# Analysis of the Relationship between Scleritis andT Cell Activation in Patients with Hepatocellular Carcinoma Treated with PD-1 Carrelizumab

Authors: Yazhen Yuan; Shuaixi Pan; Changpeng Zou; Yan Guo; Changwang Zhang; Zheng Zheng; Ninghan Wu; Na Li
Journal: Contrast Media & Molecular Imaging
 (2022)

---

## Abstract

In order to explore the function of inhibiting the immune effect, the relationship between programmed death receptor 1 (PD-1) carrelizumab in the treatment of hepatocellular carcinoma-induced scleritis and T cell activation is investigated. A total of 120 patients with primary liver cancer treated in the department of oncology of our hospital from July 2020 to January 2022 are selected and treated with carrelizumab. According to the occurrence of PD-1 carrelizumab treatment, the patients are divided into the scleritis group and nonscleritis group. The levels of T cells, PD-1, PD-L1 proteins, and serum inflammatory factors at different time points are compared. The experimental results show that the occurrence of scleritis after liver cancer treatment with PD-1 carrelizumab is closely associated with Treg cells, the percentage of Th17 cells, the expression of PD-1, PD-L1 proteins, and inflammatory factors. It is clearly evident that PD-1 carrelizumab can increase the risk of scleritis by affecting T cell activation.


---

## Body

## 1. Introduction

Primary liver cancer is one of the common malignant tumors in China, which is involved by many factors and plays a synergistic role. Among them, hepatitis B is the main factor inducing liver cancer. The number of people carrying the hepatitis virus is nearly 100 million in China, which is a high-risk group of primary liver cancer. Therefore, actively preventing the occurrence of liver cancer is an important issue that needs to be solved urgently in Chinese medicine [1]. Clinical surgical treatment requires a high liver function reserve, and it is easy to relapse after operation. Patients with liver cancer have poor sensitivity to radiotherapy, so radiofrequency ablation and chemoembolization can be used for local treatment. This treatment has a certain clinical effect, but it is only used in cases of small nodular lesions and a small number of lesions. The effect of comprehensive treatment of patients with advanced liver cancer is not obvious, which fails to meet clinical expectations [2]. Immune checkpoint inhibitors are new targets for clinical cancer therapy. It can inhibit the immune activity of checkpoints and activate the tumor immune response effect of T cells, thus playing a certain antitumor effect. Among them, PD-1 carilizhu as a new drug has made some achievements in clinical tumor treatment in recent years, with less adverse reactions. However, PD-1 carrelizumab treatment can cause certain toxic reactions, including the skin, eyes, and heart. Scleritis is one of the ocular toxic reactions after PD-1 carrelizumab treatment in patients with liver cancer, and its pathological features include inflammation of the scleral matrix layer caused by cell infiltration, collagen destruction, and vascular reconstruction. Scleritis is caused by mediated vasculitis and is related to immune system diseases [3]. Currently, there is no unified conclusion on the mechanism of interaction between programmed cell death-1 (PD-1) carrelizumab in the treatment of scleritis induced by primary liver cancer and T cell activation [4]. Therefore, this study further analyzed the relationship between scleritis induced by PD-1 carrelizumab treatment and T cell activation in primary liver cancer, providing a new target for subsequent clinical treatment of liver cancer.The rest of the paper is organized as follows: Section2 discusses related studies, and Section 3 presents the general information and experimental method. Section 4 describes the experimental results and analysis. The conclusions are provided in Section 5.


## 2. Related Work

As a transmembrane protein, PD-1 is widely expressed in T cells and B cells. TheT cell receptor (TCR) and cytokines can activate PD-1 on T cells. PD-1 can bind to T cells and inhibit the cremation of its proximal myase during the activation of T cells, so as to inhibit the immune effect [5]. As a natural ligand of PD-1, PD-L1 can inhibit the immune-mediated killing process of cancer cells by transmitting antiapoptotic signals of cancer cells. Therefore, because of the high expression of PD-1, PD-L1 can cause the immune escape of cancer cells, thus aggravating the development of the disease [6]. Lyon et al. [7] indicate that the expression of PD-1 and PD-L1 proteins in both groups showed a significant downward trend, which may be due to the existence of two common antigens between tumor cells and scleral fibroblasts. One is that the muscle antigens of tumor antigens are homologous with different TCR targets and the other is specific with different TCR targets. PD-1 and PD-L1 protein expressions are decreased when PD-1 carrelizumab treatment is performed, and the drug may target the common antigen, resulting in scleral injury and scleritis.Th17 cells are a kind of helper T cells that are different from Th1 and Th2 cells. Th17 cells can secrete pro-inflammatory factors such as IL-17, IL-6, and TNF-α to participate in the occurrence and development of various inflammatory diseases [8]. Il-17 secreted by Th17 cells can induce and promote macrophage synthesis, inflammatory factor secretion, and complement C3 synthesis [9]. Treg cells can inhibit the production of IL-13, IL-5, and other proinflammatory factors, thus inhibiting the increase of eosinophils. In addition, Treg cells can secrete IL-10, which is an anti-inflammatory factor. When the body's immunity is activated, it can inhibit the activation of relevant cells and the generation of proinflammatory cells in the process of immune response. IL-10 can effectively control inflammation by reducing the expression of reactivity, and inhibit the secretion and generation of IL-6 and other proinflammatory factors. Treg cells regulate the body's antigen immune response through this pathway [10, 11]. Further analysis of the changes of Treg cell population, IL-10, Th17 cell population, Th17/Treg, IL-6, TNF-α, and other indicators in the scleritis group indicates that the correlation between the activation of T cells and the expression of related inflammatory factors in scleritis after PD-1 carrelizumab treatment should be explored in-depth. The reason may be that ruili is resistant to PD-1 single treatment and can play an active role by blocking the immune signaling pathway targeted by PD-1/PD-L1, enhancing the immune system function, activating T cells, and improving inflammation. It should be noted that the immune function of the tumor microenvironment of selective may selectively restore immune deficiency induced by tumors, which can cause immune system imbalance and immune tolerance [12]. From the perspective of IFN-γ analysis, IFN-γ is the main cytokine secreted by immune Th1 and can be conductive to promote the differentiation of T cells and B cells and clearing human pathogens. Proliferation of CD8+T cells after overactivation of T cells expressing IFN-γ will result in abnormal increase of IFN-γ. Activated myeloid cells express TNF-α and IL-6 inflammatory substances and cxCL9/10 chemokines, which further induce peripheral T cells to infiltrate the intestine and cause inflammation [13].


## 3. General Information and Experimental Method

### 3.1. General Information

A total of 120 patients with primary liver cancer treated in the Department of Oncology, First Hospital of Hebei Medical University, from July 2020 to January 2022 are selected. According to the occurrence of PD-1 carrelizumab treatment, the patients are divided into the scleritis group (15 cases) and nonscleritis group (105 cases), respectively. The age ranged from 45 to 75 years, with an average of 60.41 ± 4.32 years, including 76 males and 44 females, 49 stage III and 71 stage IV. The inclusion criteria are as follows: (1) combined CT/MRI and tumor marker (AFP) detection of primary liver cancer confirmed clinically or by preoperative biopsy and pathology; (2) there is no indication for surgical resection, or the patient refuses surgery; (3) Child–Pugh A or B; (4) estimated survival ≥12 weeks. Besides, the exclusion criteria are as follows: (1) use of immunosuppressive drugs within 14 days before administration; (2) the presence or history of any active autoimmune disease; (3) patients with vitiligo and asthma; (4) AIDS, active hepatitis B, hepatitis C, and patients requiring antiviral treatment during the study; (5) severe infection occurred within 4 weeks before medication; (6) participated in any other drug clinical studies within 4 weeks prior to initial administration [14].


### 3.2. Experimental Method

#### 3.2.1. Treatment Plan

Carrelizumab treatment PD-1 carrelizumab 200 mg is administered intravenously for 30 minutes, with an interval of 3 weeks for 2 months.


#### 3.2.2. Detection of the Percentage of Treg Cells and Th17 Cells

Before and 2 months after treatment, 8 ml fasting venous blood is collected in the morning, and 5 ml is extracted and placed in a heparin anticoagulant tube. Complete medium 800 ml, 1 mlPMA, and 1μl lonomycin are added, repeated blowing and fully mixed, and then cultured in a 5% CO2 environment for 4 hours. PE Cy5 antihuman CD4 is added and incubated under dark conditions. After the membrane is broken, PE antihuman IL-17a, APC antihuman FOXP3, and FITC antihuman CD25 are added, and the reaction continued under dark conditions for half an hour. 2 ml PBS is added and centrifuged at 1000 r/min speed and 7 cm radius. After 10 min, the supernatant is removed, and CD4 + IL-17A + Th17 and CD4 + CD25 + Foxp3 + Treg cells are detected and analyzed by up-flow cytometry.


#### 3.2.3. Detection of PD-1 and PD-L1 Proteins by WB

Western blot is used to detect PD-1 and PD-L1 proteins from T1 to T2 [15–17]. The liver cancer tissue is thoroughly ground, 200 μl of lysis buffer is added, and an ice bath is performed for 30 min. After full lysis, the supernatant is centrifuged for 10 min at a speed of 1300 r/min and a radius of 6.5 cm at 4°C. The supernatant is separated into containers and stored at −20°C for future use. A BCA assay kit is used to detect the protein concentration. Sds-page electrophoresis is used to isolate the protein, and then, the membrane is transferred to a PVDF membrane and labeled. TBST is used for cleaning for 10 min and repeated 3 times. 5% skim milk powder is added to seal for 2 h, a primary solvent is added placed in a 37°C environment. the bed is fully shaked and mixed, and incubated overnight. Clean with TBST for 10 min and repeat 3 times. The second antibody is added and the reaction is continued for 2 hours at room temperature. Then, clean with TBST for 10 min and repeat the operation 3 times. Exposure is carried out in ECL chemiluminescence liquid obscura. Gel-pro32 software is used for gray analysis of the obtained results, and the protein expression of PD-1 and PD-L1 is calculated [18, 19].


### 3.3. Inflammatory Factors

3 ml venous blood samples are taken and centrifuged at 4°C for 10 min at a speed of 1000 r/min and a radius of 20 cm at 3000 g. The levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), interleukin-6 (IL-6) in serum are measured by ELISA. IL-6 and interferon γ (IFN-γ) are tested in strict accordance with the kit instructions.


### 3.4. Statistical Methods

SPSS 26.0 software is used for statistical analysis [20, 21]. The mean ± standard deviation x±s is used to represent the measurement data of the normal distribution. The T test and the x2 test is used to compare the count data. P<0.05 denotes that the difference is statistically significant.


## 3.1. General Information

A total of 120 patients with primary liver cancer treated in the Department of Oncology, First Hospital of Hebei Medical University, from July 2020 to January 2022 are selected. According to the occurrence of PD-1 carrelizumab treatment, the patients are divided into the scleritis group (15 cases) and nonscleritis group (105 cases), respectively. The age ranged from 45 to 75 years, with an average of 60.41 ± 4.32 years, including 76 males and 44 females, 49 stage III and 71 stage IV. The inclusion criteria are as follows: (1) combined CT/MRI and tumor marker (AFP) detection of primary liver cancer confirmed clinically or by preoperative biopsy and pathology; (2) there is no indication for surgical resection, or the patient refuses surgery; (3) Child–Pugh A or B; (4) estimated survival ≥12 weeks. Besides, the exclusion criteria are as follows: (1) use of immunosuppressive drugs within 14 days before administration; (2) the presence or history of any active autoimmune disease; (3) patients with vitiligo and asthma; (4) AIDS, active hepatitis B, hepatitis C, and patients requiring antiviral treatment during the study; (5) severe infection occurred within 4 weeks before medication; (6) participated in any other drug clinical studies within 4 weeks prior to initial administration [14].


## 3.2. Experimental Method

### 3.2.1. Treatment Plan

Carrelizumab treatment PD-1 carrelizumab 200 mg is administered intravenously for 30 minutes, with an interval of 3 weeks for 2 months.


### 3.2.2. Detection of the Percentage of Treg Cells and Th17 Cells

Before and 2 months after treatment, 8 ml fasting venous blood is collected in the morning, and 5 ml is extracted and placed in a heparin anticoagulant tube. Complete medium 800 ml, 1 mlPMA, and 1μl lonomycin are added, repeated blowing and fully mixed, and then cultured in a 5% CO2 environment for 4 hours. PE Cy5 antihuman CD4 is added and incubated under dark conditions. After the membrane is broken, PE antihuman IL-17a, APC antihuman FOXP3, and FITC antihuman CD25 are added, and the reaction continued under dark conditions for half an hour. 2 ml PBS is added and centrifuged at 1000 r/min speed and 7 cm radius. After 10 min, the supernatant is removed, and CD4 + IL-17A + Th17 and CD4 + CD25 + Foxp3 + Treg cells are detected and analyzed by up-flow cytometry.


### 3.2.3. Detection of PD-1 and PD-L1 Proteins by WB

Western blot is used to detect PD-1 and PD-L1 proteins from T1 to T2 [15–17]. The liver cancer tissue is thoroughly ground, 200 μl of lysis buffer is added, and an ice bath is performed for 30 min. After full lysis, the supernatant is centrifuged for 10 min at a speed of 1300 r/min and a radius of 6.5 cm at 4°C. The supernatant is separated into containers and stored at −20°C for future use. A BCA assay kit is used to detect the protein concentration. Sds-page electrophoresis is used to isolate the protein, and then, the membrane is transferred to a PVDF membrane and labeled. TBST is used for cleaning for 10 min and repeated 3 times. 5% skim milk powder is added to seal for 2 h, a primary solvent is added placed in a 37°C environment. the bed is fully shaked and mixed, and incubated overnight. Clean with TBST for 10 min and repeat 3 times. The second antibody is added and the reaction is continued for 2 hours at room temperature. Then, clean with TBST for 10 min and repeat the operation 3 times. Exposure is carried out in ECL chemiluminescence liquid obscura. Gel-pro32 software is used for gray analysis of the obtained results, and the protein expression of PD-1 and PD-L1 is calculated [18, 19].


## 3.2.1. Treatment Plan

Carrelizumab treatment PD-1 carrelizumab 200 mg is administered intravenously for 30 minutes, with an interval of 3 weeks for 2 months.


## 3.2.2. Detection of the Percentage of Treg Cells and Th17 Cells

Before and 2 months after treatment, 8 ml fasting venous blood is collected in the morning, and 5 ml is extracted and placed in a heparin anticoagulant tube. Complete medium 800 ml, 1 mlPMA, and 1μl lonomycin are added, repeated blowing and fully mixed, and then cultured in a 5% CO2 environment for 4 hours. PE Cy5 antihuman CD4 is added and incubated under dark conditions. After the membrane is broken, PE antihuman IL-17a, APC antihuman FOXP3, and FITC antihuman CD25 are added, and the reaction continued under dark conditions for half an hour. 2 ml PBS is added and centrifuged at 1000 r/min speed and 7 cm radius. After 10 min, the supernatant is removed, and CD4 + IL-17A + Th17 and CD4 + CD25 + Foxp3 + Treg cells are detected and analyzed by up-flow cytometry.


## 3.2.3. Detection of PD-1 and PD-L1 Proteins by WB

Western blot is used to detect PD-1 and PD-L1 proteins from T1 to T2 [15–17]. The liver cancer tissue is thoroughly ground, 200 μl of lysis buffer is added, and an ice bath is performed for 30 min. After full lysis, the supernatant is centrifuged for 10 min at a speed of 1300 r/min and a radius of 6.5 cm at 4°C. The supernatant is separated into containers and stored at −20°C for future use. A BCA assay kit is used to detect the protein concentration. Sds-page electrophoresis is used to isolate the protein, and then, the membrane is transferred to a PVDF membrane and labeled. TBST is used for cleaning for 10 min and repeated 3 times. 5% skim milk powder is added to seal for 2 h, a primary solvent is added placed in a 37°C environment. the bed is fully shaked and mixed, and incubated overnight. Clean with TBST for 10 min and repeat 3 times. The second antibody is added and the reaction is continued for 2 hours at room temperature. Then, clean with TBST for 10 min and repeat the operation 3 times. Exposure is carried out in ECL chemiluminescence liquid obscura. Gel-pro32 software is used for gray analysis of the obtained results, and the protein expression of PD-1 and PD-L1 is calculated [18, 19].


## 3.3. Inflammatory Factors

3 ml venous blood samples are taken and centrifuged at 4°C for 10 min at a speed of 1000 r/min and a radius of 20 cm at 3000 g. The levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), interleukin-6 (IL-6) in serum are measured by ELISA. IL-6 and interferon γ (IFN-γ) are tested in strict accordance with the kit instructions.


## 3.4. Statistical Methods

SPSS 26.0 software is used for statistical analysis [20, 21]. The mean ± standard deviation x±s is used to represent the measurement data of the normal distribution. The T test and the x2 test is used to compare the count data. P<0.05 denotes that the difference is statistically significant.


## 4. Results and Analysis

### 4.1. Percentage Difference between Treg Cells and Th17 Cells

The number of Treg cells on T1∼T2 in 120 patients showed an increasing trend, while the number of Th17 cells and Th17/Treg showed a decreasing trend. In addition, the number of Treg cells on T2 in the scleritis group is higher, and the number of Th17 cells and Th17/Treg are smaller. There are statistical differences in all indicators (P<0.05), as shown in Table 1 and Figure 1. The symbol “∗” means that the value is compared with T1 and P<0.05.Table 1
Percentage difference between Treg cells and Th17 cells (‾x ± s).
GroupNumber of Treg cells (%)Number of Th17 cells (%)Th17/TregT1T2T1T2T1T2The nonscleral group (n = 105)4.72 ± 1.195.49 ± 0.81∗1.59 ± 0.491.27 ± 0.39∗2.26 ± 0.691.12 ± 0.49∗Scleritis group (n = 15)4.68 ± 1.186.58 ± 0.78∗1.58 ± 0.511.03 ± 0.28∗2.29 ± 0.710.56 ± 0.29∗t−0.1224.896−0.074−2.2960.157−4.310P0.903<0.0010.9410.0230.876<0.001Figure 1
Percentage changes of Treg cells and Th17 cells.


### 4.2. Protein Expression Differences between PD-1 and PD-L1

PD-1 and PD-L1 proteins showed a decreasing trend from T1 to T2, and PD-1 and PD-L1 protein levels are lower in the scleritis group at T2, with statistical differences in all indicators (P<0.05), as shown in Table 2 and Figure 2.Table 2
Protein expression differences between PD-1 and PD-L1x±s.
GroupPD-1PD-L1T1T2T1T2Scleritis group (n = 15)288.72 ± 21.40225.49 ± 0.81∗341.59 ± 40.59271.27 ± 0.39∗Nonscleral group (n = 105)290.68 ± 21.38246.58 ± 0.78∗341.63 ± 40.61301.03 ± 0.28∗t−0.332−97.504−0.004−365.228P0.740<0.0010.997<0.001Figure 2
Protein expression differences between PD-1 and PD-L1.


### 4.3. Differences in Expression of Inflammatory Factors

The levels of IL-10 and IFN-γ increased from T1 to T2 and are lower in the scleritis group, while the levels of IL-6 and TNF-α decreased and are higher in the scleritis group, with statistical differences (P<0.05), as shown in Table 3 and Figure 3.Table 3
Expression differences of inflammatory factorsx±s.
GroupIL-10 (pg/mL)IL-6 (pg/L)TNF-α (ng/L)IFN-γ (ng/L)T1T2T1T2T1T2T1T2Scleritis group (n = 15)2.12 ± 0.913.52 ± 0.42∗54.62 ± 7.1942.29 ± 5.16∗58.82 ± 6.3145.31 ± 5.26∗633.44 ± 91.021001.22 ± 215.34∗Nonscleral group (n = 105)2.14 ± 0.894.55 ± 0.22∗54.56 ± 7.1822.28 ± 5.13∗58.86 ± 6.2821.28 ± 5.11∗630.44 ± 88.211213.45 ± 294.82∗t−0.081−14.7980.03014.121−0.02316.9770.123−2.683P0.935<0.0010.976<0.0010.982<0.0010.9030.008Figure 3
Changes in the expression of inflammatory factors.


## 4.1. Percentage Difference between Treg Cells and Th17 Cells

The number of Treg cells on T1∼T2 in 120 patients showed an increasing trend, while the number of Th17 cells and Th17/Treg showed a decreasing trend. In addition, the number of Treg cells on T2 in the scleritis group is higher, and the number of Th17 cells and Th17/Treg are smaller. There are statistical differences in all indicators (P<0.05), as shown in Table 1 and Figure 1. The symbol “∗” means that the value is compared with T1 and P<0.05.Table 1
Percentage difference between Treg cells and Th17 cells (‾x ± s).
GroupNumber of Treg cells (%)Number of Th17 cells (%)Th17/TregT1T2T1T2T1T2The nonscleral group (n = 105)4.72 ± 1.195.49 ± 0.81∗1.59 ± 0.491.27 ± 0.39∗2.26 ± 0.691.12 ± 0.49∗Scleritis group (n = 15)4.68 ± 1.186.58 ± 0.78∗1.58 ± 0.511.03 ± 0.28∗2.29 ± 0.710.56 ± 0.29∗t−0.1224.896−0.074−2.2960.157−4.310P0.903<0.0010.9410.0230.876<0.001Figure 1
Percentage changes of Treg cells and Th17 cells.


## 4.2. Protein Expression Differences between PD-1 and PD-L1

PD-1 and PD-L1 proteins showed a decreasing trend from T1 to T2, and PD-1 and PD-L1 protein levels are lower in the scleritis group at T2, with statistical differences in all indicators (P<0.05), as shown in Table 2 and Figure 2.Table 2
Protein expression differences between PD-1 and PD-L1x±s.
GroupPD-1PD-L1T1T2T1T2Scleritis group (n = 15)288.72 ± 21.40225.49 ± 0.81∗341.59 ± 40.59271.27 ± 0.39∗Nonscleral group (n = 105)290.68 ± 21.38246.58 ± 0.78∗341.63 ± 40.61301.03 ± 0.28∗t−0.332−97.504−0.004−365.228P0.740<0.0010.997<0.001Figure 2
Protein expression differences between PD-1 and PD-L1.


## 4.3. Differences in Expression of Inflammatory Factors

The levels of IL-10 and IFN-γ increased from T1 to T2 and are lower in the scleritis group, while the levels of IL-6 and TNF-α decreased and are higher in the scleritis group, with statistical differences (P<0.05), as shown in Table 3 and Figure 3.Table 3
Expression differences of inflammatory factorsx±s.
GroupIL-10 (pg/mL)IL-6 (pg/L)TNF-α (ng/L)IFN-γ (ng/L)T1T2T1T2T1T2T1T2Scleritis group (n = 15)2.12 ± 0.913.52 ± 0.42∗54.62 ± 7.1942.29 ± 5.16∗58.82 ± 6.3145.31 ± 5.26∗633.44 ± 91.021001.22 ± 215.34∗Nonscleral group (n = 105)2.14 ± 0.894.55 ± 0.22∗54.56 ± 7.1822.28 ± 5.13∗58.86 ± 6.2821.28 ± 5.11∗630.44 ± 88.211213.45 ± 294.82∗t−0.081−14.7980.03014.121−0.02316.9770.123−2.683P0.935<0.0010.976<0.0010.982<0.0010.9030.008Figure 3
Changes in the expression of inflammatory factors.


## 5. Conclusion

In this study, the relationship between programmed death receptor 1 (PD-1) carrelizumab in the treatment of hepatocellular carcinoma-induced scleritis and T cell activation is investigated. The levels of T cells, PD-1, PD-L1 proteins, and serum inflammatory factors at different time points are compared. The occurrence of scleritis after liver cancer treatment with PD-1 carrelizumab is closely associated with Treg cells, the percentage of Th17 cells, the expression of PD-1, PD-L1 proteins, and inflammatory factors. The results indicate that PD-1 carrelizumab can increase the risk of scleritis by affecting T cell activation. Also, there are still some deficiencies in this study, such as a small sample size and incomplete inclusion of immune indicators. A small sample size may lead to data bias in subsequent analysis, and there are many types of immune T cell indexes, which may be related to the occurrence of scleritis in patients with liver cancer after PD-1 carrelizumab treatment. In the future, we will expand the sample size and include more immune indicators in the follow-up research for more in-depth research.



---

*Source: 4853481-2022-09-05.xml*