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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

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.
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pone.0140672.g004: 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.

Mentions: Considering that cDCs from old mice could efficiently internalize OVA, but poorly cross-present OVA to CD8+ T cells, we then analyzed OVA persistence in whole cell lysates of splenic cDCs from young and old mice by Western blot as an approach to assessing Ag processing. After a 60-minute pulse of cDCs with OVA plus polyU/DO (0 hour), cDC lysates from both young and old mice showed an equivalent 45 kDa OVA band (Fig 4A, left). Four hours later, the 45 kDa OVA band had almost completely disappeared in cDC lysates from young mice, while cDC lysates from old mice still showed a consistent band. Under the same conditions, CD8α+ cDCs from old mice showed a similar result to that using total cDCs (Fig 4B). As shown by densitometric analysis, neither total nor CD8α+ cDCs from old mice could degrade OVA as cDCs from young mice did at the times assayed (Fig 4A and 4B right). The OVA band detected in our plots is the result of an active uptake mechanism, and not only membrane-bound OVA. OVA uptake did not occur when performed at 4°C (data not shown). We also assayed the viability of cDCs under these Ag processing experimental conditions. Using 7AAD staining, we found no significant difference in viability between young or old cDCs after stimulation (Fig 4C). Ag persistence in cDCs from old mice was not due to a decrease in their viability. Together, these results clearly demonstrate that aging altered the ability of cDCs to process exogenous Ag, which would correlate with defects in Ag cross-presentation.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608578&req=5

pone.0140672.g004: 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.
Mentions: Considering that cDCs from old mice could efficiently internalize OVA, but poorly cross-present OVA to CD8+ T cells, we then analyzed OVA persistence in whole cell lysates of splenic cDCs from young and old mice by Western blot as an approach to assessing Ag processing. After a 60-minute pulse of cDCs with OVA plus polyU/DO (0 hour), cDC lysates from both young and old mice showed an equivalent 45 kDa OVA band (Fig 4A, left). Four hours later, the 45 kDa OVA band had almost completely disappeared in cDC lysates from young mice, while cDC lysates from old mice still showed a consistent band. Under the same conditions, CD8α+ cDCs from old mice showed a similar result to that using total cDCs (Fig 4B). As shown by densitometric analysis, neither total nor CD8α+ cDCs from old mice could degrade OVA as cDCs from young mice did at the times assayed (Fig 4A and 4B right). The OVA band detected in our plots is the result of an active uptake mechanism, and not only membrane-bound OVA. OVA uptake did not occur when performed at 4°C (data not shown). We also assayed the viability of cDCs under these Ag processing experimental conditions. Using 7AAD staining, we found no significant difference in viability between young or old cDCs after stimulation (Fig 4C). Ag persistence in cDCs from old mice was not due to a decrease in their viability. Together, these results clearly demonstrate that aging altered the ability of cDCs to process exogenous Ag, which would correlate with defects in Ag cross-presentation.

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