24Chemokines in mobilising γδ T cells in biliary tract cancer and implications for γδ T cell– based adoptive cell therapy
Saikat Mandal, Manideepa Maji, Arkadeep DhaliAbstract
Background
Biliary tract cancer (BTC) has a poor prognosis with limited therapeutic options. γδ T cells represent an MHC-independent immune cell population; however, their therapeutic efficacy in solid tumours is constrained by insufficient tumour infiltration. Chemokine- mediated trafficking is fundamental to T lymphocyte recruitment; however, the chemokine landscape of the BTC tumour microenvironment (TME) remains uncharacterised.
Objective
Using single-cell RNA sequencing of BTC tissues, we delineated chemokine ligand expression patterns, stratified chemokine producers by lineage, assessed mechanisms of γδ T- cell recruitment, and identified chemokine-mediated immune escape.
Methods
Single-cell RNA-sequencing data from three GEO cohorts (GSE210066, GSE201425, GSE213452; 19 patients) encompassing intrahepatic, extrahepatic cholangiocarcinoma, and gallbladder malignancy were analysed using Seurat v5.0 in R. Quality control filtering applied stringent criteria (>200 genes, normal UMI counts, <15% mitochondrial content, <3% haemoglobin). Harmony batch correction across datasets preceded unsupervised clustering (resolution 0.9) and UMAP visualisation. Chemokine expression (17 ligands; 5 receptors) was systematically quantified across all cell types using average expression and percentage expression calculations.
Results
Analysis of 119,840 quality-checked cells identified 26 distinct populations, including immune, epithelial, and stromal compartments. Comprehensive analysis identified a multi- axis chemokine profile, with high expression of CCL5 (30%), CXCL16 (24%), and CCL4 (23%) supporting γδ T-cell recruitment, while CCL2 and modest CXCL8 supported alternative Vδ1 T- cell axes. Notably, CXCL16 expression supported epithelial γδ T-cell homing via CXCR6. Paradoxically, chemokine axes supporting γδ T cell recruitment (CCL2-CCR2, CXCL8-CXCR1, CXCL12-CXCR4) simultaneously recruit immunosuppressive populations, such as myeloid- derived suppressor cells (MDSCs), regulatory T cells (Tregs), and tumour-associated macrophages (TAMs).
Conclusion
Comprehensive single-cell analysis identified selective chemokine recruitment signatures supporting γδ T cell infiltration but revealed paradoxical co-recruitment of immunosuppressive populations in the BTC TME. Patient stratification via chemokine profiling combined with γδ T cell enrichment and targeted chemokine antagonism represents a rational therapeutic strategy.