A Ligand-Centered Framework for γδ T Cell Activation in Colorectal Cancer Revealed by Single-Cell and Transformer-Based Perturbation

Ran Ran¹Douglas K. Brubaker^1,2*

• ¹School of Medicine, Case Western Reserve University, Cleveland, United States
• ²University Hospitals Health System, Cleveland, United States

Understanding the activation mechanisms of γδ T cells in colorectal cancer (CRC) is critical for harnessing their therapeutic potential. Here, using an atlas of human CRC-infiltrating γδ T cells that we built by integrating multiple single-cell RNA-seq datasets, we developed a γδ T cell-refined ligand inference pipeline by combining differential gene expression, gene regulatory network prediction, ligand inference, and in silico perturbation analysis. This approach identified ligands, including IL-15 and TNFSF9 (4-1BBL), as candidates promoting γδ T cell effector function and highlighted NCR2 and KLRC3 (NKG2E), whose in silico overexpression was associated with γδ T cell activation. Ligand enrichment analyses further indicated that monocytes and dendritic cells are key contributors to γδ T cell activation in the tumor microenvironment. Our results also highlighted transcription factors IKZF1, FOSL2, and FOXO1 in the less activated γδ T cells and IRF1, KLF2, and BHLHE40 in the effector γδ T cells that plausibly regulated the differential activation state. Together, our results offer a systems-level view of the signaling and transcriptional programs governing γδ T cell phenotypes in CRC and provide a foundation for γδ T cell-based immunotherapies with enhanced antitumor functions.