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Article(id=1320442290158556057, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320442289126752969, articleNumber=null, orderNo=null, doi=10.16016/j.2097-0927.202512105, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1766073600000, receivedDateStr=2025-12-19, revisedDate=1773244800000, revisedDateStr=2026-03-12, acceptedDate=null, acceptedDateStr=null, onlineDate=1792747574605, onlineDateStr=2026-10-23, pubDate=1777478400000, pubDateStr=2026-04-30, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792747574605, onlineIssueDateStr=2026-10-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792747574605, creator=13701087609, updateTime=1792747574605, updator=13701087609, issue=Issue{id=1320442289126752969, tenantId=1146029695717560320, journalId=1319373162502258839, year='2026', volume='48', issue='8', pageStart='971', pageEnd='1128', issueExtLink='null', onlineDate='null', pubDate='1777478400000', pubDateStr='2026-04-30', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792747574359, creator='13701087609', updateTime=1792749820502, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1320451710187844159, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320442289126752969, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1320451710187844160, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320442289126752969, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1026, endPage=1041, ext={EN=ArticleExt(id=1320442292700304283, articleId=1320442290158556057, tenantId=1146029695717560320, journalId=1319373162502258839, language=EN, title=Notch-RBPJ axis promotes formation of Helicobacter pylori vaccine-induced gastric CD4+ tissue-resident memory T cells via transcriptional activation of CD38, columnId=1320442291748192970, journalTitle=Journal of Army Medical University, columnName=Monographic Reports, runingTitle=null, highlight=null, articleAbstract=
Objective

Tissue-resident memory T (TRM) cells play a key role as “immune sentinels” in mucosal immune protection. However, the molecular regulatory mechanisms underlying the formation and long-term residence of gastric CD4⁺ TRM cells induced by Helicobacter pylori (H. pylori) vaccination remain unclear. This study aims to explore the key molecules and signaling pathways affecting TRM formation, residence, and survival, with a particular focus on whether the Notch-RBPJ axis transcriptionally activates CD38, thereby revealing the specific molecular mechanisms underlying the formation and long-term maintenance of gastric CD4⁺ TRM cells induced by H. pylori vaccination.

Methods

Single-cell RNA sequencing (scRNA-seq) was performed on gastric tissue CD4+ T cells following vaccination. Mice were vaccinated via subserosal injection in the gastric wall, with gastric tissue and splenic lymphocytes harvested on days 21 and 28 for multicolor flow cytometry analysis. Immunofluorescence co-staining was conducted on gastric tissue sections from mice on day 28 post-vaccination. H.pylori vaccine-immunized mice were continuously administered the RBPJ-specific inhibitor RIN1, and the changes in the proportions of CD4+ T cell subsets in gastric tissue were analyzed by flow cytometry. Primary murine CD4+ T cells were isolated via magnetic bead sorting and treatedwith the Notch pathway inhibitor RG-4733 or the RBPJ inhibitor RIN1 in an in vitro culture system. The treated primary CD4+ T cells were harvested for multicolor flow cytometry, and total RNA was extracted for qRT-PCR to assess the mRNA expression levels of target genes. The PGL3-CD38 promoter luciferase reporter plasmid and an RBPJ overexpression plasmid were constructed, with overexpression efficiency validated by qRT-PCR. The CD38 promoter reporter plasmids, RBPJ overexpression plasmids, and internal control plasmids were co-transfected into 293T cells, and luciferase activity was measured in 48 h later.

Results

The Notch-RBPJ-CD38 axis was selected as the key factor influencing the establishment and residence of gastric epithelial CD4+ TRM induced by H. pylori vaccine CCF/SF. On day 21 post-subserosal immunization with the H.pylori vaccine, gastric epithelial CD69+CD103+CD4+ TRM cells exhibited significant activation of gene sets related to the Notch and Hedgehog signaling pathways and others compared to other gastric CD4+ T cell subsets, displaying the most pronounced differences. SCENIC analysis revealed a significant correlation between the core transcription factor RBPJ and CD38（P<0.05）. Beyond typical residency-associated molecules such as CD69 and CD103, elevated mRNA levels of Notch 1, RBPJ, Gzma, and Gzmb represented characteristic features distinguishing this subset from other CD4+ T cell populations. GO and pathway analyses suggested that CD4+ TRM cells might possess dynamic proliferative potential. Subserosal surgical vaccination with the H. pylori vaccine CCF/SF upregulated the local expression of Notch signaling pathway-related protein Notch 1 and promoted protein expression of the key transcription factor RBPJ（P<0.05）. Blockade of the Notch pathway significantly reduced the proportion of resident TRM cells in the stomach. In vitro experiments confirmed that Notch-RBPJ pathway inhibition decreased CD38 protein expression, thereby reducing TRM residency. RBPJ directly bound to the CD38 promoter to regulate CD38 expression（P<0.05）.

Conclusion

Following H. pylori vaccination, TRM cells exhibit activation of the Notch signaling pathway, characterized by high expression of Notch 1 and RBPJ. This study confirms that the Notch-RBPJ signaling pathway transcriptionally regulates CD38 expression, representing a critical mechanism for the residency of gastric epithelial CD4+ TRM cells.

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目的

组织驻留记忆T细胞（tissue resident memory T cells，TRM）在黏膜免疫保护中发挥关键“免疫哨兵”作用，但其在幽门螺杆菌（Helicobacter pylori，H.pylori）疫苗诱导下的胃CD4+ TRM形成与长期驻留的分子调控机制尚不明确。本研究探索影响TRM形成、驻留及生存的关键分子及信号通路，特别是Notch-RBPJ轴是否通过转录激活CD38，揭示H.pylori疫苗诱导的胃CD4+ TRM形成以及长期维持的具体分子机制。

方法

对疫苗接种后的胃组织CD4+ T细胞的单细胞转录组测序（single-cell RNA sequencing，SCRNA-seq）进行分析。在小鼠胃浆膜下层接种疫苗后，于第21天与第28天采集胃组织和脾淋巴细胞，进行多色流式细胞术检测；对疫苗接种后28 d的小鼠胃组织切片进行免疫荧光共染色；对H.pylori疫苗接种小鼠持续给予RBPJ特异性抑制剂RIN1，通过流式细胞术分析胃组织中CD4+ T细胞亚群的比例变化。通过磁珠分选获得小鼠原代CD4+ T细胞，在体外培养体系中分别加入Notch通路抑制剂RG-4733和RBPJ抑制剂RIN1进行处理。取经抑制剂处理后的原代CD4+ T细胞，进行多色流式细胞术检测；提取经抑制剂处理后的原代CD4+ T细胞总RNA，通过qRT-PCR检测基因mRNA表达水平。构建PGL3-CD38启动子荧光素酶报告质粒和RBPJ过表达质粒，通过qRT-PCR验证过表达质粒的有效性。将CD38启动子报告质粒、RBPJ过表达质粒及内参质粒共转染至293T细胞，培养48 h后检测荧光素酶活性。

结果

选择Notch-RBPJ-CD38轴作为探究影响H.pylori疫苗CCF/SF诱导的胃上皮CD4+ TRM建立与驻留的关键因素。胃浆膜层接种H.pylori疫苗21 d后，与其他类型的胃组织CD4+ T细胞亚群相比，胃上皮CD69+CD103+CD4+ TRM在Notch、Hedgehog信号通路等基因集显著激活，且与其他CD4+ T细胞亚群差异最明显，SCENIC分析显示核心转录因子RBPJ与CD38存在显著相关性（P<0.05）。除CD69、CD103等典型驻留相关分子外，Notch 1、RBPJ、Gzma、Gzmb的mRNA水平是其显著区别于其他CD4+ T细胞亚群的特征分子。GO分析及Pathway分析CD4+ TRM细胞可能具有动态增殖潜力。胃浆膜下层手术接种H.pylori疫苗CCF/SF可增加局部Notch信号通路相关蛋白Notch 1的高表达，促进Notch信号通路关键转录因子RBPJ的蛋白表达（P<0.05）。阻断Notch通路可显著降低胃部TRM细胞驻留比例，体外实验验证了Notch-RBPJ通路抑制后会降低CD38的蛋白表达，从而减少TRM的驻留，RBPJ通过直接与CD38启动子结合从而调控CD38的表达（P<0.05）。

结论

H.pylori疫苗免疫后，TRM呈现Notch、RBPJ高表达的Notch信号通路激活；证实Notch-RBPJ信号通路通过转录调控CD38的表达，是胃上皮CD4+ TRM细胞驻留的关键机制。

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邢莹莹，E-mail：
XING Yingying， E-mail：
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Keyword(id=1320442298727519182, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, orderNo=2, keyword=Notch信号通路), Keyword(id=1320442298803016655, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, orderNo=3, keyword=幽门螺杆菌疫苗)], refs=[Reference(id=1320442302292677604, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, doi=10.3389/fimmu.2020.618897, pmid=null, pmcid=null, year=2020, volume=11, issue=null, pageStart=618897, pageEnd=null, url=null, language=null, rfNumber=1, rfOrder=0, authorNames=TOKURA Y, PHADUNGSAKSAWASDI P, KURIHARA K, journalName=Front Immunol, refType=null, unstructuredReference=TOKURA Y， PHADUNGSAKSAWASDI P， KURIHARA K， et al. 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J Exp Med， 2025， 222（5）： e20231758., articleTitle=Early Notch signals from fibroblastic reticular cells program effector CD8+ T cell differentiation, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1320442297104323506, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, xref=1, ext=[AuthorCompanyExt(id=1320442297112712115, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, companyId=1320442297104323506, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1School of Life Science and Technology， China Pharmaceutical University， Nanjing， Jiangsu， China), AuthorCompanyExt(id=1320442297121100724, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, companyId=1320442297104323506, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1中国药科大学生命科学与技术学院，江苏 南京)])], figs=[ArticleFig(id=1320442298912068560, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 1, caption=Single-cell transcriptome sequencing-based quantitative correlation analysis of Notch gene set expression and CD4+ TRM activation status in gastric epithelium, figureFileSmall=p+bOH406B1c6sduc1ZWteA==, figureFileBig=RaJYl+nDfFcNaRZQTzD0gg==, tableContent=null), ArticleFig(id=1320442298983371729, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图1, caption=基于单细胞转录组测序的胃上皮Notch基因集表达与CD4+ TRM活化状态定量相关性分析

A： tSNE plot of CD4+ T cells from gastric tissue on day 21 after receiving H. pylori vaccine CCF/SF； B： Quantitative analysis of the gene set expression and activation of CD4+ TRM cells in gastric epithelium； C： tSNE plot of CD4+ T cells from gastric tissue on day 21 after receiving CCF/SF vaccine； D： The detailed distribution of Notch 1 genes； E： The activity level of each gene in the gene set of Notch signaling pathway

, figureFileSmall=p+bOH406B1c6sduc1ZWteA==, figureFileBig=RaJYl+nDfFcNaRZQTzD0gg==, tableContent=null), ArticleFig(id=1320442300703036370, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 2, caption=SCENIC analysis of transcriptional regulatory networks in gastric CD4+ TRM cells, figureFileSmall=J+SV9GzE6DBi4sCCLMelUQ==, figureFileBig=DzvhSZUOBkYqYlNOqYJztg==, tableContent=null), ArticleFig(id=1320442300799505363, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图2, caption=基于SCENIC分析的胃CD4+ TRM细胞转录因子调控网络

A： Potential binding strength of the transcription factor to the target gene， based on SCENIC analysis （top 24 high-binding targets shown）； B： Expression distribution map of CD38-related genes of transcription factors； C： The distribution of transcription factors of in whole single cells

, figureFileSmall=J+SV9GzE6DBi4sCCLMelUQ==, figureFileBig=DzvhSZUOBkYqYlNOqYJztg==, tableContent=null), ArticleFig(id=1320442300879197140, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 3, caption=SCENIC analysis-based Gene Ontology （GO） and pathway analysis of Cluster 4 TRM cells, figureFileSmall=Mi/SQejZmPa85xB6LGQPeQ==, figureFileBig=dZR6MD6LmffIf5nsn56Hew==, tableContent=null), ArticleFig(id=1320442300958888917, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图3, caption=基于SCENIC分析的Cluster 4 TRM细胞基因本体（GO）与通路富集分析

A： Enrichment analysis of top 15 GO terms； B： Enrichment analysis of top 15 pathway terms； C： Enrichment analysis of top 20 GO terms； D： Enrichment analysis of top 20 pathway terms

, figureFileSmall=Mi/SQejZmPa85xB6LGQPeQ==, figureFileBig=dZR6MD6LmffIf5nsn56Hew==, tableContent=null), ArticleFig(id=1320442301025997782, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 4, caption=Heatmap of the top 20 upregulated marker genes identified through differential screening, figureFileSmall=crLQQx5Y1czXWt5q3/++HA==, figureFileBig=nV1VNu8BI1U8lzhMJNJVyw==, tableContent=null), ArticleFig(id=1320442301097300951, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图4, caption=基于差异筛选的上调标记基因前20位绘制的热图, figureFileSmall=crLQQx5Y1czXWt5q3/++HA==, figureFileBig=nV1VNu8BI1U8lzhMJNJVyw==, tableContent=null), ArticleFig(id=1320442301156021208, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 5, caption=Transcriptional regulation of vaccine-induced CD4+ TRM cells in gastric epithelium, figureFileSmall=8DS8+gho5OXvM2MGDXCN3Q==, figureFileBig=zqFViDme/jrUAWzo2wJgqw==, tableContent=null), ArticleFig(id=1320442301244101593, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图5, caption=胃上皮组织中疫苗诱导CD4+ TRM的转录调控机制

A： Expression distribution of high-expression transcription factors of TRM and subsets； B： Expression distribution of low-expression transcription factors of TRM in all clusters； C： Granzymes and cytokines of CD4+ T cells from gastric tissue after receiving CCF/SF vaccine

, figureFileSmall=8DS8+gho5OXvM2MGDXCN3Q==, figureFileBig=zqFViDme/jrUAWzo2wJgqw==, tableContent=null), ArticleFig(id=1320442301302821850, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 6, caption=Detection of Notch-related signaling pathways in TRM cells, figureFileSmall=cDpen7aUdkM22HBCXJ+30Q==, figureFileBig=kJUf5v1KLehVejbgPwOzDQ==, tableContent=null), ArticleFig(id=1320442301374125019, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图6, caption=TRM细胞中Notch相关信号通路的检测

A： Representative flow cytometry histograms depicting Notch 1 expression in gastric and splenic CD4+ T cell populations on day 21 post-vaccination； B： Quantitative analysis of Notch 1+ cell frequency among CD103+CD4+ versus CD103-CD4+ T cells in gastric tissues on day 21； C： Histogram profiles showing Notch 1 expression patterns in indicated CD4+ T cell subsets on day 28 post-vaccination； D： Comparative quantification of Notch 1+ cell proportions between CD103+CD4+ and CD103-CD4+ T cell populations on day 28

, figureFileSmall=cDpen7aUdkM22HBCXJ+30Q==, figureFileBig=kJUf5v1KLehVejbgPwOzDQ==, tableContent=null), ArticleFig(id=1320442301453816796, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 7, caption=Detection of Notch-related signaling pathways in TRM cells, figureFileSmall=rP+/HjIJFH/DwC8Mx3tKeA==, figureFileBig=njmEWoL+1d93RRchbAqNKA==, tableContent=null), ArticleFig(id=1320442301520925661, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图7, caption=TRM细胞中Notch相关信号通路的检测

A： Colocalization of CD4， CD69， CD103 and RBPJ in gastric tissues of mice on day 28 post-immunization； B： Schematic diagram of continuous administration of RIN1； C： Representative flow plots demonstrating CD69 and CD103 co-expression on gastric CD4+ T cells； D： Quantitative comparison of CD4⁺ TRM cell frequencies in gastric tissues between RIN1-treated and untreated mice following CCF/SF vaccination

, figureFileSmall=rP+/HjIJFH/DwC8Mx3tKeA==, figureFileBig=njmEWoL+1d93RRchbAqNKA==, tableContent=null), ArticleFig(id=1320442301583840222, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 8, caption=Verification of regulation of CD4+ T cells by Notch signal pathway in vitro, figureFileSmall=KwDPBrJk7SoPLUHq+3ke0Q==, figureFileBig=qo0Wzrb9tMg+XaN+QlPujg==, tableContent=null), ArticleFig(id=1320442301709669343, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图8, caption=Notch信号通路体外调控CD4+ T细胞的验证

A： Schematic diagram illustrating isolation and activation of CD4+ T cells； B： Flow cytometry histogram of CD38 expression profiles in activated CD4+ T cells following RG-4733 stimulation； C： The protein expression level of RBPJ on activated CD4+ T cells after RG-4733 stimulation； D and E： The mRNA expression levels of RBPJ， CD38 and HES5； F： Flow cytometry histogram analysis of CD38 expression； G： The mRNA expression levels of RBPJ， CD38 and HES5 on activated CD4+ T cells after RIN1 stimulation； H： One putative binding site of RBPJ with the highest scores on CD38 promoter was shown via JASPAR database

, figureFileSmall=KwDPBrJk7SoPLUHq+3ke0Q==, figureFileBig=qo0Wzrb9tMg+XaN+QlPujg==, tableContent=null), ArticleFig(id=1320442301789361120, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Figure 9, caption=Regulatory mechanism of RBPJ on CD38 by luciferase reporter assay, figureFileSmall=pM38AvdeVT1v/74AQeoQmA==, figureFileBig=MkQHkprpURabN0rkYox4Eg==, tableContent=null), ArticleFig(id=1320442301873247201, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=图9, caption=采用荧光素酶报告基因实验探究RBPJ对CD38的调控机制

A： Sketch map of CD38 promoter vector； B： Sketch map of RBPJ overexpression vector； C： The mRNA expression levels of RBPJ in HEK293T cells； D： Relative luciferase activity of CD38 luciferase reporter plasmids in HEK293T cells with RBPJ expression

, figureFileSmall=pM38AvdeVT1v/74AQeoQmA==, figureFileBig=MkQHkprpURabN0rkYox4Eg==, tableContent=null), ArticleFig(id=1320442302103933922, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=EN, label=Table 1, caption=

Primer sequences for qRT-PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
GenePrimer sequence （5’ → 3’）
GAPDHUpstream：GAGCTGAACGGGAAGCTCAC
Downstream：AGTGTAGCCCAAGATGCCCT
RBPJUpstream：GGAGAGGAGTTCACAGTTAG
Downstream：AATGCCGTCTGCTTATCAA
HES5Upstream：CGCATCAACAGCAGCATAGAG
Downstream：TGGAAGTGGTAAAGCAGCTTC
CD38Upstream：AGTGGGGCGTAGTCTTCTCT
Downstream：ACTGGAGAGCCTACCACGAA
), ArticleFig(id=1320442302192014307, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320442290158556057, language=CN, label=表1, caption=

qRT-PCR引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
GenePrimer sequence （5’ → 3’）
GAPDHUpstream：GAGCTGAACGGGAAGCTCAC
Downstream：AGTGTAGCCCAAGATGCCCT
RBPJUpstream：GGAGAGGAGTTCACAGTTAG
Downstream：AATGCCGTCTGCTTATCAA
HES5Upstream：CGCATCAACAGCAGCATAGAG
Downstream：TGGAAGTGGTAAAGCAGCTTC
CD38Upstream：AGTGGGGCGTAGTCTTCTCT
Downstream：ACTGGAGAGCCTACCACGAA
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Notch-RBPJ轴通过转录激活CD38促进幽门螺杆菌疫苗诱导的胃CD4+组织驻留记忆T细胞形成
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卜令怡 , 佟金哲 , 祝俊彦 , 邢莹莹
陆军军医大学学报 | 论著·专题报道 2026,48(8): 1026-1041
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陆军军医大学学报 |论著·专题报道 2026 , 48 (8) : 1026 -1041
Notch-RBPJ轴通过转录激活CD38促进幽门螺杆菌疫苗诱导的胃CD4+组织驻留记忆T细胞形成
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卜令怡, 佟金哲, 祝俊彦, 邢莹莹
作者信息
  • 1中国药科大学生命科学与技术学院，江苏 南京
通讯作者:
邢莹莹，E-mail：
XING Yingying， E-mail：
Notch-RBPJ axis promotes formation of Helicobacter pylori vaccine-induced gastric CD4+ tissue-resident memory T cells via transcriptional activation of CD38
Lingyi BU, Jinzhe TONG, Junyan ZHU, Yingying XING
Affiliations
  • 1School of Life Science and Technology， China Pharmaceutical University， Nanjing， Jiangsu， China
出版时间: 2026-04-30 doi: 10.16016/j.2097-0927.202512105
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摘要
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目的

组织驻留记忆T细胞（tissue resident memory T cells，TRM）在黏膜免疫保护中发挥关键“免疫哨兵”作用，但其在幽门螺杆菌（Helicobacter pylori，H.pylori）疫苗诱导下的胃CD4+ TRM形成与长期驻留的分子调控机制尚不明确。本研究探索影响TRM形成、驻留及生存的关键分子及信号通路，特别是Notch-RBPJ轴是否通过转录激活CD38，揭示H.pylori疫苗诱导的胃CD4+ TRM形成以及长期维持的具体分子机制。

方法

对疫苗接种后的胃组织CD4+ T细胞的单细胞转录组测序（single-cell RNA sequencing，SCRNA-seq）进行分析。在小鼠胃浆膜下层接种疫苗后，于第21天与第28天采集胃组织和脾淋巴细胞，进行多色流式细胞术检测；对疫苗接种后28 d的小鼠胃组织切片进行免疫荧光共染色；对H.pylori疫苗接种小鼠持续给予RBPJ特异性抑制剂RIN1，通过流式细胞术分析胃组织中CD4+ T细胞亚群的比例变化。通过磁珠分选获得小鼠原代CD4+ T细胞，在体外培养体系中分别加入Notch通路抑制剂RG-4733和RBPJ抑制剂RIN1进行处理。取经抑制剂处理后的原代CD4+ T细胞，进行多色流式细胞术检测；提取经抑制剂处理后的原代CD4+ T细胞总RNA，通过qRT-PCR检测基因mRNA表达水平。构建PGL3-CD38启动子荧光素酶报告质粒和RBPJ过表达质粒，通过qRT-PCR验证过表达质粒的有效性。将CD38启动子报告质粒、RBPJ过表达质粒及内参质粒共转染至293T细胞，培养48 h后检测荧光素酶活性。

结果

选择Notch-RBPJ-CD38轴作为探究影响H.pylori疫苗CCF/SF诱导的胃上皮CD4+ TRM建立与驻留的关键因素。胃浆膜层接种H.pylori疫苗21 d后，与其他类型的胃组织CD4+ T细胞亚群相比，胃上皮CD69+CD103+CD4+ TRM在Notch、Hedgehog信号通路等基因集显著激活，且与其他CD4+ T细胞亚群差异最明显，SCENIC分析显示核心转录因子RBPJ与CD38存在显著相关性（P<0.05）。除CD69、CD103等典型驻留相关分子外，Notch 1、RBPJ、Gzma、Gzmb的mRNA水平是其显著区别于其他CD4+ T细胞亚群的特征分子。GO分析及Pathway分析CD4+ TRM细胞可能具有动态增殖潜力。胃浆膜下层手术接种H.pylori疫苗CCF/SF可增加局部Notch信号通路相关蛋白Notch 1的高表达，促进Notch信号通路关键转录因子RBPJ的蛋白表达（P<0.05）。阻断Notch通路可显著降低胃部TRM细胞驻留比例，体外实验验证了Notch-RBPJ通路抑制后会降低CD38的蛋白表达，从而减少TRM的驻留，RBPJ通过直接与CD38启动子结合从而调控CD38的表达（P<0.05）。

结论

H.pylori疫苗免疫后，TRM呈现Notch、RBPJ高表达的Notch信号通路激活；证实Notch-RBPJ信号通路通过转录调控CD38的表达，是胃上皮CD4+ TRM细胞驻留的关键机制。

CD4+组织驻留记忆T细胞  /  Notch信号通路  /  幽门螺杆菌疫苗
Objective

Tissue-resident memory T (TRM) cells play a key role as “immune sentinels” in mucosal immune protection. However, the molecular regulatory mechanisms underlying the formation and long-term residence of gastric CD4⁺ TRM cells induced by Helicobacter pylori (H. pylori) vaccination remain unclear. This study aims to explore the key molecules and signaling pathways affecting TRM formation, residence, and survival, with a particular focus on whether the Notch-RBPJ axis transcriptionally activates CD38, thereby revealing the specific molecular mechanisms underlying the formation and long-term maintenance of gastric CD4⁺ TRM cells induced by H. pylori vaccination.

Methods

Single-cell RNA sequencing (scRNA-seq) was performed on gastric tissue CD4+ T cells following vaccination. Mice were vaccinated via subserosal injection in the gastric wall, with gastric tissue and splenic lymphocytes harvested on days 21 and 28 for multicolor flow cytometry analysis. Immunofluorescence co-staining was conducted on gastric tissue sections from mice on day 28 post-vaccination. H.pylori vaccine-immunized mice were continuously administered the RBPJ-specific inhibitor RIN1, and the changes in the proportions of CD4+ T cell subsets in gastric tissue were analyzed by flow cytometry. Primary murine CD4+ T cells were isolated via magnetic bead sorting and treatedwith the Notch pathway inhibitor RG-4733 or the RBPJ inhibitor RIN1 in an in vitro culture system. The treated primary CD4+ T cells were harvested for multicolor flow cytometry, and total RNA was extracted for qRT-PCR to assess the mRNA expression levels of target genes. The PGL3-CD38 promoter luciferase reporter plasmid and an RBPJ overexpression plasmid were constructed, with overexpression efficiency validated by qRT-PCR. The CD38 promoter reporter plasmids, RBPJ overexpression plasmids, and internal control plasmids were co-transfected into 293T cells, and luciferase activity was measured in 48 h later.

Results

The Notch-RBPJ-CD38 axis was selected as the key factor influencing the establishment and residence of gastric epithelial CD4+ TRM induced by H. pylori vaccine CCF/SF. On day 21 post-subserosal immunization with the H.pylori vaccine, gastric epithelial CD69+CD103+CD4+ TRM cells exhibited significant activation of gene sets related to the Notch and Hedgehog signaling pathways and others compared to other gastric CD4+ T cell subsets, displaying the most pronounced differences. SCENIC analysis revealed a significant correlation between the core transcription factor RBPJ and CD38（P<0.05）. Beyond typical residency-associated molecules such as CD69 and CD103, elevated mRNA levels of Notch 1, RBPJ, Gzma, and Gzmb represented characteristic features distinguishing this subset from other CD4+ T cell populations. GO and pathway analyses suggested that CD4+ TRM cells might possess dynamic proliferative potential. Subserosal surgical vaccination with the H. pylori vaccine CCF/SF upregulated the local expression of Notch signaling pathway-related protein Notch 1 and promoted protein expression of the key transcription factor RBPJ（P<0.05）. Blockade of the Notch pathway significantly reduced the proportion of resident TRM cells in the stomach. In vitro experiments confirmed that Notch-RBPJ pathway inhibition decreased CD38 protein expression, thereby reducing TRM residency. RBPJ directly bound to the CD38 promoter to regulate CD38 expression（P<0.05）.

Conclusion

Following H. pylori vaccination, TRM cells exhibit activation of the Notch signaling pathway, characterized by high expression of Notch 1 and RBPJ. This study confirms that the Notch-RBPJ signaling pathway transcriptionally regulates CD38 expression, representing a critical mechanism for the residency of gastric epithelial CD4+ TRM cells.

CD4+ tissue-resident memory T cells  /  Notch signaling pathway  /  Helicobacter pylori vaccine
卜令怡, 佟金哲, 祝俊彦, 邢莹莹. Notch-RBPJ轴通过转录激活CD38促进幽门螺杆菌疫苗诱导的胃CD4+组织驻留记忆T细胞形成. 陆军军医大学学报, 2026 , 48 (8) : 1026 -1041 . DOI: 10.16016/j.2097-0927.202512105
Lingyi BU, Jinzhe TONG, Junyan ZHU, Yingying XING. Notch-RBPJ axis promotes formation of Helicobacter pylori vaccine-induced gastric CD4+ tissue-resident memory T cells via transcriptional activation of CD38[J]. Journal of Army Medical University, 2026 , 48 (8) : 1026 -1041 . DOI: 10.16016/j.2097-0927.202512105
正文
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组织驻留记忆T细胞（tissue resident memory T cells，TRM）作为局部组织中的关键免疫细胞，通过原位驻留、快速免疫应答、强化局部免疫屏障及持久保护效应等特点，在抗感染免疫与疫苗设计中发挥着不可或缺的作用。旨在诱导TRM细胞的疫苗接种策略，是获得长效优质免疫保护的潜在方案。当前，该领域研究的核心挑战在于如何确保TRM细胞的有效形成与稳定驻留。与循环记忆T细胞不同，TRM细胞在感染或疫苗接种后可长期存留于局部组织，并在再次暴露于病原体时无需经过血液或淋巴系统迁移即可快速启动免疫应答，因而被誉为“免疫哨兵”［1］。尽管TRM存在异质性，在不同组织中可能呈现不同的功能或表达情况，甚至在肿瘤免疫与自身免疫中具有双重作用，如抑制肿瘤进展或疾病发生，或因过度活化及微环境失调导致免疫逃逸［1-4］，但其功能与机制研究仍是局部免疫应答领域的重要方向。在相关研究中，TRM往往与疫苗设计联系起来，通过增强其在特定组织（如肺部、肠道）的驻留能力优化疫苗接种方案，以实现长期保护［5-7］。因此，探究TRM的生成与功能调控机制对疫苗开发、抗感染免疫及肿瘤免疫治疗等具有重要意义。
幽门螺杆菌（Helicobacter pylori，H.pylori）作为专性胃黏膜病原体，通过外膜蛋白作为黏附素识别宿主受体，促进其与胃上皮细胞结合，并借助天然免疫与适应性免疫逃逸机制逃避宿主免疫应答［8-9］。CD4+ T细胞介导的Th1/Th17免疫应答在清除H.pylori感染中发挥核心作用：一方面通过分泌IFN-γ等细胞因子激活巨噬细胞抗菌功能，另一方面诱导B细胞产生特异性抗体实现黏膜免疫保护［10-12］。此外，研究还证实，CD8+ TRM细胞在H.pylori感染早期会快速浸润胃黏膜。它们不仅是感染初期的重要免疫调节者，更通过针对CagA等毒力因子执行抗原特异性效应功能，成为介导病原体控制的核心环节［13］。
霍乱毒素B亚单位-脲酶融合蛋白（Cholera toxin B subunit and tandem copies of Th and B cell epitopes from Helicobacter pylori urease A and B subunits，CTB-UE）在胃肠道病原体疫苗研究中，是一种经典的黏膜疫苗设计策略，CTB-UE菌株是用于生产CTB-UE疫苗抗原蛋白的基因工程细菌。一种新型嵌合鞭毛（chimeric flagellum，CF）蛋白主要通过将H.pylori的FlaA的高突变区替换鼠伤寒沙门菌的FliC的高突变区而成。然后将CF链接到CTB-UE的C端，从而构建出一种双重抗原表位-双重佐剂的疫苗CCF，该疫苗具有H.pylori尿素酶和鞭毛蛋白两种抗原表位、霍乱毒素B亚基和鼠伤寒沙门菌鞭毛蛋白FliC两种黏膜佐剂。本研究采用了胃浆膜层接种途径来递送疫苗。虽然这是一种非常规的免疫途径，但其设计旨在最大化疫苗在胃局部淋巴组织（如胃周淋巴结）中的抗原暴露。与全身性接种相比，这种局部-区域免疫策略被广泛认为能更有效地模拟黏膜感染，并优先诱导表达组织特异性归巢受体的T细胞，从而促进其在胃黏膜中分化为TRM［14］。因此，该方法为本研究疫苗诱导的胃上皮特异性TRM应答提供了一个生理相关的模型。前期工作证实，鞭毛素（CTB-UE-CF，CCF）疫苗联合丝素蛋白（silk fibroin，SF）在小鼠模型中成功诱导了稳定的胃上皮CD4+ TRM细胞，并进一步明确CD69+CD103+CD4+ TRM表面分子CD38、P2RX7及TGF-βRIII介导了H.pylori疫苗诱导的胃上皮CD4+ TRM的形成与驻留，但其具体作用通路与机制尚未明晰［15］。因此，本研究旨在探究H.pylori疫苗CCF/SF免疫过程中，调控胃上皮组织CD4+ TRM成熟与驻留的关键分子机制，并明确其上游信号调控网络，以揭示优化黏膜疫苗策略的潜在靶点。
1　材料与方法
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1.1　实验动物
6~8周龄SPF级C57BL/6雌性小鼠（体质量18~22 g），购自杭州医学院动物实验中心与河南百奥生物工程有限公司，饲养于中国药科大学动物实验中心。所有动物实验操作经中国药科大学动物实验伦理委员会批准（YSL-202505033）。饲养环境条件：温度（22±2）℃，相对湿度（55±5）%，昼夜节律为12 h光照/12 h黑暗循环，笼具每周更换2次以保持饲养环境清洁。实验过程中，所有手术操作在无菌条件下进行，以最大限度减少动物疼痛与不适。实验期间，每日对动物进行监测；术后或干预后，监测频率增加至每日2~3次，以便及时发现并处理异常情况，当动物出现预定的严重痛苦或濒死状态时将及时实施安乐死。实验期间未见与实验相关的预期外不良事件发生。
1.2　 H.pylori疫苗CCF/SF制备
纯化的CTB-UE-CF（CCF）蛋白通过携带pET-28a-CCF质粒的大肠杆菌Rosetta（DE3）表达，采用镍亲和层析法（GE Healthcare，Amersham）纯化蛋白，并通过考马斯亮蓝染色确认CCF纯度。将1.5 mg/mL CCF蛋白与40 mg/mL冻干丝素蛋白混合，随后加入等体积的90%PEG 400，即得H.pylori疫苗CCF/SF，现配现用。
1.3　小鼠胃浆膜下疫苗注射手术
按50 mg/kg剂量注射戊巴比妥钠麻醉小鼠腹腔，使C57BL/6小鼠充分麻醉，仰卧位固定，常规备皮消毒。沿左上腹做长约1 cm切口，逐层分离组织，轻轻将胃托出至无菌纱布上。于胃大弯血管稀少区，用微量注射器将10 μL CCF/SF疫苗缓慢注入浆膜下，以形成边界清晰的透亮小泡且无液体渗漏为成功标准。注射后停留数秒拔针并轻压针眼，将胃归位，用无菌生理盐水冲洗腹腔后依次缝合肌层与皮肤，再次消毒。术后将小鼠置于加热垫上直至苏醒，连续3 d监测其体质量、活动及进食情况。
1.4　胃组织淋巴细胞分离
在50 mL规格离心管中加入含有10%胎牛血清的15 mL胃组织T细胞提取液，胃组织T细胞提取液以RPMI 1640基础培养基为溶剂，每500 mL添加乙二胺四乙酸0.58 g（终浓度为4 mmol/L）、4-羟乙基哌嗪乙磺酸1.191 5 g（终浓度为10 mmol/L）、二硫苏糖醇0.04 g（终浓度为0.5 mmol/L），配制完成后，使用盐酸或氢氧化钠调节（pH=7.2）。每1管盛有1~2只疫苗注射21 d后C57BL/6小鼠的胃组织，37 ℃、0.8 ×g振荡消化35 min，涡旋震荡1 min后经无菌70 μm细胞筛过筛。剩余组织剪碎后同法处理，缩短消化时间至20 min，合并2次滤液，605 ×g、4 ℃离心5 min收集细胞。使用现配现用的无菌44%Percoll重悬细胞，约1 200 μL重悬液经300目筛网过滤至流式管，并于底部小心使用1 mL注射器注射67%Percoll，966 ×g（升速3/降速3）、4 ℃离心25 min。吸取中间层，收集细胞至1.5 mL EP管中。再次离心，605 ×g、4 ℃离心5 min收集细胞。
1.5　脾脏淋巴细胞悬液制备
无菌摘取小鼠脾脏，置于预冷PBS的培养皿中，附上300目细胞筛网；用注射器平头单向轻柔碾压覆盖300目筛网的脾脏组织，仅从脾脏一端向筛网方向推动，保证脾脏包膜严重破损，可看到明显的红细胞与淋巴细胞液。61 ×g、4 ℃离心5 min，收集细胞，弃上清，加入预冷的5 mL红细胞裂解液重悬轻柔吹匀，冰上静置10 min并检查裂解状态；61 ×g、4 ℃离心5 min收集细胞，弃上清，加入预冷的PBS再次重悬洗涤离心1次备用。
1.6　单细胞测序以及生信分析
免疫后第21天，对C57BL/6小鼠实施安乐死，并制备其胃组织淋巴细胞的单细胞悬液，单细胞测序由上海烈冰生物医药科技有限公司提供。采用CellRanger软件对原始单细胞测序数据进行质量统计及基因组比对，通过识别Barcode与UMI标签实现转录组准确定量，获取细胞数、基因中位值及测序饱和度等质控指标；随后使用Seurat软件包进行深度过滤与标准化处理。基于Seurat筛选前3 000个高变基因进行主成分分析，并利用t分布随机邻域嵌入（tSNE）算法将结果投影至二维空间进行可视化，通过无监督聚类划分细胞亚群，结合差异筛选算法计算各亚群Marker基因以推测其生物学身份。差异表达基因的筛选阈值为P<0.05且差异倍数>2，并基于超几何分布检验进行GO与KEGG通路富集分析。利用方差膨胀因子算法对各亚群的KEGG及GSEA基因集进行富集度打分，系统比较不同细胞亚群间的功能通路激活程度差异。
1.7　免疫小鼠胃组织免疫荧光
C57BL/6小鼠于CCF/SF疫苗胃黏膜下接种后第28天，按50 mg/kg剂量注射戊巴比妥钠麻醉腹腔使其麻醉，从免疫小鼠取胃组织。样本胃组织经4%多聚甲醛固定后，免疫荧光检测CD4、CD69、CD103、RBPJ的表达水平，由赛维尔生物（武汉）完成，所得数据采用CaseViewer软件分析。
1.8　免疫小鼠淋巴细胞流式细胞术
对免疫小鼠胃组织及脾脏淋巴细胞悬液调整细胞浓度，细胞于冰上避光条件下与抗体孵育20 min，所有抗体购自美国BioLegend公司。完成表面染色后，为分析线粒体功能，细胞重悬于37 ℃预热的培养基，并使用MitoTellTM Orange荧光染料（Cell MeterTM橙色线粒体膜电位检测试剂盒，AAT Bioquest）在37 ℃、5% CO₂培养箱中避光孵育20 min，以评估线粒体膜电位。流式细胞术以检测第21天以及28天免疫小鼠胃组织和脾脏Notch 1的表达变化、第28天免疫小鼠胃组织中CD4+ T细胞亚群的比例变化、定量分析CD38蛋白的表达水平，所有样本数据经由FACS Celesta流式细胞仪（BD Biosciences）采集，并运用FlowJo软件（v10.0.7）进行后续分析。
1.9　HEK293T细胞系培养、转染、处理及双荧光素酶报告基因检测
HEK293T细胞购自武汉普诺赛，培养于含10%胎牛血清和1%青霉素-链霉素的DMEM培养基中。待细胞密度达70%时，按说明书步骤使用Jet prime（Poly Plus）进行转染。将RBPJ过表达质粒与PGL3-CD38启动子荧光素酶报告质粒（金斯瑞生物科技）共转染至HEK293T细胞。转染48 h后，依照制造商说明书使用Duo-Lite Luciferase Assay System（诺唯赞）检测相对荧光素酶活性。通过i3x多功能酶标仪在560 nm（萤火虫荧光素酶）和480 nm（海肾荧光素酶）波长下检测CD38启动子驱动的荧光素酶活性。
1.10　体内药物治疗
为探究Notch通路对体内TRM形成的影响，将小鼠随机分为以下2组，第0天对C57BL/6小鼠进行单剂量H.pylori疫苗CCF/SF免疫，第2、5、8、11、14、17、20、23天实验组腹腔注射溶于DMSO的RIN1（0.6 mg/kg，MedChemExpress），对照组腹腔注射等体积的DMSO，于第28天处死小鼠，取胃上皮组织，通过多色流式细胞术检测胃上皮组织CD4+ TRM细胞。
1.11　体外T细胞培养与药物处理
从6~8周龄SPF级C57BL/6小鼠总脾细胞中，按照说明书步骤使用MojoSortTM小鼠CD4+ T细胞分选试剂盒（BioLegend）纯化初始CD4+ T细胞。将CD4+ T细胞在包被抗小鼠CD3（5 μg/mL，克隆号145-2C11，BioLegend）和可溶性抗小鼠CD28（3 μg/mL，克隆号37.51，BioLegend）的24孔板中过夜刺激。将刺激过夜后的活化CD4+ T细胞细胞接种于包被抗CD3和可溶性抗CD28的24孔板中，实验组1加入25 μmol/L的RG-4733（MedChemExpress），实验组2加入50 μmol/L的RG-4733（MedChemExpress），实验组3加入2 μmol/L的RIN1（MedChemExpress），对照组加入等体积的DMSO检测活化CD4+ T细胞中CD38的表达水平，各组细胞于培养箱中继续培养24~48 h，用于后续实验检测。
1.12　细胞RNA提取及逆转录
从培养箱取出24孔板，收取CD4+ T细胞。4 ℃，61 ×g离心5 min，添加2 mL PBS洗涤细胞1次，加入1 mL TRIzol室温裂解细胞2~3 min，移液枪吸取至1.5 mL离心管中。每管加入200 µL三氯甲烷，剧烈振荡混匀2 min，室温静置3 min，转移至4 ℃离心机内，转速2 415 ×g离心15 min，小心吸取上层水相至无酶离心管中，加入500 μL的异丙醇，-20 ℃环境下静置20 min。4℃，2 415 ×g离心15 min，弃上清，保留白色RNA沉淀管，1 mL 75%的乙醇洗涤沉淀。4℃，2 415 ×g离心3 min，移走上清，开盖室温放置2 min。DEPC水溶解。使用预混液形式的qPCR专用反转录试剂盒进行mRNA反转录。
1.13　qRT-PCR检测RBPJ、HES5和CD38转录水平
根据目的基因和内参基因设计上下游引物，见表1。3 µL DEPC水、5 µL ChamQ Universal SYBR、0.5 µL上游引物、3 µL cDNA、0.5 µL下游引物为1个反应体系，反应程序为预循环95 ℃ 3 min；热循环（40次）95 ℃ 10 s、60 ℃ 30 s；熔解阶段95 ℃ 15 s、60 ℃ 60 s、95 ℃ 15 s，以分析RBPJ、Notch经典靶基因HES5以及CD38的转录水平变化。
1.14　质粒转化与提取
将DH5α感受态细胞冰上解冻，加入1 µL质粒DNA（100 ng/µL）至感受态细胞中，轻柔混匀，冰浴30 min后进行42 ℃热激45 s，立即冰浴2 min；加900 µL无抗LB培养基，37 ℃振荡培养1 h；取30~50 µL培养物涂布于含抗性LB平板，37 ℃培养至单菌落形成，根据质粒提取说明操作，取15 mL菌液，2 834 ×g，离心1 min收集菌体，弃上清；离心收集菌体，依次加入P1、P2、E3缓冲液处理，离心后上清过Endo-Remover FM柱，加入异丙醇沉淀DNA，使用Spin Columns DL柱纯化，先后用Buffer PS、PW洗涤，最后用Endo-Free Buffer EB洗脱，测定浓度后-20 ℃保存。
1.15　Western blotting实验检测RBPJ蛋白表达水平
按上海雅酶凝胶试剂盒说明书配制，1∶1加入下层胶溶液和下层胶缓冲液，并加入适量促凝剂混匀；异丙醇压胶15 min；加入1.5 mL上层胶溶液、1.5 mL上层胶缓冲液和30 µL改良型促凝剂的混合液并插入10孔梳齿；待上层胶凝固后加入（1×）电泳缓冲液拔去梳齿进行电泳操作。蛋白Marker和蛋白样品分别混匀上样；80 V恒压电泳30 min，随后调整至100 V恒压电泳1 h；按蛋白分子量大小切胶；按胶条大小裁剪相应的PVDF膜并标记贴合于胶条表面，转移至“三明治”夹中；200或400 mA转膜相应的时间。转膜结束后取出PVDF膜；将其置于事先配置好的封闭液中封闭2~3 h。封闭结束后用（1×）TBST溶液清洗3次，每次10 min；将PVDF膜于稀释配制好的一抗中于冰箱4 ℃孵育过夜；用（1×）TBST溶液清洗PVDF膜3次，每次10 min；加入配置好的二抗溶液室温孵育45 min；（1×）TBST溶液清洗3次，每次10 min。PVDF膜上加入曝光液放置于凝胶成像仪中曝光条带。
1.16　统计学分析
采用GraphPad Prism软件进行统计学分析。两组间比较采用独立样本t检验；多组间比较采用单因素方差分析，并以Tukey法进行事后多重比较。针对单细胞转录组测序数据，采用Wilcoxon秩和检验进行差异表达分析，筛选标准设定为：|Log₂（FC）|>0.25、Benjamini-Hochberg校正P<0.05、最小表达细胞比例>10%），需同时满足上述3项条件。双侧P<0.05为差异具有统计学意义。
2　结果
收起
2.1　Notch信号通路的激活是疫苗诱导胃上皮CD69+CD103+CD4+ TRM的典型特征
确定Cluster 4为CD4+ TRM细胞的目标群体（图1A）。与其他胃组织CD4+ T细胞亚群相比，以Cluster 4为代表的TRM细胞显示出显著更高的Notch信号通路、Hedgehog信号通路及神经活性配体-受体相互作用通路活性（图1B），表明CD4+ TRM细胞仍受增殖与分化等通路的影响。对Notch通路相关基因的进一步分析显示，在tSNE图中，Notch通路中标志性Marker Notch 1在Cluster 4中的表达高于其他细胞簇（图1C）。不同细胞簇Notch 1表达的气泡图可视化结果证实，目标群体Cluster 4呈现显著的Notch 1上调，且有较多与Notch 1高表达的相关基因（图1D），证明Notch信号激活是疫苗诱导胃上皮CD69+CD103+CD4+ TRM细胞的特征之一。此外，通过分析Notch通路基因集的激活水平，发现免疫球蛋白κJ区重组信号结合蛋白（recombination signal binding protein for immunoglobulin kappa j region，RBPJ）表达最为显著（图1E）。作为Notch通路的核心转录共激活因子，RBPJ不仅激活Notch信号，并作为转录因子介导下游靶基因。综上所述，以上结果证明Notch-RBPJ信号通路在TRM细胞中呈现特异性激活模式，提示该Notch-RBPJ信号轴可能调控该细胞亚群的发育、分化及长期组织驻留，从而在H.pylori疫苗接种后的黏膜免疫中发挥关键作用。
2.2　SCENIC分析揭示RBPJ与CD38存在显著相关性
通过单细胞调控网络推断与聚类（SCENIC）分析，鉴定细胞亚型特异性的转录调控网络，结果显示，CD38是Notch-RBPJ信号轴的潜在关键下游靶基因（图2A）。通过对比分析不同细胞簇中RBPJ的表达，发现RBPJ在TRM细胞（Cluster 4）中特异性高表达，表明其在TRM分化中具有独特调控功能（图2B、C）。相比之下，其他与CD38可能具有较强结合潜在的转录因子——包括Nr4a2（甾体-甲状腺激素-维甲酸受体超家族成员）、Trim30a（TRIM蛋白家族成员）及Ahr（芳烃受体，碱性螺旋-环-螺旋转录因子家族成员）在TRM亚群中均未呈现显著差异。这些结果进一步印证了Notch-RBPJ信号在TRM细胞中的不可替代性，提示CD38的表达可能受RBPJ的转录调控，以维持TRM的存活与组织驻留。
2.3　胃部CD4+ TRM细胞在黏膜下接种后可能具有动态增殖潜能
通过对SCENIC调控网络预测与功能富集结果的整合分析，本研究系统表征了胃组织Cluster 4细胞亚群的基因表达模式。通过基因本体（gene ontology，GO）三大类别——分子功能（molecular function，MF）、生物过程（biological process，BP）与细胞组分（cellular component，CC）的差异基因功能注释，并同步识别差异表达基因中显著富集的信号通路。GO分析（图3A、C）与通路分析（图3B、D）共同表明，Cluster 4中受转录因子调控的差异表达基因主要富集于细胞周期调控、细胞分裂及细胞因子-细胞因子受体相互作用等通路。这些发现提示，在转录水平上，黏膜下接种21 d后的胃CD4+ TRM细胞具有动态增殖潜能。细胞因子相关通路的显著激活进一步表明，TRM细胞可能依赖特定细胞因子（如IL-15、TGF-β）维持其驻留表型并实现长期存活。
为解析疫苗诱导胃上皮CD4+CD69+CD103+ TRM细胞的特征，本研究通过差异表达分析筛选细胞亚群特异性标志基因。热图展示了各亚群前20个最显著上调的标志基因，并通过细线划分不同细胞簇。颜色梯度直观呈现表达水平（黄色表示高表达，深红色表示低表达），清晰展现了Cluster 4相较于其他T细胞亚群的特异性转录特征（图4）。Cluster 4高表达经典TRM标志转录本，如Itgae（编码CD103），CD103与上皮E-钙黏蛋白相互作用从而实现组织保留；与之相反，鞘氨醇-1-磷酸受体1（S1PR1）呈现低表达，以减弱淋巴细胞的迁移能力、阻止TRM从组织中迁出。除已确认的TRM标志物（Itgae、S1PR1、RBPJ、CD38）外，可以观察到CCR5、CCL5与CCL4的高表达，这些趋化因子受体家族成员可能引导后续T细胞迁移，促进TRM前体细胞的招募与分化。TRM的异质性涉及多层次动态调控，不同器官特异性TRM亚群通过独特转录组特征执行效应功能。
为进一步探究TRM生存机制，本研究通过小提琴图展示Cluster 4与其他亚群的转录因子表达模式对比（图5A、B）。Runx1、Prdm1、Rb1、Stat1、Lef1与Tcf7都是报道过与TRM相关的转录因子，结果显示Runx1的转录水平在Cluster 4中表达较高；Prdm1、Rb1与Stat1则高于部分亚群但低于Cluster 7或8呈中等表达；Lef1与Tcf7在Cluster 4中相对其他亚群低表达。Tcf7和Lef1是Wnt/β-catenin信号通路的关键效应分子，通常与中央记忆T细胞的生成相关。该表达模式提示在CD4+ TRM分化过程中，中央记忆T细胞的发育可能被抑制，从而促进TRM的分化或维持，这对理解TRM的形成与驻留机制具有重要意义。单细胞测序数据的功能性基因热图分析显示，颗粒酶Gzma、Gzmb的转录水平显著升高（图5C），佐证了细胞因子-细胞因子受体相互作用的发现，进一步凸显了TRM细胞强大的效应潜能。
2.4　 H.pylori 疫苗诱导胃上皮CD103+CD4+ TRM细胞发育中Notch 1信号通路上调
基于scRNA-seq测序结果，可以发现在胃上皮CD69+CD103+CD4+ TRM细胞中Notch信号通路显著激活，其中RBPJ与Notch 1基因表达尤为突出。本研究采用多色流式细胞术动态监测H.pylori疫苗接种后Notch 1的表达变化。在小鼠胃浆膜下层接种疫苗后，分别于第21天与第28天采集胃组织与脾淋巴细胞。流式细胞分析显示：第21天后，胃部CD69+CD103+CD4+ TRM细胞中Notch 1表达量始终高于脾naïve T细胞与CD69+CD103-CD4+ TRM细胞（图6A）；且在CD69+CD103+CD4+ TRM中Notch 1显著高于CD69+CD103-CD4+ TRM细胞（图6B，P<0.05）。至第28天后，胃CD69+CD103+CD4+ TRM细胞中Notch 1表达量仍高于脾naïve T细胞，而CD69+CD103-CD4+ TRM与CD69+CD103+CD4+ TRM细胞的表达水平趋于相近，无显著差异（图6C）；Notch 1比例亦不再受CD103表达影响（图6D）。这些结果提示，在TRM形成早期阶段，Notch 1优先表达于CD69+CD103+CD4+ TRM上，随时间推移，其表达逐渐从CD103+亚群扩展至整个CD69+CD4+ TRM群体，此现象提示Notch信号通路在TRM早期分化阶段可能与CD103的表达相关。
H.pylori疫苗接种28 d后对小鼠胃组织进行针对DAPI、CD4、CD69、CD103与RBPJ的免疫荧光染色，证实RBPJ在胃TRM细胞中有所表达（图7A）。为进一步验证TRM发育受Notch信号通路调控的猜想，采用RBPJ抑制剂RIN1阻断下游Notch信号（图7B）。结果显示：与对照组相比，持续给予RIN1（图7C）导致CD4+CD69+CD103+ TRM细胞或总CD4+CD69+ TRM细胞比例下降（图7D、E，P<0.05），表明TRM的形成或驻留受Notch-RBPJ信号调控。抑制Notch及RBPJ通路会损害胃上皮TRM的产生，这些发现凸显了Notch-RBPJ在疫苗诱导胃上皮CD4+ TRM发育与维持中的关键作用。
2.5　RBPJ转录激活CD38表达
为明确Notch-RBPJ轴是否调控CD38表达，本研究通过磁珠分选获得小鼠原代CD4+ T细胞，在体外分别使用Notch通路抑制剂RG-4733和RIN1进行处理（图8A），采用流式细胞术检测CD38蛋白表达水平，qRT-PCR分析RBPJ及Notch经典靶基因HES5的mRNA表达。结果显示：RG-4733处理使得CD38蛋白表达显著下调（图8B），并降低CD4+ T细胞中RBPJ蛋白水平（图8C）；同时，RBPJ与HES5的mRNA水平同步下降（图8D、E），表明Notch通路活性被有效抑制，且证实RBPJ的转录依赖于Notch信号。
为进一步验证RBPJ对CD38表达的调控作用，本研究用RBPJ特异性抑制剂RIN1处理原代CD4+ T细胞，分别通过流式细胞术与qRT-PCR检测CD38蛋白与mRNA水平，并同步分析HES5 mRNA表达。结果显示：RIN1处理导致CD38蛋白与mRNA水平同步下降（图8F、G，P<0.05），激活RBPJ对CD38有调控作用；HES5 mRNA的下调（图8G）进一步证实RBPJ作为Notch信号下游效应子，介导靶基因网络的转录调控。为深入阐明RBPJ调控CD38的具体机制，本研究通过JASPAR数据库（http://jaspar.genereg.net/）对CD38启动子区（转录起始点-2 000 bp至+500 bp）进行生物信息学分析。利用保守的RBPJ结合基序（TGGGAA）扫描发现，CD38启动子内存在高置信度的RBPJ结合位点（图8H）。
为验证此发现，本研究构建了PGL3-CD38启动子荧光素酶报告质粒（图9A）与RBPJ过表达质粒（图9B），通过qRT-PCR确认转染后细胞中RBPJ mRNA水平显著升高（图9C），证实过表达质粒的有效性。在293T细胞中进行双荧光素酶报告基因共转染实验，将上述质粒与Renilla荧光素酶对照载体（pRL-TK）共转染。48 h后检测显示，RBPJ过表达可显著增强CD38启动子驱动的荧光素酶活性（图9D，P=0.003 2）。这些结果证明RBPJ通过特异性结合CD38启动子区域，实现对其转录激活的直接调控。
3　讨论
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3.1　TRM细胞与疫苗研究现状
当前研究已明确，TRM是疫苗在黏膜组织中提供早期持久免疫保护的关键介质。相较于传统的肌肉注射，鼻内等黏膜接种途径能更有效地将抗原递送至门户组织，从而在局部富集并分化出TRM细胞。目前研究还致力于解析TRM的分化机制，并据此设计新型疫苗。这包括利用TGF-β等关键细胞因子、筛选能激活特定信号通路（如TLR3）的新型佐剂，以及优化抗原设计，以驱动T细胞向TRM谱系分化。目前，这些策略已在结核病、流感等疾病的临床前模型中取得显著成果，证实通过黏膜接种疫苗能诱导强大的肺TRM群体，其保护效力甚至优于系统免疫。
3.2　Notch-RBPJ轴可能通过CD38驱动胃部CD4+ TRM细胞形成并维持其增殖能力
本研究聚焦于阐明CD4+ TRM细胞的形成机制，通过SCENIC分析发现Notch-RBPJ可能是CD38的上游调控通路［16］。基于此，本研究进一步揭示了关键因子的调控机制，并识别出细胞簇特异性转录因子。对转录因子调控基因的GO功能注释和通路预测分析显示，这些基因主要富集于细胞周期、细胞分裂及细胞因子-细胞因子受体相互作用等通路，提示胃部CD4+ TRM细胞在感染后第21天仍可能具有增殖潜能。
3.3　Notch-RBPJ信号在疫苗诱导的胃上皮CD4+ TRM发育中的关键作用
目前Notch信号通路的研究主要集中于肿瘤、胚胎发育及成体组织稳态维持等领域［17］。尽管该通路也参与CD4+ T细胞的Th1/Th2分化调控，但其在记忆T细胞生物学中的作用尚待深入探索。有研究证实Notch通路可通过RBPJ介导的糖代谢维持记忆CD4+ T细胞的存活［18］。此外，在疫苗接种或感染后3 d内，成纤维网状细胞来源的Delta-like1/4配体通过传递早期Notch信号，决定CD8+ T细胞的最终分子与功能命运［19］。这与本研究发现相契合，本研究证明Notch信号在早期免疫应答中刺激T细胞快速扩增，并通过促进组织驻留表型增强免疫保护，并进一步证明Notch-RBPJ正向调控TRM发育，其中RBPJ作为Notch的核心下游效应分子直接介导CD38的转录激活。值得注意的是，RBPJ活性与CD38表达水平呈正相关。在疫苗开发领域，本研究为设计高效黏膜疫苗提供了新策略。通过在疫苗配方中引入Notch信号激动剂，或可定向增强T细胞的早期扩增与组织驻留能力，从而建立长效的胃黏膜免疫屏障。同时，CD38作为Notch-RBPJ信号的直接靶点，可作为评估候选疫苗诱导TRM应答效能的潜在生物标志物，加速新型疫苗的临床评价。
本研究初始设计聚焦于纵向追踪疫苗免疫后TRM细胞群的表型、功能与信号通路变化，因此将资源和实验集中于疫苗组的深度分析。疫苗组内TRM与非TRM的比较构成内部对照，结果显示Notch-RBPJ-CD38信号分子特异性上调于TRM亚群，提示该信号通路可能参与调控TRM的分化与维持，并在疫苗接种后被特异性启动。尽管缺乏与未免疫组的直接比较，但现有数据中的严格内部对照及因果性功能实验共同指向该信号通路在疫苗诱导的TRM中特异性激活并发挥功能。因从胃组织中分选足够数量和高纯度的原代CD4+ TRM细胞面临技术挑战，故选用初始态或活化后的总细胞作为体外模型。在总CD4+ T细胞中，本研究证实Notch-RBPJ信号直接调控CD38转录，表明该调控关系在CD4+ T细胞谱系中有效。结合该信号轴在TRM中的特异性激活，提示Notch-RBPJ可能通过作用于CD38启动子上的RBPJ结合位点，驱动CD38在TRM中的上调表达。
综上所述，本研究明确了TRM细胞在黏膜疫苗免疫保护中的核心价值，揭示了Notch-RBPJ-CD38轴对胃部CD4+ TRM细胞形成、增殖及发育的关键调控机制，为开发可增强T细胞功能与持久性的新型疫苗策略提供了重要的理论依据。未来将利用CD38⁻/⁻小鼠模型开展体内实验，直接评估CD38缺失对TRM细胞形成及驻留的影响，进一步完善该调控机制，为新型疫苗的研发奠定坚实基础。

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doi: 10.16016/j.2097-0927.202512105
  • 接收时间：2025-12-19
  • 首发时间：2026-10-23
  • 出版时间：2026-04-30
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  • 收稿日期：2025-12-19
  • 修回日期：2026-03-12
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    1中国药科大学生命科学与技术学院，江苏 南京

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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
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红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
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光柄菇属 Pluteus 5 2.39
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