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Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

Zacca ER, Crespo MI, Acland RP, Roselli E, Núñez NG, Maccioni M, Maletto BA, Pistoresi-Palencia MC, Morón G - PLoS ONE (2015)

Bottom Line: Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established.Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation.Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Bioquímica Clínica e Inmunología, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0140672.g001: 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.

Mentions: We recently reported that polyU complexed with DOTAP (polyU/DO) is a potent inducer of cytotoxic immune responses in a TLR7-dependent fashion [18]. With this in mind, in this study we first evaluated whether aging affects an Ag-specific cytotoxic response induced by TLR7 stimulation with polyU/DO. We intravenously immunized young and old mice with ovalbumin (OVA) as a monitor Ag coated to polystyrene beads (OVA beads) plus polyU/DO. Seven days later, we determined the presence of CTLs by an in vivo killing assay. To this end, OVA257–264-pulsed naïve syngeneic splenocyte targets (CFSEhigh-labeled cells) were intravenously injected into immunized mice. As an internal control, equal numbers of nonpulsed naïve syngeneic splenocytes (CFSElow-labeled cells) were injected. The number of CFSE+ cells remaining in the spleen after 24 hours was determined by flow cytometry. Immunization with OVA beads plus polyU/DO led to a potent cytotoxic response in young mice, whereas old mice did not develop a CTL response against OVA (Fig 1A). In addition to Ag-specific killing, we also determined IFN-γ secretion in the supernatants of splenocytes of these groups after being restimulated in vitro with whole OVA or OVA257–264 during 72 hours. We found that splenocytes from young immunized mice secreted higher levels of IFN-γ than splenocytes from old immunized mice (Fig 1B).


Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

Zacca ER, Crespo MI, Acland RP, Roselli E, Núñez NG, Maccioni M, Maletto BA, Pistoresi-Palencia MC, Morón G - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608578&req=5

pone.0140672.g001: 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.
Mentions: We recently reported that polyU complexed with DOTAP (polyU/DO) is a potent inducer of cytotoxic immune responses in a TLR7-dependent fashion [18]. With this in mind, in this study we first evaluated whether aging affects an Ag-specific cytotoxic response induced by TLR7 stimulation with polyU/DO. We intravenously immunized young and old mice with ovalbumin (OVA) as a monitor Ag coated to polystyrene beads (OVA beads) plus polyU/DO. Seven days later, we determined the presence of CTLs by an in vivo killing assay. To this end, OVA257–264-pulsed naïve syngeneic splenocyte targets (CFSEhigh-labeled cells) were intravenously injected into immunized mice. As an internal control, equal numbers of nonpulsed naïve syngeneic splenocytes (CFSElow-labeled cells) were injected. The number of CFSE+ cells remaining in the spleen after 24 hours was determined by flow cytometry. Immunization with OVA beads plus polyU/DO led to a potent cytotoxic response in young mice, whereas old mice did not develop a CTL response against OVA (Fig 1A). In addition to Ag-specific killing, we also determined IFN-γ secretion in the supernatants of splenocytes of these groups after being restimulated in vitro with whole OVA or OVA257–264 during 72 hours. We found that splenocytes from young immunized mice secreted higher levels of IFN-γ than splenocytes from old immunized mice (Fig 1B).

Bottom Line: Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established.Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation.Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Bioquímica Clínica e Inmunología, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

No MeSH data available.


Related in: MedlinePlus