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Unimpaired dendritic cells can be derived from monocytes in old age and can mobilize residual function in senescent T cells
Vaccine 18 (2000) 1606±1612
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Unimpaired dendritic cells can be derived from monocytes in old age and can mobilize residual function in senescent T cells T.L. Lung a, M. Saurwein-Teissl a, W. Parson b, D. SchoÈnitzer c, B. Grubeck-Loebenstein a,* a
Institute for Biomedical Aging Research of the Austrian Academy of Sciences, Rennweg 10, A-6020, Innsbruck, Austria b Institute of Legal Medicine, University of Innsbruck, Innsbruck, Austria c Central Institute for Blood Transfusion and Immunological Department, General Hospital, University Clinics Innsbruck, Innsbruck, Austria
Abstract Dendritic cells (DC) are powerful antigen presenting cells, which have the unique capacity to stimulate naive T cells. In spite of the well-known decline of T cell function in old age, little information is available on whether DC are also aected by the aging process. This is mainly due to problems with the isolation and puri®cation of DC. Rapid progress in the characterization of DC has been made in recent years, as simple methods to generate large numbers of DC from precursors have been developed. It was the aim of the present study to compare monocyte derived DC from old and young healthy persons. The generation of DC from blood monocytes in response to GM-CSF and IL-4 treatment was similar in cells from young and old persons. The DC population thus obtained had a typical dendritic morphology and expressed DC surface markers, such as HLA class II, CD1a, CD11c, CD54, CD80 and CD86, but not CD14 for a period of up to three weeks in culture. DC from young and old persons produced IL-12 and TNF-a and responded equally well to maturation-inducing stimuli. DC maturation was stimulated by puri®ed protein derivative (PPD) of Mycobacterium tuberculosis, whole inactivated in¯uenza virus and by in¯uenza split vaccine, but not by puri®ed viral RNA. When tested for their antigen-presenting capacity, DC from young and old persons were capable of stimulating the proliferation and the cytokine production of T cells. It was of particular interest that CD45RA+ as well as CD45RO+ T cells from aged donors were unable to respond to stimulation with in¯uenza proteins presented by monocytes, but were triggered to proliferate and to produce cytokines when antigen was presented by DC. The results demonstrate that DC from old persons (a) may still function as powerful antigen-presenting cells provided the right dierentiation and maturation stimuli are present; (b) are capable of mobilizing residual capacity in senescent T cells and (c) may therefore represent a potent tool for immunotherapy and vaccines in old age. # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Dendritic cells; T lymphocytes; Aging
1. Introduction Dendritic cells (DC) are identi®ed by a triad of criteria: Morphologically, they exhibit pronounced cytoplasmic veils which are mobile as can easily be
* Corresponding author. Tel.: +43-512-583919-14; fax: +43-512583919-8. E-mail address: [email protected] (B. Grubeck-Loebenstein).
observed under a phase contrast microscope [1,2]. These veils become apparent only in the mature state. Phenotypically, they express high levels of MHC (class I and II) as well as of adhesion (CD11c, CD54, CD58), and costimulatory (CD80, CD86, CD40) molecules on their surface. They also express CD1a and CD83, two DC cell markers, but lack CD14. Functionally, they are potent stimulators of resting T lymphocytes. DC derived from various tissues have been shown to undergo a complex maturation process during which their morphology, phenotype and func-
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T.L. Lung et al. / Vaccine 18 (2000) 1606±1612
tion change. DC of myeloid as well as of lymphoid origin have been described [3±5]. DC take up and process antigen and consecutively present antigenic peptides in the context of MHC molecules. Upon appropriate stimulation they undergo further maturation and migrate to secondary lymphoid tissues where they present antigens to T cells. Recent knowledge on DC has been summarized in excellent reviews [4,6±9]. In contrast to the rapidly increasing amount of knowledge on DC cell physiology in young individuals relatively little is still known on DC in aged humans and animals. Langerhans cells (LC), the epidermal equivalent of DC, have been studied in some detail in aged mice, the most obvious question being whether the numbers of LC change with age. Several authors found a decline of LC densities in the epidermis with age [10±12]. The extent of LC reduction was about one third. This was also observed with LC in the oral mucosa: a reduction by 30±60% was reported in the mouse [13]. No detailed studies are available concerning age-related changes in the numbers and distribution of DC from sources other than the skin. Similar to the results on experimental animals, early studies on aged human skin have also demonstrated a decreased density of LC [14,15]. This may, however, partly be due to UV irradiation, as signi®cantly fewer LC were observed in exposed vs. covered skin in old individuals, while no such disparity was noted in younger subjects. Whether the low LC density in old age is the result of lower DC progenitor numbers or of an impaired migration of progenitors from the marrow to the target tissues is not yet known [10]. The latter possibility seems, however, more likely, as recent results on humans demonstrate that higher numbers of DC can be derived from puri®ed monocytes of aged than of young persons [16]. No consensus has yet been reached whether DC function is impaired in old age. Whereas some studies suggest reduced function [10±12,17,18], other more recent studies tend to agree that the function of monocytes and monocyte derived cells are not aected by the aging process [16,19±23]. Further work will be needed to obtain entire clarity on this subject. It was the aim of the present study to focus on monocyte derived human DC and to analyze their phenotype and their responsiveness to maturation inducing stimuli as well as their capacity to stimulate senescent T cells. 2. Materials and methods 2.1. Reagents, monoclonal antibodies and serum IL-4 and GM-CSF were kindly provided by Sandoz
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Pharma AG (Basel, Switzerland). Mouse MoAbs directed against the following cell surface determinants were used: MHC class II (HLA-D, An der Grub GmbH, Kaumberg, Austria, FITC-conjugated), CD1a (Serotec, FITC-conjugated), CD14 (Serotec, PE-conjugated), CD11c (Pharmingen, PE-conjugated), CD54 (Pharmingen, PE-conjugated), CD80 (Becton Dickinson, PE-conjugated), CD86 (Pharmingen, FITC-conjugated) and CD83 (Pharmingen, PE-conjugated). Fetal calf serum (FCS) was purchased from Schoeller Pharma (Vienna, Austria). 2.1.1. Antigens An intact virion in¯uenza vaccine inactivated by propiolactone and in¯uenza subunit vaccine were kindly provided by the Swiss Serum & Vaccine Institute (Berne, Switzerland). Both vaccine contained the following three in¯uenza strains: A/Beijing/262/95 (H1N1), A/Sydney/5/97, (H3N2) and B/Harbin/7/94. In¯uenza split vaccine containing the same in¯uenza strains was provided by Pasteur MeÂrieux Connaught (Marcy l'Etoile, France). Both vaccines were in the following referred to as FLU. PPD (150 mg/ml) was a gift from the Behring Institute (Vienna, Austria). For the isolation of RNA, in¯uenza life virus was used (kindly provided by the Swiss Serum & Vaccine Institute) and isolated by resuspending the pellet in Trizol reagent (Gibco BRL). As a control total RNA from human tissues was additionally isolated. Transfection of DC with in¯uenza and control RNA was carried out with DOTAP liposomal transfection reagent (Boehringer Mannheim, Vienna, Austria). 2.2. Puri®cation of DC Peripheral blood mononuclear cells (PBMC) were obtained from old (>65 years) and young (<30 years) healthy individuals. DC were prepared from peripheral blood monocytes, as previously described [16,24]. In brief, PBMC were resuspended in RPMI-1640 (BIO Whittaker, Verviers, Belgium), 10% FCS and 1% Penicillin/Streptomycin (P/S; Gibco) as culture medium (CM) and allowed to adhere to six-well plates (9 106 cells/well; Falcon, Becton Dickinson, Franklin Lakes, NJ, USA). After 2 h at 378C, non-adherent cells were removed and the adherent monocyte enriched population (85 2 2% CD14+ cells) cultured in CM supplemented with 800 U GM-CSF and 1000 U IL-4/ml. Cells were then fed every second day with fresh CM containing 800 U GM-CSF and 300 U IL-4/ ml and analyzed after 1±3 weeks of culture. 2.3. Preparation of DC for surface marker and cytokine secretion analysis After 1 week in culture, DC, which were at that
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time point mostly non-adherent, 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 immuno¯uorescence staining and FACScan analysis [16]. Cells were then counted and incubated at 106 cells per tube in fresh CM at 378C, 5% CO2 in tissue culture tubes (Greiner, KremsmuÈnster, Austria) in the absence or presence of stimuli. After 24 h supernatants were harvested, centrifuged and stored at ÿ208C. Cells were washed and analyzed by immuno¯uorescence staining, as described below. 2.4. Immuno¯uorescence staining and FACScan analysis Cells were transferred into round bottom tubes (105 cells/tube; Falcon) and washed at 48C in PBS containing 0.1% FCS. Antibodies were added and the cells left to incubate at 48C. After 40 min the cells were washed twice in PBS. Analysis was performed on a Becton Dickinson FACScan. Five thousand scattergated cells were analyzed in each sample. The frequency and ¯uorescence pro®les of the cells were determined with logarithmic signal ampli®ers.
carried out in triplicates. Results were expressed as counts per minute (CPM). 2.9. TCR repertoire Puri®ed CD4+CD45RO+/RA+ cells from young and old persons were stimulated with in¯uenza subunit vaccine (1 mg/ml) presented by either autologous irradiated DC or PBMC and left to incubate for 1 week. RNA, prepared from T cells was reverse transcribed and TCR Vb transcripts were ampli®ed by PCR using primers speci®c for each of the human Vb families and a speci®c primer for the constant region of the b chain (labeled with the ¯uorescent dye marker 6-FAM (6carboxy¯uorescein) coupled with an aminohexyl linker; Applied Biosystems, Weiterstadt, Germany). Sequences of Vb primers were as in GeneveÂe et al. [25]. PCRs were prepared in a ®nal volume of 50 ml and contained 0.5 mM of each oligonucleotide primer in Taq-PCR Master Mix (Quiagen, Hilden, Germany). The PCR reactions were started with an initial heating at 948C for 30 s followed by 35 cycles of ampli®cation by denaturation at 948C for 45 s, annealing of primers at
2.5. Puri®cation of CD4+CD45RO+ and of CD4+CD45RA+T cells T cell subsets were separated with magnetic MicroBeads according to manufacturers protocol: CD4 MultiSort Kit and CD45RO MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). 2.6. Cocultures of DC, PBMC and T cells CD45RO+ and CD45RA+ T cells (105 or 5 105 cells/well) were cocultured with and without irradiated (35 Gy) autologous DC (3 104 or 1,5 105) or PBMC (105 or 5 105) in the presence or absence of in¯uenza antigen (1 mg/ml). Cells were cultured for 1 week in 24- or 96-well ¯at-bottom plates (Falcon). 2.7. Cytokine determinations by ELISA Cytokine concentrations in conditioned supernatants were determined by commercially available ELISA kits: TNF-a, IL-12 and IFN-g (Endogen Inc., Cambridge, MA, USA). 2.8. Proliferation assays After the respective incubation periods cells were pulsed with 1 mCi of [3H]-thymidine (ICN Pharmaceuticals, CA, USA) and [3H]-thymidine incorporation was assessed by scintillation counting. All assays were
Fig. 1. Dendritic cell dierentiation from monocytes is unimpaired in old age. Puri®ed monocytes from young and aged persons were stimulated with GM-CSF and IL-4. CD14 and CD1a surface expression was monitored by immuno¯uorescence analysis during 1 week (x-2SEM; n = 3 in each group).
T.L. Lung et al. / Vaccine 18 (2000) 1606±1612
728C for 45 s and an extension step at 728C for 10 min. An aliquot of the PCR product was diluted in 16 ml deionized formamide and 1.2 fmol internal lane standard GeneScan-350 Tamra (Perkin Elmer, Norwalk, CT, USA). The samples were denatured at 908C for 2 min. and snap cooled on ice prior to loading on a CE 310 Genetic Analyser. Each sample was injected for 5 s at 15 kV and electrophoresed for 24 min at 10 kV using a 36 cm capillary and POP4 (Perkin Elmer, Norwalk, CT). Analysis of the raw data was performed applying the GeneScan 2.1 analysis software package (PE, ABD) using the Local Southern method for fragment size estimation. Analyzed data were imported into Genotyper 2.0 and labeled according to the estimated fragment size. 3. Results 3.1. Generation and propagation of DC from monocytes CD14+ monocytes were puri®ed from the peripheral blood of young and aged persons. These cells were stimulated with IL-4 and GM-CSF as described in Section 2. In both age groups CD14 expression disap-
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peared rapidly upon cytokine treatment whereas CD1a appeared on the surface of the cells (Fig. 1). After a week of culture 93 2 1% of the cells expressed CD1a whereas only low percentages of CD14 cells were found. The results demonstrate that the generation of DC from monocytic precursor cells is unaected by the aging process provided the right stimuli (cytokines) are present. 3.2. Monocyte derived DC have an unimpaired phenotype and an unimpaired cytokine production pattern DC from young and aged persons were consecutively analyzed for their surface molecule expression. Immuno¯uorescence staining demonstrated a similar expression pattern of CD54, MHC class II, CD86, CD80, CD1a and CD11c in DC derived from young and aged persons over a time span of three weeks in culture (Fig. 2). It was of interest that CD86 reached its maximal expression in the third week of culture when CD1a expression was rapidly lost. In spite of their decreased CD1a expression, cells from young and aged persons still had a pronounced stimulatory activity in the allogeneic mixed lymphocyte reaction
Fig. 2. Surface molecule expression is similar in DC derived from young and old persons over prolonged culture periods. Surface molecule expression was assessed by immuno¯uorescence staining (x-2SEM; n = 3 in each group).
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did not induce maturation. DC maturation could still be stimulated by split vaccine, indicating that in¯uenza nucleoproteins may have a non-speci®c stimulatory eect on the innate immune system. Expectedly, PPD was an excellent stimulator of DC maturation [27] and worked equally well on ``young'' and ``old'' DC (data not shown). 3.4. DC from aged persons can induce proliferation and cytokine production in senescent T cells Fig. 3. Cytokine production is similar in DC from young and aged persons. DC were stimulated with whole inactivated in¯uenza virus and their conditioned supernatants analyzed for the presence of IL12 and TNF-a following a 24 h incubation (x-2SEM; n = 15 in each group).
(MLR) at this late time point (data not shown). DC from young and aged persons also had a similar capacity to secrete cytokines in the resting state as well as following stimulation (Fig. 3). 3.3. DC from aged persons have an unimpaired responsiveness to maturation inducing stimuli The capacity of the following agents to induce maturation was tested on DC from young and aged persons: whole inactivated in¯uenza virus, in¯uenza split vaccine which contains surface proteins and nucleoproteins but no viral RNA, puri®ed in¯uenza virus RNA and PPD. The increase in the number of CD83+ cells, as well as in the intensity of CD54 and CD86 expression were analyzed to assess DC maturation. In accordance with previous results from our group [19,26] whole inactivated in¯uenza virus was an excellent stimulator of DC maturation and was equally eective on DC from aged and young persons (Table 1). As previous results from our laboratory had demonstrated that in¯uenza surface protein had no stimulatory eect on DC [26], the eect of the whole virus was obviously due to the presence of core components. Viral RNA per se was not eective, as the transfection of DC with puri®ed in¯uenza virus RNA
Previous results from our laboratory have shown that DC from aged persons are capable of stimulating the clonal expansion and of postponing the clonal elimination of antigen speci®c T cell populations in an in vitro senescence model [28]. We therefore analyzed whether DC were also capable of stimulating freshly isolated T cells from aged persons. For this reason, puri®ed CD4+CD45RO+ and CD4+CD45RA+ T cells from aged persons were stimulated with in¯uenza antigen in combination with either irradiated autologous PBMC or corresponding numbers of DC. T cell proliferation and IFN-g production were used as read out parameters of T cell function. While T cell responsiveness was very low when PBMC were used as antigen presenting cells, proliferation as well as IFN-g production could be strongly increased when DC were used as APC (Fig. 4). It was of interest that the increase in the proliferation/IFN-g production of T cells induced by DC was even more pronounced when CD45RA+ T cells were used as responder cells. DC aected the magnitude but not the clonal composition of the response, which was demonstrated by CDR3 size analysis (Fig. 5). The clonal composition of responding T cells from dierent Vb families was the same whether PBMC or DC were used as presenter cells. A typical experiment is depicted in Fig. 5.
4. Discussion Our results demonstrate that monocytes from aged
Table 1 Changes in the surface expression of CD83, CD54 and CD86 in DC from young and old healthy individuals following stimulationa CD83 D % p.c.
Whole virus FLU Split vaccine FLU Viral RNA Human RNA a
CD54 D % MFI
CD86 D % MFI
Young
Old
Young
Old
Young
Old
+2582171% +57225% ND ND
+6852466% +145230% ND ND
+108288% +79222% +521% +122%
+108281% +50215% +025% +024%
+168238% +2925% +922% +021%
+121213% +826% +523% +822%
Data are expressed as mean2SEM in each group (n = 3±6); MFI, mean¯uorescence; % p. c., percent positive cells.
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Fig. 4. DC from aged persons can trigger proliferation and IFN-g production in autologous T cells. CD4+CD45RO+ and CD4+CD45RA+ T cells were cocultured with antigen in combination with either irradiated autologous PBMC or DC. Proliferation and IFN-g production were assessed following a 7 day culture. Proliferation is expressed as cpm, changes in the production of IFN-g following stimulation as percentual increases (X 2SEM; n = 4 in each group).
persons have an unaltered capacity to dierentiate into DC providing the presence of the right stimuli. IL-4 and GM-CSF rapidly induced the loss of monocytic markers and the appearance of DC surface molecules. The DC population thus generated had unimpaired phenotypic and functional properties and was even capable of stimulating senescent T cells which were hardly able to respond when stimulated with conventional antigen presenting cells. This demonstrates that DC are capable of mobilizing residual functional capacity in senescent T cells. The responsiveness of CD45RA+ was hereby more pronounced than that of CD45RO+ cells. This indicates that age-related intrinsic changes of naive T cells are relatively easy to overcome with simple measurements, such as the provision of the right type of antigen presenting cell, while mem-
Fig. 5. The usage of DC as APC does not change the clonal composition of the T cell response following stimulation with in¯uenza antigen. CDR3 size analysis was performed as described in Section 2. A characteristic experiment is shown.
ory T cell populations may be terminally dierentiated by many rounds of divisions and thus more dicult to restimulate. In this context, it was also of interest that DC from aged persons responded equally well to maturation inducing stimuli as their young counterparts. They responded to PPD as well as to GM-CSF (not shown), but also to whole inactivated in¯uenza virus. The latter response was obviously due to the stimulatory eect of in¯uenza core components. Although the exact nature of the stimulatory agent has yet to be de®ned, our results suggest that in¯uenza nucleoproteins may play a role. Thus, split vaccine, which contains nucleoproteins and surface proteins but no or very little RNA was stimulatory while in¯uenza surface components [26] and puri®ed viral RNA had no eect. Whole inactivated in¯uenza virus was still more stimulatory than split vaccine, suggesting that additional agents may be operative. Thus, it seems possible that viral RNA is stimulatory in RNA/nucleoprotein complexes in analogy to the immunostimulatory eects of short bacterial DNA sequences [29]. Nucleoproteins may hereby be necessary to stabilize the RNA. This would explain why puri®ed RNA on its own had no eect. Whether nucleoproteins as such also have stimulatory properties or whether split vaccine eects are due to small RNA quantities present in the vaccine is presently being investigated in our laboratory. Viral nucleoproteins and/or nucleoprotein/RNA complexes might thus be capable of stimulating non-speci®c innate immunity and be useful as adjuvants in vaccines. Stimulation of DC maturation by adjuvants may be of particular relevance in the aging immune system, where properly stimulated DC might be capable of migrating into the
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draining lymph nodes to ensure the appropriate stimulation of senescent T cells. Summarizing, our results demonstrate that unimpaired DC can be derived from monocytes in aged persons. This does obviously not guarantee that the stimuli needed for the dierentiation process are always present in the aged microenvironment. It may therefore be an important goal of future vaccination strategies to provide the right adjuvants and cytokines to ensure DC dierentiation, maturation and migration in situ. Thus, it may be possible to mobilize residual T cell function and to overcome some of the features of immunosenescence. Acknowledgements This work was supported by the Austrian National Bank (project 6851). We are grateful to Esther Asch, Roswitha MuÃhlmann and Anna Ko-nig for excellent technical assistance. References [1] Steinman RM, The dendritic cell system and its role in immunogenicity, Annu Rev Immunol 1991;9271±96. [2] Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997;90(9):3245± 87. [3] Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med 1996;183(4):1789±96. [4] Steinman RM, Pack M, Inaba K, Dendritic cells in the T cell areas of lymphoid organs, Immunol Rev 1997;15625±37. [5] Kronin V, Vremec D, Winkel K, et al. Are CD8+ dendritic cells (DC) veto cells? The role of CD8 on DC in DC development and in the regulation of CD4 and CD8 T cell response. Int Immunol 1997;9(7):1061±4. [6] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6673):245±52. [7] Schuler G, Thurner B, Romani N. Dendritic cells: from ignored cells to major players in T cell-mediated immunity. Int Arch Allergy Immunol 1997;112(4):317±22. [8] Stingl G, Bergstresser PR. Dendritic cells: a major story unfolds. Immunol Today 1995;16(7):330±3. [9] Caux C, Liu YJ, Banchereau J. Recent advances in the study of dendritic cells and follicular dendritic cells. Immunol Today 1995;16(1):2±4. [10] Sprecher E, Becker Y, Kraal G, et al. Eect of aging on epidermal dendritic cell populations in C57BL/6J mice. J Invest Dermatol 1990;94(2):247±53. [11] Belsito DV, Epstein SP, Schultz JM, Baer RL, Thorbecke GJ. Enhancement by various cytokines or 2-beta-mercaptoethanol of Ia antigen expression on Langerhans cells in skin from normal aged and young mice. Eect of cyclosporine A. J Immunol 1989;143(5):1530±6. [12] Choi KL, Sauder DN. Epidermal Langerhans cell density and contact sensitivity in young and aged BALB/c mice. Mech Ageing Dev 1987;39(1):69±79.
[13] Rittman BR, Hill MW, Rittman GA, Mackenzie IC. Age-associated changes in Langerhans cells of murine oral epithelium and epidermis. Arch Oral Biol 1987;32(12):885±9. [14] Thiers BH, Maize JC, Spicer SS, Cantor AB. The eect of aging and chronic sun exposure on human Langerhans cell populations. J Invest Dermatol 1984;82(3):223±6. [15] Gilchrest BA, Murphy GF, Soter NA. Eect of chronologic aging and ultraviolet irradiation on Langerhans cells in human epidermis. J Invest Dermatol 1982;79(2):85±8. [16] Steger MM, Maczek C, Grubeck-Loebenstein B. Morphologically and functionally intact dendritic cells can be derived from the peripheral blood of aged individuals. Clin Exp Immunol 1996;105(3):544±50. [17] Haruna H, Inaba M, Inaba K, et al. Abnormalities of B cells and dendritic cells in SAMP1 mice. Eur J Immunol 1995;25(5):1319±25. [18] Villadsen JH, Langkjer ST, Ebbesen P, Bjerring P. Syngrafting skin among mice of similar and dierent ages increases the number of Langerhans cells and decreases responsiveness to 1,4dinitro¯uorobenzene. Compr Gerontol [A] 1987;1(2):78±9. [19] Saurwein-Teissl M, SchoÈnitzer D, Grubeck-Loebenstein B. Dendritic cell responsiveness to stimulation with in¯uenza vaccine is unimpaired in old age. Exp Gerontol 1998;33(6):625±31. [20] Perkins EH, Massucci JM, Glover PL. Antigen presentation by peritoneal macrophages from young adult and old mice. Cell Immunol 1982;70(1):1±10. [21] Komatsubara S, Cinader B, Muramatsu S. Functional competence of dendritic cells of ageing C57BL/6 mice. Scand J Immunol 1986;24(5):517±25. [22] Schwab R, Hausman PB, Rinnooy-Kan E, Weksler ME. Immunological studies of ageing. X. Impaired T lymphocytes and normal monocyte response from elderly humans to the mitogenic antibodies OKT3 and Leu 4. Immunology 1985;55(4):677±84. [23] Fagiolo U, Cossarizza A, Scala E, et al. Increased cytokine production in mononuclear cells of healthy elderly people. Eur J Immunol 1993;23(9):2375±8. [24] Sallusto F, Lanzavecchia A. Ecient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994;179(4):1109±18. [25] Genevee C, Diu A, Nierat J, et al. An experimentally validated panel of subfamily-speci®c oligonucleotide primers (V alpha 1w29/V beta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur J Immunol 1992;22(5):1261±9. [26] Saurwein-Teissl M, Zisterer K, Schmitt TL, et al. Whole virus in¯uenza vaccine activates dendritic cells (DC) and stimulates cytokine production by peripheral blood mononuclear cells (PBMC) while subunit vaccines support T cell proliferation. Clin Exp Immunol 1998;114(2):271±6. [27] Thurnher M, Ramoner R, Gastl G, et al. Bacillus CalmetteGuerin mycobacteria stimulate human blood dendritic cells. Int J Cancer 1997;70(1):128±34. [28] Steger MM, Maczek C, Grubeck-Loebenstein B. Peripheral blood dendritic cells reinduce proliferation in in vitro aged T cell populations. Mech Ageing Dev 1997;93(1±3):125±30. [29] Oxenius A, Martinic MM, Hengartner H, Klenerman P. CpGcontaining oligonucleotides are ecient adjuvants for induction of protective antiviral immune responses with T cell peptide vaccines. J Virol 1999;73(5):4120±6.
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Unimpaired dendritic cells can be derived from monocytes in old age and can mobilize residual function in senescent T cells T.L. Lung a, M. Saurwein-Teissl a, W. Parson b, D. SchoÈnitzer c, B. Grubeck-Loebenstein a,* a
Institute for Biomedical Aging Research of the Austrian Academy of Sciences, Rennweg 10, A-6020, Innsbruck, Austria b Institute of Legal Medicine, University of Innsbruck, Innsbruck, Austria c Central Institute for Blood Transfusion and Immunological Department, General Hospital, University Clinics Innsbruck, Innsbruck, Austria
Abstract Dendritic cells (DC) are powerful antigen presenting cells, which have the unique capacity to stimulate naive T cells. In spite of the well-known decline of T cell function in old age, little information is available on whether DC are also aected by the aging process. This is mainly due to problems with the isolation and puri®cation of DC. Rapid progress in the characterization of DC has been made in recent years, as simple methods to generate large numbers of DC from precursors have been developed. It was the aim of the present study to compare monocyte derived DC from old and young healthy persons. The generation of DC from blood monocytes in response to GM-CSF and IL-4 treatment was similar in cells from young and old persons. The DC population thus obtained had a typical dendritic morphology and expressed DC surface markers, such as HLA class II, CD1a, CD11c, CD54, CD80 and CD86, but not CD14 for a period of up to three weeks in culture. DC from young and old persons produced IL-12 and TNF-a and responded equally well to maturation-inducing stimuli. DC maturation was stimulated by puri®ed protein derivative (PPD) of Mycobacterium tuberculosis, whole inactivated in¯uenza virus and by in¯uenza split vaccine, but not by puri®ed viral RNA. When tested for their antigen-presenting capacity, DC from young and old persons were capable of stimulating the proliferation and the cytokine production of T cells. It was of particular interest that CD45RA+ as well as CD45RO+ T cells from aged donors were unable to respond to stimulation with in¯uenza proteins presented by monocytes, but were triggered to proliferate and to produce cytokines when antigen was presented by DC. The results demonstrate that DC from old persons (a) may still function as powerful antigen-presenting cells provided the right dierentiation and maturation stimuli are present; (b) are capable of mobilizing residual capacity in senescent T cells and (c) may therefore represent a potent tool for immunotherapy and vaccines in old age. # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Dendritic cells; T lymphocytes; Aging
1. Introduction Dendritic cells (DC) are identi®ed by a triad of criteria: Morphologically, they exhibit pronounced cytoplasmic veils which are mobile as can easily be
* Corresponding author. Tel.: +43-512-583919-14; fax: +43-512583919-8. E-mail address: [email protected] (B. Grubeck-Loebenstein).
observed under a phase contrast microscope [1,2]. These veils become apparent only in the mature state. Phenotypically, they express high levels of MHC (class I and II) as well as of adhesion (CD11c, CD54, CD58), and costimulatory (CD80, CD86, CD40) molecules on their surface. They also express CD1a and CD83, two DC cell markers, but lack CD14. Functionally, they are potent stimulators of resting T lymphocytes. DC derived from various tissues have been shown to undergo a complex maturation process during which their morphology, phenotype and func-
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T.L. Lung et al. / Vaccine 18 (2000) 1606±1612
tion change. DC of myeloid as well as of lymphoid origin have been described [3±5]. DC take up and process antigen and consecutively present antigenic peptides in the context of MHC molecules. Upon appropriate stimulation they undergo further maturation and migrate to secondary lymphoid tissues where they present antigens to T cells. Recent knowledge on DC has been summarized in excellent reviews [4,6±9]. In contrast to the rapidly increasing amount of knowledge on DC cell physiology in young individuals relatively little is still known on DC in aged humans and animals. Langerhans cells (LC), the epidermal equivalent of DC, have been studied in some detail in aged mice, the most obvious question being whether the numbers of LC change with age. Several authors found a decline of LC densities in the epidermis with age [10±12]. The extent of LC reduction was about one third. This was also observed with LC in the oral mucosa: a reduction by 30±60% was reported in the mouse [13]. No detailed studies are available concerning age-related changes in the numbers and distribution of DC from sources other than the skin. Similar to the results on experimental animals, early studies on aged human skin have also demonstrated a decreased density of LC [14,15]. This may, however, partly be due to UV irradiation, as signi®cantly fewer LC were observed in exposed vs. covered skin in old individuals, while no such disparity was noted in younger subjects. Whether the low LC density in old age is the result of lower DC progenitor numbers or of an impaired migration of progenitors from the marrow to the target tissues is not yet known [10]. The latter possibility seems, however, more likely, as recent results on humans demonstrate that higher numbers of DC can be derived from puri®ed monocytes of aged than of young persons [16]. No consensus has yet been reached whether DC function is impaired in old age. Whereas some studies suggest reduced function [10±12,17,18], other more recent studies tend to agree that the function of monocytes and monocyte derived cells are not aected by the aging process [16,19±23]. Further work will be needed to obtain entire clarity on this subject. It was the aim of the present study to focus on monocyte derived human DC and to analyze their phenotype and their responsiveness to maturation inducing stimuli as well as their capacity to stimulate senescent T cells. 2. Materials and methods 2.1. Reagents, monoclonal antibodies and serum IL-4 and GM-CSF were kindly provided by Sandoz
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Pharma AG (Basel, Switzerland). Mouse MoAbs directed against the following cell surface determinants were used: MHC class II (HLA-D, An der Grub GmbH, Kaumberg, Austria, FITC-conjugated), CD1a (Serotec, FITC-conjugated), CD14 (Serotec, PE-conjugated), CD11c (Pharmingen, PE-conjugated), CD54 (Pharmingen, PE-conjugated), CD80 (Becton Dickinson, PE-conjugated), CD86 (Pharmingen, FITC-conjugated) and CD83 (Pharmingen, PE-conjugated). Fetal calf serum (FCS) was purchased from Schoeller Pharma (Vienna, Austria). 2.1.1. Antigens An intact virion in¯uenza vaccine inactivated by propiolactone and in¯uenza subunit vaccine were kindly provided by the Swiss Serum & Vaccine Institute (Berne, Switzerland). Both vaccine contained the following three in¯uenza strains: A/Beijing/262/95 (H1N1), A/Sydney/5/97, (H3N2) and B/Harbin/7/94. In¯uenza split vaccine containing the same in¯uenza strains was provided by Pasteur MeÂrieux Connaught (Marcy l'Etoile, France). Both vaccines were in the following referred to as FLU. PPD (150 mg/ml) was a gift from the Behring Institute (Vienna, Austria). For the isolation of RNA, in¯uenza life virus was used (kindly provided by the Swiss Serum & Vaccine Institute) and isolated by resuspending the pellet in Trizol reagent (Gibco BRL). As a control total RNA from human tissues was additionally isolated. Transfection of DC with in¯uenza and control RNA was carried out with DOTAP liposomal transfection reagent (Boehringer Mannheim, Vienna, Austria). 2.2. Puri®cation of DC Peripheral blood mononuclear cells (PBMC) were obtained from old (>65 years) and young (<30 years) healthy individuals. DC were prepared from peripheral blood monocytes, as previously described [16,24]. In brief, PBMC were resuspended in RPMI-1640 (BIO Whittaker, Verviers, Belgium), 10% FCS and 1% Penicillin/Streptomycin (P/S; Gibco) as culture medium (CM) and allowed to adhere to six-well plates (9 106 cells/well; Falcon, Becton Dickinson, Franklin Lakes, NJ, USA). After 2 h at 378C, non-adherent cells were removed and the adherent monocyte enriched population (85 2 2% CD14+ cells) cultured in CM supplemented with 800 U GM-CSF and 1000 U IL-4/ml. Cells were then fed every second day with fresh CM containing 800 U GM-CSF and 300 U IL-4/ ml and analyzed after 1±3 weeks of culture. 2.3. Preparation of DC for surface marker and cytokine secretion analysis After 1 week in culture, DC, which were at that
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time point mostly non-adherent, 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 immuno¯uorescence staining and FACScan analysis [16]. Cells were then counted and incubated at 106 cells per tube in fresh CM at 378C, 5% CO2 in tissue culture tubes (Greiner, KremsmuÈnster, Austria) in the absence or presence of stimuli. After 24 h supernatants were harvested, centrifuged and stored at ÿ208C. Cells were washed and analyzed by immuno¯uorescence staining, as described below. 2.4. Immuno¯uorescence staining and FACScan analysis Cells were transferred into round bottom tubes (105 cells/tube; Falcon) and washed at 48C in PBS containing 0.1% FCS. Antibodies were added and the cells left to incubate at 48C. After 40 min the cells were washed twice in PBS. Analysis was performed on a Becton Dickinson FACScan. Five thousand scattergated cells were analyzed in each sample. The frequency and ¯uorescence pro®les of the cells were determined with logarithmic signal ampli®ers.
carried out in triplicates. Results were expressed as counts per minute (CPM). 2.9. TCR repertoire Puri®ed CD4+CD45RO+/RA+ cells from young and old persons were stimulated with in¯uenza subunit vaccine (1 mg/ml) presented by either autologous irradiated DC or PBMC and left to incubate for 1 week. RNA, prepared from T cells was reverse transcribed and TCR Vb transcripts were ampli®ed by PCR using primers speci®c for each of the human Vb families and a speci®c primer for the constant region of the b chain (labeled with the ¯uorescent dye marker 6-FAM (6carboxy¯uorescein) coupled with an aminohexyl linker; Applied Biosystems, Weiterstadt, Germany). Sequences of Vb primers were as in GeneveÂe et al. [25]. PCRs were prepared in a ®nal volume of 50 ml and contained 0.5 mM of each oligonucleotide primer in Taq-PCR Master Mix (Quiagen, Hilden, Germany). The PCR reactions were started with an initial heating at 948C for 30 s followed by 35 cycles of ampli®cation by denaturation at 948C for 45 s, annealing of primers at
2.5. Puri®cation of CD4+CD45RO+ and of CD4+CD45RA+T cells T cell subsets were separated with magnetic MicroBeads according to manufacturers protocol: CD4 MultiSort Kit and CD45RO MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). 2.6. Cocultures of DC, PBMC and T cells CD45RO+ and CD45RA+ T cells (105 or 5 105 cells/well) were cocultured with and without irradiated (35 Gy) autologous DC (3 104 or 1,5 105) or PBMC (105 or 5 105) in the presence or absence of in¯uenza antigen (1 mg/ml). Cells were cultured for 1 week in 24- or 96-well ¯at-bottom plates (Falcon). 2.7. Cytokine determinations by ELISA Cytokine concentrations in conditioned supernatants were determined by commercially available ELISA kits: TNF-a, IL-12 and IFN-g (Endogen Inc., Cambridge, MA, USA). 2.8. Proliferation assays After the respective incubation periods cells were pulsed with 1 mCi of [3H]-thymidine (ICN Pharmaceuticals, CA, USA) and [3H]-thymidine incorporation was assessed by scintillation counting. All assays were
Fig. 1. Dendritic cell dierentiation from monocytes is unimpaired in old age. Puri®ed monocytes from young and aged persons were stimulated with GM-CSF and IL-4. CD14 and CD1a surface expression was monitored by immuno¯uorescence analysis during 1 week (x-2SEM; n = 3 in each group).
T.L. Lung et al. / Vaccine 18 (2000) 1606±1612
728C for 45 s and an extension step at 728C for 10 min. An aliquot of the PCR product was diluted in 16 ml deionized formamide and 1.2 fmol internal lane standard GeneScan-350 Tamra (Perkin Elmer, Norwalk, CT, USA). The samples were denatured at 908C for 2 min. and snap cooled on ice prior to loading on a CE 310 Genetic Analyser. Each sample was injected for 5 s at 15 kV and electrophoresed for 24 min at 10 kV using a 36 cm capillary and POP4 (Perkin Elmer, Norwalk, CT). Analysis of the raw data was performed applying the GeneScan 2.1 analysis software package (PE, ABD) using the Local Southern method for fragment size estimation. Analyzed data were imported into Genotyper 2.0 and labeled according to the estimated fragment size. 3. Results 3.1. Generation and propagation of DC from monocytes CD14+ monocytes were puri®ed from the peripheral blood of young and aged persons. These cells were stimulated with IL-4 and GM-CSF as described in Section 2. In both age groups CD14 expression disap-
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peared rapidly upon cytokine treatment whereas CD1a appeared on the surface of the cells (Fig. 1). After a week of culture 93 2 1% of the cells expressed CD1a whereas only low percentages of CD14 cells were found. The results demonstrate that the generation of DC from monocytic precursor cells is unaected by the aging process provided the right stimuli (cytokines) are present. 3.2. Monocyte derived DC have an unimpaired phenotype and an unimpaired cytokine production pattern DC from young and aged persons were consecutively analyzed for their surface molecule expression. Immuno¯uorescence staining demonstrated a similar expression pattern of CD54, MHC class II, CD86, CD80, CD1a and CD11c in DC derived from young and aged persons over a time span of three weeks in culture (Fig. 2). It was of interest that CD86 reached its maximal expression in the third week of culture when CD1a expression was rapidly lost. In spite of their decreased CD1a expression, cells from young and aged persons still had a pronounced stimulatory activity in the allogeneic mixed lymphocyte reaction
Fig. 2. Surface molecule expression is similar in DC derived from young and old persons over prolonged culture periods. Surface molecule expression was assessed by immuno¯uorescence staining (x-2SEM; n = 3 in each group).
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did not induce maturation. DC maturation could still be stimulated by split vaccine, indicating that in¯uenza nucleoproteins may have a non-speci®c stimulatory eect on the innate immune system. Expectedly, PPD was an excellent stimulator of DC maturation [27] and worked equally well on ``young'' and ``old'' DC (data not shown). 3.4. DC from aged persons can induce proliferation and cytokine production in senescent T cells Fig. 3. Cytokine production is similar in DC from young and aged persons. DC were stimulated with whole inactivated in¯uenza virus and their conditioned supernatants analyzed for the presence of IL12 and TNF-a following a 24 h incubation (x-2SEM; n = 15 in each group).
(MLR) at this late time point (data not shown). DC from young and aged persons also had a similar capacity to secrete cytokines in the resting state as well as following stimulation (Fig. 3). 3.3. DC from aged persons have an unimpaired responsiveness to maturation inducing stimuli The capacity of the following agents to induce maturation was tested on DC from young and aged persons: whole inactivated in¯uenza virus, in¯uenza split vaccine which contains surface proteins and nucleoproteins but no viral RNA, puri®ed in¯uenza virus RNA and PPD. The increase in the number of CD83+ cells, as well as in the intensity of CD54 and CD86 expression were analyzed to assess DC maturation. In accordance with previous results from our group [19,26] whole inactivated in¯uenza virus was an excellent stimulator of DC maturation and was equally eective on DC from aged and young persons (Table 1). As previous results from our laboratory had demonstrated that in¯uenza surface protein had no stimulatory eect on DC [26], the eect of the whole virus was obviously due to the presence of core components. Viral RNA per se was not eective, as the transfection of DC with puri®ed in¯uenza virus RNA
Previous results from our laboratory have shown that DC from aged persons are capable of stimulating the clonal expansion and of postponing the clonal elimination of antigen speci®c T cell populations in an in vitro senescence model [28]. We therefore analyzed whether DC were also capable of stimulating freshly isolated T cells from aged persons. For this reason, puri®ed CD4+CD45RO+ and CD4+CD45RA+ T cells from aged persons were stimulated with in¯uenza antigen in combination with either irradiated autologous PBMC or corresponding numbers of DC. T cell proliferation and IFN-g production were used as read out parameters of T cell function. While T cell responsiveness was very low when PBMC were used as antigen presenting cells, proliferation as well as IFN-g production could be strongly increased when DC were used as APC (Fig. 4). It was of interest that the increase in the proliferation/IFN-g production of T cells induced by DC was even more pronounced when CD45RA+ T cells were used as responder cells. DC aected the magnitude but not the clonal composition of the response, which was demonstrated by CDR3 size analysis (Fig. 5). The clonal composition of responding T cells from dierent Vb families was the same whether PBMC or DC were used as presenter cells. A typical experiment is depicted in Fig. 5.
4. Discussion Our results demonstrate that monocytes from aged
Table 1 Changes in the surface expression of CD83, CD54 and CD86 in DC from young and old healthy individuals following stimulationa CD83 D % p.c.
Whole virus FLU Split vaccine FLU Viral RNA Human RNA a
CD54 D % MFI
CD86 D % MFI
Young
Old
Young
Old
Young
Old
+2582171% +57225% ND ND
+6852466% +145230% ND ND
+108288% +79222% +521% +122%
+108281% +50215% +025% +024%
+168238% +2925% +922% +021%
+121213% +826% +523% +822%
Data are expressed as mean2SEM in each group (n = 3±6); MFI, mean¯uorescence; % p. c., percent positive cells.
T.L. Lung et al. / Vaccine 18 (2000) 1606±1612
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Fig. 4. DC from aged persons can trigger proliferation and IFN-g production in autologous T cells. CD4+CD45RO+ and CD4+CD45RA+ T cells were cocultured with antigen in combination with either irradiated autologous PBMC or DC. Proliferation and IFN-g production were assessed following a 7 day culture. Proliferation is expressed as cpm, changes in the production of IFN-g following stimulation as percentual increases (X 2SEM; n = 4 in each group).
persons have an unaltered capacity to dierentiate into DC providing the presence of the right stimuli. IL-4 and GM-CSF rapidly induced the loss of monocytic markers and the appearance of DC surface molecules. The DC population thus generated had unimpaired phenotypic and functional properties and was even capable of stimulating senescent T cells which were hardly able to respond when stimulated with conventional antigen presenting cells. This demonstrates that DC are capable of mobilizing residual functional capacity in senescent T cells. The responsiveness of CD45RA+ was hereby more pronounced than that of CD45RO+ cells. This indicates that age-related intrinsic changes of naive T cells are relatively easy to overcome with simple measurements, such as the provision of the right type of antigen presenting cell, while mem-
Fig. 5. The usage of DC as APC does not change the clonal composition of the T cell response following stimulation with in¯uenza antigen. CDR3 size analysis was performed as described in Section 2. A characteristic experiment is shown.
ory T cell populations may be terminally dierentiated by many rounds of divisions and thus more dicult to restimulate. In this context, it was also of interest that DC from aged persons responded equally well to maturation inducing stimuli as their young counterparts. They responded to PPD as well as to GM-CSF (not shown), but also to whole inactivated in¯uenza virus. The latter response was obviously due to the stimulatory eect of in¯uenza core components. Although the exact nature of the stimulatory agent has yet to be de®ned, our results suggest that in¯uenza nucleoproteins may play a role. Thus, split vaccine, which contains nucleoproteins and surface proteins but no or very little RNA was stimulatory while in¯uenza surface components [26] and puri®ed viral RNA had no eect. Whole inactivated in¯uenza virus was still more stimulatory than split vaccine, suggesting that additional agents may be operative. Thus, it seems possible that viral RNA is stimulatory in RNA/nucleoprotein complexes in analogy to the immunostimulatory eects of short bacterial DNA sequences [29]. Nucleoproteins may hereby be necessary to stabilize the RNA. This would explain why puri®ed RNA on its own had no eect. Whether nucleoproteins as such also have stimulatory properties or whether split vaccine eects are due to small RNA quantities present in the vaccine is presently being investigated in our laboratory. Viral nucleoproteins and/or nucleoprotein/RNA complexes might thus be capable of stimulating non-speci®c innate immunity and be useful as adjuvants in vaccines. Stimulation of DC maturation by adjuvants may be of particular relevance in the aging immune system, where properly stimulated DC might be capable of migrating into the
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draining lymph nodes to ensure the appropriate stimulation of senescent T cells. Summarizing, our results demonstrate that unimpaired DC can be derived from monocytes in aged persons. This does obviously not guarantee that the stimuli needed for the dierentiation process are always present in the aged microenvironment. It may therefore be an important goal of future vaccination strategies to provide the right adjuvants and cytokines to ensure DC dierentiation, maturation and migration in situ. Thus, it may be possible to mobilize residual T cell function and to overcome some of the features of immunosenescence. Acknowledgements This work was supported by the Austrian National Bank (project 6851). We are grateful to Esther Asch, Roswitha MuÃhlmann and Anna Ko-nig for excellent technical assistance. References [1] Steinman RM, The dendritic cell system and its role in immunogenicity, Annu Rev Immunol 1991;9271±96. [2] Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997;90(9):3245± 87. [3] Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med 1996;183(4):1789±96. [4] Steinman RM, Pack M, Inaba K, Dendritic cells in the T cell areas of lymphoid organs, Immunol Rev 1997;15625±37. [5] Kronin V, Vremec D, Winkel K, et al. Are CD8+ dendritic cells (DC) veto cells? The role of CD8 on DC in DC development and in the regulation of CD4 and CD8 T cell response. Int Immunol 1997;9(7):1061±4. [6] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6673):245±52. [7] Schuler G, Thurner B, Romani N. Dendritic cells: from ignored cells to major players in T cell-mediated immunity. Int Arch Allergy Immunol 1997;112(4):317±22. [8] Stingl G, Bergstresser PR. Dendritic cells: a major story unfolds. Immunol Today 1995;16(7):330±3. [9] Caux C, Liu YJ, Banchereau J. Recent advances in the study of dendritic cells and follicular dendritic cells. Immunol Today 1995;16(1):2±4. [10] Sprecher E, Becker Y, Kraal G, et al. Eect of aging on epidermal dendritic cell populations in C57BL/6J mice. J Invest Dermatol 1990;94(2):247±53. [11] Belsito DV, Epstein SP, Schultz JM, Baer RL, Thorbecke GJ. Enhancement by various cytokines or 2-beta-mercaptoethanol of Ia antigen expression on Langerhans cells in skin from normal aged and young mice. Eect of cyclosporine A. J Immunol 1989;143(5):1530±6. [12] Choi KL, Sauder DN. Epidermal Langerhans cell density and contact sensitivity in young and aged BALB/c mice. Mech Ageing Dev 1987;39(1):69±79.
[13] Rittman BR, Hill MW, Rittman GA, Mackenzie IC. Age-associated changes in Langerhans cells of murine oral epithelium and epidermis. Arch Oral Biol 1987;32(12):885±9. [14] Thiers BH, Maize JC, Spicer SS, Cantor AB. The eect of aging and chronic sun exposure on human Langerhans cell populations. J Invest Dermatol 1984;82(3):223±6. [15] Gilchrest BA, Murphy GF, Soter NA. Eect of chronologic aging and ultraviolet irradiation on Langerhans cells in human epidermis. J Invest Dermatol 1982;79(2):85±8. [16] Steger MM, Maczek C, Grubeck-Loebenstein B. Morphologically and functionally intact dendritic cells can be derived from the peripheral blood of aged individuals. Clin Exp Immunol 1996;105(3):544±50. [17] Haruna H, Inaba M, Inaba K, et al. Abnormalities of B cells and dendritic cells in SAMP1 mice. Eur J Immunol 1995;25(5):1319±25. [18] Villadsen JH, Langkjer ST, Ebbesen P, Bjerring P. Syngrafting skin among mice of similar and dierent ages increases the number of Langerhans cells and decreases responsiveness to 1,4dinitro¯uorobenzene. Compr Gerontol [A] 1987;1(2):78±9. [19] Saurwein-Teissl M, SchoÈnitzer D, Grubeck-Loebenstein B. Dendritic cell responsiveness to stimulation with in¯uenza vaccine is unimpaired in old age. Exp Gerontol 1998;33(6):625±31. [20] Perkins EH, Massucci JM, Glover PL. Antigen presentation by peritoneal macrophages from young adult and old mice. Cell Immunol 1982;70(1):1±10. [21] Komatsubara S, Cinader B, Muramatsu S. Functional competence of dendritic cells of ageing C57BL/6 mice. Scand J Immunol 1986;24(5):517±25. [22] Schwab R, Hausman PB, Rinnooy-Kan E, Weksler ME. Immunological studies of ageing. X. Impaired T lymphocytes and normal monocyte response from elderly humans to the mitogenic antibodies OKT3 and Leu 4. Immunology 1985;55(4):677±84. [23] Fagiolo U, Cossarizza A, Scala E, et al. Increased cytokine production in mononuclear cells of healthy elderly people. Eur J Immunol 1993;23(9):2375±8. [24] Sallusto F, Lanzavecchia A. Ecient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994;179(4):1109±18. [25] Genevee C, Diu A, Nierat J, et al. An experimentally validated panel of subfamily-speci®c oligonucleotide primers (V alpha 1w29/V beta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur J Immunol 1992;22(5):1261±9. [26] Saurwein-Teissl M, Zisterer K, Schmitt TL, et al. Whole virus in¯uenza vaccine activates dendritic cells (DC) and stimulates cytokine production by peripheral blood mononuclear cells (PBMC) while subunit vaccines support T cell proliferation. Clin Exp Immunol 1998;114(2):271±6. [27] Thurnher M, Ramoner R, Gastl G, et al. Bacillus CalmetteGuerin mycobacteria stimulate human blood dendritic cells. Int J Cancer 1997;70(1):128±34. [28] Steger MM, Maczek C, Grubeck-Loebenstein B. Peripheral blood dendritic cells reinduce proliferation in in vitro aged T cell populations. Mech Ageing Dev 1997;93(1±3):125±30. [29] Oxenius A, Martinic MM, Hengartner H, Klenerman P. CpGcontaining oligonucleotides are ecient adjuvants for induction of protective antiviral immune responses with T cell peptide vaccines. J Virol 1999;73(5):4120±6.