ABSTRACT

CTLA-4 blocking therapeutic antibodies are currently under investigation in head and neck squamous cell carcinoma (HNSCC). A better understanding of the epigenetic regulation of the CD28 superfamily members CD28, CTLA-4, and ICOS and their B7 ligands, CD80 and CD86, could support the development of biomarkers for response prediction to anti-CTLA-4 immunotherapy.

We investigated methylation of the encoding genes CD28, CTLA4, ICOS, CD80, and CD86 at single CpG resolution (51 CpG sites) in a cohort of HNSCC (N = 528) and normal adjacent tissue samples (N = 50) provided by The Cancer Genome Research Atlas, in isolated blood leukocytes from healthy individuals (N = 28), and HNSCC cell lines (N = 39). We analysed methylation levels with regard to mRNA expression, overall survival, mutational load, interferon-γ signature, and signatures of immune cell infiltrates.

Depending on the location of the CpG sites (promoter, promoter flank, gene body, and intergenic sites), we found significant differences in methylation levels among isolated leukocytes, between leukocytes and HNSCC cell lines, and among HNSCCs. Methylation of all analysed genes correlated inversely or positively with mRNA expression, depending on the CpG site. CD28, CTLA4, and ICOS revealed almost identical correlation patterns. Furthermore, we found significant correlations with survival and features of response to immunotherapy, i.e. interferon-γ signature, signatures of tumour infiltrating immune cells, and mutational load.

Our results suggest CD28, CTLA4, ICOS, CD80, and CD86 expression levels are epigenetically co-regulated by DNA methylation. This study provides rationale to test their DNA methylation as potential biomarker for prediction of response to CTLA-4 immune checkpoint inhibitors.

摘要

目前正在头颈部鳞状细胞癌（HNSCC）中研究阻断CTLA-4的治疗性抗体。更好地理解CD28超家族成员CD28，CTLA-4和ICOS及其B7配体CD80和CD86的表观遗传学调控，可以支持生物标记物的发展，从而预测抗CTLA-4免疫疗法的反应。

我们调查了HNSCC（N =  528）和癌症患者提供的正常邻近组织样本（N =  50）中单个CpG分辨率（51 CpG位点）的编码基因CD28，CTLA4，ICOS，CD80和CD86的甲基化基因组研究图集，来自健康个体（N =  28）和HNSCC细胞系（N = 39）的分离的血液白细胞。我们分析了甲基化水平有关的mRNA表达，总体生存，突变负荷，干扰素-γ签名和免疫细胞浸润的签名。

根据CpG位点的位置（启动子，启动子侧翼，基因体和基因间位点），我们发现分离的白细胞之间，白细胞和HNSCC细胞系之间以及HNSCC之间的甲基化水平存在显着差异。根据CpG位点，所有分析基因的甲基化与mRNA表达成反比或成正比。CD28，CTLA4和ICOS显示出几乎相同的相关模式。此外，我们发现与存活率和对免疫疗法的反应特征显着相关，即干扰素-γ标记，肿瘤浸润性免疫细胞的标记和突变负荷。

我们的结果表明CD28，CTLA4，ICOS，CD80和CD86的表达水平在表观遗传上受DNA甲基化的共同调节。这项研究为测试其DNA甲基化作为预测CTLA-4免疫检查点抑制剂反应的潜在生物标志物提供了理论依据。