The progression of TSCC is affected by several factors, including gene mutation, dysregulation of long non-coding RNA, and changes in the tumor microenvironment [19-21].
With the development of single-cell sequencing technology, increasing numbers of immune cells signatures have been identified and immune cells in the tumor microenvironment have become characterized in more detail [13, 22, 23]. The composition of various components in the microenvironment can be analyzed using the expression profile obtained by RNA sequencing.
Various mechanisms leading to immune cell dysfunction in the tumor microenvironment have been successively identified. The immune microenvironment can directly or indirectly affect the occurrence and development of tumors. Its mechanisms include promoting tumor angiogenesis, changing the biological characteristics of tumors cells, or establishing an appropriate tumor microenvironment to promote tumor progression . Liu et al.  suggested that alterations in the Treg/Th17 balance in TSCC promote disease progression. Macrophages in the tumor microenvironment of TSCC can promote the invasion and migration abilities of tumor cells . In our study, Th2 cells were downregulated in the advanced stage samples. Th2 cells are differentiated from naïve CD4+ T cells and mainly mediate humoral immunity but the role of Th2 cells in tumors is unclear. Studies have indicated that increased Th2 cells or cytokines secreted by Th2 cells indicates a poor prognosis in colorectal cancer, esophageal cancer, and prostate cancer [9, 10, 26]. However, a study of B cell non-Hodgkin's lymphoma suggested that high expression of IL4, which is secreted by Th2 cells, is strongly correlated with reduced levels of proliferation and a greater survival duration . Another study of non-small cell lung cancer suggested that high Th2 and low Th17 levels indicate good prognosis. . Our results suggest that high Th2 levels play a protective role in TSCC.
Changes in the Th2 cell expression level are related to the recruitment and differentiation of these cells. CCL22 and CCL17 are chemokines that regulate Treg and Th2 cell recruitment via binding to their receptor CCR4. In our study, CCL17 expression did not differ between the early and advanced stage, whereas CCL22 was significantly decreased in the advanced stage. Overexpression of CCL22 in human tumors was reported to be associated with high infiltration of Treg cells, along with augmented tumor growth and poor prognosis in breast, gastric, and liver cancer [29-33]. Studies of CCL22 and Th2 cells are limited but indicate that CCL22 is a prognostic factor in breast and colorectal cancer [34, 35]. A study of TSCC suggested that high expression of CCL22 in TSCC influences the balance of M1- and M2-like macrophages and leads to worse prognosis . However, in our study, CCL22 was a protective prognostic factor, contrasting the results of previous studies. The expression of Treg cells between early and advanced stage patients did not significantly differ (Fig. S5). We hypothesized that CCL22 only affects the recruitment of Th2 cells in patients with TSCC, rather than Treg cells, suggesting that CCL22 affects the prognosis of patients and progression of TSCC by regulating Th2 cells. Our results suggest a new idea that CCL22 can be used as a treatment target in TSCC.
Gene mutation plays a very important role in the development and progression of cancer. Recent large-scale genome sequencing efforts have validated TP53 as the most common mutation in head and neck squamous cell carcinoma . A study of the mutations landscape of TSCC revealed that TP53, FAT1, CDKN2A, NOTCH1, and PIK3CA are the most frequently mutated genes . In our study, NOTCH1 mutation was significantly reduced in early stage patients. NOTCH signaling is involved in different types of malignant tumors  and has mostly been found to be altered in head and neck squamous cell carcinoma ; NOTCH can act as an oncogene or tumor suppressor depending on the cellular context . The role of NOTCH as a tumor suppressor pathway in oral squamous cell carcinoma has been suggested in several studies [37, 42, 43]. Activated NOTCH1 exerts an anti-proliferative effect in tongue tumor cells by downregulating Wnt/β-catenin signaling and inducing apoptosis and cell cycle arrest .
However, in our study, the activation level of the NOTCH1 signaling pathway did not differ betweenearly and advanced stage patients. Previous studies suggested that NOTCH1 can induce Th2 cell differentiation [17, 18]. The NOTCH-Th2 differentiation pathway was upregulated in early stage patients. These results indicate that NOTCH1 mutation affects activation of the NOTCH-Th2 differentiation pathway, thus reducing the number of Th2 cells.
According to our results, increased expression of CCL22 in TSCC may activate the recruitment of Th2 cells, whereas mutations of NOTCH1 may inhibit Th2 cell differentiation. These two mechanisms influence the expression level of Th2 cells, leading to the progression of TSCC. However, there were several limitations to this study. First, the data were obtained from a public database. Fresh tumor tissue should be analyzed to verify our results. Second, our findings were mainly based on bioinformatics and algorithms, and validation using biological experiments was not performed.
In conclusion, in TSCC, high expression of CCL22 can promote the recruitment of Th2 cells and predict better survival. Mutation of NOTCH1 inhibits the differentiation of Th2 cells. Combined with decreased Th2 cell recruitment and differentiation, these effects can lead to tumor progression.