背景:ACAP1在内吞再循环中起着关键作用,这对淋巴细胞的正常功能至关重要。然而,ACAP1在淋巴细胞中的表达和功能很少被研究。
方法:大规模基因组数据,包括多个批量RNA测序数据集,单细胞测序数据集,和免疫疗法队列,被用来全面表征ACAP1表达,regulation,和功能。使用基因集富集分析(GSEA)来揭示与ACAP1表达相关的途径。八种算法,包括TIMER,CIBERSORT,CIBERSORT-ABS,QUANTISER,xCELL,MCPCOUNTER,EPIC,和潮汐,用于估计免疫细胞的浸润水平。西方印迹,qPCR,和ChIP-PCR用于验证生物信息学分析的结果。T细胞共培养杀伤试验用于研究ACAP1在淋巴细胞中的功能。
结果:ACAP1在免疫相关组织和细胞中高表达,在其他组织中最低限度地表达。此外,肿瘤样本中的单细胞测序分析显示,ACAP1主要在肿瘤浸润淋巴细胞(TIL)中表达,包括T,B,NK细胞ACAP1表达受启动子DNA甲基化负调控,其启动子在免疫细胞中低甲基化,但在其他细胞中高甲基化。此外,SPI1与ACAP1启动子结合,并在免疫细胞中正向调节其表达。ACAP1水平与TIL的浸润水平呈正相关,尤其是CD8+T细胞,广泛的实体癌症类型。ACAP1缺乏与检查点阻断疗法(ICT)治疗的多种癌症类型的不良预后和免疫治疗反应相关。功能上,通过RNA干扰消除ACAP1显著损害T细胞介导的肿瘤细胞杀伤。
结论:我们的研究表明,ACAP1对于TILs的正常功能至关重要,它的缺乏表明对ICT具有抗性的肿瘤的免疫学“冷”状态。
BACKGROUND: ACAP1 plays a key role in endocytic recycling, which is essential for the normal function of lymphocytes. However, the expression and function of
ACAP1 in lymphocytes have rarely been studied.
METHODS: Large-scale genomic data, including multiple bulk RNA-sequencing datasets, single-cell sequencing datasets, and immunotherapy cohorts, were exploited to comprehensively characterize ACAP1 expression, regulation, and function. Gene set enrichment analysis (GSEA) was used to uncover the pathways associated with
ACAP1 expression. Eight algorithms, including TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, xCELL, MCPCOUNTER, EPIC, and TIDE, were applied to estimate the infiltrating level of immune cells. Western blotting, qPCR, and ChIP-PCR were used to validate the findings from bioinformatic analyses. A T-cell co-culture killing assay was used to investigate the function of
ACAP1 in lymphocytes.
RESULTS: ACAP1 was highly expressed in immune-related tissues and cells and minimally in other tissues. Moreover, single-cell sequencing analysis in tumor samples revealed that
ACAP1 is expressed primarily in tumor-infiltrating lymphocytes (TILs), including T, B, and NK cells. ACAP1 expression is negatively regulated by promoter DNA methylation, with its promoter hypo-methylated in immune cells but hyper-methylated in other cells. Furthermore, SPI1 binds to the ACAP1 promoter and positively regulates its expression in immune cells. ACAP1 levels positively correlate with the infiltrating levels of TILs, especially CD8+ T cells, across a broad range of solid cancer types. ACAP1 deficiency is associated with poor prognosis and immunotherapeutic response in multiple cancer types treated with checkpoint blockade therapy (ICT). Functionally, the depletion of ACAP1 by RNA interference significantly impairs the T cell-mediated killing of tumor cells.
CONCLUSIONS: Our study demonstrates that ACAP1 is essential for the normal function of TILs, and its deficiency indicates an immunologically \"cold\" status of tumors that are resistant to ICT.