Aging Inhibits Memory Immune Response of CD8+ T Cells in Lungs of C57BL/6J Mice Against Influenza A (H1N1) Virus

doi:10.12300/j.issn.1674-5817.2025.066

Abstract

Abstract:

Objective To compare functional differences of CD8⁺ T cells in lung tissues between young and aged C57BL/6J mice during the contraction phase and memory immune response phase after infection with influenza A (H1N1) virus. Methods Lung tissues from young (3-month-old) and aged (24-month-old) C57BL/6J female mice without influenza virus infection were collected to prepare single-cell suspensions, which were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin or cluster of differentiation (CD) 3/CD28 antibodies (T-cell antigen receptor/co-stimulatory signals) respectively (non-specific antigens stimulation). Flow cytometry intracellular cytokine staining (ICS) was performed on lung CD8⁺ T cells to detect their secretion capacity of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Young and aged C57BL/6J mice were infected intranasally with 490 PFU PR8 influenza virus, and reinfected with homologous influenza virus 28 days later. Lung tissues were isolated on day 28 (the contraction phase) and day 32 (the memory immune response phase) after primary infection. Influenza virus-specific MHC-Ⅰ tetramer staining was used to detect the proportion of virus-specific CD8⁺ T cells in lung tissue CD8⁺ T cells, and ICS was used to analyze TNF-α, IFN-γ, and granzyme B expression in CD8⁺CD44^high T cell subset. Results After non-specific antigen stimulation, TNF-α and IFN-γ secretion capacity in lung tissue CD8⁺ T cells of aged group mice was significantly higher than that of young group (P<0.05). After virus-specific antigen stimulation, there were no statistically significant differences in the proportion of virus-specific CD8⁺ T cells and the expression levels of TNF-α, IFN-γ, and granzyme B between the two groups of mice during the contraction phase (P>0.05), while during the memory immune response phase, the proportion of virus-specific CD8⁺ T cells and the expression levels of TNF-α, IFN-γ, and granzyme B in the aged group mice were significantly lower than those in the young group (P<0.05). Conclusion CD8⁺ T cells in aged mice maintain normal immune-related factor expression function under non-specific antigen stimulation, but show impaired immune-related factor expression function during antigen-specific memory immune response phase, suggesting that aging leads to defects in the formation or maintenance of CD8⁺ T cell immune memory.

Key words: Influenza A (H1N1) virus, CD8⁺ T cells, Immunosenescence, Memory immune response, Mice

CLC Number:

R-332

WANG Chao,LI Shun,REN Xiaonan,et al. Aging Inhibits Memory Immune Response of CD8⁺ T Cells in Lungs of C57BL/6J Mice Against Influenza A (H1N1) Virus[J]. Laboratory Animal and Comparative Medicine, 2025, 45(5): 515-523. DOI: 10.12300/j.issn.1674-5817.2025.066.

Figures/Tables 5

Note： Data gated on CD8⁺CD44^high T cell subset； PMA， Phorbol 12-myristate 13-acetate； I， Ionomycin； Control， Unstimulated group； IFN-γ， Interferon-γ； TNF-α， Tumor necrosis factor-α； SSC-H， Side scatter-height； Y， Young group； A， Aged group.
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Figure 1 Detection of IFN-γ and TNF-α expression levels in CD8⁺CD44^high T cell subset from lung tissues of aged and young mice after PMA/ionomycin stimulation by flow cytometry
Note： Data gated on CD8⁺CD44^high T cell subset； PMA， Phorbol 12-myristate 13-acetate； I， Ionomycin； Control， Unstimulated group； IFN-γ， Interferon-γ； TNF-α， Tumor necrosis factor-α； SSC-H， Side scatter-height； Y， Young group； A， Aged group.

Note： Data gated on CD8⁺CD44^high T cell subset； CD3， Cluster of differentiation 3； CD28， Cluster of differentiation 28.
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Figure 2 Detection of IFN-γ and TNF-α expression levels in CD8⁺CD44^high T cell subset from lung tissues of aged and young mice after CD3/CD28 antibodies stimulation by flow cytometry
Note： Data gated on CD8⁺CD44^high T cell subset； CD3， Cluster of differentiation 3； CD28， Cluster of differentiation 28.

Note：Data gated on CD8⁺ T cell subset； D28， 28th day after the initial infection； D32， On the fourth day after the second infection， that is， on the 32nd day after the first infection； Control， The group not infected with influenza virus； Infected， The group infected with influenza virus； Tetramer， Influenza virus-specific MHC-Ⅰ tetramer； CD44， Cluster of differentiation 44.
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Figure 3 Proportions of virus-specific CD8⁺ T cells in lung tissues of aged and young mice on day 28 after first influenza virus infection and on day 4 （the 32nd day after first infection） after secondary infection
Note：Data gated on CD8⁺ T cell subset； D28， 28th day after the initial infection； D32， On the fourth day after the second infection， that is， on the 32nd day after the first infection； Control， The group not infected with influenza virus； Infected， The group infected with influenza virus； Tetramer， Influenza virus-specific MHC-Ⅰ tetramer； CD44， Cluster of differentiation 44.

Note： Data gated on CD8⁺CD44^high T cell subset； ^*P<0.05， ^**P<0.01.
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Figure 4 Expression levels and MFI of IFN-γ and TNF-α in viral peptide-stimulated CD8?CD44^high T cells from lung tissues of aged and young mice on days 28 and 32 after influenza virus infection?
Note： Data gated on CD8⁺CD44^high T cell subset； ^*P<0.05， ^**P<0.01.

Note： Data gated on CD8⁺CD44^high T cell subset； ^**P<0.01.
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Figure 5 Expression levels and MFI of granzyme B in viral peptide-stimulated CD8?CD44^high T cells from lung tissues of aged and young mice on days 28 and 32 after influenza virus infection
Note： Data gated on CD8⁺CD44^high T cell subset； ^**P<0.01.

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