Effects of a New Tuberculosis Vaccine Strain on T Cell Immune Memory and Proliferation Capacity

doi:10.12114/j.issn.1007-9572.2021.00.159

Abstract

Abstract: Background　Tuberculosis（TB） is a global chronic infectious disease with the highest mortality caused by an infective agent.But there is a lack of safe and effective vaccines to prevent adult tuberculosis.Vaccination mainly uses body's immune memory to prevent the re-invasion of pathogens.Therefore，studying the immune memory and proliferation capacity of T cells is crucial for the development of new tuberculosis vaccines.Objective　To investigate the effects of a new tuberculosis vaccine strain（B/R strain） on T cell immune memory and proliferation capacity.Methods　In January 2019，48 SPF female C57BL/6 mice aged 6-8 weeks were randomly divided into PBS group，BCG group，H37Ra group and B/R strain group，with 12 mice in each group，and were subcutaneously inoculated，respectively.At 8，12，and 16 weeks after immunization，the splenic lymphocytes of three mice in each group were extracted and cultured in vitro，and the levels of central memory lymphocytes（TCM） and effector memory lymphocytes（TEM） were detected by flow cytometry.ELISA was used to detect antigen-specific Th1/Th2 cytokines（IL-2，IFN-γ and IL-4） levels secreted by splenic lymphocytes.CFSE labeling method was used to detect the proliferation of CD4+ and CD8+ T lymphocytes.Results　At 8，12，and 16 weeks after immunization，the TCM and TEM levels of CD4+ in BCG，H37Ra，and B/R strain groups were higher than those of PBS group（P<0.05）.At 8 weeks post-inoculation，the level of CD4+ TEM in B/R group was lower than that in H37Ra group（P<0.05）.At 16 weeks post-inoculation，B/R strain group showed higher level of TCM of CD4+ than H37Ra and BCG groups（P<0.05），and higher level of TEM of CD4+ than BCG group（P<0.05）.At 8，12，and 16 weeks after immunization，the TCM and TEM levels of CD8+ in BCG，H37Ra，and B/R strain groups were higher than those of PBS group（P<0.05）.At 12 weeks post-inoculation，the level of TEM of CD8+ in B/R strain group was higher than that in H37Ra and BCG groups（P<0.05）.At 16 weeks post-inoculation，the TCM and TEM levels of CD8+ in B/R strain group were higher than those of BCG group（P<0.05）.At 8，12，and 16 weeks post-inoculation，IL-2 and IFN-γ levels in PBS group were lower than those of other three groups（P<0.05）.At 12 weeks post-inoculation，the level of IFN-γ in B/R strain group was higher than that of BCG group（P<0.05）.At 16 weeks post-inoculation，IFN-γ and IL-2 levels in B/R strain group were higher than those of BCG and H37Ra groups（P<0.05）.The proliferation rates of CD4+ and CD8+ T lymphocytes in BCG，H37Ra and B/R strain groups were higher than those of PBS group（P<0.05）.The proliferation rates of CD4+ and CD8+ T lymphocytes in B/R strain group were higher than those of BCG and H37Ra groups（P<0.05）.Conclusion　The immunizing with the B/R strain in mice could awake up the body to produce higher levels of protective immune memory cells，mainly Th1 type immune responses，and enhance the proliferation capacity of T cells.

Key words: Tuberculosis；Vaccines；Tuberculosis vaccines；BCG Vaccine；T-Lymphocytes；Immunity, cellular

摘要： 背景　结核病是世界范围内单一致病菌感染导致死亡率最高的慢性感染性疾病，目前缺乏安全有效的疫苗预防成人结核病。而预防接种主要是利用机体具有免疫记忆的特征以阻止病原体的再次入侵，因此，研究T淋巴细胞的免疫记忆及增殖能力对新型结核病疫苗的设计至关重要。目的　探讨新型结核病疫苗菌株（简称B/R菌株）对T淋巴细胞免疫记忆及增殖能力的影响。方法　2019年1月，选取6~8周龄、SPF级雌性C57BL/6小鼠48只，随机分为磷酸盐缓冲液（PBS）组、卡介苗（BCG）组、结核分枝杆菌国际标准毒株（H37Ra）组和新型结核病疫苗（B/R）菌株组，各12只。PBS组、BCG组、H37Ra组和B/R菌株组分别皮下接种PBS、BCG、H37Ra和B/R菌株。4组分别于免疫后8周、12周、16周各取3只小鼠提取脾淋巴细胞体外刺激培养，利用流式细胞技术检测中央型记忆性淋巴细胞（TCM）及效应型记忆性淋巴细胞（TEM），酶联免疫吸附试验（ELISA）检测脾淋巴细胞分泌的抗原特异性Th1/Th2型细胞因子〔白介素2（IL-2）、干扰素γ（IFN-γ）和白介素4（IL-4）〕水平；羟基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记法检测CD4+T淋巴细胞增殖率、CD8+ T淋巴细胞增殖率。结果　免疫后8、12、16周BCG组、H37Ra组、B/R菌株组CD4+的TCM和TEM高于PBS组（P<0.05）；免疫后8周B/R菌株组CD4+的TEM低于H37Ra组（P<0.05）；免疫后16周B/R菌株组CD4+的TCM高于H37Ra组和BCG组（P<0.05）；免疫后16周B/R菌株组CD4+的TEM高于BCG组（P<0.05）。免疫后8、12、16周BCG组、H37Ra组、B/R菌株组CD8+的TCM和TEM高于PBS对照组（P<0.05）；免疫后12周B/R菌株组CD8+的TEM高于H37Ra组和BCG组（P<0.05）；免疫后16周，B/R菌株组CD8+的TCM和TEM高于BCG组（P<0.05）。免疫后8、12、16周PBS组IL-2和IFN-γ低于BCG组、H37Ra组、B/R菌株组（P<0.05）；免疫后12周，B/R菌株组IFN-γ高于BCG组（P<0.05）；免疫后16周，B/R菌株组IFN-γ和IL-2高于BCG组和H37Ra组（P<0.05）。BCG组、H37Ra组和B/R菌株组CD4+T淋巴细胞增殖率、CD8+ T淋巴细胞增殖率高于PBS组（P<0.05）；B/R菌株组的CD4+T淋巴细胞增殖率、CD8+T淋巴细胞增殖率均高于BCG组和H37Ra组（P<0.05）。结论　B/R菌株免疫小鼠后，可促进机体产生更高水平的免疫保护性记忆细胞，以Th1型细胞免疫应答为主，并可增强T淋巴细胞增殖能力。

关键词: 结核, 疫苗, 结核菌苗, 卡介苗, T淋巴细胞, 免疫, 细胞

SHAO Meng，WU Fang，ZHANG Jie，DONG Jiangtao，LIU Xiaoling，WU Jiangdong，ZHANG Le，ZHAO Haijun，ZHANG Wanjiang. Effects of a New Tuberculosis Vaccine Strain on T Cell Immune Memory and Proliferation Capacity　[J]. Chinese General Practice, 2021, 24(11): 1376-1382.

邵萌，吴芳，张杰，董江涛，柳小玲，吴江东1章乐，赵海军，张万江. 新型结核病疫苗菌株对T淋巴细胞免疫记忆及增殖能力的影响研究[J]. 中国全科医学, 2021, 24(11): 1376-1382.

References

［1］ORGANIZATION W H.Global tuberculosis report［R］．Geneva：World Health Organization，2019.
［2］MUSTAFA A D，KALYANASUNDRAM J，SABIDI S，et al．Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice［J］．BMC Biotechnol，2018，18（1）：63．DOI：10.1186/s12896-018-0461-y.
［3］BARRETO M L，CUNHA S S，PEREIRA S M，et al．Neonatal BCG protection against tuberculosis lasts for 20 years in Brazil［J］．Int J Tuberc Lung Dis，2005，9（10）：1171-1173.
［4］柳小玲，吴芳，吴江东，等．新型结核病疫苗融合菌株制备及其免疫学特性的研究［J］．中国病原生物学杂志，2016，11（1）：21-24.
［5］梁粟，吴芳，程青虹，等．新型结核病疫苗菌株在小鼠体内的毒性及安全性研究［J］．中国病原生物学杂志，2016，11（3）：193-199，214.
［6］DE MARTINO M，LODI L，GALLI L，et al．Immune response to mycobacterium tuberculosis：a narrative review［J］．Front Pediatr，2019，7：350．DOI：10.3389/fped.2019.00350．
［7］PRADEU T，DU PASQUIER L.Immunological memory：What's in a name?［J］．Immunol Rev，2018，283（1）：7-20．DOI：10.1111/imr.12652.
［8］FARBER D L，NETEA M G，RADBRUCH A，et al．Immunological memory：lessons from the past and a look to the future［J］．Nat Rev Immunol，2016，16（2）：124-128．DOI：10.1038/nri.2016.13.
［9］MAGGIOLI M F，PALMER M V，THACKER T C，et al．Increased TNF-α/IFN-γ/IL-2 and decreased TNF-α/IFN-γ production by central memory T cells are associated with protective responses against bovine tuberculosis following BCG vaccination［J］．Front Immunol，2016，7：421．DOI：10.3389/fimmu.2016.00421.
［10］VAN PINXTEREN L A，CASSIDY J P，SMEDEGAARD B H，et al．Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells［J］．Eur J Immunol，2000，30（12）：3689-3698．DOI：10.1002/1521-4141（200012）30：12&lt；3689：AID-IMMU3689&gt；3.0.CO；2-4.
［11］BLATTMAN J N，ANTIA R，SOURDIVE D J，et al．Estimating the precursor frequency of naive antigen-specific CD8 T cells［J］．J Exp Med，2002，195（5）：657-664．DOI：10.1084/jem.20001021.
［12］BORSA M，BARNSTORF I，BAUMANN N S，et al．Modulation of asymmetric cell Division as a mechanism to boost CD8+ T cell memory［J］．Sci Immunol，2019，4（34）：eaav1730．DOI：10.1126/sciimmunol.aav1730.
［13］KAVEH D A，GARCIA-PELAYO M C，HOGARTH P J.Persistent BCG bacilli perpetuate CD4 T effector memory and optimal protection against tuberculosis［J］．Vaccine，2014，32（51）：6911-6918．DOI：10.1016/j.vaccine.2014.10.041.
［14］KAVEH D A，BACHY V S，HEWINSON R G，et al．Systemic BCG immunization induces persistent lung mucosal multifunctional CD4 T（EM） cells which expand following virulent mycobacterial challenge［J］．PLoS One，2011，6（6）：e21566．DOI：10.1371/journal.pone.0021566.
［15］吴家宝，陈颖盈，季萍，等．抗结核治疗中特异性T细胞免疫反应动态分析及其临床意义［J］．现代免疫学，2017，37（3）：198-205.
WU J B，CHEN Y Y，JI P，et al．Dynamic properties and clinical significance of antigen specific T cell responses during anti——tuberculosis treatment［J］．Current Immunology，2017，37（3）：198-205.
［16］LI Q F，ZHANG H，YU L，et al．Down-regulation of Notch signaling pathway reverses the Th1/Th2 imbalance in tuberculosis patients［J］．Int Immunopharmacol，2018，54：24-32．DOI：10.1016/j.intimp.2017.10.026.
［17］张怡田，王秉翔.新型结核病疫苗及其评价方法的研究进展［J］．微生物学免疫学进展，2016，44（2）：68-74.
［18］SUGAWARA I，YAMADA H，SHI R.Pulmonary tuberculosis in various gene knockout mice with special emphasis on roles of cytokines and transcription factors ［J］．Current Respiratory Medicine Reviews，2005，1（1）：7-13.
［19］LIAO W，LIN J X，WANG L，et al．Modulation of cytokine receptors by IL-2 broadly regulates differentiation into helper T cell lineages［J］．Nat Immunol，2011，12（6）：551-559．DOI：10.1038/ni.2030.
［20］OH H S，CHOI B K，KIM Y H，et al．4-1BB signaling enhances primary and secondary population expansion of CD8+ T cells by maximizing autocrine IL-2/IL-2 receptor signaling［J］．PLoS One，2015，10（5）：e0126765．DOI：10.1371/journal.pone.0126765.
［21］POORAN A，DAVIDS M，NEL A，et al．IL-4 subverts mycobacterial containment in Mycobacterium tuberculosis-infected human macrophages［J］．Eur Respir J，2019，54（2）：1802242．DOI：10.1183/13993003.02242-2018.
［22］LILIANA P，CARMEN C，Adrian S A，et al．Flow-cytometric analysis of specific-proliferation intuberculosis using the CFSE dye dilution technique ［J］．The Journal of preventive Medicine，2003，11（1）：67-74.
［23］薛妮娜，董凯，来芳芳，等．CFSE法检测刺激剂对淋巴细胞增殖与活化［J］．哈尔滨商业大学学报（自然科学版），2017，33（2）：129-134．DOI：10.3969/j.issn.1672-0946.2017.02.001.
［24］王鹏鹏，柴长斌，汪洋.CD8^+T细胞激活和免疫记忆形成的分子调节机制［J］．中华微生物学和免疫学杂志，2015，35（7）：541-545.
［25］MOGUCHE A O，MUSVOSVI M，PENN-NICHOLSON A，et al．
Antigen availability shapes T cell differentiation and function during tuberculosis［J］．Cell Host Microbe，2017，21（6）：695-706.e5．DOI：10.1016/j.chom.2017.05.012.
［26］HOPE J L，STAIRIKER C J，BAE E A，et al．Striking a balance-cellular and molecular drivers of memory T cell development and responses to chronic stimulation［J］．Front Immunol，2019，10：1595．DOI：10.3389/fimmu.2019.01595.

[1]	MA Xiaoyu, MA Xiaomei, WU Guozhi, ZHENG Ya, GUO Qinghong. Research Progress of Neutrophil Extracellular Traps in Gastric Cancer [J]. Chinese General Practice, 2026, 29(17): 2425-2432.
[2]	CHEN Annan, YAN Zhixin, ZHANG Jian, SHEN Bo, DING Xiaoqiang, SONG Nana. Acidic Preconditioning Activates the Sodium-hydrogen Exchanger 1 to Regulate Intracellular pH Homeostasis and Reduce Renal Ischemia-reperfusion Injury [J]. Chinese General Practice, 2026, 29(12): 1607-1617.
[3]	ZHANG Shiya, MIAO Tongguo, LOU Xianzhe, ZHANG Yunjing, MA Dong, NAN Yuemin. Predictive Value of Serum HDL-C in the Occurrence of PVTT in Patients with NBNC-HCC [J]. Chinese General Practice, 2026, 29(11): 1441-1447.
[4]	NONG Jingtang, YANG Chengmin, MO Shenglong, LU Zhicheng, TANG Lina, JIAN Chongdong, SHANG Jingwei. Research Progress on Prognostic Biomarkers after Cerebral Infarction: Mechanisms and Clinical Applications [J]. Chinese General Practice, 2025, 28(29): 3711-3720.
[5]	CHEN Yijing, XU Qi, LIU Zhongdian, QIN Lingqiao, CHEN Shuping, TANG Weiting, ZHONG Qiuan. The Regulatory Role of Hexosamine Biosynthesis Pathway in Vascular Endothelial Inflammation [J]. Chinese General Practice, 2025, 28(15): 1871-1877.
[6]	LOU Xianzhe, MIAO Tongguo, ZHANG Shiya, MA Dong, NAN Yuemin. The Impact of the Monocyte-to-High-Density Lipoprotein Cholesterol Ratio on the Prognosis of Patients with Non-Viral, Non-Alcoholic Hepatocellular Carcinoma: a Retrospective Cohort Study [J]. Chinese General Practice, 2025, 28(09): 1092-1099.
[7]	MA Xiaoxuan, WANG Rongqi. Research Progress in Metabolomics for HCC Diagnosis and Treatment [J]. Chinese General Practice, 2025, 28(08): 1017-1022.
[8]	YANG Anyin, LIU Hongli, CHEN Miaoyang, ZHENG Yufeng, XU Zhiyuan, YANG Yongfeng. Mechanism and in Vitro Experiment of Wogonin in Treatment of Hepatocellular Carcinoma Based on Network Pharmacology [J]. Chinese General Practice, 2024, 27(32): 4040-4049.
[9]	CHEN Chao, CHEN Tianxiang, LIU Qianwei, ZHANG Zhi, WANG Huanhuan, WU Pingping, GAO Lei, YU Zhaoxiang. Analysis and Identification of Hub Genes in Hepatocellular Carcinoma Based on Weighted Gene Co-expression Network and Cancer Genome Atlas Clinical Data [J]. Chinese General Practice, 2024, 27(32): 4050-4059.
[10]	MAIERHABA· Maimaitiaili, ZHANG Kainan, ZHAO Hui, YAKUFU· Tuoheti, YE Jianwei, LYU Guodong. Investigating Serum PARP2 as a Potential Diagnostic Biomarker for Hepatocellular Carcinoma [J]. Chinese General Practice, 2024, 27(32): 4060-4065.
[11]	HU Lingxi, LI Mei, FU Yiwei, LU Lixia, MA Xiaoxuan, WANG Rongqi, NAN Yuemin. The Value of Serum VEGF in Evaluating the Clinical Efficacy of TACE Alone or Combined with Target Immunotherapy in Advanced Hepatocellular Carcinoma [J]. Chinese General Practice, 2024, 27(32): 4033-4039.
[12]	REN Lingxuan, LU Ziqi, QI Wei, FENG Zhijie. Functional Analysis of Macrophages in the Progression of Liver Cirrhosis and Liver Cancer [J]. Chinese General Practice, 2024, 27(29): 3654-3663.
[13]	XIAO Yuqian, BAI Yanjie, WANG Yan, SUN Kexin, WAN Jun, CHEN Shuying, CHEN Limin. Research Progress of Astrocyte-derived Extracellular Vesicles in Post-stroke Cognitive Impairment [J]. Chinese General Practice, 2024, 27(20): 2551-2556.
[14]	ZHANG Yun, CAI Xinyi, DING Jingnuo, LU Shengwei, CHEN Cuiying, WU Tingting, ZHANG Junli, ZHAO Weifeng. Study on the Value of Oligosaccharide Chain and Alpha-fetoprotein for Risk Screening and Diagnosis of Hepatitis B Virus-related Hepatocellular Carcinoma [J]. Chinese General Practice, 2024, 27(15): 1855-1860.
[15]	WANG Zuoyu, ZHOU Yang, XIONG Mingxia, ZHAO Shasha, YANG Junwei. Advances in Metabolic Reprogramming of Osteoblasts with the Development of Early Renal Bone Disease [J]. Chinese General Practice, 2024, 27(15): 1904-1910.