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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:1039-1047 (2006)
© 2006 The Gerontological Society of America

Effect of Aging on Bone Marrow-Derived Murine CD11c+CD4–CD8{alpha}– Dendritic Cell Function

Annabelle Grolleau-Julius, Monika R. Garg, RuRan Mo, Lloyd L. Stoolman and Raymond L. Yung

1 Divisions of Geriatric Medicine and Rheumatology, Department of Internal Medicine, and 2 Department of Pathology, University of Michigan, Ann Arbor.
3 GRECC, Ann Arbor Veteran Affairs Medical Center, Michigan.

Address correspondence to Raymond L. Yung, MD, Room 5312 CCGC, 1500 East Medical Center Drive, Ann Arbor, Michigan, 48109-0940. E-mail: ryung{at}umich.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) are actively used as cellular adjuvant in cancer immunotherapy. However, although DC immunotherapies primarily target the elderly population, little is known about the effect of aging on DC functions. Here, we compared the T-cell stimulation, cytokine production, and tumor surveillance functions of bone marrow-derived CD11c+CD4–CD8{alpha}– DCs of old and young C57BL/6 mice. Old immature bone marrow-derived CD4–CD8{alpha}– DCs (imDCs) were 4 times less effective than were young DCs in stimulating syngeneic CD4+ T-cell proliferation. Old imDCs also have decreased DC-specific/intracellular adhesion molecule type 3-grabbing, nonintegrin (DC-SIGN) expression compared to young DCs. Interestingly, mice treated with the ovalbumin peptide-pulsed young DCs exhibited significantly greater tumor regression than with ovalbumin peptide-pulsed old DCs. Old terminally differentiated bone marrow-derived DCs (tDC) also have increased interleukin-10, but decreased interleukin-6 and tumor necrosis factor-{alpha} production. Taken together, these results have important implications in the clinical application of DC-based tumor immunotherapy in elderly persons.

AGING is associated with a general decline in both the humoral and cell-mediated immune responses. However, the basis for the decline in immune function in aging is incompletely understood. Previous investigations in immune senescence have largely focused on the age-related decline in T-cell functions (1). Reported T-cell abnormalities in aging include defects in signaling pathway, decreased proliferation response to mitogens and antigens, and an increase in memory phenotype. However, attempts to correlate the observed T-cell defects to disease process or clinical outcome have largely been unsuccessful, suggesting that other supporting members of the immune system or the coordinated effects of immune cell disregulation may be responsible for the reduced ability to defend against infection and cancer in aging.

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) in the body and play a principal role in the priming of T-cell immune response (2). The DC system arises from the CD34+ bone marrow stem cells and is made up of a number of distinct subsets from both the myeloid (DC1) and lymphoid (DC2) lineages (3). Heterogeneity among DCs isolated from different lymphoid organs is well recognized on the basis of differences in their cell surface markers. For example, murine CD11c+ DCs are known to comprise at least three major subpopulations: CD8{alpha}+, CD8{alpha}–CD4+, and CD8{alpha}–CD4– (double negative) (4). Immature DCs (imDCs) can be generated from bone marrow progenitor cells by stimulation with granulocyte macrophage–colony-stimulating factor (GM–CSF) and interleukin-4 (IL-4) (5). These cells display high endocytic/phagocytic activity and low expression of accessory molecules for T-cell activation. After capturing microbial and tumor antigens, the imDCs begin the process of maturation and migrate to secondary lymphoid tissues where they can bring the processed antigens to naïve T cells (6). In addition, in vitro DC maturation can be induced by cytokines (e.g., tumor necrosis factor-{alpha} [TNF-{alpha}] or IL-1ß) or microbial products (e.g., lipopolysaccharides [LPS]). Mature DCs are characterized by their reduced ability to ingest antigen and enhanced T-cell costimulatory capacity. Activation of naïve T cells requires the cooperation of DCs expressing Major Histocompatibility Complex (MHC) Class I/II and costimulatory molecules including CD40, CD80, CD86, and the recently characterized DC-specific/intracellular adhesion molecule type-3-grabbing, nonintegrin (DC-SIGN) where ICAM is intercellular adhesion molecule-3. DC-SIGN is a member of the type II C-type lectin family that also functions as an endocytic receptor mediating antigen presentation (7). Finally, cytokines secreted by DCs, particularly IL-12, are instrumental in the final differentiation of T cells into Type 1 or Type 2 effector cells.

Because of the central role DCs play in priming T-cell immune responses, these cells are actively pursued as a possible tool in immune tolerance and tumor immunotherapy. DCs pulsed with defined tumor-associated peptides or proteins have been shown to elicit potent antitumor T-cell responses in both in vitro and in vivo systems (8–10). These encouraging results further warrant the clarification of several issues for optimal design of DC-based immunotherapy protocols. The complexity of the DC lineage—with diverse subsets, stages of maturation, and methods of generation—necessitates that each variable be tested independently. Because elderly persons are preferentially affected by diseases targeted by DC-directed immunotherapy it will be important to understand DC functions in this population as well.

Despite the increasingly recognized critical role of DCs in immune reactions, surprisingly little is known about the effect of aging on these powerful APCs. Most of the currently available data on the effect of aging focused on individual human (11–14) and murine (15,16) DC functions. The results are often contradictory and are difficult to compare as the origin of the cells, their culture condition, and maturation protocols vary greatly. Furthermore, given the heterogeneity of the DC subsets, it is unclear if such data can be generalized to all DCs. In this study, we sought to comprehensively examine the effect of aging on the generation and on three major functions (CD4+ T cell stimulation, cytokine production, and tumor surveillance) of carefully characterized bone marrow-derived murine DCs.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young (3–6 months) and old (21–24 months) C57BL/6 (B6, H-2b, I-Ab) mice were purchased from Harlan (Indianapolis, IN). Ovalbumin (OVA) T-cell receptor (TCR) OT-II transgenic mice C57BL/6 (H-2b) were purchased from The Jackson Laboratory (Bar Harbor, ME). As before, mice with evidence of disease were not used in these studies (17,18). The mice were maintained in a pathogen-free environment provided by the Unit for Laboratory Animal Medicine at the University of Michigan until use. Procedures involving the animals and their care were conducted in accordance with the guidelines for animal treatment at the University of Michigan.

Generation, Purification, and Maturation of Bone Marrow-Derived DCs
Erythrocyte-depleted bone-marrow cells flushed from the marrow cavities of femurs and tibiae of C57BL/6 mice were cultured at 1 x 106 cells/mL in the presence of murine recombinant GM-CSF at 20 ng/mL and murine recombinant IL-4 at 20 ng/mL (R&D Systems, Minneapolis, MN) in complete medium (RPMI-1640 with 10% heat-inactivated fetal bovine serum [FBS], 0.1 mM nonessential amino acids, 1 µM sodium pyruvate, 10 mM HEPES buffer, 2 mM glutamine, 50 µM 2-mercaptoethanol and antibiotics) as described previously (19,20). Day 5 bone marrow-derived DCs were purified by labeling with bead-conjugated anti-CD11c monoclonal antibody (mAb) followed by positive selection through paramagnetic columns (Miltenyi Biotec, Auburn, CA). The purity of the fraction, assessed by flow cytometry using phycoerythrin (PE)-conjugated anti-CD11c mAb, was consistently >90%. DC maturation was induced by culturing these immature cells in the presence of LPS at 1 µg/mL (Sigma, St. Louis, MO) for 24 hours.

Morphology and Cell Counting
Cell morphology was determined by direct examination with a light microscope (Olympus BX51; Center Valley, PA). Cells were counted using a hemocytometer.

Proliferation Assays
CD4+ T-cell proliferation response was assessed using CD11c+ bone marrow-derived DCs from young and old C57BL/6 mice as stimulator cells, and total splenocytes or purified T cells from OVA TCR transgenic (Tg) C57BL/6 mice (OT-II) mice as responders. The OT-II Tg mice have been made to express the chicken OVA-specific MHC Class II molecule on the CD4+ T cells and are therefore useful in determining CD4-specific T-cell response. Day 5 imDCs were pulsed with or without OVA-peptide(323-339) (Peptides International, Louisville, KY) at 10 µg/mL for 4 hours at 37°C in complete medium, washed two times before irradiation (3000 rads), and then incubated with freshly isolated splenocytes from the OT-II Tg mice. The responder cells (0.1 x 106 cells per well) were placed in 96-well round-bottomed plates with titrating numbers of DCs, giving responder/stimulator (R:S) ratios of 50:1, 100:1, 500:1, 1000:1, 5000:1, and 10,000:1, in a culture volume of 0.2 mL. Quadruplicate DC:splenocyte cocultures were performed and incubated in 5% CO2 at 37°C for 3 days. For controls, DCs and splenocytes were cultured separately. [3H]thymidine was added at 1 µCi/well 18 hours before cell harvest, and was quantified using a scintillation counter. In confirming experiments, negatively selected microbead (Pan T cell Isolation Kit; Miltenyi Biotec, Auburn, CA)-purified T cells from the C57BL/6 OT-II Tg mice were used in the place of total splenocytes. The capacity of the young and old bone marrow-derived DCs to stimulate proliferation of the responder cells is expressed as a proliferation index, calculated as proliferation of the responder cells (mean cpm value) divided by their background proliferation (mean cpm value). In some experiments, supernatants were collected and stored at –80°C until they were assayed with the mouse IL-4 and interferon-{gamma} (IFN-{gamma}) enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, San Diego, CA), according to the manufacturer's instructions.

Cell Surface Analysis
For phenotypic analyses, immature and mature bone marrow-derived DCs were incubated for 30 minutes at 4°C with PE-conjugated anti-H2Db, anti-I-Ab(Aßb), anti-CD40, anti-CD80, anti-CD86, FITC-conjugated anti-CD54, anti-CD4, anti-CD8{alpha}, or the appropriate isotype controls (all obtained from BD PharMingen, San Diego, CA). Alternately, the cells were incubated with biotin anti-DC-SIGN mAb followed by Streptavidin-PE (BD PharMingen). Labeled cells were then washed twice with 1% bovine serum albumin (BSA) and 0.1% sodium azide in phosphate-buffered saline (PBS; Mediatech, Inc., Herndon, VA), fixed in 1% paraformaldehyde (PFA) in PBS, and analyzed for fluorescence on a FACSCalibur (Becton Dickinson, San Jose, CA). Data analyses were done based on examination of 10,000 cells per sample and performed using Cell Quest software (Becton Dickinson).

Cytokine Production
For cytokine protein quantitation, supernatants of the bone marrow-derived DC culture were harvested after 24 hours of incubation with or without LPS stimulation. The supernatants were then simultaneously assayed for IL-6, IL-10, IL12-p70, TNF-{alpha}, and IFN-{gamma} cytokines using the mouse inflammatory Cytometric Bead Array (CBA) kit (BD Biosciences), following the manufacturer's instructions. This assay kit provides a mixture of five microbeads with distinct fluorescence intensities (FL-3) that are precoated with capturing antibodies specific for the inflammatory proteins. When the beads were incubated with the corresponding PE-conjugated detection antibodies and the test sample, sandwich complexes were formed. The fluorescence produced by the beads was measured using a FACSCalibur flow cytometer.

In Vivo Treatment of B16-OVA Tumor Cells
C57BL/6 mice (6–7 week old) were subcutaneously inoculated in their right flanks with 0.5 x 106 viable B16-OVA tumor cells in 0.2 mL of PBS using a 27-gauge needle. The B16-BL6 melanoma is a poorly immunogenic melanoma cell line. The B16-OVA tumor line has been transfected to express OVA that serves as a tumor-associated antigen (21). Six days after the tumor cell inoculation, when tumors reached 3–4 mm in diameter, the mice received injections in their left flanks with 2 x 106 OVA–peptide(257–264)-pulsed young or old bone marrow-derived imDCs in 0.2 mL of PBS or PBS alone. The imDCs were generated and purified from old and young animals, and then pulsed with OVA–peptide(257–264) (SINFEKL) (synthesized by the University of Michigan Protein Facility, Ann Arbor) at 10 µg/mL for 6 hours before injection. Tumor growth was monitored every other day by measuring in a blinded fashion two perpendicular tumor diameters using a caliper, and was recorded as the product of two orthogonal diameters (a x b).

Statistical Analysis
Results are expressed as means ± standard error of the mean (SEM). Statistical analyses were performed using Student's t test, and p ≤.05 was considered to be statistically significant. For multiple comparisons, the two-tailed Student t test was used.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Generation and Morphology of CD11c+ DCs From the Bone Marrow of Old and Young Mice
A recent report suggested that the number of CD11c-expressing cells is decreased in aged athymic nude mice (22). However, others have found that the number of peripheral blood DCs generated from older humans was higher than that from younger persons (13). To determine if aging affects the generation of DCs from bone marrow progenitor cells, we first determined the number of DCs obtained from the bone marrow of old and young C57BL/6 mice following in vitro treatment with IL-4/GM-CSF. The total number of cells and the number of CD11c+-purified DCs from the bone marrow of 20 old and 20 young individual mice were compared after 5 days of culture. As shown in Figure 1A, there was no difference between the mean number of bone marrow-derived DCs, total cells or CD11c+-purified cells, in young and old mice. The ratio of the total number of cells/CD11c+ DCs is 7.4 + 1.66 (mean + SEM) and 7.2 + 1.7 (mean + SEM) for old and young, respectively.


Figure 01
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  		Figure 1. Yield and morphology of dendritic cells (DCs) generated from the bone marrow of young and old mice. DCs were cultured for 5 days with interleukin-4 and granulocyte macrophage–colony-stimulating factor. A, Cells were harvested, and the yield of DC was assessed before and after CD11c+ purification. Results are reported as mean ± standard error of the mean of 20 old and 20 young mice, examined in 7 independent experiments. B, Morphological appearance of Day 5 CD11c+ DCs after 24 hours without (a and c) or with stimulation by lipopolysaccharide (1 µg/mL; b and d) was assessed. One representative culture from a young (a and b) and one from an old mouse (c and d) of 8 experiments are shown. Original magnification, x40 for all panels

 
The morphology of the bone marrow-derived DCs pre- and postmaturation was also examined (Figure 1B). Terminally matured DCs (tDCs) were obtained after further incubation with LPS (1 µg/mL) for 24 hours. Whereas imDCs were round with short, blunt prolongations (panels a and c), tDCs displayed multiple long processes (panels b and c). No significant change in DCs morphology with age was observed.

Costimulatory Capacity of Young and Old DCs
As professional APC, the major function of DCs is to stimulate T-cell response. We therefore sought to determine the effect of aging on the bone marrow-derived CD11c+ DCs' ability to stimulate syngeneic T lymphocyte proliferation. The titrating numbers of Day 5 imDCs derived from young and old mice were pulsed with OVA(323-339) peptide, and cocultured with syngeneic OVA-specific splenocytes for 3 days. The results show that young imDCs stimulate syngeneic splenocyte proliferation approximately 4-fold more effectively than do old DCs, at R:S ratios ranging from 50:1 to 500:1 (p <.0005, Figure 2A). This finding is consistent with that from a previous report showing that the ability of epidermal imDCs to stimulate OVA-specific T-cell proliferation was significantly lower in aging (23). In confirming experiments, we used microbead-purified T cells from OT-II Tg mice as responder cells, and the results are similar to those using total splenocytes (Figure 2B), with old DCs showing reduced syngeneic CD4+ T-cell stimulation capacity.


Figure 02
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  		Figure 2. Comparison of syngeneic CD4+ T-cell stimulation by old and young immature bone marrow-derived CD4–CD8{alpha}– dendritic cells (imDCs). Total splenocytes (A) or microbead-purified T cells (B) from ovalbumin (OVA) T-cell receptor–transgenic mice were stimulated with irradiated OVA peptide(323-339)-pulsed imDCs at the responder/stimulator (R:S) ratios indicated. In these experiments, 100,000 splenocytes or T cells were cultured with the indicated ratios of DCs. After a 48-hour incubation, [3H]-thymidine (25 µCi/well) was added. Cultures were then left at 37°C for another 18 hours, after which they were harvested and [3H]-thymidine incorporation was measured. The stimulatory effect was expressed as proliferation index, mean values ± standard error of the mean of 6 (A) or 2 (B) independent experiments with at least 2 mice in each age group. *p <.0005

 
Production of IFN-{gamma} (a Th-1 cytokine) and IL-4 (a Th-2 cytokine) was assessed following a 3-day culture of syngeneic OVA-specific T cells with either young or old OVA peptide-pulsed imDCs. Whereas IFN-{gamma} production was strongly increased when OVA peptide-pulsed imDCs were used as APCs (Figure 3A), we observed a low level of IL-4 in both groups (Figure 3B). No effect of aging was observed in T-cell IFN-{gamma} or IL-4 production.


Figure 03
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  		Figure 3. Total splenocytes from the ovalbumin (OVA) T-cell receptor transgenic mice were cultured with irradiated ovalbumin peptide(323-339)-pulsed immature bone marrow-derived CD4–CD8{alpha}– dendritic cells (PP-DCs) at the R:S ratios of 50:1. After 3 days, production of interferon-{gamma} (IFN-{gamma}) (A) and interleukin-4 (IL-4) (B) was assessed using enzyme-linked immunosorbent assay (ELISA) kits. Results are expressed as means + standard error of the mean of 3 experiments with 2 or 3 mice per group

 
Expression of Molecules Involved in DC:T-Cell Interaction
Contact between DCs and resting T cells is essential to initiate a primary immune response. CD4 and CD8 T cells respond to peptide antigen displayed on MHC Class II and MHC Class I molecules, respectively. In addition, DCs express selected accessory molecules that interact with receptors on T cells to enhance adhesion and signaling, including CD40, CD86, and CD80 (24). We analyzed the cell surface protein expression of CD40, CD80, CD86, CD54, and MHC Class I and II of the bone marrow-derived imDCs (Figure 4) and tDCs (Figure 5A and B). As expected, LPS treatment causes the terminal differentiation of the bone marrow-derived DCs, with upregulation of CD40, CD54, CD80, and CD86 (25,26). However, even the "immature" bone marrow-derived DCs express a significant amount of MHC Class I and Class II molecules, suggesting that the cells are already partially activated by the combined IL-4/GM-CSF and before the LPS treatment. Compared to their younger cohort, old bone marrow-derived imDCs have similar expression of the CD40, CD54, CD80, and CD86, consistent with other human reports showing that these surface markers of blood monocyte–derived DCs do not differ significantly in aging (11,13). However, we found that both the proportion of positive cells and the level of expression of CD80 were reduced in the young tDCs (p <.025 and p <.01, respectively). Although we also detected a small increase in MHC Class I expression (mean fluorescent intensity) in the bone marrow-derived tDCs from old mice (p <.005), there is no change in the number of MHC Class I positive tDCs, and the functional significance of this small change is unclear. To exclude the possibility that any observed changes in the bone marrow-derived DC surface protein expression in aging is due to a difference in DC subset composition, we examined the distribution of the three major murine CD11c+ DC subsets (CD8{alpha}+, CD8{alpha}–CD4+, and CD8{alpha}–CD4–) (4) by determining the expression of CD4 and CD8 on the young and old DCs. We found that the murine bone marrow-derived immature and mature DCs uniformly belong to the CD11c+CD4–CD8{alpha}– subset, and aging does not affect the CD4 or CD8 expression on the DCs (data not shown) The results suggest that there is no significant age-related difference in bone marrow-derived DC subsets.


Figure 04
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  		Figure 4. Phenotype of immature bone marrow-derived CD4–CD8{alpha}– dendritic cells (imDCs) from young and old mice. Day 5 CD11c+ imDCs were stained with different monoclonal antibodies (mAbs) or isotype-matched control mAbs, as described in Materials and Methods. The percentage of positive cells and their mean fluorescence intensity (MFI) over isotype-matched control are shown. Results represent the means ± standard error of the mean (n = 20 individual mice per group). MHC, Major Histocompatibility Complex

 

Figure 05
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  		Figure 5. Phenotype of terminally bone marrow-derived DCs (tDCs) from young and old mice. Cells were stained with different monoclonal antibodies (mAbs) or isotype-matched control mAbs, as described in Materials and Methods. A, Percentage of positive cells and their mean fluorescence intensity (MFI) over isotype-matched controls are shown. Results represent the means ± standard error of the mean (n = 20 individual mice per group). B, Histogram of one representative experiment showing Major Histocompatibility Complex (MHC) Class I and CD80 staining. Black histograms show the background with isotype-matched control antibody; gray histograms show the immunofluorescence staining of tDCs for the indicated mAbs. *p <.025; **p <.005; ***p <.01

 
DC-SIGN is a recently described mannose-specific C-type lectin expressed by DCs. DC-SIGN binds to ICAM-3 on T lymphocytes, and plays an important role in T-cell activation (27,28). We next determined the effect of aging on the expression of DC-SIGN on young and old DCs. Cell surface expression of DC-SIGN on bone marrow-derived imDCs obtained from 20 individual mice (10 old and 10 young) was analyzed by FACS. As shown in Figure 6, young DCs express a higher level of DC-SIGN compared to old DCs (mean fluorescence intensity = 43.4 ± 5.78 vs 20.5 ± 3.11; p <.0025). We also examined the effect of aging on DC-SIGN expression on DCs after antigen loading. DCs were pulsed with the OVA-peptide(323-339) for 6 hours, and DC-SIGN expression was determined by flow cytometry. The results showed a similar decrease in DC-SIGN in aging (data not shown), suggesting that antigen loading does not affect the expression of DC-SIGN. IL-4 has been reported to increase DC-SIGN expression in human monocyte-derived DCs (29,30). However, we did not detect any difference in DC-SIGN expression in our murine bone marrow-derived DCs when we cultured the cells with and without IL-4 (data not shown).


Figure 06
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  		Figure 6. Expression of DC-specific/intracellular adhesion molecule type 3-grabbing, nonintegrin (DC-SIGN) on immature bone marrow-derived CD4–CD8{alpha}– dendritic cells (imDCs). Day 5 CD11c+ imDCs were stained with anti-DC-SIGN monoclonal antibody (mAb) as described in Materials and Methods. A, Histogram of representative experiment. Filled histograms show the background with isotype-matched control antibody; open histograms show the immunofluorescence staining of imDCs. B, Expression of DC-SIGN in old and young DCs. The results represent the mean of mean fluorescence intensity (MFI) ± standard error of the mean of 10 independent experiments (1 young and 1 old mouse in each experiment)

 
Cytokine Profile of Bone Marrow-Derived DCs
Recent data suggest that selected stimuli, including LPS, induce DCs to produce cytokines such as IFN-{gamma}, IL-10, and IL-12 that promote naïve T cells toward Th1 cell response (31–34). Furthermore, IL-10 has been reported to inhibit IL-12 production by DC and to present pre-Th cells from entering the Th1 pathway (35). Aging has also been reported to be associated with the increase in Th1 cytokine responses (1), although the underlying mechanisms for this are unclear. To determine if aging affects DC cytokine production that may in turn affect Th1 response in aging, we compared the capacity of DCs of young and old mice to produce selected cytokines. Cytometric bead arrays were used to determine the cytokine protein production in the culture supernatants from young and old DCs. The level of all the cytokines produced by imDCs from young and old mice was either undetectable or very low (Figure 7). The cytokine production was significantly enhanced in both young and old DCs following LPS stimulation. There was no statistical difference in the levels of IL-12p70 and IFN-{gamma} produced by young and old tDCs. However, young tDCs showed higher levels of TNF-{alpha} (22,019 ± 665 vs 5774 ± 1537 ng/mL; p <.0005) and IL-6 (134,683 ± 12,051 vs 71,033 ± 8895 ng/mL; p <.0005) than old tDCs. In contrast, IL-10 production was higher in old than in young tDCs (p <.025).


Figure 07
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  		Figure 7. Analysis of dendritic cells' supernatants for cytokine secretion. Indicated cytokines in 24-hour supernatants of immature bone marrow-derived CD4–CD8{alpha}– dendritic cells (imDCs) or terminally bone marrow-derived DCs (tDCs) were determined by using the Cytometric Bead Array kit. Results are expressed as means ± standard error of the mean (n = 15 in each group). *p <.0005; **p <.025. IFN-{gamma}, interferon-{gamma}; TNF-{alpha}, tumor necrosis factor-{alpha}; IL, interleukin

 
DC Treatment of Established Tumor
DCs elicit potent immune responses to tumors through their capacity to process and present tumor-associated antigens. To determine if the in vitro changes in DC function in aging have in vivo functional significance, we next compared the in vivo therapeutic efficacy of young and old OVA peptide (257-264)-pulsed DCs. For this purpose, we established subcutaneously tumor in B6 mice and began treatment with young and old antigen-pulsed DCs 6 days later (Figure 8A). Figure 8B shows the results of a representative experiment of three performed. When OVA peptide-pulsed young DCs were administered, B16 melanoma growth was inhibited by ≥70% compared to mice receiving PBS alone (p <.001). In contrast, injection of OVA peptide-pulsed old DCs was markedly less effective (27% inhibition, p <.001). At day 19, the mean tumor size in the mice receiving the young DCs was significantly smaller than the mice receiving OVA peptide-pulsed old DCs (young = 173.3 ± 39.2 vs old = 454.2 ± 27.5; p <.0005).


Figure 08
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  		Figure 8. Treatment of established tumor. A, Vaccination protocol. B6 mice were given 1 x 105 viable B16-ovalbumin (OVA) melanoma cells subcutaneously in the right flank. Six days later, when the tumor size reached 3–4 mm in diameter, young and old OVA–peptide(257–264) (SINFEKL)-pulsed immature bone marrow-derived CD4–CD8{alpha}– dendritic cells (PP-DCs), obtained as described in Materials and Methods, or phosphate-buffered saline (PBS) were injected once subcutaneously in the left flank. Tumor size was determined over time by measuring perpendicular dimensions with a Vernier caliper and recorded as tumor area (in mm2). B, Tumor growth over time. Data are representative of 3 experiments with similar results, and are reported as the mean tumor area ± standard error of the mean of 5 or 6 mice per group. *p <.001: young PP-DCs vs PBS control, old PP-DCs vs PBS control; **p <.0005: young PP-DCs vs old PP-DCs

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
DCs play a critical role in both the innate and adaptive immune responses. However, relatively little is known about the effect of aging on DCs' ability to support T-cell functions. Most of the current knowledge of DC functions in aging represents reports in scientific meetings, with relatively few studies appearing in the published literature. These studies into the effect of aging on DC functions have so far yielded conflicting results. For example, some investigators reported relative preservation of DC function in aging, and proposed that this may compensate for the age-dependent decline in T-cell function (3).

Decreased number of epidermal DCs (Langerhans cells) in aging (15,23,36) has been attributed to a deficiency in bone marrow progenitor cells (14). Other than skin-derived DCs, very little is known about the effect of aging on the number or distribution of DCs. Bone marrow cell CD11c expression appears to be decreased in aged athymic nude mice (22). Whether the frequency of DC progenitors in bone marrow is altered in normal aging and whether these cells have the same capability of producing peripheral DCs as in the young remain unanswered. Others have reported that aging does not impair the maturation response of human monocyte-derived DCs (37). Our results indicate that aged rodents are capable of generating at least equal number of immature bone marrow DCs as their younger cohort.

Age-dependent increase in splenic DC ability to induce lymphocyte proliferation has also been reported in mixed lymphocyte reactions (38). In contrast, splenic DCs from a substrain of senescence-accelerated mouse (SAMP1) have reduced stimulatory activity in mixed lymphocyte reactions in aging (39). Old murine follicular DCs have also been reported to be less capable of supporting B-cell immunoglobulin production (40,41). Others have found that human monocyte-derived DCs from healthy young and old individuals are equally capable of supporting antigen-specific T-cell proliferation (13,37). Recognition of MHC–peptide complexes on DCs by antigen-specific TCRs constitutes the "signal one" in DC–T-cell interaction. This initial phase is strengthened by the high expression of several adhesion molecules. Interactions of these and other accessory molecules expressed on DCs (CD80, CD86, and CD40) with their counterreceptors on T cells, constituting "signal two," are required to sustain T-cell activation. For example, Salomon and Bluestone (42) reported that LFA-1 (lymphocyte function associated antigen-1) interaction with ICAM-1 and ICAM-2 is important in OVA peptide TCR–specific Th1 T-cell proliferation response to splenic DCs. Recent studies have also shown that DC-SIGN expression on DCs is critical in DC–T-cell interaction (28,43) by mediating the transient adhesion with T cells. As antibodies against DC-SIGN inhibit DC-induced proliferation of resting T cells, these findings suggest that DC-SIGN enables TCR engagement by stabilization of the DC/T-cell contact zone (43,44). In addition, DC-SIGN also interacts with ICAM-2 in regulating chemokine-induced transmigration of DCs across both resting and activated endothelium (27). The effect of aging on these interactions is at present unclear. Our current results provide the first evidence that aging is associated with the decreased expression of DC-SIGN in DCs. Our results also showed that old imDCs pulsed with the OVA peptide have reduced ability to induce syngeneic T-cell proliferation. Given the established role of DC-SIGN in T-cell–DC interaction, it is possible that the observed impaired DC-SIGN expression in aged imDCs may contribute to the impaired T-cell response. However, it should be pointed out that we have not proven that there is a direct relationship between decreased DC-SIGN expression and impaired DC function in aging. Future experiment will have to be done to determine if overexpressing DC-SIGN in aged DCs will restore the observed functional defects.

Upon stimulation, DCs produce cytokines that are important in linking the innate and adaptive responses. DCs also provide T cells with the cytokine microenvironments that determine the specific Th1 or Th2 response. The induced pattern of T-cell cytokine secretion is dependent on the production of specific DC cytokines, including IL-12 (45). In this report, we only examined the effect of young and old DCs on the T-cell production of one Th1 (IFN-{gamma}) and one Th2 (IL-4) cytokine. It remains possible that young and old DCs may stimulate T cells to produce other cytokines that can contribute to the observed functional changes. We also showed that aged DCs secrete a higher level of IL-10, but less IL-6 and TNF-{alpha} than do their young counterparts following LPS maturation. Interestingly, level of IL-10 (a cytokine that can suppress cell-mediated immunity) has also been reported to be elevated in elderly individuals (46).

The unique ability of DCs to induce and sustain primary immune responses makes them prime candidates in vaccination protocols in cancer therapy. DCs loaded with the appropriate tumor-associated antigens can induce protective/rejection-immune responses in animals models (9,47,48) and in humans (49–51). There have been relatively few reports examining the effect of aging on antitumor activity, with varying results. Norian and Allen (52) examined the ability of enriched DC populations from the spleen and lymph nodes of old mice to prime T-cell response against the CMS5 fibrosarcoma cells, and found that these cells could initiate antitumor T-cell responses to the same degree as those from young mice. Furthermore, IL-10 is capable of converting DC-APC function to the induction of antigen-specific anergy, thus leading to the state of tolerance against tumor tissue (53). In the current study, we showed that the growth of established B16–OVA tumors was significantly inhibited by a single immunization using OVA peptide-pulsed bone marrow-derived DCs. However, in contrast to the previous report using fibrosarcoma cells (49), we found that mice receiving old bone marrow-derived DCs achieved considerably less potent antitumor immunity than did their young counterparts. The reasons for the differences are unclear, but the origin and purity of the DCs, as well as the specific tumor cell targeted may explain some of the differences. The mechanisms for the age-dependent impaired DC antitumor activity are also unclear. Potential mechanisms include defective old DC migration to secondary lymphoid organs to initiate T-lymphocyte responses, or impaired DC/T-cell stimulatory capacity. Of interest, it was recently reported that the ability of Langerhans cells to migrate to regional lymph nodes declines with age (54). Similarly, recruitment of airway DCs to draining mediastinal lymph nodes may also diminish in aged mice (55). Furthermore, a recent study reported that imDCs interact in a DC-SIGN-dependent manner with colorectal cancer cells to control tumor cell spread (56). Data provided by Tacken and colleagues (57) also identified DC-SIGN as an important target molecule for DC-based vaccination in cancer therapy. Our findings of impaired DC-SIGN expression in bone marrow-derived DCs in aging, coupled with the reduced ability to fight against melanoma tumor cells, suggest that DC-based tumor immunotherapy may potentially be a less effective strategy in the elderly population. It will be important to test if improving DC-SIGN function in DCs can restore the age-dependent decline in tumor surveillance. Finally, because we only examined the effect of aging on bone marrow-derived DCs from C57BL/6 mice, these results will need to be confirmed in other DC subsets as well.


   Acknowledgments
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by Public Health Service Grants 1RO1 AG020628 and 1RO1 AI42753, and by the Geriatrics Research, Education, and Clinical Center of the Ann Arbor Veterans Affair Medical Center.


   Footnotes
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: James R. Smith, PhD

Received September 23, 2005

Accepted March 27, 2006


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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