突变型<i>PSMB5</i>通过下调非小细胞肺癌抗原加工呈递功能介导抗PD-1免疫治疗耐药

韩解非; 陈恺; 王策; 陈峰; 李文斌

doi:10.12019/j.issn.1671-5144.2023.01.008

突变型PSMB5通过下调非小细胞肺癌抗原加工呈递功能介导抗PD-1免疫治疗耐药

Acquired Resistance to Anti-PD-1 Therapy Induced by Mutant PSMB5 Down-Regulation Antigen Processing and Presentation in Non-Small Cell Lung Cancer

摘要

摘要:
目的探讨突变型编码蛋白酶体B5亚基的基因（proteasome subunit beta 5，PSMB5）介导非小细胞肺癌抗程序性死亡受体1（programmed death 1，PD-1）免疫治疗继发耐药的作用及机制。
方法对6例接受免疫治疗的继发PSMB5 p.A79T或p.M104I突变的非小细胞肺癌患者治疗前及耐药后肿瘤组织进行RNA测序，对比基因表达差异。利用I-Mutant 2.0评估PSMB5突变对蛋白稳定性的影响。对抗PD-1免疫治疗前后的肿瘤组织通过免疫组化检测CD8表达水平。流式分选患者免疫治疗期间的PD-1⁺CD8⁺ T细胞，并进行T细胞受体（T cell receptor，TCR）测序，结合外周血游离细胞DNA（cell free DNA，cfDNA）测序结果进行克隆进化分析。利用肿瘤细胞与T细胞共培养体系，明确突变型PSMB5对T细胞应答的影响。
结果 PSMB5 p.A79T或p.M104I突变可导致蛋白稳定性降低，基因信号通路富集分析（gene set enrichment analysis，GSEA）提示突变型PSMB5相比免疫治疗前野生型PSMB5抗原呈递能力下降，同时微环境中细胞溶解评分也低于基线水平。免疫组化结果指出，突变型PSMB5肿瘤组织中CD8表达较野生型显著减少。通过外周血cfDNA及TCR动态监测及克隆分析，随着PSMB5克隆的出现，PD-1⁺CD8⁺ TCR并未呈现出相应的丰度变化，提示未发生有效免疫应答。在突变型/野生型PSMB5肿瘤细胞与T细胞共培养的过程中，突变型PSMB5未能刺激T细胞的扩增，同时流式细胞术检测突变型PSMB5 4-1BB CD8 T细胞的比例明显低于野生型。
结论突变型PSMB5可能下调肿瘤细胞抗原加工呈递功能，介导T细胞的应答障碍从而导致免疫治疗继发耐药。

Abstract:
Objective To explore the role of mutant PSMB5 in required resistance to immunotherapy in non-small cell lung cancer (NSCLC).
Methods Comparison of RNA expression was performed on six pairs of pre- and post-treatment tumor tissues from patients who received immunotherapy. CD8 expression levels were detected by immunohistochemistry in paired tumor samples. PD-1⁺CD8⁺ T cells were sorted through flow cytometry for TCR sequencing. Cell-free DNA (cfDNA) was extracted for genetic analysis. We co-cultured tumor cells with mutant or wide PSMB5 with T cells to explore the impact of PSMB5 on the immune response.
Results Gene set enrichment analysis (GSEA) showed that the antigen presentation ability of mutant PSMB5 from baseline was lower than wild-type PSMB5 after resistance to immunotherapy, as well as the cytolytic activity score of the tumor microenvironment. The immunohistochemical results showed that CD8 expression in mutant PSMB5 tumors was significantly reduced compared with wild type. Clonal analysis of dynamic cfDNA and PD-1⁺CD8⁺ TCR showed immune ignoring with the emergence of PSMB5 clones. In vitro coculture of immune cells with mutant or wide-type PSMB5 tumor cells showed the mutant PSMB5 failed to stimulate the expansion of T cells, following flow cytometry analysis indicated a lower proportion of 4-1BB in the mutant PSMB5 stimulated CD8 T cells.
Conclusions We identified that mutant PSMB5 was associated with the dysfunction of the proteasome B5 subunit and impaired the function and quantity of lymphocytes in the tumor microenvironment.

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