Andreas Behren

Intraepithelial lymphocytes (IELs), including αβ and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/ TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

Conventional immunohistochemistry methods though once fundamental for the individual staining of cell markers, have now been superseded by multispectral immunohistochemistry (mIHC). mIHC enables simultaneous detection of multiple cell markers in situ using single formalin-fixed paraffin-embedded (FFPE) tissue sections. In addition to conserving patient tissue specimens, the ability to visualise more than one marker on individual cells allows for further refining of cell phenotypes, and provides insight into cell-to-cell interactions and spatial arrangements across single tissue sections. Here, a comprehensive protocol is described for the in situ interrogation of γδ T cells and phosphoantigen-presenting butyrophilin (BTN) molecules (BTN2A1 and BTN3A1) in human FFPE tissue using Opal™ tyramide signal amplification (TSA)-based mIHC. It is demonstrated that an effectively optimised Opal™-TSA 7-marker [CD3, Pan-γδ T cell receptor (TCR), granzyme B, BTN2A1, BTN3A1, tumour marker, 4’,6-diamidino-2-phenylindole (DAPI)] mIHC panel can be used to define the presence, localisation, and activation status of γδ T cells and the BTN2A1 and BTN3A1 ligands.