Integrated Single-Cell Transcriptomic Analysis from Human Colorectal Cancer Studies Reveals Limited Pro-tumor IL-17 Secretion Potential in γδ T Cells

Introduction: Gamma delta T cells (γδ T) are a distinct subset of T cells enriched in the intestinal mucosa. Unlike conventional alpha-beta T cells (αβ T), γδ T cells can be activated independently of the major histocompatibility complex (MHC), enabling them to recognize a variety of tumor-associated antigens. Although γδ T cells are clinically observed to constitute a major lymphocyte population infiltrating colorectal cancers, where they play significant roles in the antitumor immune response, there is debate over the potential pro-tumoral activities of human γδ T cells regarding their production of interleukin-17 (IL-17), which has been implicated in promoting epithelial-mesenchymal transition, enhancing tumor survival, and recruiting myeloid-derived suppressor cells (MDSCs) to create an immunosuppressive environment that favors tumor growth. Given that tumor-promoting γδ T17 cells are mostly reported in mice but not vastly in humans, we analyzed 16 published human CRC whole-tissue scRNA-seq datasets and integrated all identifiable γδ T to evaluate their subtype signatures, tissue adherence, cytotoxic effects, and most importantly, their IL-17 secreting potential.

Materials and

Methods: Human CRC whole-tissue scRNA-seq datasets were obtained from Gene Expression Omnibus (GEO) Series GSE178341, GSE200997, GSE221575, GSE196964, GSE232525, GSE245552, GSE231559, GSE188711, GSE161277, GSE183916, GSE201348, GSE108989, GSE146771, GSE164522, GSE178318 and also PubMed PMID35773407. If processed data is not provided, Seurat and Scanpy were used for quality control, normalization, transformation, and downstream processing. In each study, cell clusters that highly co-express CD3D, CD3E, CD3G, CD247, and TRDC were identified as γδ T. Raw counts of cells in the clusters of all studies were integrated using Seurat. Differentially expressed gene analysis was done by using Monocle3 in R.

Results, Conclusions, and Discussions: A total of 18,686 γδ T cells were identified from the tumor and adjacent normal tissue of 165 human CRC patients in 9 different studies. These cells could be further delineated into 5 subtypes: central memory T cells (TCM), tissue-resident memory T cells (TRM), exhausted T cells (Tex), and effector T cells, which could be divided further based on their expression level of tissue residency related genes. All γδ T cells exhibited limited transcript of IL17A-F, RORC, and previously defined IL17-secreting related signatures like IL23R, CCR6, MAF, AHR, CLEC7A, SLAMF1, and DPP4. The Tex cells, which co-express several exhaustion markers (PDCD1, CTLA4, LAG3, TIGIT, HAVCR2), still show the transcription of cytotoxic molecules GZMA, GZMB, PRF1, NKG7, and IFNG, indicating a potentially maintained effector function which differs from the IL-17 producing phenotype identified in mice exhausted γδ T. DEG analysis revealed that tumor-infiltrating γδ T’s phenotype significantly differs from γδ T in the normal tissue of CRC patients; notably, the unexhausted γδ effector T cells found in tumor show higher cytotoxicity by their GZMH and IFNG expression and more mobility by their KLF2 level. Together, our findings indicate a potential cross-species discrepancy that the pro-tumor γδ T17 prevalent in mice CRC model may not be transcriptomically identifiable in the human CRC.

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