Fig 1.
Effect of aging on the induction of CTL response.
(A and B) Young and old C57BL/6 mice were immunized with a single intravenous injection of 2.5x109 OVA-beads in 100 μg polyU/DO. Additional young and old mice were injected with saline as control. Seven days later, CTL response was determined by an in vivo killing assay. (A) Data show the percentage of specific in vivo killing of each individual mouse and the bars indicate the mean of each group. (B) IFN-γ content in culture supernatants of splenocytes from immunized mice determined by ELISA. Spleen cells were recovered and cultured for 72 hours in the presence of OVA or OVA257–264. (C and D) cDCs purified from the spleen of young and old C57BL/6 mice were incubated with 20 mg/mL OVA in 20 μg/mL polyU/DO, or with RPMI alone (control) for 90 minutes and then washed twice. One million cDCs per age group were intravenously injected into young C57BL/6 mice. Seven days later, CTL was determined by in vivo killing assay. (C) Representative flow cytometry histograms gated on CFSE+ cells are shown. (D) Data show the percentage of specific in vivo killing values, expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Results are representative of 3 independent experiments (4 mice/age group/experiment). In all cases, young and old control groups gave similar results, and only the results of the young control group are depicted.
Fig 2.
Aged splenic cDCs have impaired ability to cross-prime naïve CD8+ T cells in vitro.
Total (A-D) or CD8α+ (E) cDCs purified from young and old mice were incubated with 1 mg/mL OVA mixed with 20 μg/mL polyU/DO for 90 minutes. Additional cDCs from young and old mice were incubated with RPMI or OVA as control. cDCs were then washed and cultured for 3 days with CFSE-labeled CD8β+ T cells isolated from the spleen of OT-I mice at different DC:T cell ratios. After culture, T cell proliferation and CD25 expression were analyzed by flow cytometry. (A) Representative histograms of T cell proliferation are shown from 1:1 ratio. (B, E) Percentages of proliferating T cells, (C) CD25 expression and (D) IFN-γ content in culture supernatants, determined by ELISA. Data show the mean ± SEM. *p < 0.05, **p < 0.01. Results are representative of 3 independent experiments (3–4 mice/age group/experiment).
Fig 3.
Ag presentation on MHC I molecules is affected in cDCs from old mice but Ag uptake is preserved.
(A) In vitro Ag presentation assay. cDCs purified from the spleen of young and old mice were incubated with several OVA concentrations forming IC-OVA for 4 hours and then they were washed and incubated with B3Z cells overnight. B3Z stimulation, monitored by colorimetric bulk determination of β-galactosidase, is expressed as optical density (OD) at λ = 595. (B, C) In vitro Ag capture assay. (B) Spleen cells from young and old mice were recovered and incubated for 90 minutes with soluble OVA-FITC (upper) or IC-OVA-FITC (lower). Then, spleen cells were labeled with anti-CD11c Ab. Results are expressed as mean ± SEM of MFI in FITC channel. (C) CD8α+ cDCs purified from young and old mice were incubated for 90 minutes with soluble OVA-AF647. Results are expressed as mean ± SEM of MFI in AF647 channel. Results are representative of 3 independent experiments (3–4 mice/age group/experiment).
Fig 4.
Ag degradation in cDCs is affected by aging.
Persistence of OVA protein in cell lysates of total (A) or CD8α+ (B) cDCs purified from young and old mice was determined by Western blot after 1 hour pulse loading with 0.625 mg/mL OVA plus 20 μg/mL polyU/DO (time 0, 0h) and 4h chase. Actin was used for loading control. The control line contains total cell lysates of untouched splenic DCs. Densitometric analysis of Western blots (right) is expressed as the ratio of integrated optical density (IOD) at time 0 relative to IOD at chase time. (C) Percentages of total live and dead cDCs after 1 hour pulse loading and 4h chase. Data represent the mean ± SEM of duplicate cultures and are representative of 2 independent experiments. ***p < 0.001.
Fig 5.
DC maturation is affected by aging.
(A and B) Young and old mice were intravenously injected with 100 μg polyU/DO. Eighteen hours later, expression levels of CD86, CD40, MHC II (A) and PDL-1 (B) were determined in DC subsets by flow cytometry. Total (C) or CD8α+ (D) cDCs purified from the spleen of young and old mice were stimulated with 20 μg/mL polyU/DO and then supernatants were assayed for cytokine production by ELISA. (D) Spleen DC subsets from young and old mice were purified and total RNA of 1x106 DCs was extracted. Relative mRNA levels for Tlr7 were quantified by qPCR and normalized to Hprt1. Data show the mean ± SEM. Results are representative of 3 independent experiments (3–4 mice/age group/experiment). *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 6.
Differential phosphorylation of IκB-α in cDCs from young and old mice after TLR7 stimulation.
(A) Pooled splenic cDC isolated from 3 young or old mice were incubated with 20 μg/mL polyU/DO for the indicated time. Cells were then lysed, and the level of phosphorylation of IκB-α was evaluated by Western blot using an anti-pIκB-α antibody. β-actin levels were used for loading control. (B) IOD of phosphorylated IκB-α normalized to β-actin. Data represent mean ± SEM of 2 pooled independent experiments.