Experimental Gerontology, Vol. 33, No. 6, pp. 625– 631, 1998
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SHORT COMMUNICATION

DENDRITIC CELL RESPONSIVENESS TO STIMULATION WITH
INFLUENZA VACCINE IS UNIMPAIRED IN OLD AGE

M. SAURWEIN-TEISSL,1 D. SCHONITZER,2 and B. GRUBECK-LOEBENSTEIN1
¨
1

Institute for Biomedical Aging Research of the Austrian Academy of Sciences, Innsbruck, Austria, and 2Central
Institute for Blood Transfusion and Immunological Department, General Hospital and University Clinics, Innsbruck,
Austria

Abstract—We have previously demonstrated that whole virus influenza vaccine can activate
dendritic cells (DC). In the present study we analyzed whether DC activation was affected by
the aging process. For this reason the expression of immunoregulatory molecules and the
production of cytokines were compared in blood-derived DC from old and young healthy
individuals following stimulation with inactivated influenza virus. Unstimulated DC from
young and old individuals had a similar surface expression of MHC class II and CD54 and
secreted moderate amounts of IL-12 and TNF-␣. Stimulation with influenza vaccine led to a
marked increase in the production of surface molecules and cytokines. These changes were
equally pronounced in cells from young and old individuals. Our results demonstrate that DC
responsiveness to stimulation with a viral vaccine is unimpaired in old age. DC may,
therefore, represent a potent tool for immunotherapy and may increase the efficacy of
vaccines in the elderly. © 1998 Elsevier Science Inc.
Key Words: dendritic cells, aging, cytokines, influenza, surface marker, vaccines

INTRODUCTION
DENDRITIC CELLS (DC) are crucial for the initiation of immune responses of both helper and
cytotoxic T lymphocytes, and thus act as “nature’s adjuvant” (Schuler and Steineman, 1997).
This is due to the remarkable ability of DC to present antigen, to provide costimulatory signals,
and to modulate immune responses by producing cytokines such as for instance IL-12, which
induces IFN-␥ production and facilitates the development of Th1 responses (Caux et al., 1995).
Due to their central role in immunology, DC have been considered as useful tools for immunotherapy, in particular, as carriers for tumor vaccines (Nair et al., 1997). Recent studies have
Correspondence to: Beatrix Grubeck-Loebenstein, Institute for Biomedical Aging Research of the Austrian Academy
of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. Tel: ϩ43-512-583919-14; Fax: ϩ43-512-583919-8; E-mail:
beatrix.grubeck@oeaw.ac.at
(Received 9 April 1998; Accepted 20 April 1998)
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shown that DC-based tumor vaccines lead to tumor regression and to a prolonged survival in
mice (Song et al., 1997; Specht et al., 1997). Clinical trials in humans are presently in progress
(Banchereau and Steinman, 1998). In view of their successful application as presenter cells for
tumor vaccines, DC might also be considered as carrier cells for other immunization regimes.
This could be of particular relevance in elderly subjects, whose response to primary and
secondary immunization is frequently impaired (Stein, 1994; Steger et al., 1996b). Usage of DC
as vaccine carriers in old age may be a promising approach, as a recent study from our
laboratory demonstrated that unstimulated DC derived from the peripheral blood of aged
individuals were unimpaired by the aging process (Steger et al., 1996a). In the absence of
stimuli these cells had a normal capacity to express MHC and costimulatory molecules and to
present antigen to tetanus-specific T cell clones. They could also reinduce proliferation in in
vitro aged T cells (Steger et al., 1997). In these previous studies DC were, however not tested
for their adaptability to cellular stress situations such as the uptake of bacteria (Henderson et al.,
1997), viruses (Schnorr et al., 1997), and viral vaccines (Schnorr et al., 1997; Saurwein-Teissl
et al., in press). Uptake of inactivated influenza virus triggers the maturation of DC by inducing
the upregulation of immunoregulatory molecules and by stimulating the secretion of IL-12 in the
young (Saurwein-Teissl et al., in press). No information is yet available of whether DC from
aged individuals are equally prone to respond to the stimulatory challenge of pathogens or
vaccines. It was, therefore, the aim of the present study to analyze immunoregulatory molecules
and the production of cytokines following stimulation with a whole virus influenza vaccine in
DC from aged persons.
MATERIALS AND METHODS
Reagents, monoclonal antibodies, and serum
IL-4 and GM-CSF were kindly provided by Sandoz Pharma AG (Basel, Switzerland). Mouse
MoAbs directed against the following cell surface determinants were used: MHC class II
(HLA-DR; An der Grub GmbH, Kaumberg, Austria; FITC-conjugated), CD54 (Monosan, Uden,
The Netherlands; PE-conjugated), CD14 and CD19 (Dako A/S, Glostrup, Denmark; PEconjugated), and CD3 (SeraLab, Crawley Down, UK; FITC-conjugated). Fetal calf serum (FCS)
was purchased from Schoeller Pharma (Vienna, Austria). An intact virion influenza vaccine
inactivated by propiolactone was generously provided by the Swiss Serum- & Vaccine Institute
(Berne, Switzerland). It will, in the following, be referred to as influenza vaccine. The vaccine
contained the following three influenza strains: A/Singapore/6/86 (H1N1), A/Wuhan/359/95
(H3N2), and B/Beijing/184/93.
Purification of DC
Peripheral blood mononuclear cells (PBMC) were obtained from old (Ͼ65 years; n ϭ 15) and
young (Ͻ30 years; n ϭ 15) healthy individuals. DC were prepared from PBMC, as previously
described (Steger et al., 1996a). In brief, PBMC were resuspended in RPMI-1640 (Gibco, Grand
Island, NY), 10% FCS, and 1% Penicillin/Streptomycin (P/S; Gibco) as culture medium (CM)
and allowed to adhere to six-well plates (Falcon; 9 ϫ 106 cells per well). After two hours at
37°C, nonadherent cells were removed and adherent cells cultured in CM supplemented with
800 U GM-CSF and 1000 U IL-4 per mL. Cells were then fed every other day with fresh CM
containing 800 U GM-CSF and 300 U IL-4 per mL.

DENDRITIC CELL RESPONSIVENESS IN OLD AGE

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Preparation of DC for surface marker and cytokine secretion analysis
After one week in culture, DC, which were at that time point mostly nonadherent, were
removed from the plate and washed twice in RPMI. The purity of the population was demonstrated by the complete lack of CD3-, CD19-, and CD14-positive cells, as assessed by immunofluorescence staining and FACScan analysis (Steger et al., 1996a). Cells were then counted
and incubated at 106 cells per tube in fresh CM at 37°C, 5% CO2 in tissue culture tubes (Greiner,
Kremsmunster, Austria) in the absence or presence of influenza vaccine (1 ␮g/mL). After 24 h
¨
supernatants were harvested, centrifuged, and stored at Ϫ20°C. Cells were washed and analyzed
by immunofluorescence staining, as described below. The optimal vaccine concentration and
incubation time had been defined in pilot experiments.
Immunofluorescence staining and FACScan analysis
Cells were transferred into round-bottom tubes (105 cells/tube; Becton Dickinson, Mountain
View, CA) and washed at 4°C in PBS containing 0.1% FCS. Antibodies were added and the
cells left to incubate at 4°C. After 40 min the cells were washed twice in PBS. Analysis was
performed on a Becton Dickinson FACScan. Five thousand scatter-gated cells were analyzed in
each sample. The frequency and fluorescence profiles of the cells were determined with
logarithmic signal amplifiers.
Cytokine determinations by ELISA
Cytokine concentrations in conditioned supernatants were determined by commercially available ELISA kits: IL-12 and TNF-␣ (Endogen Inc., Cambridge, MA).
Statistical analysis
Student’s t-test was used for statistical evaluation.
RESULTS
In accordance with a previous study (Steger et al., 1996a) the surface expression of CD54 and
of MHC class II was similar in unstimulated DC from young and old persons. Incubation of DC
with influenza vaccine for 24 h led to a marked increase in MHC class II and CD54 in both
groups ( p Ͻ 0.05, Fig. 1 and Table 1). Following stimulation, the intensity of the staining did
not differ between DC from young and old individuals.
To analyze whether DC from aged persons had an intact capacity to secrete cytokines, DC
were stimulated with influenza vaccine and their conditioned supernatants were analyzed for the
presence of IL-12 and TNF-␣. Unstimulated DC from young and aged individuals produced
similar amounts of IL-12 and TNF-␣ (Fig. 2). Influenza vaccine increased the secretion of IL-12
and TNF-␣ in both groups ( p Ͻ 0.01). The concentrations of cytokines following stimulation
did not differ between cultures conditioned by DC populations derived from young and old
individuals.
DISCUSSION
Adequate stimulation and maturation of DC is necessary for the presentation of antigen to T
cells. This is a prerequisite for the induction of cytotoxicity, T helper function, and consecutively, the production of antibodies. The success of immunization regimes therefore greatly

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M. SAURWEIN-TEISSL et al.

FIG. 1. Influenza vaccine stimulates the surface expression of MHC class II and CD54 in DC from
young and old individuals. Immunofluorescence staining of purified DC incubated for 24 h in the
presence or absence of a whole inactivated influenza virus vaccine. The figure shows one characteristic experiment in which DC from one young (a) and from one old (b) person were used.

depends on the state of activation of DC. Vaccination is frequently hampered in the elderly by
a decline in the reactivity of the immune system with age (Miller, 1996; Wick and GrubeckLoebenstein, 1997). Due to the involution of the thymus, T cells are hereby more severely
affected than B cells (George and Ritter, 1996). Age-dependent changes in T cell reactivity may
be compensated by the augmentation of the intensity of the activating stimulus (Candore et al.,
1992). Activation of DC leads to an upregulation of costimulatory molecules as well as to the
production of cytokines (Banchereau and Steinman, 1998). Improved costimulation and cytokine production could mobilize residual activity in aged T lymphocytes. DC activation can be
induced by various pathogens (Henderson et al., 1997, Schnorr et al., 1997; Thurnher et al.,
1997). Attenuated or inactivated agents are hereby almost equally effective as their wild-type
counterparts (Schnorr et al., 1997). In the young, whole inactivated influenza virus can, for

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DENDRITIC CELL RESPONSIVENESS IN OLD AGE

TABLE 1. MHC II AND CD54 SURFACE STAINING INTENSITY
(MEAN CHANNEL FLUORESCENCE) IN DC FROM YOUNG (N ϭ 15) AND OLD
(N ϭ 15) HEALTHY INDIVIDUALS BEFORE AND AFTER STIMULATION WITH
WHOLE INACTIVATED INFLUENZA VIRUS

MHC class II

CD54

Unstimulated
Young
Old

Stimulated

Unstimulated

Stimulated

278 Ϯ 35
311 Ϯ 52

411 Ϯ 75a
470 Ϯ 109a

439 Ϯ 80
580 Ϯ 188

659 Ϯ 124a
893 Ϯ 263a

Data are expressed as mean Ϯ SEM in each group.
a
p Ͻ 0.05 vs. unstimulated cells.

example, stimulate DC maturation, trigger Th-1 activity, and support the propagation of CD8ϩ
cells (Saurwein-Teissl et al., in press). DC stimulatory vaccines might thus be a tool to increase
T cell activity and the success rate of vaccination in the elderly. No information was yet
available whether DC responsiveness to stimulation was affected by the aging process. The
present study for the first time demonstrates that DC from aged individuals are indeed capable
of responding to the stimulatory effect of a whole virus vaccine in a similar way as young DC.
High MHC class II and CD54 expression improves the contact between antigen presenting cell
and T cell. IL-12 and TNF-␣ trigger Th-1 activity and the recruitment of cytotoxic T cells, which
can protect elderlies from disease even in the absence of sufficient antibody titers (SaurweinTeissl et al., 1998).
In conclusion, our results suggest that DC are not only suitable carriers for tumor vaccines in
the elderly but could also improve the efficiency of conventional vaccines in old age. Vaccine
preparations with the capacity to stimulate DC may be a useful tool to compensate failing T cell
reactivity in aged persons and to ensure adequate protection.
Acknowledgments—We are grateful to Georg Wick for his continuous encouragement and support. This study was
supported by a grant from the Austrian National Bank (Grant No. 6851).

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