Down‐regulation of EOMES drives T‐cell exhaustion via abolishing EOMES‐mediated repression of inhibitory receptors of T cells in liver cancer

T‐cell exhaustion is one of the hallmarks in cancer, but the mechanisms underlying T‐cell dysregulation remains unclear. Here, we reported that down‐regulation of transcription factor EOMES contributed to increased levels of inhibitory receptors in T cell among the tumour tissues and resulted in the poor prognosis of hepatocellular carcinoma (HCC). By analysing the correlation between EOMES in tumour‐infiltrating T cells and the clinical features, we demonstrated that the EOMES was related to the advanced stage and poor prognosis of HCC. Further mechanistic studies revealed that the EOMES mainly expressed in the CD8+ T cells and were down‐regulated in tumour samples. Moreover, we demonstrated that the EOMES directly bound at the transcriptional regulatory regions of the key inhibitory factors including PD‐1, CTAL‐4 and CD39, and lower levels of EOMES contributed to overexpression of these factors in T cells. Together, our studies provide new insight into the transcriptional deregulation of the inhibitory receptors on T cells during the tumorigenesis.

cells and its related immune cells has been applied in many malignant tumours including HCC. Some patients have achieved a successful response after therapy. 3 Immune escape is one of the hallmarks of cancer. 4 The immune cells lost the ability to clearance of the tumour cells or even promote the growth and invasion of tumour cells, such as the activation of the inhibitory between T cells and tumour cells. 3 The cell surface receptors in T cells including PD-1, CTAL-4 and CD39 interact with the ligand expressed on the tumour cells and trigger a 'do-not-eat me' signal, which protected the tumour cells from T-cell-mediated clearance. 5,6 Targeting these immune blockades by using antibodies such as anti-PD-1 or anti-PD-L1 reactivated the effects of T cells on the tumour cells and achieved great successes in some patients with cancer including HCC. [7][8][9] Still, many patients do not benefit from such therapy due to some unclear reasons. Thus, systematical unveiling the detailed mechanisms underlying the dysregulation of T cells in tumours is needed for developing new strategies of immunotherapy.

Eomesodermin (EOMES) is a T-box transcription factor originally
identified as a regulator of mesodermal development. 10,11 It initiates the transcriptional network governing the formation of endoderm via direct interaction with SMAD2/3 and transcription repressed the mesoderm genes. 12 Lines of evidence also reveal that the EOMES regulates the maturation and function of CD8 + T cells, 13 γδT cells 14 and NK cells 15  Here, in our study, we analysed the expression of EMOES in the tumour-infiltrated lymphocytes in 342 patients with HCC and found the high levels of EMOES expression were correlated with favourable prognosis of HCC. These observations were also supported by the TCGA cohorts. Further single-cell transcriptomic analysis reveals the EMOES mainly expressed in the CD8 + T cells and dysregulated in tumour-infiltrated CD8 + T cells as compared to the T cells in normal liver tissue. Moreover, ChIP-seq re-analysis revealed that the EMOES directly bound to the enhancer or promoter regions inhibitory receptors including PD-1 (PDCD1), CD39 (ENTPD1) and CTLA-4 (CTLA4). The Barcelona Clinic Liver Cancer (BCLC) staging system was applied for clinical staging. 16 Tumour differentiation was graded by the Edmond-son-Steiner grading system. 17 Time to recurrence (TTR) and overall survival (OS) were defined as the interval from primary surgical resection to the first recurrence or death, respectively. The follow-up was censored on 31 March, 2012.

| Immunohistochemistry
Tissue microarray (TMA) was constructed as previously described. 18 Triplicates of 1-mm cores from two different areas (tumour centre and non-tumour tissues that at least 1 cm away from tumour margin) were obtained for each case to ensure the reproducibility of the staining. The immunohistochemistry of the TMA chips was performed as previously described. 18 Briefly, the TMA chip was deparaffinized in xylene and then hydrated with a graded alcohol series. The antigen retrieval was carried out in a pressure   19 ) in three visual fields in each 1-mm cylinder was totally counted by two experienced pathologists who were blinded to the clinicopathological data of the patients. The assessments were performed independently, and disagreements were solved by discussion. Cell counts were expressed as the mean value of all nine fields.

| Analysis of the single-cell transcriptome data
The single-cell transcriptome data of T cells isolated from patients with HCC or normal donors were retrieved from http://hcc.cance r-pku.cn/. 20 The expression of EOMES across the T cells, as well as the expression of EOMES in tumour-infiltrating or normal T cells, was plotted according to the websites' instructions.

| ChIP-seq re-analysis
The ChIP-seq of EOMES was retrieved from the Gene Expression Omnibus (GEO) database with the accession of GSM640691. 21 The raw data were downloaded using the fastq-dump algorithm. The fastq files were mapped to the hg38 reference genome using the bowtie2 software. The visualized files were generated by the homer suite.

| Analysis of the expression of PD-1, CTAL-4 and CD39 in T cells with EOMES overexpression
The microarray data of the T cells with lentiviral transduction of EOMES were retrieved from the GEO database (GSE11 0545). 22 The expression of the PD-1, CTAL-4 and CD39 was extracted using the R program under the 'affy' package.

| Statistical analysis
Normally distributed continuous data were expressed as mean ± standard deviation (SD) and analysed using the Student's t test. Categorical data were presented as frequencies and analysed using the chi-square test. Survival curves were computed using the Kaplan-Meier method and analysed using the log-rank test. All analyses were conducted using SPSS 17.0 (IBM, Armonk, NY, USA). Twotailed P-values <.05 were considered statistically significant.

| EOMES-positive T cells were decreased in advanced stages of HCC
To investigate the role of EOMES in T cells, we first examined the expression of EOMES in tumour and peritumoral samples of HCC in a cohort containing 384 patients with HCC. The EOMES was mainly expressed in the nucleus of lymphocytes and the EOMES-positive cells in peritumoral samples were significantly higher than those in tumour samples ( Figure 1A-B). Next, we determined the correlation between the EOMES-positive score in tumour and peritumoral samples and the tumour stages of HCC. As shown in Figure 1C,D, the EOMES-positive score was significantly decreased in advanced stages of HCC, but not significantly change in different stages of peritumoral samples. Also, we performed flow cytometry analysis in HCC samples and adjacent normal tissues and found the EOMES mainly expressed in the CD8-positive T cells, and the EOMES levels in HCC samples were lower as compared to adjacent normal tissues ( Figure S1).
Next, we collected the expression profiling of HCC samples in TCGA cohorts and determined the expression of EOMES in the RNAseq data of HCC samples as well as the normal control. In accordant with our observation using immunohistochemistry staining, the expression of EOMES was significantly decreased in tumour samples and stage IV HCC showed the lowest EOMES expression ( Figure 1E).
We also determined the correlation between EOMES expression in tumour-associated lymphocytes and the clinicopathologic characteristics of HCC patients. We found lower EOMES score was significantly enriched in tumour size >5 cm tumour tissues (P < .001) and stage III-V patient samples (P = .005) ( Table 1). But these were no significant correlation between EOMES score and gender, age, HBV infection, AFP score, cirrhosis and tumour number (Table 1). Suggesting down-regulation of EOMES in the lymphocytes might contribute to HCC progression.

| Low levels of EOMES correlated with poor prognosis of HCC
To further investigate the prognostic value of EOMES in the tumour sample of HCC. We separate the HCC into two groups according to the EOMES score. We found that the lower EOMES score in tumour samples shown to be associated with poor prognosis of HCC ( Figure 2A). Further analysis using the TCGA cohort further supported our finding ( Figure 2B). Together, these observations suggested that the lower levels of EOMES-positive cells might contribute to the development and progression of HCC and the expression of EOMES might serve as a marker for the prognosis of HCC.

| EOMES regulates the expression of inhibitory receptors of immune checkpoints
Since EOMES functions as a transcription factor in gene regulation, we speculate that the deregulated EOMES might also contribute to the deregulation of genes important in T cells of HCC. We thus retrieved the ChIP-seq data of EOMES in the GEO database (GSM640691). Interestingly, we found that the EOMES directly bound to many genes involved in T cells regulation ( Figure 4A).
We found EOMES directly bound to the regulatory region of PD-1

| D ISCUSS I ON
T-cell exhaustion is one of the hallmarks of cancer, but the mechanisms underlying T-cell dysfunction remain unclear. In this study, we level of immune checkpoint protein, such as PD-1 and CTLA4, in tumour-infiltrated T cells remain largely unexplored. We here reported that the EOMES gene was down-regulated and contributed to highly expressed PD-1 and CTLA4 in tumour-infiltrated T cells.
Noteworthy, several studies also reported that the higher levels of PD-1 in T cells are a poor prognostic factors for patients without immune therapy in many cancers including liver cancer. [25][26][27][28] We here reported the lower levels of EOMES might contribute to the higher levels of PD-1 and resulted in an unfavourable prognosis of liver cancer.
EOMES is a transcription factor mainly expressed in T cells and NK cells. Normally, EOMES enhances the expression of IFNγ in CD8 + T cells. 13,29,30 In addition, the EOMES expression is in- EOMES has been reported to control the type of adaptive immune responses within the tumour site via promoting Th1 fate and suppressing Th17 differentiation. 13,39 It suppresses the promoter activity of Rorc and IL-17a through directly transcriptional regulation. 40 We showed that EOMES expression was higher in per- suggested to play an important part in controlling CD8 + T-cell activity and lymph node metastasis in human colorectal cancer. 37 Dielmann et al 41 showed that high EOMES expression was associated with better survival of patients with renal cell cancer treated with sorafenib, suggesting that lower levels of EOMES might be a common signature for cancer with poor prognosis. Still, additional studies are necessary to examine the exact mechanisms involved in these observations.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.