Shigella flexneri activates the mature DC-Tfh axis to drive tertiary lymphoid structure neogenesis and maturation in colorectal tumour microenvironments

Background Tertiary lymphoid structures (TLSs) serve as critical antitumour immune mediators within the tumour microenvironment and are associated with favourable immunotherapy outcomes and prognosis. Therefore, inducing intratumoural TLS formation represents a promising therapeutic approach. However, the mechanisms underlying their formation in colorectal cancer (CRC) remain unclear. Objective This study aims to characterise the immune landscape of TLS-enriched CRC and to identify TLS-associated microbes along with their mechanisms of action as TLS inducers. Design 16S rRNA sequencing identified TLS-associated microbial signatures. Immunocompetent mouse models validated microbial effects on TLS formation and tumour growth. Single-cell RNA sequencing, multiplex immunofluorescence and flow cytometry characterised the immune landscape. In vitro infection models assessed microbial effects on immune cells. Results Microbiome sequencing identified Escherichia coli and Shigella as TLS-correlated intratumoural microbes in CRC. In vivo models demonstrated that only Shigella flexneri (S. flexneri) colonisation promoted the formation of TLSs and significantly suppressed tumour growth. Core TLS-associated immune subsets were identified: T follicular helper cells (Tfh), germinal centre B cells and mature dendritic cells (mature DCs). Mechanistically, S. flexneri activated type I interferon signalling to drive DC maturation. Mature DCs recruit T cells via CCL19 and CXCL10, and Tfh cells further recruit B cells through CXCL13, thereby establishing structured TLSs. Conclusion This study is the first to demonstrate that S. flexneri promotes the organised formation of TLSs in CRC via a mature DC-Tfh axis-mediated chemokine network. Therapeutic strategies targeting this bacterium represent a novel paradigm for TLS induction and may contribute to immunotherapy in CRC.