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. 2024 Apr 1;42(2):192-206.
doi: 10.7518/hxkq.2024.2023280.

Verification of the expression trend and interaction prediction of innate immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis

[Article in English, Chinese]
Kaiyu Li  1 , Lijuan Shi  1 , Linxin Liu  1 , Jie Wang  1 , Minhai Nie  1 , Xuqian Liu  1
Affiliations

Verification of the expression trend and interaction prediction of innate immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis

[Article in English, Chinese]
Kaiyu Li et al. Hua Xi Kou Qiang Yi Xue Za Zhi. .

Abstract
in English, Chinese

Objectives: This study aimed to explore the expression trends of innate immune cells and immune-checkpoint molecules validated by data calculation in the process of oral mucosal carcinogenesis, as well as to explore methods of suppressing oral mucosal carcinogenesis based on immunotherapy by predicting their interactions. Me-thods 1) The cancer genome atlas (TCGA) database comprehensively scores immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis and screens out intrinsic immune cells and immune-checkpoint molecules that interfere with tumor immune escape. 2) Clinical patient blood routine data were collected for the statistical analysis of peripheral blood immune cells during the progression of oral mucosal carcinogenesis. Immune cells in peripheral blood that may affect the progression of oral mucosal carcinogenesis were screened. 3) Immunohistochemical staining was performed on intrinsic immune cells and immune-checkpoint molecules validated based on data calculation in various stages of oral mucosal carcinogenesis. 4) Special staining was used to identify innate immune cells in various stages of oral mucosal carcinogenesis based on data-calculation verification. 5) Survival analysis was conducted on intrinsic immune cells and immune-checkpoint molecules validated based on data calculation during the process of oral mucosal carcinogenesis. The association of intrinsic immune cells and immune-checkpoint molecules with the prognosis of oral squamous cell carcinoma was verified.

Results: The expression of monocytes and neutrophils increased during the process of oral mucosal carcinogenesis. The expression of eosinophils showed a single peak trend of up and down. The expression of mast cells decreased. In the process of oral mucosal carcinogenesis, the expression of the immune-checkpoint molecules cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death-ligand (PD-L1) increased. The expression trends of monocytes, neutrophils, and eosinophils were positively correlated with those of CTLA4 and PD-L1 immune-checkpoint molecules. The expression trend of mast cells was negatively correlated with the expression of CTLA4 and PD-L1. Monocytes, neutrophils, and eosinophils may promote tumor immune escape mediated by CTLA4 and/or PD-L1, thereby accelerating the progression of oral mucosal carcinogenesis. Mast cells may inhibit tumor immune escape mediated by CTLA4 and/or PD-L1, delaying the progression of oral mucosal carcinogenesis.

Conclusions: Therefore, interference with specific immune cells in innate immunity can regulate the expression of CTLA4 and/or PD-L1 to a certain extent, inhibit tumor immune escape, and delay the progression of oral mucosal carcinogenesis.

目的: 研究口腔黏膜癌变进程中基于数据计算验证的固有免疫细胞和免疫检查点分子的表达趋势，并通过预测其交互作用，探索免疫治疗抑制口腔黏膜癌变进程的方法。方法: 1）利用癌症基因组图谱对口腔黏膜癌变进程中的免疫细胞和免疫检查点分子进行全面评分，筛选出干扰肿瘤细胞免疫逃逸的固有免疫细胞和免疫检查点分子；2）收集血常规资料，对口腔黏膜癌变进程中外周血免疫细胞进行统计学分析，筛选外周血中可能影响口腔黏膜癌变进程的免疫细胞；3）对口腔黏膜癌变进程各阶段中基于数据计算验证的固有免疫细胞和免疫检查点分子进行免疫组织化学染色；4）采用特殊染色鉴定口腔黏膜癌变进程各阶段中基于数据计算验证的固有免疫细胞；5）对口腔黏膜癌变进程中基于数据计算验证的固有免疫细胞和免疫检查点分子进行生存分析，验证固有免疫细胞和免疫检查点分子与口腔鳞状细胞癌预后间的关联。结果: 在口腔黏膜癌变进程中，单核细胞、中性粒细胞表达呈上升趋势；嗜酸性粒细胞表达呈升降单峰趋势；肥大细胞表达呈下降趋势；免疫检查点分子细胞毒性T淋巴细胞相关蛋白4（CTLA4）和细胞程序性死亡-配体1（PD-L1）的表达呈上升趋势。单核细胞、中性粒细胞和嗜酸性粒细胞表达趋势与CTLA4和PD-L1免疫检查点分子的表达趋势正相关；肥大细胞表达趋势与CTLA4和PD-L1免疫检查点分子的表达趋势负相关。单核细胞、中性粒细胞和嗜酸性粒细胞可能促进CTLA4和（或）PD-L1介导的肿瘤细胞免疫逃逸，加速口腔黏膜癌变进程；肥大细胞可能抑制CTLA4和（或）PD-L1介导的肿瘤细胞免疫逃逸，延缓口腔黏膜癌变进程。结论: 干扰固有免疫中特定免疫细胞可在一定程度上调控CTLA4和（或）PD-L1的表达，抑制肿瘤细胞免疫逃逸，延缓口腔黏膜癌变进程。.

Keywords: immune checkpoint molecules; immune escape; immunotherapy; inherent immune cells; malignant transformation of oral mucosa.

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Conflict of interest statement

利益冲突声明：作者声明本文无利益冲突。

Figures

图 1
图 1. HNSCC组织和正常组织中免疫细胞评分的分布情况
Fig 1 The distribution of immune cell scores in HNSCC tissues and normal tissues A：活化髓样树突状细胞、B淋巴细胞、CD4+记忆T淋巴细胞、CD4+幼稚T淋巴细胞、CD4+非调节性T细胞、CD4+中枢记忆T淋巴细胞、CD4+效应记忆T细胞、CD8+幼稚T淋巴细胞、CD8+T淋巴细胞、CD8+中枢记忆T淋巴细胞；B：CD8+效应T淋巴细胞、转换记忆B淋巴细胞、普通髓系祖细胞、淋巴样祖细胞、髓样树突状细胞、内皮细胞、嗜酸性粒细胞、粒-单核细胞祖细胞、造血干细胞、巨噬细胞；C：M1巨噬细胞、M2巨噬细胞、肥大细胞、记忆B淋巴细胞、单核细胞、幼稚B淋巴细胞、中性粒细胞、NK细胞、NK-T淋巴细胞、浆细胞样树突状细胞；D：B淋巴细胞基质、T淋巴细胞、CD4+辅助性T细胞1、CD4+辅助性T细胞2、调节性T细胞、免疫评分、基质评分、免疫微环境评分。横坐标代表免疫细胞浸润类型，纵坐标代表该免疫浸润评分在不同组的分布情况。*P<0.05，**P<0.01，***P<0.001，-差异无统计学意义。
图 2
图 2. 免疫检查点分子相关基因的表达热图
Fig 2 Immunological checkpoint molecule related gene expression heatmap 不同颜色代表在不同样本中的表达趋势，***P<0.001。
图 3
图 3. HNSCC组织和正常组织中免疫检查点分子基因的表达分布情况
Fig 3 The expression distribution of immune checkpoint molecular genes in HNSCC tissue and normal tissue ***P<0.001。
图 4
图 4. CYH、MCP1、P29和PRG2的IHC染色结果
Fig 4 IHC results of CYH, MCP1, P29 and PRG2 箭头示各免疫细胞的胞质显棕黄色，为阳性表达。第1、3、5、7行 × 200，第2、4、6、8行 × 400。
图 5
图 5. CYH、MCP1、P29和PRG2的IHC吸光光密度值分析结果
Fig 5 IHC absorbance optical density analysis of CYH, MCP1, P29 and PRG2 A：CYH在口腔黏膜癌变进程中的表达趋势；B：CYH在OSCC各分化阶段中的表达趋势；C：MCP1在口腔黏膜癌变进程中的表达趋势；D：MCP1在OSCC各分化阶段中的表达趋势；E：P29在口腔黏膜癌变进程中的表达趋势；F：P29在OSCC各分化阶段中的表达趋势；G：PRG2在口腔黏膜癌变进程中的表达趋势；H：PRG2在OSCC各分化阶段中的表达趋势。*P<0.05，**P<0.01，***P<0.001，ns差异无统计学意义。
图 6
图 6. CTLA4、PD-L1、PD-L2的IHC染色结果
Fig 6 IHC staining results of CTLA4, PD-L1, PD-L2 箭头示各免疫细胞胞质显棕黄色，为阳性表达。第1、3、5、7行 × 200，第2、4、6、8行 × 400。
图 7
图 7. CTLA4、PD-L1、PD-L2的IHC吸光光密度值分析结果
Fig 7 IHC absorbance optical density analysis of CTLA4, PD-L1, PD-L2 A：CTLA4在口腔黏膜癌变进程中的表达趋势；B：CTLA4在OSCC各分化阶段中的表达趋势；C：PD-L1在口腔黏膜癌变进程中的表达趋势；D：PD-L1在OSCC各分化阶段中的表达趋势；E：PD-L2在口腔黏膜癌变进程中的表达趋势；F：PD-L2在OSCC各分化阶段中的表达趋势。*P<0.05，**P<0.01，***P<0.001，ns差异无统计学意义。
图 8
图 8. 特殊染色结果
Fig 8 Special staining results 第1、2行：甲苯胺蓝染色法，箭头示肥大细胞细胞核呈蓝色，细胞颗粒呈紫红色；第3、4行：Carbol 2R染色法，箭头示嗜酸性粒细胞细胞核呈蓝色，细胞颗粒呈粉红色。第1、3行 × 200，第2、4行 × 400。
图 9
图 9. CYH、MCP1、P29和PRG2的生存分析图
Fig 9 Survival analysis of CYH, MCP1, P29 and PRG2 A：CYH总生存期；B：CYH无病生存期；C：MCP1总生存期；D：MCP1无病生存期；E：P29总生存期；F：P29无病生存期；G：PRG2总生存期；H：PRG2无病生存期。
图 10
图 10. CTLA4、PD-L1和PD-L2的生存分析图
Fig 10 Survival analysis of CTLA4, PD-L1 and PD-L2 A：CTLA4总生存期；B：CTLA4无病生存期；C：PD-L1总生存期；D：PD-L1无病生存期；E：PD-L2总生存期；F：PD-L2无病生存期。
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