Purpose

Epstein-Barr virus associated gastric cancer (EBVaGC) often exhibits a favorable prognosis that correlates with highly methylated viral and host genes and significant immune cell infiltration compared to EBV-negative gastric cancers (GCs). Previously, it has been reported that expression of the IL-15 receptor α (IL-15Rα) is down-regulated in EBVaGC via promoter hypermethylation. In the present study, we offer a novel explanation for this puzzle by associating IL-15Rα expression with infiltration of lymphocytes in GC lesions.

Methods

We investigated the expression of IL-15Rα by RT-PCR, Western-blotting and immunohistochemistry in GC cell lines and primary tissues, respectively. IL-15Rα promoter methylation was analyzed using genomic methylation sequencing. The growth behavior of GC cells was analyzed using MTT, flow cytometry, colony formation, transwell invasion and scratch wound healing assays. Demethylation of IL-15Rα was carried out using 5-Aza-CdR, and rIL-15 was added to evaluate growth promoting effects of the IL-15/IL-15Rα complex. Human peripheral blood mononuclear cells (PBMCs) were co-cultured with GC cells with/without the addition of rIL-15, after which the phosphorylation of STAT5 in PBMCs was evaluated using flow cytometry to estimate the activation of these immune cells through IL-15 binding to IL-2Rβ/γ receptors by in trans presentation.

Results

We found that EBV-positive GC cells (AE) expressed IL-15Rα at a significantly lower level than EBV-negative GC cells (AGS) due to promoter hypermethylation. In the absence of immune cells, IL-15Rα on the cancer cell surface induced a malignant phenotype, including augmented cell growth, migration and invasion, and decreased apoptosis. 5-Aza-CdR reverted AE cells to a more malignant phenotype similar to AGS cells, which may be attributed to activation of the STAT1, STAT3 and ERK1/2 pathways. However, when PBMCs were added to the GC cell cultures, these immune cells were activated as detected by increased pSTAT5 levels. Also, more GC cells underwent apoptosis. These effects were enhanced by the addition of rIL-15 and, subsequently, confirmed in EBVaGC patient samples exhibiting increased expression of T cell surface markers and activation of immune co-stimulating pathways.

Conclusions

Our findings suggest a mechanistic explanation for the clinical association of EBVaGC with a lower IL-15Rα expression, a better prognosis and an increased lymphocyte infiltration. We propose that in highly infiltrated GCs the IL-15/IL-15Rα complex on the GC cell surface may present IL-15 in trans to IL-2Rβ/γ-expressing immune cells to activate these cells in the tumor microenvironment.

中文翻译：

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肿瘤细胞表达的 IL-15Rα 驱动对胃癌进展和免疫控制的拮抗作用，并在 EBV 阳性胃癌中受到表观遗传调节。

目的

与 EBV 阴性胃癌 (GC) 相比，Epstein-Barr 病毒相关胃癌 (EBVaGC) 通常表现出良好的预后，这与高度甲基化的病毒和宿主基因以及显着的免疫细胞浸润相关。以前，有报道称 IL-15 受体 α (IL-15Rα) 的表达通过启动子高甲基化在 EBVaGC 中下调。在本研究中，我们通过将 IL-15Rα 表达与 GC 病变中淋巴细胞的浸润联系起来，为这个难题提供了一个新的解释。

方法

我们分别通过 RT-PCR、蛋白质印迹和免疫组织化学在 GC 细胞系和原代组织中研究了 IL-15Rα 的表达。使用基因组甲基化测序分析 IL-15Rα 启动子甲基化。GC 细胞的生长行为使用 MTT、流式细胞术、集落形成、transwell 侵袭和划痕伤口愈合试验进行分析。使用 5-Aza-CdR 进行 IL-15Rα 的去甲基化，并添加 rIL-15 以评估 IL-15/IL-15Rα 复合物的生长促进作用。人外周血单个核细胞 (PBMC) 与 GC 细胞在添加/不添加 rIL-15 的情况下共培养，然后使用流式细胞术评估 PBMC 中 STAT5 的磷酸化，以估计这些免疫细胞通过 IL-15 的激活通过反式呈递与 IL-2Rβ/γ 受体结合。

结果

我们发现，由于启动子高甲基化，EBV 阳性 GC 细胞 (AE) 表达的 IL-15Rα 水平显着低于 EBV 阴性 GC 细胞 (AGS)。在没有免疫细胞的情况下，癌细胞表面的 IL-15Rα 诱导恶性表型，包括增强的细胞生长、迁移和侵袭，以及减少的细胞凋亡。5-Aza-CdR 将 AE 细胞恢复为类似于 AGS 细胞的更恶性表型，这可能归因于 STAT1、STAT3 和 ERK1/2 通路的激活。然而，当 PBMC 被添加到 GC 细胞培养物中时，这些免疫细胞被激活，如通过增加的 pSTAT5 水平所检测到的。此外，更多的GC细胞经历了细胞凋亡。这些作用通过添加 rIL-15 得到增强，随后，

结论

我们的研究结果表明，EBVaGC 与较低的 IL-15Rα 表达、更好的预后和淋巴细胞浸润增加的临床关联的机制解释。我们提出，在高度浸润的 GC 中，GC 细胞表面的 IL-15/IL-15Rα 复合物可能将 IL-15 呈反式呈现给表达 IL-2Rβ/γ 的免疫细胞，以激活肿瘤微环境中的这些细胞。