Notch-RBPJ axis promotes formation of Helicobacter pylori vaccine-induced gastric CD4+ tissue-resident memory T cells via transcriptional activation of CD38

Abstract
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.