ABSTRACT: Papillary thyroid cancer (PTC) is the most common thyroid malignancy. Although PTC patients usually show a favorable prognosis, some still have a high rate of recurrence and a relatively low survival rate. We aimed to reveal the mechanisms involved in the development of thyroid cancer using single-cell RNA sequencing (scRNA-seq).scRNA-seq data was collected from 15 samples, including primary tumors of PTC, metastatic lymph nodes (LNs), and adjacent normal tissues. The results from scRNA-seq data were further validated with flow cytometry, proliferation, invasion, and migration assays, and bulk RNA-seq data.A novel CD4+T cell subset was identified, termed as EGR1+CD4+T cells. It produced high levels of IL-10 and IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin, and showed a distinct molecular pathway activity compared to CD4+Tregs. CD4+T cell-specific over-expression of EGR1 in vitro enhanced tumor cell proliferation, invasion, and migration when co-cultured with PTC cell lines. The expression profiling analysis of immune checkpoint molecules indicated that CD96 was commonly up-regulated in EGR1+CD4+T cells, CD4+Tregs, and other T cell subsets, especially in tumor tissues. Single CD96 blockade significantly increased the levels of IFN-γ, IL-17a, and IL-10 in EGR1+CD4+T cells, and inhibited the proliferation of PTC tumor cells. Co-blockade of CD96/TIGIT significantly enhanced the production of IFN-γ, TNF-α, and IL-17a in both EGR1+CD4+T and CD3+CD4+T cells, which also suppressed cell proliferation, invasion, and migration when co-cultured with PTC tumor cells. These findings indicated that EGR1+CD4+T cell was a novel CD4+T cell subset with specific functions. Single CD96 blockade or co-blockade of CD96/TIGIT enhanced antitumor immunity of EGR1+CD4+T cells, which might be promising therapeutic strategies in PTC treatment.
