A Role for KSHV in the Pathogenesis of Malignancies

我们主要关注艾滋病患者原发性积液性淋巴瘤的研究。原发性积液性淋巴瘤（PEL）是一种卡波西肉瘤疱疹病毒（KSHV）诱导的淋巴瘤，通常发生在hiv感染患者的体腔中。PEL细胞常与eb病毒（EBV）共感染。“pel样”淋巴瘤是一种与kshv无关的淋巴瘤，发生在hiv阴性患者的体腔中。“pel样淋巴瘤”有时是ebv阳性。PEL/“类PEL”细胞的来源尚不清楚。为了研究PEL的发病机制，我们从23例患者的体腔积液中培养间皮细胞。这些患者包括伴有或不伴有PEL的艾滋病患者，并伴有体腔积液，以及伴有恶性或非恶性积液的卵巢癌患者。检测细胞增殖、细胞因子分泌、标志物表型、KSHV/EBV感染及克隆性。采用qPCR和免疫印迹法检测基因表达。采用小鼠PEL模型评价其致瘤性。我们发现6例hiv感染PEL或其他KSHV相关疾病患者的渗出液中含有罕见的KSHV+或EBV+间皮细胞。经过长时间培养（16-17周），一些间皮细胞经历了反分化过程，产生了类似于“b1细胞”的淋巴型CD45+/B220+、CD5+、CD27+、CD43+、CD11c+和CD3-细胞，这些细胞最常见于小鼠体腔。这些“b1样”细胞寿命很短。然而，长期的KSHV+EBV-和EBV+KSHV-克隆细胞系从两名患者的间皮培养中出现，它们与患者原始积液中发现的单克隆或多克隆b细胞群在克隆上不同。目前的研究为PEL和“PEL样”淋巴瘤形成提供了一种新颖而统一的见解。这些研究得出了三个观察结果。首先，我们发现间皮细胞单层经历了“间皮到淋巴细胞”的转变（MLT），导致“淋巴型”细胞的出现。这一发现扩展了间皮功能的范围，超越了润滑剂的分泌、表面完整性的维持和修复能力。MLT类似于卵黄囊内皮和背主动脉造血细胞的出现，被称为“内皮向造血过渡”（EHT）。内皮细胞和间皮细胞可以通过“内皮-间充质”（EMT）和“间皮-间充质”转化（MMT）经历表型和功能的改变。我们的第二个发现是，从间皮培养中产生的淋巴细胞具有“b1样”表型，支持人类“b1型”细胞的间皮起源。B1淋巴细胞是小鼠体腔中主要的b细胞群，但在其他地方很少见。人类“b1样”淋巴细胞已在脐带血和外周血中发现，但据我们所知，在体腔中没有发现。尽管它们在免疫防御中很重要，但“b1型”细胞的起源尚不清楚。在小鼠发育过程中，在胚胎日(E) 8.0-8.5时，在主动脉旁中胚层中首次检测到B1淋巴细胞，之后才出现了来自背主动脉的造血干细胞（HSC），这表明B1细胞的起源与HSC无关。体腔周围的所有间皮细胞都起源于主动脉旁胸膜间胚层。因此，“b1型”细胞和间皮层具有共同的发育起源，这就提出了一种可能性，即在成年间皮层中持续存在的中胚层前体赋予了这些间皮层向b1细胞分化的潜力。第三，我们观察到间皮细胞可以被KSHV和EBV感染。从间皮培养中出现的单克隆b系细胞系表明，PEL和PEL样淋巴瘤可能来源于感染KSHV或ebv的间皮细胞。值得注意的是，这样得到的3个细胞系中有两个与最初在患者体内发现的细胞有克隆性区别。这些细胞系仍有可能是原始积液中罕见的PEL或ebv感染细胞克隆生长的结果。然而，MLT转化和克隆分析的证据表明，一个更可能的解释是，独特的克隆系来源于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细胞系）的“不确定”表面表型与“b1样”细胞的主要表面表型相似。此外，单克隆KSHV-/EBV+克隆与KSHV-“pel样”淋巴瘤相似，表现出成熟的b细胞表型和EBV感染，这提高了从间皮培养中恢复的CD20+细胞亚群是“pel样淋巴瘤”来源的可能性。从这个角度来看，体腔相关的PEL和“PEL样”恶性肿瘤可能具有共同的间皮起源。正在进行的和未来的研究将集中在确定是否可以利用这种对PEL发病机制的新认识来预防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、在小鼠体腔内的自然历史、“B1”型细胞的潜在出现以及PEL的潜在出现。由于艾滋病患者体腔内常见炎症，我们将探讨实验性炎症在人b1型细胞和PEL出现中的作用。我们已经获得了在培养中扩增的pel来源的原代间皮细胞，并将其移植到免疫缺陷小鼠（NSG小鼠）的腹腔。腹膜腔的宏观检查显示局灶性结节结构和腹膜弥漫性增厚。我们没有检测到腹腔内漂浮的pel型细胞。我们目前正在进一步评估这些病变及其与KSHV疾病的关系。在我们之前强调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Bevazizumab联合阿霉素脂质体的试点研究。该临床研究的评估表明，晚期KS患者对阿霉素脂质体联合贝伐单抗有反应。未来的研究将需要直接评估在晚期KS患者中，联合用药是否比单独使用阿霉素脂质体产生更好的结果。在一项合作研究中，我们进一步研究了病毒IL-6在HSHV感染中的作用。我们现在已经发现，vIL-6通过表观遗传沉默CAV1的表达，通过调节STAT3-DNMT1复合物的形成来促进血管生成和肿瘤发生。这一新发现将为今后KHSV感染内皮细胞引起的表观遗传改变的研究提供指导。

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. In a collaborative study, we have examined further the role of viral IL-6 in HSHV infection. We have now discovered that vIL-6 epigenetically silences caveolin-1 (CAV1) expression to promote angiogenesis and tumorigenesis by regulating the formation of STAT3-DNMT1 complex. This new information will guide future studies on epigenetic changed induced by KHSV infection of endothelial cells.