Dysregulated Th1 cells in lung squamous cell carcinoma.

e20566 Background: Lung squamous cell carcinoma (LUSC) is one of the most common subtypes of lung cancer which lacks of effective treatments compared to lung adenocarcinoma. The Th1 subset in CD4+ T cells plays an indispensable role in antitumor immune responses. However, there are few studies on Th1 cells in LUSC. Methods: Firstly, we used single-cell RNA-seq to analyze the gene expression in 8 LUSC patients’ tissues, including tumor tissues, paracancerous tissues, normal tissues, lymphoid tissues, pre-treatment blood samples and pre-operative blood samples. A total of qualified 44969 CD4+ T cells and 22971 Th1 candidate cells (Because STAT1, STAT4 and T-bet can promote IFN-γ expression. We define CD4+ T cells with one or more of above four indicators as Th1 candidate cells (Th1 CDD cells)) were identified. Then, we validated Th1 CDD cells and CD8+ T cells of tumor sites in 32 patients with LUSC by using multiplex immunofluorescence staining and immunohistochemistry. Finally, we used flow cytometry to detect the IFN-γ of CD4+ T cells in human PBMCs which were co-incubated with the supernatant derived from LUSC cell lines to simulate the tumor inhibitory microenvironment. Results: Single cell RNA-seq results showed IFN-γ+ Th1 CDD cells only account for about 25.28% of the total Th1 CDD cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially expressed genes (DEGs) between IFN-γ+ Th1 CDD cells and IFN-γ- Th1 CDD cells demonstrated that the decreased level of IFN-γ expression was associated with ribosomes, antigen presentation, cell-to-cell interactions, apoptosis, endoplasmic reticulum stress (ER stress) and so on. According to multiplex immunofluorescence staining and immunohistochemistry, there was a positive correlation between the IFN-γ+ STAT1+ T-bet+ Th1 CDD cells and CD8+ T cells in tumor stromal compartment. Flow cytometry showed decreased level of IFN-γ in Th1 cells co-incubated with LUSC-derived supernatant. Conclusions: The function of Th1 CDD cells is suppressed in LUSC. In our study, we found that the decreased function of Th1 CDD cells is associated with ER stress by single cell RNA-seq, which requires further research. Overall, this may highlight a new target for the treatment of LUSC.