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Abstract：
BACKGROUND: The immune checkpoint HERV-H LTR-associating 2 (HHLA2) is expressed in kidney cancer and various other tumor types. Therapeutics targeting HHLA2 or its inhibitory receptor KIR3DL3 are being developed for solid tumors, including renal cell carcinoma (RCC). However, the regulation of HHLA2 expression remains poorly understood. A better understanding of HHLA2 regulation in tumor cells and the tumor microenvironment is crucial for the successful translation of these therapeutic agents into clinical applications.
METHODS: Flow cytometry and quantitative real-time PCR were used to analyze HHLA2 expression in primary kidney tumors ex vivo and during in vitro culture. HHLA2 expression in A498 and 786-O ccRCC cell lines was examined in vitro and in subcutaneous tumor xenografts in NSG mice. Monocytes and dendritic cells were analyzed for HHLA2 expression. We tested a range of cytokines and culture conditions, including hypoxia, to induce HHLA2 expression.
RESULTS: Analysis of HHLA2 expression revealed that HHLA2 is expressed on tumor cells in primary kidney tumors ex vivo; however, its expression gradually diminishes during a 4-week in vitro culture period. A498 and 786-O ccRCC tumor cell lines do not express HHLA2 in vitro, but HHLA2 expression was observed when grown as subcutaneous xenografts in NSG immunodeficient mice. Induction experiments using various cytokines and culture conditions failed to induce HHLA2 expression in A498 and 786-O tumor cell lines in vitro. Analysis of HHLA2 expression in monocytes and dendritic cells demonstrated that only IL-10 and BMP4, along with IL-1β and IL-6 to a lesser extent, modestly enhanced HHLA2 protein and mRNA expression.
CONCLUSIONS: HHLA2 expression is induced on kidney cancer cells in vivo by a tumor microenvironmental signal that is not present in vitro. HHLA2 expression is differentially regulated in kidney cancer epithelial cells and monocytes. Cytokines, particularly IL10, that induce HHLA2 expression in monocytes fail to upregulate HHLA2 expression in tumor cell lines in vitro. These findings underscore the importance of the interplay between tumor cell and tumor microenvironmental signals in the regulation of HHLA2. Further investigation is warranted to elucidate the mechanisms involved in HHLA2 regulation and its implications for therapeutic development.
METHODS: Flow cytometry and quantitative real-time PCR were used to analyze HHLA2 expression in primary kidney tumors ex vivo and during in vitro culture. HHLA2 expression in A498 and 786-O ccRCC cell lines was examined in vitro and in subcutaneous tumor xenografts in NSG mice. Monocytes and dendritic cells were analyzed for HHLA2 expression. We tested a range of cytokines and culture conditions, including hypoxia, to induce HHLA2 expression.
RESULTS: Analysis of HHLA2 expression revealed that HHLA2 is expressed on tumor cells in primary kidney tumors ex vivo; however, its expression gradually diminishes during a 4-week in vitro culture period. A498 and 786-O ccRCC tumor cell lines do not express HHLA2 in vitro, but HHLA2 expression was observed when grown as subcutaneous xenografts in NSG immunodeficient mice. Induction experiments using various cytokines and culture conditions failed to induce HHLA2 expression in A498 and 786-O tumor cell lines in vitro. Analysis of HHLA2 expression in monocytes and dendritic cells demonstrated that only IL-10 and BMP4, along with IL-1β and IL-6 to a lesser extent, modestly enhanced HHLA2 protein and mRNA expression.
CONCLUSIONS: HHLA2 expression is induced on kidney cancer cells in vivo by a tumor microenvironmental signal that is not present in vitro. HHLA2 expression is differentially regulated in kidney cancer epithelial cells and monocytes. Cytokines, particularly IL10, that induce HHLA2 expression in monocytes fail to upregulate HHLA2 expression in tumor cell lines in vitro. These findings underscore the importance of the interplay between tumor cell and tumor microenvironmental signals in the regulation of HHLA2. Further investigation is warranted to elucidate the mechanisms involved in HHLA2 regulation and its implications for therapeutic development.
摘要：
背景：免疫检查点HERV-HLTR相关2（HHLA2）在肾癌和各种其他肿瘤类型中表达。靶向HHLA2或其抑制性受体KIR3DL3的治疗剂正在开发用于实体瘤，包括肾细胞癌（RCC）。然而,对HHLA2表达的调控仍知之甚少。更好地理解肿瘤细胞和肿瘤微环境中的HHLA2调节对于将这些治疗剂成功转化为临床应用至关重要。
方法：流式细胞术和定量实时PCR用于分析离体和体外培养期间原发性肾肿瘤中的HHLA2表达。在体外和NSG小鼠的皮下肿瘤异种移植物中检查了A498和786-OccRCC细胞系中的HHLA2表达。分析单核细胞和树突细胞的HHLA2表达。我们测试了一系列细胞因子和培养条件，包括缺氧,以诱导HHLA2表达。
结果：对HHLA2表达的分析显示，HHLA2在离体原发性肾肿瘤的肿瘤细胞上表达；然而，在4周的体外培养期间，其表达逐渐减少。A498和786-OccRCC肿瘤细胞系在体外不表达HHLA2，但是当在NSG免疫缺陷小鼠中作为皮下异种移植物生长时观察到HHLA2表达。使用各种细胞因子和培养条件的诱导实验未能在体外诱导A498和786-O肿瘤细胞系中的HHLA2表达。对单核细胞和树突状细胞中HHLA2表达的分析表明，只有IL-10和BMP4，以及IL-1β和IL-6在较小程度上，适度增强HHLA2蛋白和mRNA表达。
结论:HHLA2表达在体内肾癌细胞上由体外不存在的肿瘤微环境信号诱导。HHLA2表达在肾癌上皮细胞和单核细胞中被差异调节。细胞因子,特别是诱导单核细胞中HHLA2表达的IL10不能在体外上调肿瘤细胞系中的HHLA2表达。这些发现强调了肿瘤细胞和肿瘤微环境信号之间相互作用在HHLA2调节中的重要性。需要进一步的研究来阐明HHLA2调节的机制及其对治疗发展的影响。
方法：流式细胞术和定量实时PCR用于分析离体和体外培养期间原发性肾肿瘤中的HHLA2表达。在体外和NSG小鼠的皮下肿瘤异种移植物中检查了A498和786-OccRCC细胞系中的HHLA2表达。分析单核细胞和树突细胞的HHLA2表达。我们测试了一系列细胞因子和培养条件，包括缺氧,以诱导HHLA2表达。
结果：对HHLA2表达的分析显示，HHLA2在离体原发性肾肿瘤的肿瘤细胞上表达；然而，在4周的体外培养期间，其表达逐渐减少。A498和786-OccRCC肿瘤细胞系在体外不表达HHLA2，但是当在NSG免疫缺陷小鼠中作为皮下异种移植物生长时观察到HHLA2表达。使用各种细胞因子和培养条件的诱导实验未能在体外诱导A498和786-O肿瘤细胞系中的HHLA2表达。对单核细胞和树突状细胞中HHLA2表达的分析表明，只有IL-10和BMP4，以及IL-1β和IL-6在较小程度上，适度增强HHLA2蛋白和mRNA表达。
结论:HHLA2表达在体内肾癌细胞上由体外不存在的肿瘤微环境信号诱导。HHLA2表达在肾癌上皮细胞和单核细胞中被差异调节。细胞因子,特别是诱导单核细胞中HHLA2表达的IL10不能在体外上调肿瘤细胞系中的HHLA2表达。这些发现强调了肿瘤细胞和肿瘤微环境信号之间相互作用在HHLA2调节中的重要性。需要进一步的研究来阐明HHLA2调节的机制及其对治疗发展的影响。
