Landscape of Immune Cell Inltration in Cervical Cancer

Background Cervical cancer is a life-threatening cancer among women. It is the second most prevalent malignant tumor in women. It ranks high in cancer deaths among women worldwide, including in the United States. Immune checkpoint inhibitors have emerged as an important therapeutic approach to treat several cancers, including cervical cancer. Notably, the development and progress of cervical cancer may be related to sustained immune response. This underlines the need to clarify immune cell inltration (ICI) in cervical cancer tissues. Methods In this study, disease-related information of 964 cervical cancer patients was rst retrieved from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) database. We utilized bioinformatics data to analyze the expression proles of immune genes in cervical cancer tissues. Results Patients were divided into high and low groups according to ICI score. High ICI scores corresponded with activation of immune signaling pathways and high tumor mutation burden (TMB), which was related to better prognosis of G1-2 cervical cancer. In addition, most immune checkpoints and immuno-related genes such as CD274, CD8A, CXCL10, etc. were over-expressed in the high ICI group. This study demonstrated that ICI score can accurately predict the prognosis of cervical cancer. Understanding ICI patterns will deepen our understanding of tumor microenvironment (TME) of cervical cancer, which may create the foundation for the development of ecient immunotherapeutic strategies against the cancer.


Analysis Of The Ici Score
The cervical Patients were strati ed into high or low ICI score groups based on the median ICI score ( Table 6). The distribution of patients with the four gene clusters in ICI score is shown in Fig. 3A. Genes strongly associated with immunoactivity included SOX2, CD274, CD8A, CXCL10, TBX2, TLR9, CTLA4, CD28, GZMB, IFNG, CXCL9, FOXP3 and TNF genes. Except SOX2, TBX2 and TLR9, the rest of immune checkpoint and immune activity related genes were overexpressed in individuals in the high ICI score group (Fig. 3B). Gene set enrichment analysis (GSEA) revealed that the B cell receptor, JAK-STAT and T cell receptor signaling pathways were most dysregulated in the high ICI score group (Figs. 3C; Table 7).
Further analyses revealed that high ICI score correlated with better prognosis, while the survival time of patients with low ICI score was only 8.48 years (p = 0.041, Fig. 3D).
Relationship Between Ici Score And Tmb TMB is one of the greatest clinical factors that in uences the prognosis of cancers. In this study, TMB data of cervical cancer was obtained from the TCGA database. We found TMB was directly proportional to the ICI score (Wilcoxon test p = 0.046, Fig. 4A). Based on TMB and immune score, patients were also divided into discrete subgroups. Overall, there was a strong positive correlation between ICI score and TMB ( Fig. 4B; R = 0.14; P = 0.017), consistent with the above analysis.
High TMB was also associated with better prognosis of cervical cancer ( Fig. 4C; p = 0.042). Analysis shows that ICI score is an independent predictor of cervical cancer prognosis. Under the classi cation of ICI score, there was a signi cant difference in survival rate between the low and high TMB subgroups. Here, four subgroups could be recognized; TMB high and ICI high (HH), TMB high and ICI low (HL), TMB low and ICI low (LH) and nally TMB low and ICI low (LL) (Fig. 4D, p = 0.012).
Signi cant genes in the low ICI group and high ICI group were also investigated (Figs. 4E and 4F). Analysis of TCGA data revealed signi cant differences in OBSCN, DNAH9, MXRA5 and CUBN gene mutations between low and high ICI score groups (Table 8). Overall, ndings of this study lay the foundation for the development and assessment of response to cervical cancer immunotherapies.

Discussion
In recent years, signi cant progress has been made in the eld of cancer immunotherapy []  In this study, we quanti ed the tumor immune environment for cervical cancer. The expression pro le of ICI can accurately predict the prognosis of cervical cancer. Accordingly, the establishment of ICI score may be used in developing immunotherapies against cervical cancer.
Accumulating evidences show that in ltration of immune cells in TME affects survival and proliferation of cervical cancer cells [26] . Therefore, there is need to monitor the immune response of these groups of patients. Herein, we analyzed the expression pro le of ICI in 964 cervical cancer tissues. On this basis, cervical cancer is divided into three different immune subtypes (A-C). Patients in ICI Cluster B exhibited the longest survival time. Notably, Cluster B was linked to high in ltration of CD8 + T cells, resting NK cells, follicular helper T cells, memory activated CD4 + T cells. Mice models revealed that under expression of Treg cells inhibit the proliferation and metastasis of cervical cancer cells [] , the in ltration of activated CD8 + T cells promotes the participation of IL-10 in the immune response and has the characteristics of cervical cancer [] . TME of cervical cancer tissues comprises of numerous cytokines, chemokines and other host derived components, which re ects its immune ability.
Unraveling immune-related genes associated with expression of ICI can uncover strategies for developing personalized treatment for cervical cancer. The expression pro le of immune-related genes in cervical cancer tissues was downloaded from the GEO and TCGA databases. Based on the gene expression, cervical cancer patients were divided into four ICI gene clusters. ICI gene cluster A and D displayed lower immune and stromal scores, but exhibited high expression of Macrophages M0, Neutrophils, activated Mast cells, activated dendritic cells, resting memory CD4 + T cells, and naive B cells. Conversely, the immune scores of the other two groups were signi cantly higher. High stromal score in both groups was associated with high in ltration of M1 macrophages, activated CD4 + memory T cells. In addition, expression of cluster B and C ICI genes was associated with shorter survival of cervical cancer patients. Contrarily, expression of cluster A and D ICI genes was associated with longer survival. Given that PD-L1 is highly expressed in ICI gene clusters B and C, we speculate that overactivated cells in these two groups may mediate immune escape. Indeed, previous studies show that immune escape promotes disease development, thus in uencing the prognosis. Pro ling of immune genes related to ICI can help in the development of more accurate immunotherapy.
Classi cation of cancer patients is motivated by cancer heterogeneity. In this study, according to ICI score, cervical cancer patients were divided into high rating group and low rating group, and a model of quantifying ICI with ICI score was established. JAK-STAT signal is involved in a variety of cellular regulatory For instance, T cell receptor (TCR) mediates functions of activated T Reg (AT Reg) cells, which also play a central role in immune tolerance of Foxp3 + regulatory T (T Reg) cells [] . GSEA analysis revealed the over-expression of genes associated with immunosuppressive pathways such as B cell receptor, JAK-STAT and T cell receptor signaling pathways in the high ICI score groups.
New studies have shown that genetic mutations occur in glycosylation sites of G protein in Respiratory syncytial virus (RSV), and maybe participate in immune escape [] . Herein, mutations in genes are to some extent related to the body's immunity. The results of this study show that ICI score was related to the degree of genetic mutations in multiple genes. Particularly, TMB positively correlated with ICI scores (0.017). Sub-group analysis further revealed that ICI score and TMB affected cervical cancer patients prognosis. Speci cally, high TMB corresponded with longer overall survival. It is generally believed that before the occurrence of tumors, gene mutations rst appear as preneoplastic lesions [,] . However, recent gene sequencing efforts have uncovered millions of genetic mutations in numerous tumor tissues, suggesting that mutations are present in normal tissues and that does not necessarily lead to adverse outcomes [] .
Surprisingly, high genetic mutations may bene t cervical cancer patients. Therefore, TMB can indirectly re ect the ICI score. Even so, in-depth researches are needed to fully unravel this relationship.

Conclusions
To sum up, we made a comprehensive and detailed analysis of ICI of cervical cancer, evaluated it from many aspects and levels, and painted a clear picture of immune response to cervical cancer. In addition, ICI patterns are closely related to the degree of tumor lesion. Findings of this study lay the foundation for identifying ideal biomarkers for developing cervical cancer immunotherapies. This study is of great signi cance to clinical work.