IL21 is predominantly produced by a CXCL13 associated CD4⁺ T cell subset and shapes the immune microenvironment in...

Interleukin-21 (IL21) is an important immunoregulatory cytokine involved in antitumor immune response; however, its cellular source, regulatory program, and functional significance in colorectal cancer remain incompletely understood.

We analyzed tumor-infiltrating T cells from the public single-cell RNA sequencing dataset to characterize IL21 expression patterns in colorectal cancer. External validation was performed using a single-cell cohort of colorectal cancer cells treated with anti-PD-1 neoadjuvant therapy. Multiplex immunofluorescence staining was performed on colorectal cancer tissue microarrays to validate the spatial distribution and cellular origin of IL21. Correlation analysis was used to assess the relationship between IL21 and immune-related cell subsets. SCENIC analysis was performed to identify candidate transcription factors associated with CXCL13-associated CD4⁺T cell subsets. Furthermore, tumor tissues from an AOM/DSS-induced mouse colorectal cancer model were cultured in vitro (with or without IL21) and batch RNA sequenced to explore IL21-induced transcriptional changes.

Single-cell analysis showed that IL21 expression was restricted to a limited fraction of tumor-infiltrating T cells and was predominantly enriched in CD4⁺T cell subsets, particularly CXCL13⁺CD4⁺T cell populations, whereas IL21R was more broadly expressed. Analysis of the PD-1 anti-neoadjuvant therapy cohort further confirmed that IL21 is also preferentially expressed in specialized CD4⁺ T cell subsets, including CXCL13-associated cell populations. Tissue-level multiplex immunofluorescence confirmed that IL21, IL21⁺CD4⁺T cells, and CXCL13⁺IL21⁺CD4⁺T cells were all significantly increased in tumor tissues compared with adjacent normal tissues. Correlation analysis further suggested that IL21 expression was positively associated with CD4⁺, CXCL13⁺, IL21⁺CD4⁺, and CXCL13⁺IL21⁺CD4⁺T cell populations. SCENIC analysis identified a distinct regulon landscape in the CD4_C09-CXCL13 subset, with STAT1, IRF9, IRF7, TBX21, and IRF4 emerging as candidate key transcription factors. Functionally, exogenous IL21 induced marked transcriptional remodeling in murine colorectal tumor tissues, characterized by activation of immune- and cytokine-related pathways and suppression of multiple Wnt-associated genes.

Our study identifies CXCL13-associated CD4⁺T cells as a major IL21-producing population in colorectal cancer and links this axis to an immune-active tumor microenvironment. Although direct mechanistic validation remains necessary, our findings provide a cellular and transcriptional framework for future investigation of IL21-mediated immune regulation in colorectal cancer.