Studies of Receptor Interactions and Effects of Alarmins

受体相互作用和警报素作用的研究

• 批准号: 10262039
• 负责人: JOOST J OPPENHEIM
• 金额: $ 131.19万
• 依托单位: DIVISION OF BASIC 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/10262039
• 关键词: Accounting; Acute; Adjuvant; Antigens; Binding Proteins; Biopsy Specimen; CT26; Cancer Vaccines; Cell Line; Cell surface; Cells; Chemicals; China; Chinese Traditional Medicine; Chronic; Collaborations; Colon; Colonic Neoplasms; Cyclophosphamide; Dendritic Cells; Development; Dose; Endosomes; Enteric Nervous System; Epithelial Cells; Exhibits; Genes; Genetically Engineered Mouse; Growth; HMGN Proteins; HMGN1 gene; Hepatocyte; Hospitals; Human; IRF3 gene; Immune; Immune checkpoint inhibitor; Immune response; Immunization; Immunologic Adjuvants; Immunologics; Immunotherapeutic agent; In Vitro; Infection; Infiltration; Inflammatory; Injections; Interferons; Interleukin-12; Intestines; Intravenous; Kidney; Knockout Mice; Ligands; Liver; Longevity; Lung; MAP Kinase Gene; Mediating; Mitogen-Activated Protein Kinases; Mononuclear; Mus; Mutation; NF-kappa B; Neurodegenerative Disorders; Nuclear; Nucleosomes; Organism; Parkinson Disease; Pathway interactions; Patients; Phagocytes; Play; Process; Production; Proteins; Protocols documentation; Reagent; Recombinants; Recruitment Activity; Resistance; Role; Signal Transduction; Site; System; T-Lymphocyte; T-bet protein; TLR4 gene; TLR7 gene; TNF gene; Technology; Testing; Therapeutic; Thymoma; Tumor Immunity; Tumor-Derived; Vaccines; adaptive immune response; alpha synuclein; anti-CTLA4 antibodies; anti-PD-L1; antimicrobial peptide; chemokine; cytokine; cytotoxic; extracellular; immune function; improved; in vivo; interest; melanoma; microbial; monocyte; nanoGold; neoplastic cell; neutrophil; overexpression; phenotypic biomarker; professor; receptor; recruit; resiquimod; response; success; therapeutic vaccine; tissue injury; transcription factor; transcriptome sequencing; tumor; tumor growth; vaccine trial

Alarmins are characterized by having in vitro chemotactic or in vivo recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors resulting in the activation of immature dendritic cells (iDC) into mature antigen-presenting capable of interacting with T lymphocytes.This enables the DC to present antigens they have phagocytized and processed to T lymphocytes and results in adaptive immune responses. Consequently, the in vitro stimulant effects on Dc can be used to predict the capacity of an alarmin to promote immune responses in vivo. Thus, these alarmins, if administered together with an antigen result in considerable augmentation of both in vivo cellular and humoral immune responses. We previously showed that HMGN-1 knockout mice exhibit reduced resistance to tumor (EG-7 or EL-4) challenge. Conversely, tumor cells (EG-7 or EL-4) when transfected to overexpress HMGN1 showed a marked reduction in the rate of growth in normal mice. These observations indicated that HMGN1 is capable of augmenting tumor immunity. . We therefore injected a recombinant HMGN1 protein directly intratumorally into CT26 colon tumors in mice to proximate the adjuvant and antigen. This therapeutic vaccine trial did slow the tumor growth and prolonged the survival of mice, but did not cure any of the mice. We therefore improved the potency of the tumor vaccine by employing combinations of immunotherapeutic antitumor reagents to cure mice with larger tumors .We screened all the TLR ligands to identify which cooperated best with HMGN1 our TLR4 ligand. This led to the identification of R848, a TLR7/8 ligand as capable of maximally synergizing with TLR4 in stimulating the maturation of dendritic cells to markedly increase their production of IL-12 and TNF. We have been able to show that intratumoral injections of these two TLR ligands together with various checkpoint inhibitors can cure mice with five different of tumors and results in their subsequent resistance to re-challenge with these tumors. The combination of immunotherapeutic agents consisting of HMGN1, R848 (Resiquimod), a checkpoint inhibitor such as anti PDL-1 or anti CTLA4 antibody or a low dose of cytoxan successfully cured large (1cm diam.) tumors of the colon (CT26), kidney (RENCA), thymoma (EG7) lung (Lewis Lung) and liver (Hepa1-6) in mice. We have termed this combination of antitumor therapeutics, "TheraVac". We have also developed a means of delivering the HMGN1 and R848 on gold nanoparticles intravenously with success in curing large colon and hepatic cell line derived tumors located in their flanks. We have also treated genetically derived spontaneous tumors of mice with TheraVac and succeeded in slowing their growth and prolonging their life span significantly. In addition, we have been able to cure 70% of mice bearing resistant B16/F10 melanoma tumors by adding cGAMP, a a ligand of the STING pathway to our TheraVac antitumor protocol. We also investigated the mechanisms accounting for the synergistic stimulatory effects of HMGN1 and R848. It is well known that stimulation of cell surface TLR4 activates MyD88 and TRIF dependent pathways resulting in the activation of MAP kinases, NFKB and the production of type I IFNs . Although stimulation of TLR7/8 by R848 occurs intracellularly in the endosome and does not stimulate the TRIF pathway, it also activates the MyD88 pathway, MAP kinases, NFKB and IRF7 resulting in type I IFN production. We showed that simultaneous stimulation by HMGN1 and R848 resulted in synergistic activation of NFKB, MAPK, IRF3 and IRF7 transcription factors mediating the synergistic production of proinflammatory cytokines and type I IFNs as well as markedly upregulating the phenotypic markers indicative of maturation of activated DC's. Synergistic production of cytokines such as IL-12 enhanced the production of IFNgamma, which was associated with enhanced expression of the T-bet transcription factor and Th1 polarization favoring cellular antitumor immunity. Furthermore, we investigated the effects if HMGN1 and R848 alone and in combination using RNAseq technology. This has identified unique genes activated by these ligands and marked increases in the expression of many proinflammatory genes stimulated by just one of the TLR ligands by giving these TLR ligands together. In collaboration with Professor Lin and her colleagues at the Traditional Chinese Medicine (TCM) Guang'anmen Hospital, Beijing, China we have tested a number of TCM's over the past decade in an effort to detect any with immunostimulant effects. We finally determined that a purified and chemically synthesized TCM known as Cryptotanshinone (CT) not only had cytotoxic effects on tumor cells, but also activated dendritic cells in vitro to mature and produce cytokines (e.g. IL-12 and TNF). We also were able to show that CT had the capacity to cure mice with large (1 cm diam.) colon tumors, when injected either intratumorally or intravenously together with a checkpoint inhibitor. We were recently also able to show that CT required expression of the MyD88 pathway and TLR7 receptor to activate maturation of Dendritic Cells suggesting its mechanism of action is like that of R848. In collaboration with Dr. Michael Zasloff, we showed that alpha-synuclein (alpha-S) was a potent chemotactic protein for neutrophils and monocytes. This result helped to understand the high expression of alpha-S within the human enteric nervous system in biopsy specimens from patients with a variety of inflammatory conditions of the intestinal wall characterized by infiltrations of acute neutrophil and chronic mononuclear inflammatory cells. Subsequently we also demonstrated that alpha-S was also a potent activator of dendritic cells and therefore functioned as an alarmin. This is of particular interest since mutations of alpha-S are associated with the development of familiar Parkinson's Disease. Mice with deletion of the Alpha-S genes have deficient cellular and humoral immune responses, indicative of the important immunological function of Alpha-S. Thus, our findings show Alpha-s, which is causally associated with Neurodegenerative Diseases as having potent proinflammatory immunological effects.

警报素的特点是对表达 GiPCR 的细胞具有体外趋化或体内募集活性，以及​​与其他受体相互作用的能力，从而将未成熟的树突状细胞 (iDC) 激活为能够与 T 淋巴细胞相互作用的成熟抗原呈递细胞。这使得 DC 能够将其吞噬和加工的抗原呈递给 T 淋巴细胞，并导致 适应性免疫反应。因此，对 Dc 的体外刺激作用可用于预测警报素促进体内免疫反应的能力。因此，这些警报素如果与抗原一起施用，会导致体内细胞和体液免疫反应的显着增强。我们之前表明，HMGN-1 敲除小鼠对肿瘤（EG-7 或 EL-4）攻击的抵抗力降低。相反，当转染过表达 HMGN1 的肿瘤细胞（EG-7 或 EL-4）时，正常小鼠的生长速度显着降低。这些观察结果表明 HMGN1 能够增强肿瘤免疫力。 。因此，我们将重组 HMGN1 蛋白直接瘤内注射到小鼠 CT26 结肠肿瘤中，以接近佐剂和抗原。这项治疗性疫苗试验确实减缓了肿瘤的生长并延长了小鼠的生存期，但没有治愈任何小鼠。因此，我们通过采用免疫治疗抗肿瘤试剂组合来治愈具有较大肿瘤的小鼠，从而提高了肿瘤疫苗的效力。我们筛选了所有 TLR 配体，以确定哪些与我们的 TLR4 配体 HMGN1 配合最佳。这导致了 R848 的鉴定，R848 是一种 TLR7/8 配体，能够最大程度地与 TLR4 协同作用，刺激树突状细胞的成熟，从而显着增加其 IL-12 和 TNF 的产生。我们已经能够证明，瘤内注射这两种 TLR 配体与各种检查点抑制剂可以治愈患有五种不同肿瘤的小鼠，并导致它们随后对这些肿瘤的再次攻击产生抵抗力。由 HMGN1、R848（瑞西莫德）、检查点抑制剂（例如抗 PDL-1 或抗 CTLA4 抗体）或低剂量环磷酰胺组成的免疫治疗药物组合，成功治愈了大（直径 1 厘米）结肠肿瘤 (CT26)、肾肿瘤 (RENCA)、胸腺瘤 (EG7)、肺肿瘤 (Lewis Lung) 和 小鼠肝脏 (Hepa1-6)。我们将这种抗肿瘤治疗组合称为“TheraVac”。我们还开发了一种将 HMGN1 和 R848 静脉注射到金纳米颗粒上的方法，成功治愈了位于其侧翼的大结肠和肝细胞系来源的肿瘤。我们还用 TheraVac 治疗了小鼠的遗传性自发性肿瘤，并成功地减缓了它们的生长并显着延长了它们的寿命。此外，通过在 TheraVac 抗肿瘤方案中添加 cGAMP（STING 通路的配体），我们已经能够治愈 70% 患有耐药性 B16/F10 黑色素瘤的小鼠。我们还研究了 HMGN1 和 R848 协同刺激作用的机制。众所周知，细胞表面 TLR4 的刺激会激活 MyD88 和 TRIF 依赖性途径，从而导致 MAP 激酶、NFKB 的激活和 I 型 IFN 的产生。虽然 R848 对 TLR7/8 的刺激发生在细胞内的内体中，并且不会刺激 TRIF 途径，但它也会激活 MyD88 途径、MAP 激酶、NFKB 和 IRF7，从而导致 I 型 IFN 的产生。我们发现，HMGN1 和 R848 的同时刺激导致 NFKB、MAPK、IRF3 和 IRF7 转录因子的协同激活，介导促炎细胞因子和 I 型 IFN 的协同产生，并显着上调表明活化 DC 成熟的表型标记。 IL-12 等细胞因子的协同产生增强了 IFNγ 的产生，这与 T-bet 转录因子的表达增强和有利于细胞抗肿瘤免疫的 Th1 极化有关。此外，我们使用 RNAseq 技术研究了 HMGN1 和 R848 单独使用和组合使用的效果。这已经鉴定出由这些配体激活的独特基因，并且通过将这些TLR配体放在一起，仅由其中一种TLR配体刺激的许多促炎基因的表达显着增加。在过去的十年里，我们与中国北京广安门中医医院的林教授和她的同事合作，测试了多种中药，以期发现任何具有免疫刺激作用的中药。我们最终确定，一种纯化的化学合成中药隐丹参酮（CT）不仅对肿瘤细胞具有细胞毒作用，而且还能在体外激活树突状细胞成熟并产生细胞因子（例如IL-12和TNF）。我们还能够证明，当与检查点抑制剂一起瘤内或静脉注射时，CT 有能力治愈患有大（直径 1 厘米）结肠肿瘤的小鼠。我们最近还发现 CT 需要 MyD88 通路和 TLR7 受体的表达来激活树突状细胞的成熟，这表明其作用机制类似于 R848。我们与 Michael Zasloff 博士合作，证明 α-突触核蛋白 (α-S) 是中性粒细胞和单核细胞的有效趋化蛋白。这一结果有助于了解患有各种肠壁炎症性疾病（以急性中性粒细胞和慢性单核炎症细胞浸润为特征）的患者的活检标本中，人类肠神经系统中α-S的高表达。随后我们还证明α-S也是树突状细胞的有效激活剂，因此起到警报素的作用。这是特别令人感兴趣的，因为α-S突变与常见的帕金森病的发展有关。 Alpha-S 基因缺失的小鼠细胞和体液免疫反应不足，表明 Alpha-S 具有重要的免疫功能。因此，我们的研究结果表明 Alpha-s 与神经退行性疾病有因果关系，具有有效的促炎免疫作用。