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OA

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 treated with 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.