Objective:

To investigate the expression pattern, cellular source, regulatory program, and potential biological role of IL21 in colorectal cancer.

Approach:

• Single-cell RNA sequencing analysis: Analyzed tumor-infiltrating T cells from public datasets to characterize IL21 expression patterns.
• External validation: Used a single-cell cohort of colorectal cancer cells treated with anti-PD-1 neoadjuvant therapy.
• Multiplex immunofluorescence staining: Performed on colorectal cancer tissue microarrays to validate spatial distribution and cellular origin of IL21.
• Correlation analysis: Assessed the relationship between IL21 and immune-related cell subsets.
• SCENIC analysis: Identified candidate transcription factors associated with CXCL13-associated CD4+ T cell subsets.
• In vitro culture and RNA sequencing: Cultured tumor tissues from an AOM/DSS-induced mouse colorectal cancer model to explore IL21-induced transcriptional changes.

Key Findings:

• IL21 expression was restricted to a limited fraction of tumor-infiltrating T cells, predominantly in CXCL13+CD4+ T cell populations.
• IL21R was more broadly expressed across T cell populations.
• Tissue-level multiplex immunofluorescence confirmed increased IL21 and IL21+CD4+ T cells in tumor tissues compared to adjacent normal tissues.
• Correlation analysis indicated a positive association between IL21 expression and various CD4+ T cell populations.
• SCENIC analysis identified STAT1, IRF9, IRF7, TBX21, and IRF4 as candidate key transcription factors in the CD4_C09-CXCL13 subset.
• Exogenous IL21 induced transcriptional remodeling in murine colorectal tumor tissues, activating immune-related pathways.

Interpretation:

CXCL13-associated CD4+ T cells are identified as a major IL21-producing population in colorectal cancer.

Limitations:

• Direct mechanistic validation of findings remains necessary.

Conclusion:

The study provides a cellular and transcriptional framework for future investigation of IL21-mediated immune regulation in colorectal cancer.