Studies of Receptor Interactions and Effects of Alarmins

警报器的特点是对表达GiPCR的细胞具有体外趋化和（或）体内募集活性，同时具有与其他受体相互作用的能力，导致未成熟的树突状细胞（iDC）被激活，发育成能够与T淋巴细胞相互作用的成熟抗原呈递细胞。这使得DC能够将它们吞噬的抗原呈递到T淋巴细胞，并产生适应性免疫反应。因此，我们利用体外对DC的刺激作用来预测警报素在体内促进免疫反应的能力。这些警报，如果与抗原一起使用，会导致体内细胞和体液免疫反应的显著增强。我们之前的研究表明，HMGN1敲除小鼠对肿瘤（EG-7或EL-4）的抗性降低。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明显降低。这些观察结果表明，HMGN1能够增强抗肿瘤免疫。因此，我们将重组HMGN1蛋白直接注射到小鼠CT26结肠肿瘤中，以接近佐剂和CT26相关抗原。这种治疗性疫苗试验确实减缓了肿瘤的生长，延长了小鼠的生存时间，但没有治愈任何小鼠。因此，我们通过联合使用免疫治疗抗肿瘤试剂来治疗具有较大肿瘤的小鼠，从而提高了肿瘤疫苗的效力。我们筛选了所有的TLR配体，以确定哪些配体与HMGN1在DCs的激活上合作最好。这导致鉴定出R848，一个TLR7/8配体，能够最大限度地与HMGN1协同刺激DC成熟，显著增加其IL-12和TNF的产生。我们已经能够证明，肿瘤内注射由HMGN1、R848 （Resiquimod）、抗PDL-1或抗CTLA4抗体等检查点抑制剂或低剂量环环酰胺组成的免疫治疗剂组合，成功治愈了小鼠大（直径1cm）的结肠（CT26）、肾脏（RENCA）、胸腺瘤（EG7）、肺（Lewis lung）和肝脏（Hepa1-6）肿瘤，导致对同一肿瘤的再攻击产生免疫抵抗。我们将这种联合方案称为“治疗性疫苗接种”，简称为“TheraVac”。我们还用TheraVac治疗了因基因修饰而自发产生肿瘤的小鼠，并成功地减缓了它们的生长速度，显著延长了它们的寿命。此外，我们已经能够用TheraVac和cGAMP （STING途径的天然激动剂）联合治疗80%的耐药B16/F10黑色素瘤小鼠。我们还研究了HMGN1、R848和cGAMP协同刺激作用的机制。HMGN1、R848和cGAMP在DC中触发各种信号通路，导致各种转录因子（如NF-kB、MAPK、IRF3和IRF7）的协同激活，进而导致促炎细胞因子（如IL-12、TNF和I型ifn）的协同诱导，并显著上调指示DC成熟的表型标记。协同产生IL-12增强Th1极化有利于细胞抗肿瘤免疫。我们以前报道过，我们可以切割HMGN1分子，并表明N端结构域保留了免疫刺激作用。与Kouji Matsushima教授领导的小组合作，我们最近发现了HMGN1 N端具有生物活性的较小肽成分。这种所谓的“MinP”肽与抗pd - l1（一种免疫检查点阻断抗体）联合在小鼠中显示出免疫介导的抗肿瘤作用。为了克服TheraVac在肿瘤内给药的局限性，我们一直在尝试开发一种TheraVac功能化的纳米颗粒，以便在静脉给药时优先携带TheraVac到肿瘤。我们最近成功地设计和合成了一种用TheraVac （MSN@TheraVac）功能化的ros敏感介孔二氧化硅纳米颗粒（MSN），当静脉注射时，该颗粒选择性地积聚在肿瘤中，更重要的是治愈了100%携带ct26的小鼠。我们在实验室取得的另一项成就是，我们通过用FSL-1取代R848或在原来的TheraVac方案中添加cGAMP，开发出了更有效的TheraVac版本。除了我们之前的文章表明α -突触核蛋白（aS）是一种有效的吞噬细胞趋化蛋白外，我们最近还证明aS也是一种有效的树突状细胞激活剂，因此作为一种对宿主免疫至关重要的警报蛋白。这是特别有趣的，因为aS的突变与常见的帕金森病的发展偶然相关。aS基因缺失的小鼠细胞和体液免疫反应不足，表明aS具有重要的免疫功能。因此，我们的研究结果表明，aS除了参与包括帕金森病在内的神经退行性疾病的发展外，还具有强大的促炎/免疫作用。

Alarmins are characterized by having in vitro chemotactic and 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) to develop into mature antigen-presenting cells 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 is used by us to predict the capacity of an alarmin to promote immune responses in vivo. 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 HMGN1 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 antitumor immunity. We therefore injected a recombinant HMGN1 protein directly intratumorally into CT26 colon tumors in mice to proximate the adjuvant and CT26-associated antigen(s). 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 on the activation of DCs. This led to the identification of R848, a TLR7/8 ligand as capable of maximally synergizing with HMGN1 in stimulating DC maturation to markedly increase their production of IL-12 and TNF. We have been able to show that intratumoral injections of 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, resulting in the generation of immune resistance to re-challenge with the same tumor. We have termed this combinational regimen as 'therapeutic vaccination', shortened "TheraVac". We have also treated mice bearing tumors spontaneously arising from genetically modifications with TheraVac and succeeded in slowing their growth and prolonging their life span significantly. In addition, we have been able to cure 80% of mice bearing resistant B16/F10 melanoma tumors with a combination of TheraVac and cGAMP, a natural agonist of the STING pathway. We also investigated the mechanisms accounting for the synergistic stimulatory effects of HMGN1, R848, and cGAMP. Trigger of various signaling pathways in DCs by HMGN1, R848, and cGAMP results in synergistic activation of various transcription factors (e.g. NF-kB, MAPK, IRF3 and IRF7), which, in turn, accounts for synergistic induction of proinflammatory cytokines (e.g. IL-12, TNF, and type I IFNs) and marked upregulation of the phenotypic markers indicative of DC maturation. Synergistic production of IL-12 enhanced Th1 polarization favoring cellular antitumor immunity. We have previously reported that we can cleave the HMGN1 molecule and show that the N terminal domain preserve the immunostimulating effects. In collaboration with a group led by Prof. Kouji Matsushima, we recently identified a smaller peptide component of the N terminus of HMGN1 that is biologically active. This so-called "MinP" peptide in conjunction with anti-PD-L1 (an immune checkpoint blockade antibody) has demonstrated immune mediated antitumor effect in mice. To overcome the limitation of administration of TheraVac using intratumoral route, we have been trying to develop a TheraVa-functionalized nanoparticle to carry TheraVac preferentially to tumors upon intravenous administration. We have recently succeeded in doing so after we successfully designed and synthesized a ROS-sensitive mesoporous silica nanoparticle (MSN) functionalized with TheraVac (MSN@TheraVac), which when injected intravenously selectively accumulated in the tumors and more importantly cured 100% of the CT26-bearing mice. Another achivement we made in the laboratory is that we have developed more potent TheraVac versions by replacing R848 with FSL-1 or by adding cGAMP into the original TheraVac regimen. In addition to our previous publication showing that alpha-synuclein (aS) is a potent chemotactic protein for phagocytes, we have recently demonstrated that aS is also a potent activator of dendritic cells and therefore functions as an alarmin that is critical for host immunity. This is of particular interest since mutations of aS are casually associated with the development of familiar Parkinson's Disease. Mice with deletion of the aS genes have deficient cellular and humoral immune responses, indicative of the important immunological function of aS. Thus, our findings show aS, in addition to its involvement in the development of neurodegenerative diseases including Parkinson's disease, has potent proinflammatory/immunological effects.