Single-cell transcriptomic profiling reveals CD27(+) cytotoxic T Cell heterogeneity and exhaustion dynamics in colorectal cancer tumor microenvironment.

CD27+ cytotoxic T cells play critical roles in anti-tumor immunity, yet their heterogeneity and functional states within the colorectal cancer (CRC) tumor microenvironment remain poorly characterized. Understanding the molecular mechanisms underlying T cell exhaustion is essential for developing effective immunotherapeutic strategies. We performed comprehensive single-cell RNA sequencing analysis of CD27+ cytotoxic T cells from CRC tumor tissues and matched normal adjacent tissues using the GSE144735 dataset. Multiple dimensional reduction techniques, trajectory inference algorithms, and functional characterization approaches were employed to dissect cellular heterogeneity, differentiation trajectories, and exhaustion dynamics.

In vitro co-culture experiments using HCT116 and RKO (MSI-H), HCT15 and SW480 (MSS) colorectal cancer cell lines with activated PBMCs were conducted to validate computational findings. UMAP and t-SNE analyses identified 11 distinct CD27+ T cell clusters with substantial phenotypic heterogeneity, including exhausted terminal, effector, and memory-like populations. Pseudotime trajectory analysis revealed progressive exhaustion pathways with three distinct differentiation fates. Exhausted CD27+ T cells exhibited significantly elevated expression of checkpoint molecules (PDCD1, LAG3, HAVCR2, CTLA4) and reduced cytotoxic capacity compared to effector populations.

RNA velocity and PAGA connectivity analysis demonstrated that exhaustion represents a terminal differentiation state with limited plasticity. Gene regulatory network analysis identified key transcription factors governing effector-to-exhausted transitions.

Notably, exhausted CD27+ T cells concentrated in tumor core regions, while effector populations distributed peripherally. Co-culture experiments confirmed that MSI-H cell lines (HCT116 and RKO) induced 2-3 times higher exhaustion marker expression compared to MSS cell lines (HCT15 and SW480). For MSI-H cells, PDCD1 showed 5.2-fold (HCT116) and 4.8-fold (RKO) upregulation, while LAG3 showed 4.8-fold (HCT116) and 4.2-fold (RKO) upregulation. In contrast, MSS cells showed lower induction: PDCD1 at 2.1-fold (SW480) and 1.9-fold (HCT15), LAG3 at 1.8-fold (SW480) and 1.6-fold (HCT15).

Exploratory survival analysis (HR = 0.505, p = 0.673) did not reach statistical significance and is presented as hypothesis-generating only, highlighting the need for prospective validation in larger cohorts. PDCD1 and LAG3 were identified as prioritized immunotherapy targets based on their significant overexpression in exhausted T cell populations.

This study provides a comprehensive single-cell atlas of CD27+ cytotoxic T cell heterogeneity in CRC, revealing exhaustion dynamics, regulatory networks, and spatial organization patterns.

Our findings highlight the differential immunogenic capacity between MSI-H and MSS tumors and identify potential therapeutic targets for reversing T cell exhaustion in colorectal cancer.