衰老抑制C57BL/6J小鼠肺部CD8+ T细胞对甲型H1N1流感病毒的记忆性免疫应答

doi:10.12300/j.issn.1674-5817.2025.066

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (5): 515-523.DOI: 10.12300/j.issn.1674-5817.2025.066

• 人类疾病动物模型 • 下一篇

衰老抑制C57BL/6J小鼠肺部CD8⁺ T细胞对甲型H1N1流感病毒的记忆性免疫应答

王超(), 李顺, 任晓楠, 杨华, 陈丽香, 徐春华, 周晓辉()()

复旦大学附属公共卫生临床中心, 上海 201508

收稿日期:2025-04-29 修回日期:2025-06-05 出版日期:2025-10-25 发布日期:2025-10-23
通讯作者:
周晓辉（1973—），男，博士，研究员，研究方向：新发传染病动物模型构建与免疫机制研究。E-mail： zhouxiaohui@shphc.org.cn。ORCID：0000-0002-7491-628X
作者简介:王超（1992—），男，硕士，技师，研究方向：感染与免疫。E-mail：584264751@qq.com。ORCID：0009-0007-6475-3940
基金资助:
国家重点研发计划-基础科研条件与重大科学仪器设备研发专项“单/多病因传染病动物模型的研制及应用”(2023YFF0724800，2023YFF0724801);上海市公共卫生临床中心项目“流感相关脑病发生机制研究”(KY-GW-2025-22)

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

WANG Chao(), LI Shun, REN Xiaonan, YANG Hua, CHEN Lixiang, XU Chunhua, ZHOU Xiaohui()()

Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China

Received:2025-04-29 Revised:2025-06-05 Published:2025-10-25 Online:2025-10-23
Contact: ZHOU Xiaohui (ORCID: 0000-0002-7491-628X), E-mail: zhouxiaohui@shphc.org.cn

摘要/Abstract

摘要：

目的比较年轻与老年C57BL/6J小鼠感染甲型H1N1流感病毒后肺组织中CD8⁺ T细胞在收缩期及记忆性免疫应答期的功能差异。方法摘取未行流感病毒感染的年轻（3月龄）与老年（24月龄）雌性C57BL/6J小鼠肺组织，制备单细胞悬液，分别用佛波酯（phorbol 12-myristate 13-acetate，PMA）/离子霉素或分化簇（cluster of differentiation，CD）3/CD28抗体（T细胞抗原受体/共刺激信号）进行非特异性抗原刺激，通过流式细胞术对肺CD8⁺T细胞行细胞内细胞因子染色（flow cytometry intracellular cytokine staining，ICS）以检测其分泌肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和干扰素-γ（interferon-γ，IFN-γ）的能力。以490 PFU PR8流感病毒滴鼻感染年轻和老年C57BL/6J小鼠，28天后进行同源流感病毒的二次感染，于初次感染后第28天（收缩期）、第32天（记忆性免疫应答期）分离肺组织，采用流感病毒特异性MHC-Ⅰ四聚体染色法检测病毒特异性CD8⁺ T细胞在肺组织CD8⁺ T细胞中的比例，ICS法分析CD8⁺CD44^high T细胞亚群TNF-α、IFN-γ及颗粒酶B（granzyme B）的表达。结果非特异性抗原刺激后，老年组小鼠的肺组织CD8⁺ T细胞中TNF-α、IFN-γ分泌能力显著高于年轻组（P＜0.05）。病毒特异性抗原刺激后，收缩期的两组小鼠间病毒特异性CD8⁺ T细胞比例以及TNF-α、IFN-γ、granzyme B的表达水平差异均无统计学意义（P＞0.05）；而在记忆性免疫应答期，老年组小鼠的病毒特异性CD8⁺ T细胞比例以及TNF-α、IFN-γ、granzyme B的表达水平均显著低于年轻组（P＜0.05）。结论老年小鼠肺部CD8⁺ T细胞在非特异性抗原刺激下免疫相关因子表达功能正常，但在抗原特异性记忆免疫应答阶段的免疫相关因子表达功能受损，提示衰老导致CD8⁺ T细胞免疫记忆形成或维持发生缺陷。

关键词: 甲型H1N1流感病毒, CD8⁺ T细胞, 免疫衰老, 记忆性免疫应答, 小鼠

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

中图分类号:

R-332

王超,李顺,任晓楠,等. 衰老抑制C57BL/6J小鼠肺部CD8⁺ T细胞对甲型H1N1流感病毒的记忆性免疫应答[J]. 实验动物与比较医学, 2025, 45(5): 515-523. DOI: 10.12300/j.issn.1674-5817.2025.066.

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.

图/表 5

图1 流式细胞术检测PMA/离子霉素刺激老年和年轻小鼠肺组织中CD8+CD44high T细胞亚群的IFN-γ、TNF-α表达水平注：数据圈门于CD8+CD44high T细胞亚群；PMA，佛波酯；I，离子霉素；Control，未刺激组；IFN-γ，干扰素-γ；TNF-α，肿瘤坏死因子-α；SSC-H，侧向散射光-高度；Y，年轻组；A，老年组。

Figure 1 Detection of IFN-γ and TNF-α expression levels in CD8+CD44high T cell subset from lung tissues of aged and young mice after PMA/ionomycin stimulation by flow cytometryNote： Data gated on CD8+CD44high 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.

图2 流式细胞术检测CD3/CD28抗体刺激老年和年轻小鼠肺组织中CD8+CD44high T细胞亚群的IFN-γ、TNF-α表达水平注：数据圈门于CD8+CD44high T细胞亚群；CD3，分化簇3；CD28，分化簇28。

Figure 2 Detection of IFN-γ and TNF-α expression levels in CD8+CD44high T cell subset from lung tissues of aged and young mice after CD3/CD28 antibodies stimulation by flow cytometryNote： Data gated on CD8+CD44high T cell subset； CD3， Cluster of differentiation 3； CD28， Cluster of differentiation 28.

图3 流感病毒第一次感染后第28天和第二次感染后第4天（第一次感染后第32天）老年和年轻小鼠肺组织中病毒特异性CD8+ T细胞比例注：数据圈门于CD8+ T细胞群；D28，初次感染后第28天；D32，二次感染后第4天，即初次感染后第32天；Control，未感染流感病毒组；Infected，流感病毒感染组；Tetramer，流感病毒特异性MHC-Ⅰ四聚体；CD44，分化簇44。

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 infectionNote：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.

图4 流感病毒感染第28天和第32天后老年和年轻小鼠肺组织中CD8+CD44high T细胞在病毒肽段刺激后IFN-γ、TNF-α的表达水平和MFI值注：数据圈门于CD8+CD44high T细胞亚群；*P<0.05，**P<0.01。

Figure 4 Expression levels and MFI of IFN-γ and TNF-α in viral peptide-stimulated CD8?CD44high T cells from lung tissues of aged and young mice on days 28 and 32 after influenza virus infection?Note： Data gated on CD8+CD44high T cell subset； *P<0.05， **P<0.01.

图5 流感病毒感染第28天和第32天后老年和年轻小鼠肺组织中CD8+CD44high T 细胞在病毒肽段刺激后颗粒酶B的表达水平和MFI值注：数据圈门于CD8+CD44high T细胞亚群；**P<0.01。

Figure 5 Expression levels and MFI of granzyme B in viral peptide-stimulated CD8?CD44high T cells from lung tissues of aged and young mice on days 28 and 32 after influenza virus infectionNote： Data gated on CD8+CD44high T cell subset； **P<0.01.

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衰老抑制C57BL/6J小鼠肺部CD8⁺ T细胞对甲型H1N1流感病毒的记忆性免疫应答

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

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