A Role for KSHV in the Pathogenesis of Malignancies

KSHV 在恶性肿瘤发病机制中的作用

• 批准号: 10262710
• 负责人: Giovanna Tosato
• 金额: $ 62.55万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262710
• 关键词: AIDS related cancer; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Adult; Antibodies; Aorta; B-Lymphocytes; CD3 Antigens; Cause of Death; Cell Differentiation process; Cell Line; Cell Lineage; Cell Proliferation; Cells; Clinical; Clinical Research; Clonality; Clone Cells; Derivation procedure; Development; Diffuse; Disease; Dorsal; Drug Combinations; Drug Targeting; Embryo; Endothelial Cells; Endothelium; Engraftment; Environment; Environmental Risk Factor; Epstein-Barr Virus Infections; Future; Gene Expression; Gene Proteins; Genes; Goals; Greater sac of peritoneum; HIV; HIV Infections; HIV Seronegativity; Hematopoietic; Hematopoietic stem cells; Hodgkin Disease; Human; Human Herpesvirus 4; Human Herpesvirus 8; ITGAX gene; Immune; Immunity; Immunoblotting; Immunodeficient Mouse; Inflammation; Inflammatory; Kaposi Sarcoma; Laboratory Study; Lesion; Link; Liposomal Doxorubicin; Lubricants; Lymphocyte; Lymphoid; Lymphoma; Lymphoma cell; Lymphomagenesis; MS4A1 gene; Maintenance; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mature B-Lymphocyte; Measures; Mesenchymal; Mesoderm; Mesothelial Cell; Mesothelium; Molecular; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; Natural History; Outcome; Ovarian Carcinoma; PTPRC gene; Pathogenesis; Patients; Peritoneal; Peritoneum; Phenotype; Pilot Projects; Play; Population; Process; Research; Role; Signal Transduction; Source; Structure; Surface; Syndrome; T-Lymphocyte; Therapeutic; Tumor Angiogenesis; Tumorigenicity; Umbilical Cord Blood; VEGFA gene; Viral; Virus Diseases; Work; Yolk Sac; bevacizumab; body cavity; cancer type; cell growth; cell type; cytokine; effusion; hematopoietic stem cell emergence; insight; interest; monolayer; mouse development; mouse model; neoplastic cell; novel; patient population; peripheral blood; phenotypic biomarker; prevent; primary effusion lymphoma; repaired; research clinical testing; success; transdifferentiation; tumor; tumor progression; tumorigenic

We have focused primarily on the study of primary effusion lymphoma in patients with AIDS. Primary effusion lymphoma (PEL) is a Kaposi's sarcoma herpes virus (KSHV)-induced lymphoma that typically arises in body cavities of HIV-infected patients. PEL cells are often co-infected with Epstein-Barr virus (EBV). "PEL-like" lymphoma is a KSHV-unrelated lymphoma that arises in body cavities of HIV-negative patients. "PEL-like lymphoma" is sometimes EBV-positive. The derivation of PEL/"PEL-like" cells is unclear. To study PEL pathogenesis, mesothelial cells were cultured from body cavity effusions of 23 patients. These included patients with AIDS with or without PEL presenting with effusions in body cavities, and patients with ovarian carcinoma with malignant or not malignant effusions. Cell proliferation, cytokine secretion, marker phenotypes, KSHV/EBV infection and clonality were evaluated. Gene expression was measured by qPCR and immunoblotting. A mouse model of PEL was used to evaluate tumorigenicity. We found that mesothelia derived from 6 effusions of HIV-infected patients with PEL or other KSHV-associated diseases contain rare KSHV+ or EBV+ mesothelial cells. After extended culture (16-17 weeks), some mesothelial cells underwent a trans-differentiation process generating lymphoid-type CD45+/B220+, CD5+, CD27+, CD43+, CD11c+ and CD3- cells resembling "B1-cells", most commonly found in mouse body cavities. These "B1-like" cells were short-lived. However, long-term KSHV+EBV- and EBV+KSHV- clonal cell lines emerged from mesothelial cultures from two patients, which were clonally distinct from the monoclonal or polyclonal B-cell populations found in the patient's original effusion. The current study provides a novel and unifying insight into PEL and "PEL-like" lymphomagenesis. Three observations emerged from these studies. First, we discovered that mesothelial cell monolayers undergo a "mesothelial-to-lymphoid" transition (MLT)" resulting in the emergence of "lymphoid-type" cells. This discovery extends the spectrum of mesothelial functional capabilities, beyond secretion of lubricants, maintenance of surface integrity and ability to repair. MLT resembles the emergence of hematopoietic cells from the endothelium of the yolk sac and the dorsal aorta identified as "endothelial-to-hematopoietic transition" (EHT). Endothelial and mesothelial cells can undergo phenotypic and functional change through "endothelial-to-mesenchymal" (EMT) and "mesothelial-to-mesenchymal" transitions (MMT). The second discovery we made is that the lymphocytes emerging from mesothelial cultures have a "B1-like" phenotype, supporting a mesothelial origin of human "B1-type" cells. B1 lymphocytes are the main B-cell population in murine body cavities but are rare elsewhere. Human "B1-like" lymphocytes have been identified in cord and peripheral blood, but to our knowledge not in body cavities. Despite their importance in immune defense, the origin of "B1-type" cells is unclear. During mouse development, B1 lymphocytes are first detected at embryonic day (E) 8.0-8.5 in the para-aortic mesoderm prior to the emergence of hematopoietic stem cells (HSC) from the dorsal aorta, suggesting an HSC-independent origin of B1 cells. All mesothelia that line body cavities derive from the para-aortic splanchnopleural mesoderm. Thus, "B1-type" cells and mesothelia have a common developmental derivation, raising the possibility that persistence of mesodermal precursors within adult mesothelia confers B1-cell differentiation potential to these mesothelia. The third observation we made is that mesothelial cells can be infected by KSHV and EBV. The emergence of monoclonal B-lineage cell lines from mesothelial cultures suggests that PEL and PEL-like lymphoma may derive from KSHV or EBV-infected mesothelial cells. It is noteworthy that two of the 3 lines so derived were clonally distinguishable from the cells originally found in the patient. It remains possible that these lines may have emerged as a result of outgrowth from rare clones of PEL or EBV-infected cells in the original effusion. However, the evidence of MLT transition and clonal analysis suggest that a more likely explanation is that the unique clonal lines derived from the KSHV or EBV-infected mesothelial cells. Interestingly, the "indeterminate" surface phenotype of PEL cells and cell lines, including the KSHV+ 81 lines, resembles the predominant surface phenotype of the "B1-like" cells. In addition, the monoclonal KSHV-/EBV+ clone resembles the KSHV- "PEL-like" lymphoma in showing a mature B-cell phenotype and EBV infection, raising the possibility that the CD20+ cell subset recovered from mesothelial cultures is a source of "PEL-like lymphoma". From this perspective, body cavity-associated PEL and "PEL-like" malignancies would have a common mesothelial derivation. Ongoing and future studies will be focused on determining if this new understanding of PEL pathogenesis can be exploited to prevent PEL development and treat it by targeting the mesothelium. To pursue this goal, we are developing a mouse model in which cultures of human mesothelium infected or not infected with KSHV are explanted into immunodeficient mice. We want to observe the localization of peritoneal explants of mesothelium, its natural history in the mouse body cavity, the potential emergence of "B1"-type cells and the potential emergence of PEL. Since inflammation within the body cavities is commonly detected in patients with AIDS, we will explore the role of experimental inflammation in the emergence of human B1-type cells and PEL. We have obtained engraftment of PEL-derived primary mesothelial cells expanded in culture and transferred into the peritoneal cavity of immunodeficient mice (NSG mice). Macroscopic examination of the peritoneal cavity showed the presence of focal nodular structures and more diffuse thickening of the peritoneum. We did not detect the presence of PEL-type cells floating in the peritoneal cavity. We are currently further evaluating these lesions and their relationship with KSHV diseases. Following up on our previous laboratory studies highlighting the role of VEGFA in the pathogenesis of KS, we have collaborated with the clinical group of HAMB in a pilot study of the anti-VEGF antibody Bevazizumab combined with liposomal doxorubicin in a group of patients with advanced KS. Evaluation of this clinical study showed that patients with advanced KS are responsive to the combination of liposomal doxorubicin and Bevazizumab. Future studies will need to directly assess if the drug combination yields a better outcome than liposomal doxorubicin alone in patients with advanced KS.

我们主要关注艾滋病患者原发性渗出性淋巴瘤的研究。原发性渗出性淋巴瘤 (PEL) 是卡波西肉瘤疱疹病毒 (KSHV) 诱导的淋巴瘤，通常出现在 HIV 感染者的体腔中。 PEL 细胞经常与 Epstein-Barr 病毒 (EBV) 共同感染。 “PEL 样”淋巴瘤是一种与 KSHV 无关的淋巴瘤，发生于 HIV 阴性患者的体腔中。 “PEL 样淋巴瘤”有时是 EBV 阳性。 PEL/“PEL样”细胞的来源尚不清楚。为了研究 PEL 发病机制，我们从 23 名患者的体腔积液中培养了间皮细胞。这些患者包括伴有或不伴有体腔积液的 PEL 的 AIDS 患者，以及伴有恶性或非恶性积液的卵巢癌患者。评估细胞增殖、细胞因子分泌、标记表型、KSHV/EBV 感染和克隆性。通过 qPCR 和免疫印迹测量基因表达。 PEL 小鼠模型用于评估致瘤性。我们发现，来自 6 例患有 PEL 或其他 KSHV 相关疾病的 HIV 感染患者的积液中的间皮含有罕见的 KSHV+ 或 EBV+ 间皮细胞。经过长期培养（16-17 周）后，一些间皮细胞经历了转分化过程，产生类似于“B1 细胞”的淋巴型 CD45+/B220+、CD5+、CD27+、CD43+、CD11c+ 和 CD3- 细胞，最常见于小鼠体腔。这些“B1样”细胞的寿命很短。然而，长期 KSHV+EBV- 和 EBV+KSHV- 克隆细胞系是从两名患者的间皮培养物中产生的，这些细胞系在克隆上与患者原始积液中发现的单克隆或多克隆 B 细胞群不同。目前的研究为 PEL 和“PEL 样”淋巴瘤发生提供了新颖且统一的见解。这些研究得出了三项观察结果。首先，我们发现间皮细胞单层经历“间皮细胞到淋巴”转变（MLT），导致“淋巴型”细胞的出现。这一发现扩展了间皮功能能力的范围，超越了润滑剂的分泌、维持MLT 的表面完整性和修复能力类似于卵黄囊和背主动脉内皮细胞的出现。 “内皮-造血转变”（EHT） 内皮和间皮细胞可以通过“内皮-间质”（EMT）和“间皮-间质”转变（MMT）发生表型和功能变化。得出的结论是，从间皮培养物中产生的淋巴细胞具有“B1样”表型，支持间皮细胞人类“B1 型”细胞的起源 B1 淋巴细胞是小鼠体腔中的主要 B 细胞群，但在其他地方已发现人类“B1 样”淋巴细胞，但据我们所知，在脐带血和外周血中尚未发现。体腔。尽管“B1 型”细胞在免疫防御中很重要，但其起源尚不清楚。在小鼠发育过程中，B1 淋巴细胞首先在胚胎日 (E) 8.0-8.5 时在主动脉旁中胚层中检测到，然后背主动脉出现造血干细胞 (HSC)，这表明 B1 细胞的起源不依赖于 HSC。体腔内的所有间皮均源自主动脉旁内脏胸膜中胚层。因此，“B1型”细胞和间皮具有共同的发育衍生，这提高了成体间皮内中胚层前体的持续存在赋予这些间皮B1细胞分化潜力的可能性。我们的第三个观察结果是间皮细胞可以被 KSHV 和 EBV 感染。间皮培养物中单克隆 B 系细胞系的出现表明 PEL 和 PEL 样淋巴瘤可能源自 KSHV 或 EBV 感染的间皮细胞。值得注意的是，如此衍生的 3 个细胞系中的 2 个与最初在患者体内发现的细胞在克隆上有区别。这些细胞系仍有可能是原始渗出液中 PEL 或 EBV 感染细胞的稀有克隆生长出来的结果。然而，MLT 转变和克隆分析的证据表明，更可能的解释是，独特的克隆系源自 KSHV 或 EBV 感染的间皮细胞。有趣的是，PEL 细胞和细胞系（包括 KSHV+ 81 系）的“不确定”表面表型类似于“B1 样”细胞的主要表面表型。此外，单克隆 KSHV-/EBV+ 克隆在显示成熟 B 细胞表型和 EBV 感染方面与 KSHV-“PEL 样”淋巴瘤相似，这提高了从间皮培养物中回收的 CD20+ 细胞亚群是“PEL 样”淋巴瘤来源的可能性。 -类似淋巴瘤”。从这个角度来看，体腔相关的 PEL 和“PEL 样”恶性肿瘤具有共同的间皮来源。正在进行和未来的研究将集中于确定是否可以利用对 PEL 发病机制的新理解来预防 PEL 的发展并通过靶向间皮来治疗它。为了实现这一目标，我们正在开发一种小鼠模型，将感染或未感染 KSHV 的人间皮培养物移植到免疫缺陷小鼠体内。我们想要观察腹膜间皮外植体的定位、其在小鼠体腔中的自然历史、“B1”型细胞的潜在出现和PEL的潜在出现。由于艾滋病患者通常会检测到体腔内炎症，因此我们将探讨实验性炎症在人类 B1 型细胞和 PEL 出现中的作用。我们已经获得了 PEL 来源的原代间皮细胞的植入，这些细胞在培养物中扩增并转移到免疫缺陷小鼠（NSG 小鼠）的腹膜腔中。腹膜腔的宏观检查显示存在局灶性结节结构和腹膜更弥漫性增厚。我们没有检测到腹膜腔中漂浮的 PEL 型细胞的存在。我们目前正在进一步评估这些病变及其与 KSHV 疾病的关系。继我们之前强调 VEGFA 在 KS 发病机制中的作用的实验室研究之后，我们与 HAMB 临床组合作，在一组晚期 KS 患者中进行了抗 VEGF 抗体贝伐珠单抗 (Bevazizumab) 联合脂质体阿霉素的初步研究。这项临床研究的评估表明，晚期 KS 患者对脂质体阿霉素和贝伐珠单抗的联合治疗有反应。未来的研究需要直接评估对于晚期 KS 患者，药物组合是否比单独使用脂质体阿霉素能产生更好的结果。