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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Aug 1, 2009.
Published in final edited form as:
PMCID: PMC2575813
NIHMSID: NIHMS71803

Impaired Dendritic Cell Function in Aging Leads to Defective Antitumor Immunity

Abstract

We recently reported that bone marrow-derived dendritic cells (DCs) are less effective than their young counterparts in inducing the regression of B16-ovalbumin (OVA) melanomas. To examine the underlying mechanisms, we investigated the effect of aging on DC tumor antigen presentation and migration. Although aging does not affect the ability of DCs to present OVA peptide (257−264), DCs from aged mice are less efficient than those from young mice in stimulating OVA-specific T cells in vitro. Phenotypic analysis revealed a selective decrease in DC-specific/intracellular adhesion molecule type 3-grabbing nonintegrin (DC-SIGN) level in aged DCs. Adoptive transfer experiments showed defective in vivo DC trafficking in aging. This correlates with impaired in vitro migration and defective CCR7 signaling in response to CCL21 in aged DCs. Interestingly, vaccination of young mice using old OVA-peptide(257−264) pulsed DCs (OVA PP-DCs) resulted in impaired activation of OVA-specific CD8+ T cells in vivo. Effector functions of these T cells, as determined by IFN-γ production and cytotoxic activity, were similar to those obtained from mice vaccinated with young OVA PP-DCs. A decreased influx of intra-tumor CD8+ T cells was also observed. Importantly, although defective in vivo migration could be restored by increasing the number of old DCs injected, the aging defect in DC tumor surveillance and OVA-specific CD8+ T cell induction remained. Taken together, our findings suggest that defective T cell stimulation contributes to the observed impaired DC tumor immunotherapeutic response in aging.

Keywords: Aging, Dendritic Cells, Migration, Antigen Presentation, Cancer

Introduction

Increased susceptibility to malignancies, infections, autoimmune diseases, and a poor response to immunization in the elderly have been taken as indicative of declining immune function in aging. Immunosenescence, the progressive deterioration in immune function that accompanies aging, results from the alteration of both adaptive and innate immunity. With advancing age, T cells undergo major changes including a shift from the naive to memory phenotype, a reduction in the proliferation response and impaired cytolytic activity (1, 2). However, the recognized changes in T lymphocyte function may not completely explain the defect in immune responsiveness observed in old age, and other members of the immune system may play an equally important role in contributing to immunosenescence.

Dendritic cells (DCs) form a distinct heterogeneous hematopoietic lineage of antigen-presenting cells (APCs) with unique abilities to stimulate naïve T lymphocytes (3, Palucka, 1999 #94, 4). DCs are derived from bone marrow progenitors and reside in peripheral tissues or in circulation as phagocytic immature precursors. DCs acquire a terminally mature phenotype (tDCs) upon uptake of antigen (Ag) and in response to stimuli such as cytokines, necrotic cells, and microbial products, enabling them to migrate to secondary lymphoid tissues where they present Ag to T cells and induce Ag-specific responses. Due to their central role in immunology, DCs represent a potent adjuvant for use in tumor vaccination protocols (5), (6). Since the elderly are preferentially affected by diseases targeted by DC-directed immunotherapy, it will be critical to understand how aging affects DC functions as well. Data on qualitative and quantitative alterations of human and murine DCs in aging have recently been summarized (7-9), and have shown that the results of such studies are often contradictory and difficult to compare since the origin of the cells, their culture conditions and maturation protocols vary greatly.

Recent studies comparing the efficacy of young-derived DC vaccines in young and old mice suggested that the aging micro-environment affects the DC anti-tumor response (10, 11). Using young hosts, we reported that ovalbumin peptide pulsed-DCs (OVA PP-DCs) from old C57BL/6 mice were less effective than their young counterparts in inducing the regression of B16 melanomas expressing OVA (B16-OVA) (12). This implies that an intrinsic functional defect(s) also exists in aged DCs. Effective DC vaccination involves first the appropriate uptake and processing of tumor-associated Ags, then DC trafficking to regional draining lymph nodes (LNs), and finally interaction and activation of Ag-specific CTLs. In the present study we therefore examined the effect of aging on these three specialized DC functions.

Materials and Methods

Animals

Young (3−6 months) and old (18−20 months) C57BL/6 mice were purchased from Harlan Laboratory (Indianapolis, IN), and OVA–specific MHC-I–restricted, T-cell receptor (TCR)–transgenic young C57BL/6 mice (OT-I mice) from Jackson Laboratory (Bar Harbor, ME).

Generation and Purification of DCs

Erythrocyte-depleted bone-marrow cells flushed from femurs and tibiae of mice were cultured at 1 × 106 cells/ml in the presence of 20ng/ml murine recombinant GM-CSF and IL-4 (R&D Systems; Minneapolis, MN) in complete medium RPMI-1640 as before (13). DCs were purified from 5-day cultures using CD11c+ microbeads (Miltenyi Biotec, Auburn, CA). To generate mature cells, DCs were replated with 1μg/ml lipopolysaccharide (LPS) (Sigma, St Louis, MO) for 24 hrs.

FITC-OVA Uptake

DCs (5 × 105 cells) were loaded with 0.1 and 1mg/ml FITC-OVA protein (Sigma, St Louis, MO) or 10μg/ml FITC-OVA–peptide(257−264) (University of Michigan Protein Facility Core, Ann Arbor, MI) for the indicated time at 37°C or at 4°C (background). Cells were stained with PE-anti-MHC class I Ab (BD Pharmingen, San Diego, CA) and analyzed by flow cytometry.

Analysis of DC Migration In Vivo

Yound and old tDCs were stained with CMPTX or CFSE dyes (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Equal number or indicated ratio of cells from each age group were mixed together, and injected subcutaneously into the footpad of young mice. Draining LNs were harvested 24 hrs later, embedded in OCT compound (Sigma, St Louis, MO) and snap-frozen. Serial 5μm sections were made and the frequency of stained cells assessed in a blinded fashion by fluorescence microscopy (Olympus BX61 microscope, Orangeburg, NY). Results are expressed as the percent of injected DCs present per microscope field.

Chemotaxis Assay

DCs (2.5 × 105 cells in 0.1ml) were plated in the upper chamber of a 5μm pore size transwell (Costar, Cambridge, MA). Indicated concentrations of recombinant mCCL21 (R&D Systems, Minneapolis, MN) were added to the lower wells of the chamber. Plates were incubated at 37°C for 2 hrs, and 104 15-μm beads (Bangs Laboratories, Fishers, IN) added to standardize the results. Cells and beads were quantitated by flow cytometry, and the number of cells in each sample calculated using the formula: (number of cell events/number of bead events) × 104. The chemotaxis index was calculated as the ratio between cell number migrated in response to CCL21 and the cell number migrated to the medium alone.

Cell Surface Analysis

Analysis of DC surface markers was done by a FACSCalibur (Becton Dickinson, San Jose, CA) as previously described (12), using the following antibodies: I-Ab-PE, CD40-PE, CD80-PE, CD54-FITC, CD86-PE, CCR7-PE and DC-SIGN-biotin followed by Streptavidin-PE (BD Pharmingen, San Diego, CA). Data analysis was performed using FCS Express (De Novo software, Ontario, Canada).

Western Immunoblotting

Young and old tDCs were exposed to 500 ng/ml mCCL21. At indicated times, aliquots were taken and dissolved in 1% SDS, 1 mmol/L PMSF, 1 mmol/L EDTA, 1 mmol/L N-ethylmaleimide, and 2 mmol/L sodium orthovanadate. The proteins were separated by SDS-PAGE on 10% acrylamide gels and transferred to nitrocellulose membranes. Western blots using the global anti-phosphotyrosine Ab 4G10 (Millipore, Temecula, CA) were realized as previously described (14).

Proliferation Assays

DCs were pulsed with or without (background) OVA-peptide(257−264) at 10 μg/ml for 6 hrs at 37°C, irradiated (3000 rads), and titrating numbers incubated with 0.1 × 106 cells freshly isolated CD8+ T cells from the OT-I mice. Proliferation of T cells was measured by uptake of [3H]thymidine (1μCi/well; DuPont-NEN, Boston, MA), pulsed for 18 hrs after 3 days of culture.

Pentamer and Intracellular IFN-γ Staining

Splenocytes from immunized mice were depleted of CD19 positive cells using microbeads (Miltenyi Biotec, Auburn, CA). FACS staining was performed using SIINFELK Kb pentamers (OVA-Pent, Proimmune, Bradenton, FL) according to the manufacturer's instructions. Briefly, cells were stained with PE-OVA-Pent for 30 min at room temperature, washed and then incubated with RPE-Alexa Fluor 647-anti-mouse CD8α or RPE-Alexa Fluor 647-control mAbs (BD Biosciences, San Diego, CA). For IFN-γ intracellular staining, cells were stimulated or not with OVA-peptide(257−264) in the presence of brefeldin (10μg/ml), fixed with 4% paraformaldehyde, washed with buffer containing 0.1% saponin, 1% FBS and 0.1 % sodium azide, and stained with PE-OVA-Pent along with FITC-CD8 and PE-Cy7-anti-IFNγ Abs (BD Biosciences, San Diego, CA). Cells to be activated were stained with the OVA-Pent Ab before activation. Samples were analyzed by flow cytometry, gating on live lymphocytes and CD8+ cells, and acquiring 150,000 events per sample.

Enrichment of IFN-γ+ cells

Selective enrichment of IFN-γ-producing CD8+ T cells was performed using the IFN-γ secretion assay from Miltenyi Biotec (San Jose, CA) according to the manufacturer's instructions. Briefly, day 6 cultured splenocytes from immunized mice were challenged with OVA-peptide(257−264) for 6 hrs and then labeled for 5 min at 4°C with a bi-specific Ab-Ab conjugate directed against CD45 and IFN-γ. Afterward, cells were allowed to secrete IFN-γ, at 37°C for 45 min before staining with PE-anti-IFN-γ mAb. Cells were then labeled for 15 min at 4°C with anti-PE Ab microbeads and PE- IFN-γ+ cells were enriched by magnetic cell sorting and further cultured for 4 days in the presence of recombinant mIL-2 to obtain sufficient cell numbers.

Cytotoxicity Assay

At day 14 after tumor injection, splenocytes (40 × 106) were pooled from 2 mice immunized with young and old OVA PP-DCs or with Dulbecco's phosphate buffered saline (DPBS) and restimulated in vitro with 2 × 106 mitomycin C-treated B16-OVA cells in the presence of recombinant mIL-2 (5ng/ml; Peprotech, Rocky Hill, NJ) for 6 days. The cells were then harvested, subjected or not to enrichment of IFN-γ+ cells and tested for cytotoxicity against B16 or B16-OVA target cells using the CytoTox 96 assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Specific lysis was expressed as the percentage of lysis in B16-OVA targets minus the percentage of lysis in B16 targets, according to the following formula: % cytotoxicity = 100 × [(experimental – effector spontaneous- target spontaneous)/ (target maximum release - target spontaneous release)].

Cytometric Bead Array

Aliquots of splenocytes (2 × 106 cells/ml) were cultured for 48 hrs with 2 × 105 UVB-irradiated B16-OVA as previously described (15). Culture supernatants were collected for measurements of IL-6, IL-10, IL-12, MCP-1, TNF-α or IFN-γ cytokine secretion by Cytometric Bead Array assay (BD Pharmingen, San Jose, CA), according to the manufacturer's instructions. Cytokines were quantified on a FACSCalibur with FACSComp software (BD Pharmingen).

Analysis of In Vivo Immune Cell Infiltrate

Individual tumor cell suspensions were prepared by mechanical disaggregation (Medimachine, BD Biosciences) and FACS analyzed for immune infiltrate using the following antibodies: CD45-PerCP, CD4-FITC, CD8-FITC, CD11c-FITC, Ly-6G-FITC, NK1.1-PE, and CD14-PE (all from BD Biosciences). 15-μm beads (5 × 105 beads/ml were added to all samples and 100,000 events acquired. The number of infiltrating cells/tumor was determined by the following equation: (# of double stained events/# beads events) × (5 × 105 beads/ml) × cell sample volume (in ml). Because the tumors were of different sizes, the data were normalized to the tumor weight by dividing the total number of infiltrating cells in each sample by their respective tumor weight.

Statistical analysis

Results are expressed as means ± SEM. Statistical analyses were performed using Student's t-test, and p ≤ 0.05 was taken as statistically significant. For multiple comparisons, 2 tailed student t-test was used.

Results

Aged DCs have impaired ability to stimulate CD8+ T Cell Proliferation

We have previously reported that old DCs were less effective than young DCs in stimulating the proliferation of OVA-specific CD4+ T lymphocytes (12). The B16-OVA tumor model suggests that old DCs may also be less capable of supporting cytotoxic CD8+ T cell function. We therefore compared the proliferation response of OVA-specific T cells from OT-I mice to OVA-peptide(257−264) PP-DCs derived from young and old mice. Figure 1A shows that young DCs induce a greater T cell proliferation than old DCs (p<0.0025), confirming that DC capacity to stimulate OVA-specific CD8+ T cells is also impaired in aging.

Figure 1
Effect of aging on DC:T-cell interaction. (A) CD8+ T cells from OT-I transgenic mice were stimulated for 3 days with irradiated class I OVA-peptide(257−264) - pulsed young and old DCs at the R/S ratios indicated. Proliferation was determined as ...

DC-dependent CD8 + T cell response depends on the interaction of processed Ag with the T-cell receptor in the context of MHC class I molecules, and the interaction of co-stimulatory molecules with their respective cell surface receptors. We first examined the effect of aging on tumor antigen uptake and presentation in the context of MHC class I molecule using whole OVA protein and OVA peptide(257−264). As shown in Figure 1 B, phagocytosis of FITC-OVA protein was equally efficient in young and old DCs. Using FITC-OVA peptide(257−264), we also found that both young and old DCs express similar number of MHC I-peptide complexes on their cell surface, with 50.27 +/− 9.99 and 53.51 +/− 5.96 percent of double positive cells respectively (Figure 1C). Similar results were obtained using FITC-OVA protein (data not shown), suggesting that aging does not significantly impact DC presentation of OVA peptide in the context of MHC class I molecule. Next, we analyzed DCs for the expression of markers relevant to presentation of Ag to T cells. Overall, there was little or no significant difference between young and old OVA PP-DCs in MHC class I, CD80, CD86, CD54 and CD40 expression (Figure 1D). Interestingly, a decrease in DC-SIGN (CD209) expression was observed in aging (p < 0.01). This finding is consistent with our previous report showing that impaired DC-SIGN expression in aging may contribute to impaired CD4+ T cell proliferation in the OT-II model (12).

DC In Vivo Trafficking to Draining LNs is Impacted by Aging

Migration of DCs to the secondary lymphoid organs is essential for the cells to exert their T cell regulatory function. To determine in vivo DC trafficking, young and old tDCs were labeled with CMPTX and CFSE respectively, and equal number of cells mixed together before injection into the footpad of young mice to exclude the influence of the aged micro-environment. Twenty-four hours later the draining popliteal LN was harvested, and fluorescence assessed by fluorescent microscopy. Figure 2A shows representative images with young DCs in red and old DCs in green. We found that half as many aged DCs were present in the popliteal LNs (p < 0.025) (Fig. 2B). Interestingly, the number of old DCs migrating is the same as young DCs in animals injected with 2 times more old DCs than young DCs (Figure 2C). No additional beneficial effect was observed when 3 times more old DCs were injected.

Figure 2
In vivo lymph node DC migration in aging. Groups of young mice (n=3) received 50 μl of equal numbers (2 × 106) of CMPTX-labeled young DC and CFSE-labeled old DCs into their footpad hind. Popliteal draining LNs were excised 24 hrs later ...

Aged DCs Have Impaired CCR7 Signaling and Function

Engagement of CCR7 chemokine receptor on DCs to its ligands CCL19 (ELC/MIP-3ß) and CCL21 (SLC/6Ckine) in LNs has been identified as the critical event in DC lymphoid homing (16, 17). To determine whether the impaired migratory function in aged DCs correlates to a decrease in CCR7 function, the chemotactic response of young and old tDCs to CCL21 was determined. Figure 3A shows that CCL21 induced a weaker chemotaxis response by old DCs compared to young DCs (p< 0.025). We next examined CCR7 expression of young and old tDCs by FACS. Somewhat surprisingly, the age-dependent reduced CCL21 migration response did not correspond to any significant change in CCR7 surface expression on old tDCs with 60.2 +/− 5.48 positive cells as compared to 53.4 +/− 7.6 for their young counterparts (Figure 3B). Comparison of DC CCR7 signaling revealed that CCL21 stimulation induces a greater level of tyrosine phosphorylation of proteins of molecular weight 46−97 kDa in young tDCs as compared to old tDCs (Fig. 3C), suggesting that the age-dependent impaired CCR7 response may be in part secondary to the DC signaling defect in aging.

Figure 3
Effect of aging on CCR7 expression and function. (A) Transwell migration of DCs in response to CCL21 was determined as described in Materials and Methods. Values represent the average of three independent experiments, each of them with at least 3 mice ...

Aged DCs Stimulate a Weaker T Cell Immune Response

We next examined the immune function of T cells in mice treated with DCs. Mice with a 7 day old B16-OVA tumor were inoculated with either young or old OVA PP-DCs as described previously (12). Spleens were harvested on day 14 for immune function analysis. First, we measured the OVA-peptide-specific CD8+ T cell frequency by flow cytometry. For this purpose, splenocytes were depleted of CD19+ cells, as the presence of B cells has been shown to interfere with pentamer staining, and stained with anti-CD8-RPE Alexa Fluor 647 and OVA-Pent-PE mAbs. As shown in Figure 4A, OVA-specific CD8+ T cells were detectable in both groups of immunized mice. However, the frequency of those T cells decreased by more than 2.5 fold in mice that received the old PP-DC vaccine, compared to mice receiving the young PP-DC vaccine (0.39 ± 0.1 vs 1.04 ± 0.22; mean % double positive cells of 3 independent experiments, p<0.025).

Figure 4
The effect of young and old DCs on T cell functions. (A) In vivo detection of OVA-specific CD8+ T cells by MHC pentamer staining. Day 7 tumor-bearing B6 mice were vaccinated with young or old OVA PP-DCs. 7 days later, total splenocytes were harvested, ...

Cytotoxic assays using splenocytes obtained from the same vaccinated mice were next performed. As shown in Figure 4B, CTL cells generated from the spleens of young PP-DC immunized mice showed 68% cytotoxic activity against the B16-OVA cells at an effector/target ratio of 25/1. In contrast, CTLs obtained from the spleens of old PP-DC immunized mice showed only 26% cytotoxic lysis (p < 0.025). These results confirm that the enhancement of the antitumor effect in mice immunized with young PP-DC vaccine corresponds to augmentation of the cytolytic activity. Interestingly, assessment of cytokine release of activated CTL cells in response to tumor stimulation revealed that T cells from mice receiving the young DCs produced greater amounts of IFN-γ (p < 0.005), TNF-α (p < 0.001), IL-10 (p < 0.05), and IL-6 (p < 0.001) (Figure 4C).

Decreased ability to induce OVA-specific T cells but not Impaired Migration Leads to Deficient Antitumor Immunity

The results presented in Figure 4 suggest that old DCs stimulate a weaker T cell response when compared to young DCs. The experiments in Figure 5 were designed to determine if the lack of proper immune response after stimulation with old DCs is a direct consequence of their inability to migrate to the LNs, and if it reflects a difference in the quality rather that in the quantity of the T cells after young and old DC stimulation. We have shown in Figure 3C that the in vivo age-associated defect in DC migration could be restored by injecting animals with 2 times more old DCs. We investigated if the age-associated defect in DC tumor surveillance and T cell induction could be restored by increasing the number of DCs injected. Young tumor-bearing mice were immunized with either DPBS, 2 × 106 young or old PP-DCs as before or with 4 × 106 old PP-DCs. We found that mice immunized with 2 times more old DCs had similar tumor size (Figure 5A) and similar number of OVA-specific CD8+ T cells (Figure 5B) as those receiving 1× old DCs. Those values remain significantly lower than those observed in mice receiving young DCs. The results suggest that the defective antigen specific T cell induction after stimulation with old DCs is not the consequence of impaired migratory properties of those cells, but is at least in part related to a qualitative effect on T cell stimulation.

Figure 5
Effect of increasing number of old DCs on tumor growth and Ag-specific induction and function. Mice bearing 7-day established subcutaneous B16-OVA tumors were treated with 2× 106 young PP-DCs (1×), 2 × 106 old PP-DCs (1×) ...

To further test this hypothesis, we wanted to see if the weaker T cell response we observed, as defined by reduced cytotoxic activity and IFN-γ production (Figure 4B and 4C) was due to decreased OVA-specific CD8+ T cell induction by old DCs. As shown in Figure 5B, total splenic CD8+ T cells obtained from mice immunized with old PP-DCs and restimulated in vitro with OVA peptide have lower IFN-γ production as measured by intracellular staining, with a frequency of 1.46 +/− 0.16% IFN-γ positive cells as compared to 3.03 +/− 0.35 % in CD8+ T cells from mice immunized with young PP-DCs. However, when we assessed the IFN-γ production among OVA specific CD8+ T cells, by gating on OVA-Pent+ CD8+ T cells (elliptical gate in Figure 5B), we found no age difference in the ability of those cells to produce IFN-γ.

Becker et al. have shown that IFN-γ+ but not IFN-γ- T cells from tumor-immunized mice were cytolytic and mediate tumor rejection upon adoptive transfer (18). The same authors demonstrated that those cells could be enriched using an IFN-γ capture assay. Using the same technique, we recovered IFN-γ-producing CD8+ T cells from 6 day cultures of splenocytes from mice immunized with young and old PP-DC vaccines, and repeated the cytotoxic assays using same numbers of those cells. We found that the ability of IFN-γ-producing CD8+ T cells obtained from mice immunized with young DCs to induce lysis of B16-OVA cells was only slightly better (not statistically different) than those obtained from mice immunized with old DCs (Figure 5D). This result suggests that the quality of the T cells after old DC stimulation on a per cell basis is largely intact and that the defective cytokine production and cytotoxic activity presented in figure 4B and 4C are mainly due to the fact that old DCs induce half many as OVA specific T cells than young DCs and therefore that less OVA-specific T cells were added to the assays. In both experiments, increasing the number of old DCs injected did not have any effect on T cell functions.

Reduced Number of Intratumor CD8+ T Cells in Mice Receiving OVA peptide-pulsed Old DCs

Tumor rejection is initiated by CD8 cytotoxic T lymphocytes which infiltrate solid tumors. We analyzed by flow cytometry single-cell suspensions from B16-OVA melanoma for the presence of infiltrating leukocytes (CD45+ cells) at 7 days after the initiation of young and old OVA PP-DC treatment. For comparison, we also analyzed tumors from mice treated with DPBS. Figure 6A shows the typical data obtained of one untreated, one young and one old OVA PP-DC (1×) and (2×)-treated mice. Figure 6B summarizes the tumor data from a total of 6 experiments. Nearly half of the immune cells contained in OVA PP-DC-treated tumors were T cells, whereas in DPBS-treated tumors, NK1.1+ cells were the most abundant immune cell subset represented. Interestingly, although both young and old OVA PP-DC treatments could induce the influx of T cells, young OVA PP-DC administration resulted in higher numbers of infiltrating CD8+T cells, compared to old OVA PP-DC-treated mice (p < 0.005). Approximately 66% of the T cell infiltrates were CD8+ cells in the young DC immunized group whereas the proportion of CD8+ cells decreased by 20 % in the old DC immunized group, with no change in the total number of lymphocytes. Increasing the number of old DCs injected had no significant effect on the tumor infiltrate composition. Both DC-treated groups also induced an influx of CD11c+ cells to the tumor sites, although the results did not reach statistical significance.

Figure 6
FACS analysis of immune cell subsets infiltrating B16-OVA tumors. Tumors were harvested seven days after DPBS, young or old OVA PP-DC injection and used to prepare a single cell suspension for FACS analysis. The live population was gated on, and the percentage ...

Discussion

Despite numerous recent advances in the molecular and cell biology of DCs, only very few groups have addressed the topic of DC function and aging. Some studies have reported no age-related differences between the DC numbers, phenotype, morphology and maturation in human monocyte-derived DCs from young and aged subjects (7, 19, 20). Others, however have indicated that migration and phagocytosis of the same DC subset were impaired with aging (21). A progressive loss of circulating plasmaytoid DCs numbers and a decreased density of Langerhans cells (LCs) with aging have also been reported (22, 23, 24). These reports suggest that the aging-associated changes in DCs may vary with the subsets of DC studied, their tissue of residence and environmental signals. Examining different in vivo models may provide important insight into the effect of age on DC function.

The ultimate goal of tumor DC immunotherapy is to induce strong tumor-specific T-cell mediated immunity that can block the growth and metastasis of malignant tumor cells in tumor-bearing hosts. In most cases, the development of antitumor immunity requires strategies capable of stimulating CD8+ CTLs. Using the B16-OVA melanoma tumor model, we recently demonstrated that old DCs loaded with specific tumor antigen are less able to control tumor growth. The scope of the present study was to investigate the underlying mechanism responsible for the defective DC anti-tumor function observed in aging.

The tumor model requires the Ag-loaded DCs to migrate to the lymphoid tissue in order to stimulate T lymphocytes. Evidence for impaired DC migration in aging includes a recent report showing that recruitment of airway DCs to draining mediastinal LNs may diminish in aged mice (25). Similarly, the ability of LCs to migrate to regional LNs declines with age (26, 27). In the present study, we reported that the vigor of DC migration and subsequent influx of DC into draining LNs is also impacted by aging. We have also shown that aged DCs have impaired capacity to migrate in vitro in response to the CCR7 ligand CCL21. This was not due to a defect in CCR7 protein expression, but rather by a defect in signal transduction as demonstrated by comparison with the pattern of tyrosine phosphorylation using young and old DCs stimulated by CCL21. This is consistent with the recent paper from the Gupta group, who reported a decreased in vitro migration towards CCL19, another CCR7 ligand, of monocyte-derived LPS-stimulated DCs from elderly subjects as compared to young subjects, despite similar levels of expression of CCR7 (21). They attributed their results to defects in the downstream signaling pathway, possibly in the PI3K pathway. However, the exact mechanism is unknown and further studies will be needed to unravel it. We found that although the impaired in vivo migration of aged DCs can be restored to a level comparable to that seen in young DCs by increasing the quantity of cells injected, the age-associated impairment in tumor surveillance as defined by tumor growth and Ag-specific T cell induction remains, suggesting that DC migration through CCR7-CCL21 interaction is not the primary mechanism for the observed aging defects.

In mouse models using T cells reactive to defined tumor antigens, tumor regression correlated with an early and sustained influx of CD8 T cells. We found that the frequency of splenic Ag-specific CD8+ T cells is altered during aging, with a significant decrease in the influx of CD8+ T cells into tumors 7 days after injection of old PP-DCs, as compared to young PP-DC-injected mice. Importantly, effector functions of these Ag-specific T cells, as determined by IFN-γ production and cytotoxic activity, were similar to those obtained from mice vaccinated with young OVA PP-DCs on a per cell basis. This suggests that the quality of those T cells is intact and that decreased T-cell number per se is sufficient to explain the reduced inhibition of tumor growth observed in mice vaccinated with old PP-DCs. One possible explanation could be the impaired in vivo T cell induction by aged DCs that we described.

For efficient antigen presentation and induction of an immune response by DCs, the number and stability of MHC I-peptide complexes is crucial (28). Our results demonstrated that there is no age effect on the uptake and surface expression of OVA peptide/MHC I complexes. Since others have shown that aged APCs may require longer periods of contact with CD8+ T cells than young APCs to initiate the same Ag-driven response (29), we performed the same experiments after 24 hrs culture, and obtained similar results (data not shown). All together, the results suggest that difference in the formation and kinetics of MHC Class I-OVA peptide complexes on DCs is not responsible for the observed defective DC-mediated anti-tumor response. Despite intact peptide presentation and no significant change in MHC class I and the classical co-stimulatory molecules, the proliferation of OT-I T cells was impaired in old DCs. Recently, Geijtenbeek and his colleagues identified DC-SIGN as a novel adhesion receptor on DCs that is essential in several key functions throughout the DC's life cycle, including interactions between DCs and T cells (30). They showed that DC-SIGN binds ICAM-3 with high affinity and that this cellular interaction establishes the first molecular interaction between DCs and resting cells (31). In another study, van Gisbergen et al. investigated the behavior of DC-SIGN in synapse formation. Using a DC-SIGN deletion mutant, they found that DC-SIGN can recruit LFA-1 to the contact site and shift from an initial transient DC-SIGN-ICAM-3 interaction to a more stable LFA-1-ICAM-3 interaction (32). Gijzen and colleagues, have demonstrated the relevance of DC-SIGN in DC-induced T cell proliferation by showing that antibodies against human DC-SIGN inhibit DC-induced proliferation of resting T cells (33). It is clear from our data that the capacity to induce T cell proliferation in vitro is reduced with aging and that this correlates with a selective reduced DC-SIGN expression. Future studies will be needed to establish the exact relationship between those two observations. We are currently in the process of breeding DC-SIGN knock-out mice. These mice will help unravel the precise role of DC-SIGN not only in DC:T cell interaction but also in other important processes in DC biology in which DC-SIGN has been shown to be involved, such as migration and Ag uptake. Additionally, focused microarray experiments that include genes involved in DC antigen uptake, processing, and presentation will enable us to discover other genes that may potentially play a role in these processes.

In summary, we report here a reduced ability of aged DCs to support the induction of antitumor T cell responses at multiple levels. Treatment of tumors with old OVA PP-DCs eliciting a modest antitumor effect resulted in a substantial decrease in the frequency but not potency of Ag-specific CD8+ T cells and a far less pronounced CD8 T-cell infiltration into the tumor mass. Defects affecting DC Ag presentation to T cells could factor into the observed impairment in antitumor immunity, a finding that has important implications for using DC-based immunotherapy in older subjects.

Acknowledgments

This work is supported by the NIH (1RO1AG020628-01A2; 3RO1 AR42525) (RY), the Ann Arbor VA Health System (VA Merit Review) (RY), and by the NIH-NIA (AG024824, University of Michigan Claude Pepper Older Americans Independence Center) (AG).

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