甲型流感病毒PR8感染小鼠早期记忆T淋巴细胞的改变及其意义

doi:10.13481/j.1671-587X.20250614

摘要/Abstract

摘要：

目的探讨早期记忆T淋巴细胞（T_M）在甲型流感病毒（H1N1）PR8感染小鼠后的变化情况，并阐明其意义。方法将32只C57BL/6小鼠随机分为对照组和流感病毒（PR8）组，每组16只。对照组小鼠鼻滴30 μL无菌磷酸盐缓冲液（PBS），PR8组小鼠给予鼻滴2 LD₅₀ PR8病毒液。于第10天处死小鼠，收集支气管肺泡灌洗液（BALF）、肺组织和淋巴结用于后续实验。HE染色观察2组小鼠肺组织病理形态表现，血凝试验、实时荧光定量PCR（RT-qPCR）法和免疫荧光法检测小鼠肺组织病毒感染情况，流式细胞术检测2组小鼠BALF、肺组织和淋巴结中T淋巴细胞、效应性T淋巴细胞、中央型T_M淋巴细胞（T_CM）和效应性T_M淋巴细胞（T_EM）及核蛋白（NP）/聚合酶酸性蛋白（PA）特异性T_EM淋巴细胞百分率。结果与对照组比较，PR8组小鼠肺组织出现明显的病理损伤，且肺组织病毒效价和NP蛋白表达水平均明显升高（P<0.01）。与对照组比较，PR8组小鼠BALF和肺组织中CD4⁺T淋巴细胞、CD8⁺T淋巴细胞、效应性CD4⁺T淋巴细胞、效应性CD8⁺T淋巴细胞、CD4⁺T_EM淋巴细胞和CD8⁺T_EM淋巴细胞百分率均明显升高（P<0.01）。与对照组比较，PR8组小鼠BALF和肺组织中CD4⁺T_CM、CD8⁺T_CM、CD4⁺T_EM、CD8⁺T_EM及NP/PA特异性CD4⁺T_EM和CD8⁺T_EM淋巴细胞百分率均明显升高（P<0.05或P<0.01），淋巴结中CD8⁺T_CM淋巴细胞百分率明显升高（P<0.05）。结论流感病毒感染可诱导机体T淋巴细胞增殖分化为效应性T淋巴细胞，在呼吸道黏膜局部早期建立保守表位NP/PA的特异性T_M淋巴细胞。

关键词: 流感病毒, 记忆T淋巴细胞, 中央型记忆T淋巴细胞, 效应型记忆T淋巴细胞, 保守表位

Abstract:

Objective To discuss the changes of early memory T lymphocytes （T_M） in the mice infected with influenza A virus （H1N1） PR8， and to clarify its significance. Methods A total of 32 C57BL/6 mice were randomly divided into control group and influenza virus （PR8） group， with 16 mice in each group. The mice in control group were intranasally administered 30 μL of sterile phosphate buffered saline （PBS）， and the mice in PR8 group were intranasally administered 2 LD₅₀ PR8 virus solution. The mice were sacrificed on the 10 th day， and bronchoalveolar lavage fluid （BALF）， lung tissue， and lymphnodes were collected for subsequent experiments. HE staining was used to observe the pathomorphology of lung tissue in the mice in two groups； hemagglutination assay， real-time fluorescence quantitative PCR （RT-qPCR） method， and immunofluorescence method were used to detect the viral infection in lung tissue of the mice； flow cytometry was used to detect the percentages of T lymphocytes， effector T lymphocytes， central memory T lymphocytes （T_CM）， effector memory T lymphocytes （T_EM）， and nuclear protein（NP）/pdymerase acidic protein（PA）-specific T_EM lymphocytes in BALF， lung tissue， and lymphnodes of the mice in two groups. Results Compared with control group， the lung tissue of the mice in PR8 group showed significant pathological damage， and the lung tissue virus titer and NP protein expression level were significantly increased （P<0.01）. Compared with control group， the percentages of CD4⁺ T lymphocytes， CD8⁺ T lymphocytes， effector CD4⁺ T lymphocytes， effector CD8⁺ T lymphocytes， CD4⁺ T_EM lymphocytes， and CD8⁺ T_EM lymphocytes in BALF and lung tissue of the mice in PR8 group were significantly increased （P<0.01）. Compared with control group， the positive percentages of CD4⁺ T_CM， CD8⁺ T_CM， CD4⁺ T_EM， CD8⁺ T_EM， and NP/PA-specific CD4⁺ T_EM， CD8⁺ T_EM lymphocytes in BALF and lung tissue of the mice in PR8 group were significantly increased （P<0.05 or P<0.01）， and the percentage of CD8+ T_CM lymphocytes in the lymph nodes was significantly increased （P<0.05）. Conclusion Influenza virus infection can induce the proliferation and differentiation of T lymphocytes into effector T lymphocytes， leading to the early establishment of virus-specific T_M lymphocytes targeting the conserved NP/PA epitopes in the respiratory mucosa.

Key words: Influenza virus, Memory T lymphocyte, Central memory T lymphocyte, Effector memory T lymphocyte, Conserved epitope

中图分类号:

R392

徐昆,田晶. 甲型流感病毒PR8感染小鼠早期记忆T淋巴细胞的改变及其意义[J]. 吉林大学学报(医学版), 2025, 51(6): 1584-1594.

Kun XU,Jing TIAN. Changes of early memory T lymphocytes in mice infected with influenza A virus PR8 and its significance[J]. Journal of Jilin University(Medicine Edition), 2025, 51(6): 1584-1594.

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参考文献 21

[1]	LI J L， ZHANG Y F， ZHANG X L， et al. Influenza and universal vaccine research in China［J］. Viruses， 2022， 15（1）： 116.
[2]	LIANG Y Y. Pathogenicity and virulence of influenza［J］. Virulence， 2023， 14（1）： 2223057.
[3]	KOSTOLANSKÝ F， TOMČÍKOVÁ K， BRIESTENSKÁ K， et al. Universal anti-influenza vaccines based on viral HA2 and M2e antigens［J］. Acta Virol， 2020， 64（4）： 417-426.
[4]	SAYEDAHMED E E， ELSHAFIE N O， DOS SANTOS A P， et al. Development of NP-based universal vaccine for influenza a viruses［J］. Vaccines， 2024， 12（2）： 157.
[5]	SUN X Y， MA H W， WANG X J， et al. Broadly neutralizing antibodies to combat influenza virus infection［J］. Antiviral Res， 2024， 221： 105785.
[6]	CEN L Q， ZHANG Z， SUN Y， et al. Efficacy of MAGE-A4 long peptide as a universal immunoprevention cancer vaccine［J］. Cancer Cell Int， 2024， 24（1）： 232.
[7]	CORRADO M， PEARCE E L. Targeting memory T cell metabolism to improve immunity［J］. J Clin Invest， 2022， 132（1）： e148546.
[8]	ZHOU J L， UDDBACK I， KOHLMEIER J E， et al. Vaccine induced memory CD8⁺ T cells efficiently prevent viral transmission from the respiratory tract［J］. Front Immunol， 2023， 14： 1322536.
[9]	ESSER M T， MARCHESE R D， KIERSTEAD L S， et al. Memory T cells and vaccines［J］. Vaccine， 2003， 21（5/6）： 419-430.
[10]	DEEP D， GUDJONSON H， BROWN C C， et al. Precursor central memory versus effector cell fate and naïve CD4⁺ T cell heterogeneity［J］. J Exp Med， 2024， 221（10）： e20231193.
[11]	GANNON P O， BAUMGAERTNER P， HUBER A， et al. Rapid and continued T-cell differentiation into long-term effector and memory stem cells in vaccinated melanoma patients［J］. Clin Cancer Res， 2017， 23（13）： 3285-3296.
[12]	WELTEN S P M， ODERBOLZ J， YILMAZ V， et al. Influenza- and MCMV-induced memory CD8 T cells control respiratory vaccinia virus infection despite residence in distinct anatomical niches［J］. Mucosal Immunol， 2021， 14（3）： 728-742.
[13]	MALOULI D， TIWARY M， GILBRIDE R M， et al. Cytomegalovirus vaccine vector-induced effector memory CD4+ T cells protect cynomolgus macaques from lethal aerosolized heterologous avian influenza challenge［J］. Nat Commun， 2024， 15（1）： 6007.
[14]	FENG L， GAO Y Y， SUN M W， et al. The parallel presentation of two functional CTL epitopes derived from the O and Asia 1 serotypes of foot-and-mouth disease virus and swine SLA-2*HB01： implications for universal vaccine development［J］. Cells， 2022， 11（24）： 4017.
[15]	LIU X J， ZHAO T Y， WANG L L， et al. Strategies targeting hemagglutinin cocktail as a potential universal influenza vaccine［J］. Front Microbiol， 2022， 13： 1014122.
[16]	MCGEE M C， HUANG W S. Evolutionary conservation and positive selection of influenza A nucleoprotein CTL epitopes for universal vaccination［J］. J Med Virol， 2022， 94（6）： 2578-2587.
[17]	KIM S H， ESPAÑO E， PADASAS B T， et al. Influenza virus-derived CD8 T cell epitopes： implications for the development of universal influenza vaccines［J］. Immune Netw， 2024， 24（3）： e19.
[18]	HASSAN M S H， SHARIF S. Immune responses to avian influenza viruses in chickens［J］. Virology， 2025， 603： 110405.
[19]	PARK S C， WIEST M J， YAN V， et al. Induction of protective immune responses at respiratory mucosal sites［J］. Hum Vaccin Immunother， 2024， 20（1）： 2368288.
[20]	SCHREINER D， KING C G. CD4⁺ memory T cells at home in the tissue： mechanisms for health and disease［J］. Front Immunol， 2018， 9： 2394.
[21]	KEDZIERSKA K， VENTURI V， FIELD K， et al. Early establishment of diverse T cell receptor profiles for influenza-specific CD8⁺CD62L（hi） memory T cells［J］. Proc Natl Acad Sci U S A， 2006， 103（24）： 9184-9189.