those patients without the

need of additional mobilization. However, various

mobilizing conditions have been used to optimize

the generation of monocyte and CD34+-derived DC,

resulting in changes in the phenotype and/or function

of various cell types including T-lymphocytes NK and

DC (Gazitt, 2000; Roth et al., 2000). Stem cell factor

(SCF) mobilization in patients with breast cancer

resulted in minimal increases in BDC populations

(Menedez et al., 2001). Flt-3 ligand mobilizes BDL

in healthy volunteers (Maraskovsky et al., 2000) and

is currently being investigated by several groups for

its ability to mobilize BDC in patients.

DC are subject to various physiological influences

that may change their status of activation and/or

mobilization. We have observed that stress (surgery

and extreme exercise) prompts mobilization of BDC

without activation (Ho et al., 2001). Likewise, we


have documented changes in certain disease states

(Summers et al., 1999; Vuckovic et al., 1999; Brown

et al., 2001). Given their responses to environmental

influences, it was not inconceivable that apheresis

would change BDC differentiation/activation. Our

data on the evaluation of whole DC suggest that the

apheresis procedure does not induce the activation

state of collected BDC; a highly desirable feature

allowing for in vitro optimization of BDC function

for immunotherapy protocols. Differential activation

of a proportion of cells within certain DC subsets

(MacDonald et al., in press) may escape detection and

will be the subject of future analyses. In contrast,

maturation of Mo-DC from an apheresis product was

reported to occur earlier than for cells drawn from

fresh blood samples (Thurner et al., 1999). We have

shown here that BDC isolated from apheresis MNC

preparations exhibited similar functions to BDC

drawn by direct venipuncture.

In conclusion, this study describes critical infor-

mation required to generate sufficient, nonactivated

and fully functional BDC in a standard manner for

DC-based cancer immunotherapy studies.

Acknowledgements

We gratefully acknowledge the enthusiastic partic-

ipation of the volunteers recruited for this study; we

thank Drs. Cameron Turtle, Chris Ho, Geoff Hill and

Nick Murray for constructive discussions and Gilles

Bioley for help with the functional evaluation of

apheresis products. We acknowledge the support

Miltenyi Biotech (Bergisch Gladbach, Germany) for

immuno-isolation reagents. This work was funded by

the Mater Medical Research Institute and by GAM-

BRO BCT (Lakewood CO).

References

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu,

Y.J., Pulendran, B., Palucka, K., 2000. Immunobiology of den-

dritic cells. Annu. Rev. Immunol. 18, 767–811.

Brown, R.D., Pope, B., Murray, A., Esdale, W., Sze, D.M., Gibson,

J., Ho, P.J., Hart, D., Joshua, D., 2001. Dendritic cells from

patients with myeloma are numerically normal but functionally

defective as they fail to up-regulate CD80 (B7-1) expression

after huCD40LT stimulation because of inhibition by trans-

forming growth factor-beta(1) and interleukin-10. Blood 98,

2992–2998.

Caux, C., Dezutter-Dambuyant, C., Schmitt, D., Banchereau, J.,

1992. GM-CSF and TNF-cooperate in the generation of den-

dritic Langerhans cells. Nature 360, 258–261.

Caux, C., Vanbervliet, B., Massacrier, C., Dezutter-Dambuyant, C.,

de Saint-Vis, B., Jacquet, C., Yoneda, K., Imamura, S., Schmitt,

D., Banchereau, J., 1996. CD34+ hematopoietic progenitors

from human cord blood differentiate along two independent

dendritic cell pathways in response to GM-CSF+TNF alpha. J.

Exp. Med. 184, 695–706.

Clark, G.J., Angel, N., Kato, M., Lopez, J.A., Macdonald, K., Vuck-

ovic, S., Hart, D.N.J., 2000. The role of dendritic cells in the

innate immune system. Microbes Infect. 2, 257–272.

Dzionek, A., Fuchs, A., Schmidt, P., Cremer, S., Zysk, M., Miltenyi,

S., Buck, D.W., Schmitz, J., 2000. BDCA-2, BDCA-3, and

BDCA-4: three markers for distinct subsets of dendritic cells

in human peripheral blood. J. Immunol. 165, 6037–6046.

Egner, W., McKenzie, J.L., Smith, S.M., Beard, M.E.J., Hart,

D.N.J., 1993. Human bone marrow contains potent stimulatory

cells for the allogeneic MLR with the phenotype of dendritic

cells. Adv. Exp. Med. Biol. 329, 263–268.

Fearnley, D.B., McLellan, A.D., Mannering, S.I., Hock, B.D., Hart,

D.N.J., 1997. Isolation of human blood dendritic cells using the

CMRF-44 monoclonal antibody: implications for studies on

antigen presenting cell function and immunotherapy. Blood

89, 3708–3716.

Ferlazzo, G., Wesa, A., Wei, W.Z., Galy, A., 1999. Dendritic cells

generated either from CD34+ progenitor cells or from mono-

cytes differ in their ability to activate antigen-specific CD8+T

cells. J. Immunol. 163, 3597–3604.

Feuerstein, B., Berger, T.G., Maczek, C., Roder, C., Schreiner, D.,

Hirsch, U., Haendle, I., Leisgang, W., Glaser, A., Kuss, O.,

Diepgen, T.L., Schuler, G., Schuler-Thurner, B., 2000. A method

for the production of cryopreserved aliquots of antigen-pre-

loaded, mature dendritic cells ready for clinical use. J. Immunol.

Methods 245, 15–29.

Fong, L., Engleman, E.G., 2000. Dendritic cells in cancer immuno-

therapy. Annu. Rev. Immunol. 18, 245–273.

Fong, L., Brockstedt, D., Benike, C., Wu, L., Engleman, E.G.,

2001. Dendritic cells injected via different routes induce immun-

ity in cancer patients. J. Immunol. 166, 4254–4259.

Furuta, M., Shimizu, T., Mizuno, S., Kamiya, T., Ozawa, K., Na-

kase, T., Tadokoro, K., Takenaka, M., Ohkawa, T., Yokoyama,

S., Ogawa, Y., Kiyokawa, H., Shimizu, M., Sekine, N., Yoshi-

mura, I., 1999. Clinical evaluation of repeat apheresis donors in

Japan. Vox Sang. 77, 17–23.

Gatti, E., Velleca, M.A., Biedermann, B.C., Ma, W., Unternaehrer,

J., Ebersold, M.W., Medzhitov, R., Pober, J.S., Mellman, I.,

2000. Large-scale culture and selective maturation of human

Langerhans cells from granulocyte colony-stimulating factor-

mobilized CD34+ progenitors. J. Immunol. 164, 3600–3607.

Gazitt, Y., 2000. Immunologic profiles of effector cells and periph-

eral blood stem cells mobilized with different hematopoietic

growth factors. Stem Cells 18, 390–398.

Geiger, J., Hutchinson, R., Hohenkirk, L., McKenna, E., Chang, A.,


Mule, J., 2000. Treatment of solid tumours in children with

tumour-lysate-pulsed dendritic cells. Lancet 356, 1163–1165.

Glaser, A., Zingsem, J., Zimmermann, R., Weisbach, V., Eckstein,

R., 1999. Collection of mononuclear cells in the Spectra for the

generation of dendritic cells. Transfusion 39, 661–662.

Goxe, B., Latour, N., Chokri, M., Abastado, J.P., Salcedo, M., 2000.

Simplified method to generate large quantities of dendritic cells

suitable for clinical applications. Immunol. Invest. 29, 319–

336.

Hart, D.N.J., 1997. Dendritic cells: unique leucocyte populations

which control the primary immune response. Blood 90,

3245–3287.

Hart, D., Hill, G., 1999. Dendritic cell immunotherapy for cancer:

application to low-grade lymphoma and multiple myeloma. Im-

munol. Cell Biol. 77, 451.

Hart, D.N.J., Clark, G.J., MacDonald, K., Kato, M., Vuckovic, S.,

Lopez, J.A., Wykes, M., 2000. In: Mason, D. (Ed.), 7th Leu-

cocyte Differentiation Antigen Workshop DC Section Sum-

mary. Leucocyte Typing VII. Oxford Univ. Press, Oxford,

pp. 283–294.

Ho, C.S.K., Lopez, J.A., Vuckovic, S., Pyke, C.M., Hockey, R.L.,

Hart, D.N.J., 2001. Surgical and physical stress increase circu-

lating blood dendritic cell counts independently of monocyte

counts. Blood 98, 140–145.

Ho, C.S.K., Munster, D., Pyke, C.M., Hart, D.N.J., Lopez, J.A.,

2002. Spontaneous generation and survival of blood dendritic

cells in mononuclear cell culture without exogenous cytokines.

Blood 99, 2897–2904.

Hock, B.D., Starling, G.C., Daniel, P.B., Hart, D.N., 1994. Charac-

terization of CMRF-44, a novel monoclonal antibody to an

activation antigen expressed by the allostimulatory cells within

peripheral blood, including dendritic cells. Immunology 83,

573–581.

Hock, B.D., Fearnley, D.B., Boyce, A., McLellan, A.D., Sorg, R.V.,

Summers, K.L., Hart, D.N.J., 1999. Human dendritic cells ex-

press a 95 kDa activation/differentiation antigen defined by

CMRF-56. Tissue Antigens 53, 320–334.

Hsu, F.J., Benike, C., Fagnoni, F., Liles, T.M., Czerwinski, D.,

Taidi, B., Engleman, E.G., Levy, R., 1996. Vaccination of pa-

tients with B-cell lymphoma using autologous antigen-pulsed

dendritic cells. Nat. Med. 2, 52–57.

Kato, M., Neil, T.K., Fearnley, D.B., McLellan, A.D., Vuckovic, S.,

Hart, D.N., 2000. Expression of multilectin receptors and com-

parative FITC-dextran uptake by human dendritic cells. Int.

Immunol. 12, 1511–1519.

Kugler, A., Stuhler, G., Walden, P., Zoller, G., Zobywalski, A.,

Brossart, P., Trefzer, U., Ullrich, S., Muller, C.A., Becker, V.,

Gross, A.J., Hemmerlein, B., Kanz, L., Muller, G.A., Ringert,

R.H., 2000. Regression of human metastatic renal cell carcino-

ma after vaccination with tumor cell –dendritic cell hybrids. Nat.

Med. 6, 332–336.

Lewalle, P., Rouas, R., Lehmann, F., Martiat, P., 2000. Freezing of

dendritic cells, generated from cryopreserved leukaphereses,

does not influence their ability to induce antigen-specific im-

mune responses or functionally react to maturation stimuli. J.

Immunol. Methods 240, 69–78.

MacDonald, K.P.A., Munster, D., Clark, G.C., Dzionek, A.,

Schmitz, J., Hart, D.N.J., 2002. Characterization of human

blood dendritic cell subsets. Blood (in press).

Mackensen, A., Herbst, B., Kohlter, G., Wolff-Vorbeck, G., Rosen-

thal, F., Veelken, H., Kulmburg, P., Schaefer, H.E., Mertels-

mann, R., Lindemann, A., 1995. Delineation of the dendritic

cell lineage by generating large numbers of birbeck granule-

positive Langerhans cells from human peripheral blood progen-

itor cells in vitro. Blood 86, 2699–2707.

Maraskovsky, E., Daro, E., Roux, E., Teepe, M., Maliszewski, C.R.,

Hoek, J., Caron, D., Lebsack, M.E., McKenna, H.J., 2000. In

vivo generation of human dendritic cell subsets by Flt3 ligand.

Blood 96, 878–884.

McLellan, A.D., Starling, G.C., Hart, D.N.J., 1995. Isolation of

human blood dendritic cells by discontinuous Nycodenz gra-

dient centrifugation. J. Immunol. Methods 184, 81–89.

Mehta-Damani, A., Markowicz, S., Engleman, E.G., 1994. Genera-

tion of antigen-specific CD8+ CTLs from naive precursors. J.

Immunol. 153, 996–1003.

Menedez, P., Prosper, F., Bueno, C., Arbona, C., San Miguel, J.F.,

Garcia-Conde, J., Sola, C., Hornedo, J., Cortes-Funes, H., Or-

fao, A., 2001. Sequential analysis of CD34+ and CD34-cell

subsets in peripheral blood and leukapheresis products from

breast cancer patients mobilized with SCF plus G-CSF and

cyclophosphamide. Leukemia 15, 430–439.

Monji, M., Tynes-Petersons, J., Saund, N.J., Vuckovic, S., Hart,

D.N.J., Auditore-Hargreaves, K., Risdon, G., 2002. Dendritic cell

progenitors are CMRF-44+. Immunol. Cell Biol. 80, 216–225.

Murphy, G.P., Tjoa, B.A., Simmons, S.J., Ragde, H., Rogers, M.,

Elgamal, A., Kenny, G.M., Troychak, M.J., Salgaller, M.L.,

Boynton, A.L., 1999. Phase II prostate cancer vaccine trial:

report of a study involving 37 patients with disease recurrence

following primary treatment. Prostate 39, 54–59.

Nestle, F.O., Gilliet, M., Alljagic, S., Wiesner, W., Grabbe, S.,

Dummer, R., Burg, G., Schadendorf, D., 1997. Vaccination of

melanoma patients with peptide-pulsed dendritic cells. Melano-

ma Res. 7, S14.

Nestle, F.O., Banchereau, J., Hart, D., 2001. Dendritic cells: on the

move from bench to bedside. Nat. Med. 7, 761–765.

Olweus, J., BitMansour, A., Warnke, R., Thompson, P.A., Carballi-

do, J., Picker, L.J., Lund-Johansen, F., 1997. Dendritic cell on-

togeny: a human dendritic cell lineage of myeloid origin. Proc.

Natl. Acad. Sci. U. S. A. 94, 12551–12556.

Reid, C.D., Stackpoole, A., Meager, A., Tikerpae, J., 1992. Interac-

tions of tumour necrosis factor with granulocyte–macrophage

colony-stimulating factor and other cytokines in the regula-

tion of dendritic cell growth in vitro from early bipotent

CD34+ progenitors in human bone marrow. J. Immunol. 149,

2681–2688.

Rissoan, M.C., Soumelis, V., Kadowaki, N., Grouard, G., Briere,

F., de Waal Malefyt, R., Liu, Y.J., 1999. Reciprocal control of

T helper cell and dendritic cell differentiation. Science 283,

1183–1186.

Robinson, S.P., Patterson, S., English, N., Davies, D., Knight, S.C.,

Reid, C.D., 1999. Human peripheral blood contains two distinct

lineages of dendritic cells. Eur. J. Immunol. 29, 2769–2778.

Romani, N., Gruner, S., Brang, D., Kampgen, E., Lenz, A., Trock-

enbacher, B., Konwalinka, G., Fritsch, P.O., Steinman, R.M.,


Hansson, M., 1994. Proliferating dendritic cell progenitors in

human blood. J. Exp. Med. 180, 83–93.

Roth, M.D., Gitlitz, B.J., Kiertscher, S.M., Park, A.N., Mendenhall,

M., Moldawer, N., Figlin, R.A., 2000. Granulocyte macrophage

colony-stimulating factor and interleukin 4 enhance the num-

ber and antigen-presenting activity of circulating CD14+and

CD83+cells in cancer patients. Cancer Res. 60, 1934–1941.

Rouard, H., Leon, A., Klonjkowski, B., Marquet, J., Tenneze, L.,

Plonquet, A., Agrawal, S.G., Abastado, J.P., Eloit, M., Farcet,

J.P., Delfau-Larue, M.H., 2000. Adenoviral transduction of hu-

man ‘clinical grade’ immature dendritic cells enhances costimu-

latory molecule expression and T-cell stimulatory capacity. J.

Immunol. Methods 241, 69–81.

Sallusto, F., Lanzavecchia, A., 1994. Efficient presentation of solu-

ble antigen by cultured human dendritic cells is maintained by

granulocyte/macrophage colony-stimulating factor plus interleu-

kin 4 and downregulated by tumour necrosis factor-a. J. Exp.

Med. 179, 1109.

Sato, H., Shiobara, S., Yasue, S., Chuhjo, T., Nakao, S., 2001.

Lymphocyte collection for donor leucocyte infusion from nor-

mal donors: estimation of the minimum processed blood volume

and safety of the procedure. Vox Sang. 81, 124–127.

Schakel, K., Mayer, E., Federle, C., Schmitz, M., Riethmuller, G.,

Rieber, E.P., 1998. A novel dendritic cell population in human

blood: one-step immunomagnetic isolation by a specific mAb

(M-DC8) and in vitro priming of cytotoxic T lymphocytes. Eur.

J. Immunol. 28, 4084–4093.

Siena, S., Di Nicola, M., Bregni, M., Mortarini, R., Anichini, A.,

Lombardi, L., Ravagnani, F., Parmiani, G., Gianni, A.M., 1995.

Massive ex vivo generation of functional dendritic cells from

mobilized CD34+ blood progenitors for anticancer therapy. Exp.

Hematol. 23, 1463–1471.

Strunk, D., Rappersberger, K., Egger, C., Strobl, H., Kromer, E.,

Elbe, A., Maurer, D., Stingl, G., 1996. Generation of human

dendritic cell/Langerhans cells from circulating CD34+ hema-

topoietic progenitor cells. Blood 87, 1292–1302.

Summers, K.L., O’Donnell, J.L., Heiser, A., Highton, J., Hart, D.N.,

1999. Synovial fluid transforming growth factor beta inhibits

dendritic cell – T lymphocyte interactions in patients with

chronic arthritis. Arthritis Rheum. 42, 507–518.

Szabolcs, P., Moore, M.A.S., Young, J.W., 1995. Expansion of

immunostimulatory dendritic cells among the myeloid progeny

of human CD34+ bone marrow precursors cultured with c-kit

ligand, granulocyte–macrophage colony-stimulating factor, and

TNF-a. J. Immunol. 154, 5851–5861.

Tarte, K., Lu, Z.Y., Fiol, G., Legouffe, E., Rossi, J.F., Klein, B.,

1997. Generation of virtually pure and potentially proliferating

dendritic cells from non-CD34 apheresis cells from patients with

multiple myeloma. Blood 90, 3482–3495.

Thurner, B., Roder, C., Dieckmann, D., Heuer, M., Kruse, M.,

Glaser, A., Keikavoussi, P., Kampgen, E., Bender, A., Schuler,

G., 1999. Generation of large numbers of fully mature and stable

dendritic cells from leukapheresis products for clinical applica-

tion. J. Immunol. Methods 223, 1–15.

Vuckovic, S., Fearnley, D.B., Gunningham, S., Spearing, R.L., Pat-

ton, W.N., Hart, D.N., 1999. Dendritic cells in chronic myelo-

monocytic leukaemia. Br. J. Haematol. 105, 974–985.

Young, J.W., Szabolcs, P., Moore, M.A.S., 1995. Identification of

dendritic cell colony-forming units among normal human

CD34+ bone marrow progenitors that are expanded by c-kit

ligand and yield pure dendritic cell colonies in the presence of

granulocyte/macrophage colony-stimulating factor and tumor

necrosis factor a. J. Exp. Med. 182, 1120.

Zheng, C., Pisa, P., Stromberg, O., Blennow, E., Hansson, M., 2000.

Generation of dendritic cells from peripheral blood of patients at

different stages of chronic myeloid leukemia. Med. Oncol. 17,

270–278.


Documents

Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy