cryopreservation of immature monocyte-derived dendritic cells results in enhanced cell maturation...

Cryopreservation of immature monocyte-derived dendritic cells

results in enhanced cell maturation but reduced endocytic

activity and efficiency of adenoviral transduction

Justin John, James Hutchinson, Angus Dalgleish, Hardev Pandha*

Department of Oncology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, United Kingdom

Received 4 December 2001; received in revised form 26 July 2002; accepted 29 August 2002

Abstract

To date, phase I/II dendritic cell (DC)-based cancer vaccine trials have required repeated venesection or leukapheresis to

generate the DCs. Previous studies have suggested that DCs may be cryopreserved and revived for clinical use as sequential

immunisations. We have developed a method of cryopreserving monocyte-derived DCs, reviving the cells with minimal loss,

and have performed immunophenotypic and functional comparisons of freeze– thawed DCs with their fresh counterparts. We

found that the freeze–thawing process itself is efficient in terms of DC recovery, results in semimaturation and reduced

endocytic activity, but does not impair the capacity of the DCs to achieve full maturation. Revived cells also showed enhanced

allostimulatory activity and antigen-specific responses. After freeze– thawing, DCs produced lower levels of IL-12 p40 and

IL-1p70 on maturation compared to fresh DCs with little change in concentration over 72 h. Genetic modification of DCs by

adenoviral transduction was possible after cryopreservation albeit at a lower efficiency of gene transfer than with fresh cells.

We conclude that cryopreservation of DCs for clinical immunotherapy is feasible. Modification of cells by pulsing or genetic

transfer should take place prior to cryopreservation as the freeze–thawing process itself leads to increased maturation,

reduction in endocytic activity but enhanced allostimulatory activity and antigen-specific responses.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Dendritic cells; Cryopreservation; Immunotherapy; Adenoviral

1. Introduction

In mammals, dendritic cells (DCs) are the most

powerful antigen-presenting cells. They possess the

specialised machinery for the initiation of innate and

humoral immune responses by the processing and

presenting of antigens as peptide fragments to T

cells (Manca et al., 1994). In this way, antigen-

specific cytotoxic T lymphocytes (CTLs) are primed

against a variety of microbial antigens as well as

tumour cells (Hart, 1997; Nestle et al., 1998). DCs

modified by peptide (Celluzzi et al., 1996) or RNA

pulsation (Heiser et al., 2000) by gene transfection

(Wan et al., 1997) or the creation of DC–tumour

cell fusions (Gong et al., 1997; Scott-Taylor et al.,

2000) have been shown to elicit tumour-specific

CTLs in vitro and induce therapeutic responses in

animal models and human studies. The specific

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00430 -1

* Corresponding author. Tel.: +44-20-87251255x0809; fax:

+44-20-87250158.

E-mail address: [email protected] (H. Pandha).

www.elsevier.com/locate/jim

Journal of Immunological Methods 272 (2003) 35–48

properties of DCs that enable them to be used as

vaccines are directly related to their degree of

maturation. Immature DCs are characterised by a

high efficiency of antigen capture and processing

and, as they mature, this ability is replaced by

enhanced co-stimulatory function, migration to

regional lymphoid tissue and activation of T cells.

For effective antitumour immunotherapy, the opti-

mal number of DCs, the route of administration and

frequency of inoculation remain to be defined. The

ability to culture and cryopreserve numerous ali-

quots of identical DCs from a single venesection

would greatly facilitate human trials by allowing

their manipulation before or after freezing and their

subsequent use as sequential vaccines. In addition,

there would be the potential to utilise modified DCs

for repeated in vitro stimulation for the generation

of antigen-specific T cells. This would avoid

repeated venesection for DC generation in patients

already debilitated from their disease. In this strat-

egy, aliquots of DCs could be checked for pheno-

type and allostimulatory potential in vitro prior to

immunising the patient. Up to now, attempts to

freeze–thaw large numbers of mature DCs for this

purpose have involved complex and time-consuming

cell manipulation and elaborate freezing media

(Feuerstein et al., 2000; Taylor et al., 1990; Thurner

et al., 1999). This has also led to unreliable rates of

cell recovery, but apparently no alterations in phe-

notype and function (Lewalle et al., 2000; Makino

and Baba, 1997). In this study, we have cultured

DCs from monocyte precursors and show consis-

tently that high rates of recovery after cryopreser-

vation are possible using simple conditions and

media. We provide a systematic comparison of the

antigen-specific stimulatory capacity of DCs gener-

ated from normal volunteers when these cells are

pulsed with specific peptides. In direct comparison

to fresh DCs, thawed cells show enhanced allosti-

mulatory function but reduced endocytic function

and a reduction in gene transfer efficiency using

adenoviral transduction, which has clear implica-

tions for future gene therapy strategies. The freezing

process itself leads to activation and a differential

degree of maturation similar to when cells are

cultured in human serum (Duperrier et al., 2000).

This maturation is not complete, as there is little

upregulation of CD83, but there is a normal

response to maturation stimuli such as CD40 ligand

and interferon (IFN)-g.

2. Materials and methods

2.1. Isolation of peripheral blood mononuclear cells

Peripheral blood (50 ml) was obtained from

healthy individuals by venous puncture and collected

in sodium heparin vacutainers (Becton Dickinson,

Oxford, UK). The blood was diluted 1:1 with Hanks’

balanced salt solution (Sigma-Aldrich, Poole, UK).

Peripheral blood mononuclear cells (PBMCs) were

isolated by Histopaque-1077 (Sigma–Aldrich) den-

sity gradient centrifugation. Mononuclear cells were

collected from the interface, washed once in Hanks’

solution before being resuspended in the appropriate

assay medium.

2.2. Generation of monocyte-derived dendritic cells

PBMCs were resuspended in growth medium

(RPMI-1640 containing 10% heat-inactivated FCS,

50 U/ml penicillin and 50 Ag/ml streptomycin, all

purchased from Sigma) at a final concentration of

3� 106 cells/ml. Cells were incubated in 100 mm

plastic petri dishes for 2 h at 37jC. Nonadherent cellswere removed by vigorous pipetting and the remain-

ing adherent cells cultured in growth medium, sup-

plemented with 100 ng/ml recombinant human GM-

CSF (Leucomax, Novartis, Camberley, UK) and 50

ng/ml recombinant human interleukin (IL)-4 (Pepro-

tech EC, London, UK). Fresh cytokines were added

every 2–3 days up to day 7.

2.3. Direct surface staining for immunophenotyping

Cells were harvested and washed in wash buffer

(PBS containing 0.5% BSA and 0.1% sodium azide,

all purchased from Sigma), then resuspended at a

concentration of 1�105 cells/tube in wash buffer.

Cells were labelled with the relevant fluorochrome-

conjugated antibody (see below) at the concentration

recommended by the manufacturer for 30 min on ice

in the dark. Cells were also incubated with an irrel-

evant isotype-matched control antibody to compen-

sate for nonspecific binding. The cells were then

J. John et al. / Journal of Immunological Methods 272 (2003) 35–4836

washed in wash buffer and the cell pellet fixed with

200 Al CellFix (Becton Dickinson). Samples were

either analysed immediately or within 24 h (stored

at 4jC in the dark) on a FACScan flow cytometer

(Becton Dickinson); routinely, 10,000 events were

collected. Dead cells and debris were gated out on

the basis of their light scatter properties. The follow-

ing reagents were used: Isotype controls IgG1 FITC/

PE (Becton Dickinson) or IgG2a FITC/IgG1a PE

(Serotec, Oxford, UK); specific markers: HLA-ABC

FITC (IgG2a, Serotec), HLA-DR, -DP, -DQ FITC

(IgG2a, Serotec), CD80 PE (IgG1, Serotec), CD86 PE

(IgG1, Serotec), mannose receptor (MR) PE (IgG1,

Becton Dickinson), CD40 PE (IgG1, Becton Dick-

inson) and CD83 FITC (IgG1, Becton Dickinson).

2.4. Cryopreservation of dendritic cells

Dendritic cells were counted and the pellet chilled

on ice for 5 min. The cells were resuspended in

freezing medium (12% DMSO/44% FCS/44% RPMI

1640) at a final concentration of 3� 106 cells/ml and

aliquoted to cryovials (Greiner Labortechnik, Ger-

many). The cryovials were placed in freezing contain-

ers (Nalgene, Rochester, US) containing 2-isopropanol

and then transferred to a � 70jC freezer, where the

rate of cooling was controlled to � 1 jC/min (manu-

facturer’s information). After 72 h, the cryovials were

transferred directly to liquid-phase liquid nitrogen for

long-term storage.

Frozen DCs were recovered from storage in liquid

nitrogen by thawing in a 37jC water bath over 100 s.

The cells were washed in 15 ml of warm growth

medium before being resuspended in an appropriate

medium for experimentation.

2.5. DC survival following cryopreservation

DC viability was assessed for up to 3 days after

recovery from cryopreservation by propidium iodide

(PI) dye exclusion. DCs were either cultured in

growth medium alone, growth medium with 50

ng/ml IL-4 and 100 ng/ml GM-CSF or growth

medium with 50 ng/ml IL-4, 100 ng/ml GM-

CSF, 100 ng/ml IFN-g (R&D Systems, Oxford,

UK) and 0.2 Ag/ml CD40L/2 Ag/ml Enhancer

(Alexis, Nottingham, UK). At each time point, the

cells were harvested, washed twice with wash buffer

and resuspended in 200 Al PBS. PI was added

immediately and the cells analysed by flow cytom-

etry. Viable cells were verified and enumerated

depending on their ability to exclude PI.

2.6. Receptor-mediated endocytosis activity

This method has been adapted from that published

elsewhere (Sallusto et al., 1995). Five hundred micro-

liters of DC (3� 105/ml) in growth medium were

incubated at 37jC for 10 min to equilibrate the

temperature. Five hundred microliters of dextran

(40,000 MW, Sigma), diluted in growth medium, were

added at various concentrations and incubated at 37 or

4jC for 10 min. Finally, dextran–FITC (40,000 MW,

Sigma) at 1 mg/ml final concentration was added to

each sample and incubated for a further 60 min at the

same temperature. Washing the cells twice in ice-cold

PBS quenched the endocytic activity and removed

any free dextran–FITC. Cells were fixed in 200 Al ofCellFix, and routinely, 5000 cells were analysed by

flow cytometry. Background binding of FITC–dex-

tran (where DCs were incubated at 4jC) was alwayssubtracted from the total mean channel fluorescence

level.

2.7. Mixed lymphocyte reaction (MLR)

The stimulatory function of the DCs was assessed

by their ability to induce proliferation in allogeneic

nonadherent PBMCs in vitro. Graded numbers of

DCs resuspended in assay medium (RPMI 1640

containing 10% heat-inactivated pooled human se-

rum (UK Blood Transfusion Service, Colindale,

UK), 50 U/ml penicillin and 50 Ag/ml streptomycin)

were incubated with 106 allogeneic nonadherent

PBMCs resuspended in the same medium. Prolifer-

ation was measured on day 5 following 18 h of

pulsing with 1 ACi [3H]-Thymidine per well. Mean

values of triplicates were measured and expressed as

counts per minute (C.P.M.).

2.8. Antigen-specific response tests

Fresh DCs, generated from HLA-A*0201 donors,

were loaded with a 9-mer peptide derived from in-

fluenza matrix protein (IMP) sequence GILGFVFTL

(Alter BioSciences, Birmingham, UK) for 2 h at 37

J. John et al. / Journal of Immunological Methods 272 (2003) 35–48 37

jC under serum-free conditions. The cells were then

washed twice and either used immediately or frozen

as above. Revived DCs were washed once in Hanks’

solution. Freeze–thawed cells were allowed to re-

cover in assay medium (RPMI 1640 containing 10%

heat-inactivated pooled human serum, 50 U/ml pen-

icillin and 50 Ag/ml streptomycin) for at least 2 h at

37jC prior to being loaded with IMP peptide as

above. Autologous responder cells consisted of non-

adherent PBMCs collected at the time of initial DC

preparation and frozen for 6 days. These cells were

revived and allowed to recover in assay medium for

24 h. Both fresh and freeze– thawed, pulsed and

unpulsed, DC and responder cells were counted and

resuspended in assay medium at 105 and 106 cells/ml,

respectively. The IFN-g ELISpot assay followed the

manufacturer’s instructions (Mabtech, Nacka, Swe-

den). Briefly, Silent Screen 96-well plates with bio-

dyne membranes (Nalgene, Life Technologies, US)

were coated with 15 Ag/ml primary antibody (clone 1-

D1K) overnight at 4jC. Plates were washed once

with PBS to remove nonbound antibody before the

addition of PBS/10% AB serum for 2 h at room

temperature to block nonspecific binding. The plates

were washed once with PBS before adding 100 Al ofDC and 100 Al of autologous nonadherent PBMCs in

assay medium to each well. The cells were incubated

for 24 h at 37jC, 5% CO2 humid incubator. Cells

were removed by washing six times with PBS–

Tween 20 and the wells incubated with 1 Ag/ml

biotinylated detection antibody (clone 7-B6-1-biotin)

for 3 h at room temperature. Plates were washed six

times with PBS before incubation with Streptavidin-

alkaline phosphatase (1:1000 in PBS/0.5% FCS) for 2

h at room temperature. Following six final washes

with PBS, the plate was incubated with alkaline

phosphatase conjugate substrate as recommended by

manufacturer (Bio-Rad, California, US) for up to 30

min until spots appeared. Washing the plate in tap

water and then allowing it to dry in air stopped the

reaction. Spot-forming colonies (SFC) were enumer-

ated using the Zeiss Axioplan 2 ELISpot counter

(Image Associates, Oxford, UK).

2.9. Induction of dendritic cell maturation

Full DC maturation status was defined by either

the expression of specific cell surface markers or

the ability to produce IL-12. From published data

(Duperrier et al., 2000), it is clear that different

stimuli induce different maturation characteristics,

whilst the most potent phenotypic changes are

induced by TNF-a. This cytokine has little effect

on IL-12 production, whereas both IL-12 p40 and

IL-12 p70 can be induced by CD40L and IFN-g

(Mosca et al., 2000). To induce the phenotypic

mature status, fresh and freeze–thawed DCs were

washed once in medium and resuspended at

2� 105/ml in full DC growth medium containing

TNF-a (25 ng/ml) (R&D Systems) for 48 h. For

the production of IL-12, the cells were resuspended

at 2� 105/ml in full DC growth medium containing

IFN-g and CD40L (as outlined in Section 2.5). At

various time points, the supernatants were harvested

and centrifuged to remove cells and debris. The

supernatants were collected and stored at � 20jCfor the detection of IL-12 p40 and p70 by ELISA

(R&D Systems).

2.10. Adenoviral transduction of dendritic cells

The adenoviral vector used was the Ad5.CMV-

GFP (Quantum Biotechnologies, Montreal, Canada),

which is a first generation serotype 5 virus express-

ing the green fluorescent protein (GFP) gene under

the control of the cytomegalovirus–IE promoter/

enhancer. Day 7 fresh or freeze–thawed DCs were

harvested, washed twice and resuspended in serum-

free XVIVO-20 (BioWhittaker, Wokingham, UK) at

2� 105 cells/ml before transduction cells were

allowed to stabilise for 10 min at 37jC. Adenoviruswas diluted in warm XVIVO-20 to the required

multiplicity of infection (MOI) and gently mixed

with the cells before being co-cultured for 4 h at 37

jC. Warm growth medium containing 10% FCS,

100 ng/ml recombinant human GM-CSF and 50 ng/

ml recombinant human IL-4 was added to give a

final cell concentration of 105 cells/ml. Cells were

maintained for a further 48 h at 37jC before

analysis by flow cytometry.

2.11. Statistical analysis

Student’s paired t-test was used to detect signifi-

cant differences between data using the statistical


Fig. 1. Phenotypic surface molecule expression of day 7 immature, day 9 TNF-a matured, freeze– thawed (F/T) immature and F/T TNF-a matured DC. Open curves represent the

isotype control. Values indicate mean channel fluorescence. Data are representative of four independent experiments.

J.Johnet

al./JournalofIm

munologica

lMeth

ods272(2003)35–48

39

package in Microsoft Excel. A P value of < 0.05

was considered as significant.

3. Results

3.1. Immunophenotype and rates of cell recovery

The method used for cryopreservation was adap-

ted from a published protocol (Makino and Baba,

1997), which was applicable for both PBMCs and

dendritic cells. We confirmed that there was a sig-

nificant difference between 10% and 12% dimethy-

sulfoxide (DMSO) in the freezing medium with the

12% concentration resulting in highest cell viability

upon thawing (data not shown). In addition, the

procedure of controlled freezing by reducing the

temperature by 1 jC/min proved optimal in terms

of efficiency of cell recovery and was consistent with

data published elsewhere (Taylor et al., 1990). For

the recovery of DCs, a rapid thawing at 37jC was

critical and required, on average, 95.7F 5.2 s (n = 9)

for 500 Al samples. DCs generated by 7-day culture

of adherent PBMCs were assessed for surface marker

expression by flow cytometry. Fresh cells were

compared directly with cryopreserved cells that had

been thawed and recultured for 24 h (see Fig. 1).

There were no morphological differences between

fresh and freeze–thawed DCs. However, freeze–

thawed DCs appeared semimature upon revival com-

pared with fresh immature DCs characterised by an

increase in the expression of MHC class I, CD40 and

more significantly of HLA-DR, -DP, -DQ. There was

a significant decrease in the expression of mannose

receptor (Fig. 1). The number of DCs expressing

CD40, CD86 and CD83 also increased for freeze–

thawed DCs compared to their fresh counterparts

(see Fig. 2). Although the increase in CD40 expres-

sion in matured or freeze–thawed DCs was small in

terms of mean channel fluorescence, the percentage

of cells expressing CD40 increased. Both fresh and

Fig. 2. Percentage of day 7 immature, day 9 TNF-a matured, freeze– thawed (F/T) immature and F/T TNF-a matured DCs expressing surface

molecules. DCs were stained for expression of MHC class I, MHC class II, CD40, CD80, CD86, CD83 and mannose receptor (MR). Data are

representative of four independent experiments.

Table 1

The efficiency of recovery of DCs after thawing from cryopre-

servation as assessed by PI dye exclusion

Patient PBMCs� 106

/ml of blood

Percent yield

of DCs

Percent recovery

post freeze– thawing

1 1.24 15.3 79

2 1.64 28.9 81

3 1.54 20.26 98

4 1.22 25 86

5 0.95 22.05 72

Average 1.318 22.3 83.2

An attempt was made to recover cells frozen at 3� 106 cells/ml of

freezing medium. The results represent cell numbers recovered from

blood from five different healthy volunteers.


freeze–thawed DCs were capable of full maturation

upon exposure to TNF-a with the resulting pheno-

typic expression of cell surface markers similar in

both level of expression and numbers of positive

cells.

The efficiency of recovery of DCs after thawing

from cryopreservation was assessed by PI dye exclu-

sion. We attempted to recover cells frozen at 3� 106

cells/ml of freezing medium (see Table 1). Cells were

stored at � 70jC and then transferred to liquid nitro-

gen for at least 3 days before recovery was attempted.

DCs were defrosted and allowed to recover in culture

for 24 h before assessment. In six samples, the mean

PBMC of 1.38� 106/ml blood generated on average

22.3% DCs. The mean recovery rate was 83.2%

(range 72–98%).

3.2. Survival of fresh and freeze–thawed DCs

The maintenance of key physiological properties

of freeze– thawed DCs compared to their fresh

counterparts would be central to their potential use

for in vivo immunotherapy applications. The survival

of mature freeze–thawed DCs has been previously

shown to be equivalent to fresh DCs cultured over

the same time scale (Feuerstein et al., 2000), even in

the absence of cytokines in the culture medium

(referred to as the ‘washout test’). The process of

maturation itself may provide survival signals

(Feuerstein et al., 2000). We found that survival of

both fresh and freeze–thawed cells were extended

equally in terms of time by the addition of CD40L

and IFN-g to the culture medium. However, viability

did decrease by approximately 20% after 72 h (day

10 of culture). In our study, the DCs were cryopre-

served in an immature state, and the freeze–thawing

process resulted in semimaturation. In the presence

of cytokines (i.e. maintaining IL-4 and GM-CSF in

the culture medium), there was no significant reduc-

tion in fresh or freeze–thawed DC viability at 72 h

(Fig. 3 and Table 2). Upon withdrawal of cytokines

(a ‘washout test’), there was progressive loss of DC

viability with time resulting in approximately 20%

Fig. 3. Survival of day 7 (A) versus freeze– thawed (F/T) (B) DCs

incubated for 3 days either without IL-4 and GM-CSF (n), with IL-

4 and GM-CSF (5) or with IL-4, GM-CSF, IFN-g and CD40L (E).

Cells were harvested and stained with propidium iodide to evaluate

viability by flow cytometry. Typically, 10,000 events were collected

for analysis. The withdrawal of IL-4 and GM-CSF results in

reduced DCs survival especially for F/T DCs. Maintaining IL-4 and

GM-CSF in the culture medium prevents this cell death. The

addition of IFN-g and CD40L results in reduced viability of day 7

DCs, but has little effect on F/T DCs. Data are representative of

mean + S.D. of three independent donors.

Table 2

A comparison of survival of fresh (day 7) versus freeze– thawed

(F/T) DCs up to 72 h with and without CD40L and IFN-g

Percent viable (propidium iodide negative)

0 24 h 48 h 72 h

Fresh alone 96.0 83.9 64.9 58.7

Fresh + cytokines 96.0 94.6 89.9 89.0

Fresh + cytokines

+CD40L+ IFN-g

96.0 86.9 78.5 75.7

Frozen alone 84.3 69.5 55.1 51.5

Frozen + cytokines 84.3 79.5 75.2 78.3

Frozen + cytokines

+CD40L+ IFN-g

84.3 78.8 73.9 75.4

Cells were harvested and stained with propidium iodide to evaluate

viability by flow cytometry. The addition of CD40L and IFN-g

resulted in reduced viability of day 7 DCs, but had little effect on

F/T DCs. Data are representative of mean + S.D. of three inde-

pendent donors.


and 35% drop in survival of fresh and freeze–

thawed DCs, respectively, at 72 h (day 10 of cul-

ture). The addition of IFN-g and CD40L to the

culture medium resulted in reduced viability of fresh

DCs, presumably due to extensive activation, but had

less effect on freeze–thawed DCs.

3.3. DC maturation and endocytic activity

The maturation of DCs is associated with a

reduced capacity for antigen capture (Sallusto et

al., 1995). This process takes the form of two

mechanisms: fluid uptake by macropinocytosis,

which is constitutive, and a second mechanism

mediated through the mannose receptor, which

allows uptake and concentration of mannosylated

macromolecules, such as tumour-associated antigens

in the MHC class II compartments (Steinman and

Swanson, 1995). Preservation of antigen capture

function would be essential for clinical immunother-

apy strategies based on pulsing DCs with peptide or

tumour lysate. The uptake of specific molecules by

the process of receptor-mediated endocytosis may be

inhibited by increasing concentrations of various

competitive ligands in culture (Sallusto et al., 1995).

To test whether the endocytosis of dextran–FITC

was dependent on expression of the mannose re-

ceptor (MR) on DCs, we measured dextran–FITC

Fig. 4. FITC–dextran endocytosis is inhibited by excess levels of

free dextran in both day 7 immature and freeze– thawed (F/T)

immature DCs. DCs were incubated either at 4jC (background,

control) or 37jC with various concentrations of free dextran for 10

min. The cells were then incubated with 1 mg/ml FITC–dextran for

1 h. Endocytic activity was quenched by washing the cells in ice-

cold PBS and fixing before analysis by flow cytometry. F/T DCs

had a reduced capacity to endocytose FITC–dextran compared to

their fresh immature counterparts. The endocytosis could be

inhibited in a dose-dependent manner by free dextran. TNF-a

matured DCs, either fresh or freeze– thawed (F/T M), were unable

to endocytose FITC–dextran (n= 3).

Fig. 5. Proliferation of allogeneic T cells by fresh (5) and freeze– thawed (n) DCs as measured by the incorporation of [3H]-Thymidine (MLR).

A fixed number of allogeneic nonadherent PBMCs were co-cultured for 5 days with various concentrations of DCs. [3H]-Thymidine (1 ACi) wasadded for the final 18 h of culture. Data are means + S.D. of counts per minute (C.P.M.) for triplicate samples from four individuals.


uptake in the presence of increasing concentrations

of a competitive ligand (free dextran). Freeze–

thawed DCs had a reduced capacity to endocytose

dextran–FITC compared to their fresh immature

counterparts. The endocytosis could be inhibited in

a dose-dependent manner by free dextran for both

fresh and freeze–thawed DCs (Fig. 4). Exposure to

TNF-a resulted in downregulation of mannose recep-

tor expression (Fig. 1), and both maturation of fresh

and freeze–thawed DCs resulted in a total lack of

endocytic function, even in the absence of free

dextran (Fig. 4).

3.4. Allogeneic T-cell proliferation tests

A direct comparison of the allostimulatory capacity

of freeze–thawed versus fresh DCs was made. Fig. 5

shows three representative examples where DCs from

three different individuals were cultured with PBMCs

from the same donor (responder). Either fresh DCs

taken at day 7 or freeze–thawed cells revived for 24 h

were co-incubated with allogeneic PBMCs for 5 days,

including pulsing with tritiated thymidine for the last

18 h. Freeze–thawed DCs consistently induced a

marked allostimulatory response, which was of a

higher magnitude compared to proliferation seen in

response to fresh DCs. Proliferative responses to fully

matured DCs, as expected, were higher still (data not

shown).

3.5. Antigen-specific responses (ELISpot)

We compared the ability of freeze–thawed DCs

and their fresh counterparts to induce an antigen-

specific response to flu peptide, assuming a high

likelihood of previous exposure to flu. DCs from

HLA-A*0201 donors were generated for direct com-

parison. Influenza peptide-reactive T cells were

quantified using an IFN-g ELISpot assay. Fig. 6

shows representative data from two donors. DCs

were either pulsed at day 7, prior to freeze–thawing,

or after revival from freeze–thawing. They were co-

cultured with autologous nonadherent PBMCs at a

ratio of 1:10 for 24 h. The data shows the average

number of spots for two individuals who produced

either a high number of spots (A) or a low number

of spots (B). Regardless of the level of background

stimulation, flu peptide-pulsed DCs always induced

more spots compared to their unpulsed counterparts.

Freeze–thawed DCs induced more spots compared

to day 7 DCs with or without peptide. The high

number of spots seen with unpulsed DCs possibly

reflected their increased allostimulatory properties as

shown by the MLR (Fig. 5). Notably, we consis-

tently found that pulsing DCs with peptide prior to

freeze–thawing induced more spots compared to

DCs pulsed after freeze–thawing, which has impli-

cations in the design of DC-based immunotherapy.

Fig. 6. Quantification of flu-specific CD8+ T cells by ELISpot. DCs

were either pulsed at day 7, prior to freeze– thawing (F/T) or after

revival from F/T. They were co-cultured with autologous non-

adherent PBMCs for 24 h. The data showed the average number of

spots + S.D. for two individuals who produced large numbers of

spots (A) or small numbers of spots (B). Pulsing DCs with peptide

prior to F/T induced more spots compared to DC pulsed after F/T.

Statistical P values are shown.


3.6. IL-12 production

We compared the ability of fresh and freeze–thawed

DCs to secrete IL-12 in response to IFN-g and CD40L

in the culture medium. Fresh and freeze–thawed DCs

were cultured for up to 72 h, and supernatants were

assessed for the presence of IL-12 p70 and IL-12 p40

by ELISA. Fresh and freeze–thawed DCs were found

to secrete increasing amounts of p40 over time to reach

similar concentrations by 72 h. Fresh DCs produced IL-

12 p70 within the first 24 h, but this decreased by 72 h

whilst remaining easily detectable (Fig. 7). Freeze–

thawed DCs produced lower absolute levels of IL-12

p70 but these levels remained consistent and detect-

able throughout the experimental incubation. This

data contrasts with IL-12 p70 production from fresh

cells in our study and previous studies where IL-12

p70 production appeared to peak at up 24 h and then

subside (Kalinski et al., 1999; Vieira et al., 2000).

Intracellular IL-12 p70 expression was confirmed for

freeze–thawed DCs using FACS analysis (data not

shown).

3.7. Genetic modification using an adenoviral vector

In view of potential future clinical applications of

DCs transduced with specific antigens, it was impor-

tant to assess the susceptibility of freeze–thawed cells

to transduction using the high-efficiency gene transfer

vectors currently available. For this reason, we

assessed the susceptibility of freeze–thawed DCs to

adenoviral transduction compared to their fresh coun-

terparts. Fresh day 7 DCs and revived freeze–thawed

DCs were incubated with AdGFP at various MOIs up

to 1:5000. Fig. 8 shows representative results from

DCs generated from healthy volunteers. There were

increasing levels of green fluorescent protein (GFP) in

immature day 7 and freeze–thawed DC with increas-

Fig. 7. Secretion of IL-12 p40 (A,B) and IL-12 p70 (C,D) by DCs in response to IFN-g and CD40L stimulation over various time points. Day 7

DCs (A) and freeze– thawed (F/T) DCs (B) secreted increasing amounts of p40 over time to reach similar concentrations by 72 h. Day 7 DCs

produced p70 (C) within the first 24 h, which decreased over time. F/T DCs (D) produced lower levels of p70, detectable throughout the

experimental incubation. Data shows mean + S.D. for results from five donors, each performed in triplicate.


ing MOI of adenovirus-GFP. The efficiency of trans-

duction of fresh DCs was therefore significantly

greater compared to freeze–thawed DCs. Interest-

ingly, we found that even when thawed DCs were

given little time to recover after thawing, adenoviral

transduction efficiency was similar to DCs where cell

recovery had first taken place over 24 h. The exposure

of cells to AdGFP at MOIs of up to 1:5000 did not

result in significant cell death.

4. Discussion

The critical role of DCs in antigen presentation in

vivo has been well established. The concept of re-

peated injections of DCs modified by pulsing with

tumour protein, peptide or RNA or DCs transduced

with tumour antigen has been evaluated in animal

studies and early clinical trials. Amongst the limita-

tions revealed by these approaches is a reliable supply

of DCs modified and activated in an identical way to

provide sequential vaccines. The approach of gener-

ating large numbers of DCs and modifying them prior

to cryopreservation in numerous aliquots has obvious

advantages. In this way, aliquots of cells may be

thawed and fully tested for cell recovery, microbial

screening and allostimulatory properties, as well as

confirming surface phenotypic expression. Upon

thawing, the degree of maturation may be modulated

by culture conditions.

We have performed a systematic comparison of the

immunobiology of fresh monocyte-derived DCs with

DCs recovered after cryopreservation. The freeze–

thawing process is efficient in terms of cell recovery

with an average recovery rate of well over 80%, which

is consistent with previous studies. There was no

change in DC morphology after freeze–thawing. In

contrast to previous studies where cells were frozen

either immature (Feuerstein et al., 2000) or fully

matured (Lewalle et al., 2000), we have found that

Fig. 8. Transduction of dendritic cells with adenovirus before and after cryopreservation. DCs were either transduced as fresh day 7,

immediately following or 24 h after recovery from cryopreservation with increasing MOI. Cells were then returned to culture for 48 h before

being assessed for the expression of GFP by flow cytometry. Numbers above the marker indicate the percentage of GFP-expressing cells. Data

representative from three independent experiments.


the freeze–thawing process itself leads to partial DC

maturation. Semimature DCs have the advantage of

still being able to respond to maturation stimuli and

thereby be ‘driven’ to produce important cytokines

such as IL-12 for CTL responses in vivo. The pheno-

type of thawed cells compared to their fresh counter-

parts show evidence of semimaturation by phenotypic

markers and reduced endocytosis as demonstrated by

FITC–dextran uptake. Freeze–thawed cells are capa-

ble of full maturation upon exposure to TNF-a. In the

presence of cytokines used in routine culture (IL-4

and GM-CSF), the viability of freeze–thawed cells

after 72 h did not change significantly. Freeze–

thawed DCs were more resistant to pro-apoptotic

stimuli (CD40L and IFN-g) than their fresh counter-

parts. Further indications of the potential use of

freeze–thawed cells for clinical immunotherapy is

provided by evidence of an enhanced allostimulatory

capacity by MLR and of an antigen-specific response

to flu peptide by ELISpot. This has not been previ-

ously reported and cannot be explained simply in

terms of our use of foetal calf serum for DC culture,

as previous studies have shown greater DC activation

from exposure to human serum (Duperrier et al.,

2000). IL-12 is routinely used as an indicator of DC

activation and a surrogate marker of type 1 T-cell

responses. The importance of IL-12 in CTL priming in

vivo is still unclear. Freeze–thawed cells were capable

of expressing IL-12 (both the p70 bioactive hetero-

dimer and p40 subunits), although a recent study has

suggested that DC-derived IL-12 is not necessarily

required for the generation of cytotoxic T cells in vivo

(Wan et al., 2001). In DCs generated from healthy

volunteers, freeze–thawed cells produced lower abso-

lute levels of IL-12 p70 compared to fresh DCs, but

these levels remained consistent and detectable over

72 h. Intracellular staining of freeze–thawed DCs

further confirmed the expression of IL-12 p70. Pre-

vious studies have shown that in the absence of IL-12,

DCs loaded with gp100 melanoma antigen can still

elicit potent antitumour responses associated with a

robust Th-1 cytokine profile (Wan et al., 2001). This

may indicate that high or detectable levels of IL-12 are

not required for the development of cell-mediated

immunity, and experiments using p40-deficient mice

suggested that the absence of IL-12 did not affect the

allostimulatory function or the phenotype of DCs

(Wan et al., 2001). In cancer patients, we found a

marked variability in peak time and longevity of IL-12

p70 production, with IL-12 p70 detectable up to 4

days (n = 6, data not shown). However, the peak

production of IL-12 from cryopreserved DCs may

be earlier or later than the time frame used in this

study. Further experiments confirmed that using FACS

analysis, intracellular IL-12 p70 was expressed by

freeze–thawed DCs.

The use of adenoviral vectors in human cancer

gene therapy is becoming increasingly important. In

the context of DCs, the advantages of adenoviral

vectors have been clearly defined, both in terms of

high levels of transduction efficiency and the retention

of DC function (Zhong et al., 1999). Elements from

the adenoviral backbone itself leads to the upregula-

tion of activation markers (Korst et al., 2002).

Although a number of other viral vectors are under

consideration for transducing dendritic cells for

immunotherapy, replication-deficient adenoviruses

still have probably the longest safety record of any

viral gene transfer vehicle. It was therefore important

for us to highlight the potential for adenoviral trans-

duction of our freeze–thawed dendritic cells, as this

would be an obvious application for cancer immuno-

therapy. Although we have shown that efficiency of

transduction at equivalent MOIs is reduced for

freeze–thawed versus fresh cells, there is clear evi-

dence of gene transfer, which may be easily sufficient

for eliciting antigen-specific T-cell responses in a

clinical setting. DCs may be transduced by adenoviral

vectors before or after cryopreservation. We have

shown that genetic modification of thawed DCs using

adenoviral vectors is still possible albeit at lower

efficiency. When comparing GFP-positive DCs (fresh

versus freeze–thawed), there was very little difference

in the level of GFP expressed, except at the highest

MOIs. Interestingly, these findings appear to suggest

that strategies based on pulsing DCs with tumour

lysate, peptide or RNA may be preferable to viral

modification for subsequent clinical applications.

However, the efficiency of transduction of freeze–

thawed DCs is still higher than that observed with the

use of retroviral and nonviral vectors (Arthur et al.,

1997).

There are a number of advantages of our approach

compared with previous studies. Our protocol des-

cribes a simple freezing medium and revival process.

The use of FCS, as opposed to human serum as in


other studies, gives the monocyte-derived DCs a more

immature phenotype thereby permitting more efficient

uptake of tumour peptide/lysate prior to cryopreser-

vation for clinical applications. Thawed cells may be

matured under controlled culture conditions to bias

towards a Th-1 activation status. We have shown that

thawed DCs show the greatest degree of recovery and

survival if cytokines are maintained in the culture. The

thawed DCs showed greater antigen-specific and

allostimulatory capacity without full maturation com-

pared with previous reports. However, we found that

thawed DCs produced low levels of IL-12, although it

is unclear how much IL-12 is needed in vivo to induce

a Th-1 response.

In conclusion, cryopreservation of immature mo-

nocyte-derived DCs is feasible with high rates of cell

recovery upon thawing. The modification of these

cells is preferable before freezing, as the process itself

leads to a degree of maturation, reduced endocytic

activity and reduced transduction efficiency when

using adenoviral vectors. DCs pulsed with peptide

or tumour lysate before cryopreservation retain their

morphology, surface phenotype, IL-12 p70 production

and immunostimulatory function. Such characteristics

make them ideal vehicles for future vaccine strategies.

The potential for routine use of freeze–thawed DCs

(thereby avoiding the problems with labour, cost and

lack of quality control associated with repeated fresh

preparations) will only be realised by directly compar-

ing the efficacy of fresh DC versus freeze–thawed

DC-based vaccines.

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