腺苷诱导耗竭型CD8+T细胞影响肾癌细胞迁移及凋亡

Influence of depletion type CD8+ T cells induced by adenosine on migration and apoptosis of renal carcinoma cells

摘要:

目的 探索腺苷是否诱导耗竭型T细胞生成以及对肾透明细胞癌细胞迁移和凋亡的影响。方法 流式细胞术检测肾透明细胞癌及癌旁组织中CD8⁺T细胞及耗竭表型分子3（TIM-3）的表达。生物信息学分析肾透明细胞癌及癌旁组织中基因表达差异，寻找影响T细胞耗竭基因。利用shRNA技术构建稳定敲除该基因的肾透明细胞癌786-O细胞株，检测细胞的迁移、凋亡等生物学行为。制备不同肿瘤条件培养基，培养CD8⁺T细胞，研究敲除基因后的肾透明细胞癌细胞对耗竭型T细胞的影响。结果 肾透明细胞癌与癌旁组织相比高表达CD8⁺T细胞及TIM-3。生物信息学分析发现腺苷合成限速酶NT5E（CD73）在肾透明细胞癌中的表达高于癌旁组织。检测临床标本发现肾透明细胞癌组织中腺苷及CD73的表达高于癌旁组织。与对照组比较，敲除NT5E组的786-O细胞迁移能力下降及凋亡增加。敲除NT5E的肿瘤条件培养基培养的CD8⁺T细胞中程序性死亡蛋白1表达减少，抑制T细胞增殖能力下降。结论 肾透明细胞癌组织存在耗竭型CD8⁺T细胞、腺苷及CD73的高表达。敲除NT5E的肾透明细胞癌786-O细胞迁移能力下降、凋亡增加。敲除NT5E可减少腺苷合成，减少耗竭型T细胞的生成。

关键词:肾透明细胞癌;腺苷;耗竭型CD8⁺T细胞;NT5E（CD73）;CD8⁺T细胞;肿瘤微环境

Abstract:

Objective To investigate the induction of exhausted T cell generation by adenosine and its impact on the migration and apoptosis of renal clear cell carcinoma cells.Methods Flow cytometry was employed to assess the expression of CD8⁺ T cells and the exhaustion marker TIM-3 in renal clear cell carcinoma and adjacent tissues. Bioinformatics analysis was conducted to identify genes that influence T cell exhaustion in renal clear cell carcinoma and adjacent tissues. Stable knockdown of the gene was achieved in renal clear cell carcinoma 786-O cells using shRNA technology, and the cellular behaviors including migration and apoptosis were assessed. Various tumor-conditioned media were prepared to culture CD8⁺ T cells and investigate the impact of gene knockout in renal clear cell carcinoma cells on exhausted T cells.Results Renal clear cell carcinoma exhibited elevated expression levels of CD8⁺ T cells and TIM-3 in comparison to adjacent tissues. Bioinformatics analysis revealed increased expression of adenosine synthesis rate-limiting enzyme NT5E (CD73) in renal clear cell carcinoma. Clinical specimens exhibited higher expression levels of adenosine and CD73 in renal clear cell carcinoma tissues when compared to adjacent tissues. Knockout of NT5E in 786-O cells led to reduced migration ability and enhanced apoptosis compared to the control group. CD8⁺ T cells cultured in tumor-conditioned media with NT5E knockout exhibited diminished expression of programmed death protein 1 and decreased proliferation capacity.Conclusion Renal clear cell carcinoma tissues display exhausted CD8⁺ T cells and elevated expression of adenosine and CD73. Knockout of NT5E in renal clear cell carcinoma 786-O cells diminishes migration ability and enhances apoptosis. Knockout of NT5E can decrease adenosine synthesis and attenuate the generation of exhausted T cells.

Key words:renal clear cell carcinoma;adenosine;exhausted T lymphocytes;NT5E (CD73);CD8⁺ T cells;tumor microenvironment