Impaired dendritic cell function in aging leads to defective antitumor immunity

Abstract

We recently reported that bone marrow-derived dendritic cells (DC) from aged miced 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-DC) resulted in impaired activation of OVA-specific CD8(+) T cells in vivo. Effector functions of these T cells, as determined by IFN-gamma production and cytotoxic activity, were similar to those obtained from mice vaccinated with young OVA PP-DCs. A decreased influx of intratumor 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.

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 described in Materials and Methods. Data are representative of 4 independent experiments with at least 2 mice per age group. (B) Young and old DCs were incubated with FITC-OVA protein for the indicated times and phagocytosis analyzed by FACS. Specific uptake was assessed by subtracting the MFI of cells incubating at 4°C from the value at 37°C. (C) DCs were pulsed for 6 hrs with FITC-labeled OVA peptide_(257−264) and then stained with PE-conjugated MHC class I. Dot plots of one representative experiment per age group of 3 performed are shown. (D) Phenotype of young and old OVA PP-DCs. DCs were pulsed for 6 hrs with OVA peptide_(257−264) and stained with indicated antibodies. The percentages of positive cells over isotype-matched controls are shown. Results represent the means ± standard error of the mean (n= 10 mice per age group) of three independent experiments. * p<0.0025; ** p< 0.05; ***p < 0.01.

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 and the frequency of DC/node was assessed by fluorescent microscopy. (A) Representative photos (200X) showing young DCs in green, old DCs in red and the merge picture. (B) Close up (digital enlargement) of selected fields are also shown. Percent of red versus green DCs per field was derived from microscope examination data by counting at least 200 cells/group in randomly selected fields. (C) Similar experiments were done using 2 times (2×) and 3 times (3×) more old DCs than young DCs.

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 per group. (B) CCR7 cell surface expression on young and old tDCs. Cells were stained with PE-labeled anti-CCR7 mAb. A representative experiment is shown: open profiles show the background with isotype-matched control antibody; filled profiles show the fluorescence staining of CCR7. (C) Time-dependent tyrosine phosphorylation of young and old tDCs. Cells were treated with CCL21 for the times indicated and Western blot analysis was done using anti-phosphotyrosine antibody G410. The membranes were washed and reprobed with β-actin Ab to control for differences in gel loading. Relative density of the two most prominent bands in young and old DCs are indicated. The right panels show the band density relative to the value of the young sample with no CCL21 stimulation (arbitrarily defined as equal to 1). The tyrosine phosphorylation results shown are representative of three independent experiments.

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, depleted of CD19+ cells and stained for CD8-RPE Alexa Fluor 647 and OVA-Pent-PE. A representative figure of FACScan plots depicting the percentage of OVA-specific CD8+ T cells is shown. (B) CTL activity of splenocytes from mice receiving young and old DC vaccines. Spleens isolated from DC vaccinated or control mice (DPBS-treated tumor bearing mice) were stimulated for 6 days in vitro with mitomycin-C treated B16-OVA tumor cells and cytotoxic activities against B16-OVA target cells were measured. Data are expressed as mean specific lysis of quadruplicate values (%) ± standard error of the mean of 3 independent experiments. (C) Cytokine release of activated CTL cells in response to tumor stimulation. Splenocytes from vaccinated mice were co-cultured with irradiated B16-OVA cells. After 48 hrs, supernatants were collected for cytokine analysis by Cytometric Bead Arrays. Data are reported as the average amount of cytokine secreted ± SEM of 3 experiments. *p < 0.001, **p < 0.005, ***p < 0.05: young PP-DCs vs old PP-DCs.

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×) or 4 × 10⁶ old PP-DCs (2×) or saline (DPBS), and tumors and spleens harvested 7 days later. (A) Graph depicts tumor size (n = 6 tumors per treatment group). (B) Splenocytes were stimulated for 6 hrs with OVA peptide, labeled with OVA pent-PE, CD8-FITC and IFNγ-PE-CY7 and analyzed by flow cytometry. Dot plots show OVA-pent staining and intracellular IFN-γ production by splenic CD8+ T cells. The indicated number shows the percentage of the gated population within the total CD8+ live cells (C) The percentage of IFN-γ+ cells among OVA-specific CD8+ T cells (determined using the elliptical gate in B) was assessed in three independent experiments, and the mean +/− SEM is shown. (D) IFN-γ CD8+ T cells were purified from spleens as described in Materials and Methods and assessed for cytotoxicity using B16-OVA tumor cells as targets. Data are expressed as mean +/− SEM of 3 independent experiments.

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 of cells co-expressing CD45 and indicated cell surface molecules determined. (A) Pie charts representing the contribution of each subset tested relative to the immune infiltrate in one tumor-bearing mice immunized with DPBS, 2 × 10⁶ (1×) young OVA PP-DCs, 2 × 10⁶ (1×) and 4 × 10⁶ (2×) old PP-DCs. (B) The average total number of each subset was determined and represented as mean % double positive cell ± standard error of the mean of 6 independent experiments. *p < 0.01, ** p < 0.005: young PP-DCs vs old PP-DCs; ***p < 0.02 young and old PP-DCs vs DPBS.