Single-cell RNA sequencing reveals a novel CD4+T cell subset in papillary thyroid cancer
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-? 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-?, 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-?, TNF-a, 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. Overall design: Cells were firstly stained with two fluorescent dye, Calcein AM and Draq7, for precisely determination of cell concentration and viability via BD Rhapsody™ Scanner (BD Biosciences) before single cell multiplexing labelling.     Cell of each sample were sequentially labeled with BD Human Single-Cell Multiplexing Kit (Cat.     No. 633781) which utilizing the cell-hashing technology  mainly to provide higher sample throughput and eliminate batch effect for single cell library preparation and sequencing.     Briefly, cells from each sample were labeled by antibodies with different sample tags respectively and washed twice with Stain Buffer (FBS) (BD Biosciences, Cat.     No. 554656) before pooling all samples together.     Pooled samples were loaded in one BD Rhapsody micro-well cartridge based on Fan et al .     Cell capture beads were then loaded excessively to ensure that nearly every micro-well contains one bead, and the excess beads were washed away from the cartridge.     After lysing cells with lysis buffer, Cell capture beads were retrieved and washed prior to performing reverse transcription.     Microbeads-captured single cell transcriptome and SampleTag molecules were generated into cDNA library and SampleTag library separately containing cell labels and unique molecular identifiers (UMI) information.We then performed gene expression profilinf analysis use data obtained from scRNA-seq of 15 samples, including primary tumors of PTC, metastatic lymph nodes (LNs), and adjacent normal tissues. Please note that each sample record represents mixed-samples and the sample_metadata.txt contains the sample information for 15 samples (SAMPLE1-SAMPLE15).