Single-Cell Transcriptomic Analysis Reveals γδ T-Cell Infiltration and Presence of Immune Regulatory Molecules in...

Biliary tract cancer (BTC) remains therapeutically challenging with poor survival outcomes; systemic therapies achieving complete responses in only 2% to 5% of patients. γδ T-cell-based adoptive cell therapy represents a promising strategy due to MHC-independent activation. γδ T cells are activated through direct engagement of butyrophilin (BTN) family molecules; conversely, nonclassical HLA class-I molecules, particularly HLA-E, function as inhibitory ligands for NKG2A expressed on γδ T cells, suppressing anti-tumor immunity. Characteristics of tumor-infiltrating γδ T cells, their activity, and inhibitory interactions in BTC remain unexplored.

Single-cell RNA-sequencing data from 19 BTC tumor samples in the Gene Expression Omnibus (GEO) were analyzed to characterize γδ T-cell infiltration and the expression of regulatory molecules. MHC-I molecules, butyrophilin (BTN) molecules, intercellular adhesion molecules (ICAM), and NKG2A checkpoint receptors were quantified.

γδ T cells comprised 0% to 4.9% of all cells present in the BTC tumor samples. Expression of BTN2A1, BTN2A2, BTN3A1, BTN3A2, and ICAM1 was noted in all the tissue samples, supporting Vγ9Vδ2 T-cell activation potential. Classic HLA class-I expression was preserved (70% to 90% of cells). HLA-E was overexpressed (60% to 95.7% of cells expressing HLA-E). Around 30% of NK cells and γδ T cells exhibited NKG2A positivity (log2 expression >2).

Given the presence of regulatory molecules such as BTN and ICAM, Vγ9Vδ2 T-cell-based adoptive cell therapy appears promising. A combination of elevated HLA-E expression with high γδ T-cell NKG2A positivity establishes a potent inhibitory checkpoint axis in BTC. These findings support the rationale of investigating anti-NKG2A blockade combined with γδ T-cell-based adoptive therapy as a novel therapeutic strategy.