Activation of semi-invariant and diverse NKT cells with an adjuvant combination

摘要 有几种安全的候选佐剂可以增强接种疫苗后的有益免疫力。然而， 单一佐剂配方在刺激所需的混合佐剂方面表现相当温和 体液免疫与细胞免疫和/或Th1与Th2免疫。联合佐剂提供了一种可能的方式 但这些组合的作用机制仍未得到明确界定。因此，我们将结合 美国食品和药物管理局批准的含有CD1d结合糖脂佐剂α-半乳糖神经酰胺的明矾(铝水凝胶) (α-GC)，并描述了作用机制，特别强调CD1d限制性自然杀伤T (NKT)细胞与体液免疫明矾是安全的，能刺激优秀的Th2，但Th1免疫力较差。我们 发现Th2对明矾的反应在很大程度上取决于表达TCR的II型NKT的多样性 识别CD1d/糖脂复合体但不识别α-GC佐剂的细胞(DNKT)。α-GC 佐剂刺激半不变的表达TCR的I型NKT细胞(INKT)并导致Th1/Th2混合 回应。从癌症患者的I期临床试验中已经知道，α-GC耐受性良好， 安然无恙。此外，可以容易地采用α-GC结构的修饰来歪曲I型NKT- 依赖Th1/Th2平衡。我们这项提议的目标是检验核心假设，即 明矾和α-GC的结合导致CD1d/糖脂递呈和协调的dNKT和iNKT- 驱动针对疫苗抗原的Th1/Th2混合反应。 在特定目标1中，我们将在原代小鼠和人类细胞中进行一系列体外研究，以确定 佐剂组合对CD1d抗原提呈和激活及功能分化的影响 NKT细胞分为不同的效应器亚群。我们还将研究佐剂组合对I类和 联合给药蛋白抗原的II类呈递。 在具体目标2中，我们将在小鼠身上进行一系列体内实验，以检验其功能 佐剂组合对抗体介导的细菌毒素保护作用的后果。实验以实现 随着项目的发展，将包括分析细胞和体液免疫以及体内毒性(或 缺乏它)将通过测量促炎细胞因子以及 自身免疫力和器官损伤。 我们认为这个项目是创新的，因为它将为疫苗社区提供机械性的信息 当CD1d结合佐剂与明矾佐剂联合使用时，仔细考虑NKT细胞- 免疫反应的驱动成分。我们认为这个项目意义重大，因为它将照亮 在混合佐剂平台中包含CD1d结合糖脂的潜在途径。

ABSTRACT There are several safe candidate adjuvants that potentiate beneficial immunity following vaccination. However, single adjuvant formulations have performed quite modestly in terms of stimulating the desired blend of humoral versus cellular immunity and/or Th1 versus Th2 immunity. Combination adjuvants offer a possible way forward, but the mechanisms of action of such combinations remain poorly defined. Therefore, we will combine the FDA-approved adjuvant Alum (alhydrogel) with the CD1d-binding glycolipid adjuvant α-galactosylceramide (α-GC) and delineate the mechanisms of action with a particular emphasis on CD1d-restricted Natural Killer T (NKT) cells and humoral immunity. Alum is safe and stimulates excellent Th2 but poor Th1 immunity. We discovered that the Th2 response to Alum depends in large part upon diverse TCR-expressing Type II NKT cells (dNKT) which recognize CD1d/glycolipid complexes but do not recognize the α-GC adjuvant. The α-GC adjuvant stimulates semi-invariant TCR-expressing Type I NKT cells (iNKT) and results in a mixed Th1/Th2 response. It is already known from Phase I clinical trials in patients with cancer that α-GC is well-tolerated and safe. Furthermore, modification of the α-GC structure can readily be employed to skew the Type I NKT- dependent Th1/Th2 balance. Our objective for this proposal is to test the central hypothesis that the combination of Alum and α-GC leads to CD1d/glycolipid presentation and a coordinated dNKT and iNKT- driven mixed Th1/Th2 response against vaccine antigens. In Specific Aim 1, we will perform a series of in vitro studies in primary murine and human cells to determine how the adjuvant combination affects CD1d Ag presentation and activation and functional differentiation of NKT cells into different effector subsets. We will also examine the effect of adjuvant combination on class I and class II presentation of co-administered protein antigens. In Specific Aim 2, we will undertake a series of in vivo experiments in mice to examine the functional consequences of adjuvant combination for Ab-mediated protection against bacterial toxins. Experiments to analyze cellular as well as humoral immunity will be included as the project develops and in vivo toxicity (or lack thereof) will be determined by measurement of pro-inflammatory cytokines as well as markers of autoimmunity and organ damage. We feel this project is innovative because it will provide the vaccine community with mechanistic information when CD1d-binding adjuvants are combined with Alum adjuvant and give careful consideration to NKT cell- driven components of the immune response. We feel the project is significant because it will illuminate potential avenues for inclusion of CD1d-binding glycolipids in mixed adjuvant platforms.