Role of type II NKT cells during M. tuberculosis infection

描述(申请人提供)：自然杀伤T细胞(NKT)是进化上保守的先天T细胞亚群，识别CD1d分子呈递的脂类抗原。NKT细胞已被证明在自身免疫以及对癌症和感染的免疫中发挥关键作用。除了被广泛研究的利用V？14-J？18TCR？链并识别典型的半乳糖神经酰胺抗原的不变NKT(INKT)细胞外，还有利用各种TCRs的CD1d限制性NKT细胞(dNKT或II型NKT细胞)。现有数据表明，dNKT细胞具有与iNKT细胞不同的抗原特异性和功能。然而，目前尚不清楚dNKT细胞是否识别微生物脂肪抗原，它们是否有能力产生记忆反应，以及它们在感染期间的功能是什么。缺乏标记、抗原和工具来专门鉴定和研究dNKT细胞，在很大程度上阻碍了对这些关键问题的理解。我们的长期目标是了解dNKT细胞的抗原特异性，它们在微生物刺激下的功能，以及它们在结核分枝杆菌感染中的作用。这里的目标，也是追求这一目标的下一步，是为dNKT细胞的活体研究建立和鉴定TCR逆转录基因小鼠模型。基于先前未知的dNKT细胞识别不同的分枝杆菌脂类抗原并被结核分枝杆菌感染的APC刺激的能力，我们的中心假设是dNKT细胞在结核分枝杆菌感染期间具有保护功能。我们的策略是建立并鉴定表达结核分枝杆菌特异性dNKT细胞所使用的TCR的逆转录基因(Rg)小鼠。Rg小鼠的产生是一种利用逆转录病毒和干细胞介导的基因转移快速表达特定TCR？和？蛋白的新方法。我们已经用这种方法成功地从Mtb特异性CD8+T细胞中获得了4个表达MHC-I类限制性TCR的RG小鼠。我们计划测试我们的中心假设，从而通过建立和鉴定表达CD1d限制性Mtb特异性dNKT细胞TCRs的TCR回溯基因小鼠来实现这一应用的目标(目标1)。然后，我们将在体外和体内鉴定在抗原刺激和结核分枝杆菌感染后的逆转录基因小鼠中结核分枝杆菌特异性dNKT细胞的反应(目标2)。关于预期的结果，拟议的研究将产生一个小鼠体内系统，以使研究dNKT细胞对结核分枝杆菌抗原的反应和在结核分枝杆菌感染期间的功能。这些研究有望产生重大影响，因为它们将开发出第一个可在体内研究dNKT细胞的易处理的小动物模型，该模型可用于研究dNKT细胞在各种疾病过程中的贡献。其次，我们希望确定dNKT细胞在分枝杆菌感染过程中的作用，这可能导致识别新的抗原靶点，这些靶点可以被整合到针对结核分枝杆菌的疫苗中。这项工作将从根本上推进CD1d/dNKT细胞生物学领域的发展。

DESCRIPTION (provided by applicant): Natural Killer T (NKT) cells are an evolutionarily conserved subset of innate-like T cells that recognizes lipid antigens presented by CD1d molecules. NKT cells have been shown to play critical roles in autoimmunity, and in immunity to cancer and infection. In addition to the widely studied invariant NKT (iNKT) cells that utilize V¿14-J¿18 TCR¿-chains and recognize the prototypical ¿-galactosylceramide antigen, CD1d-restricted NKT cells utilizing diverse TCRs (dNKT or type II NKT cells) exist. Available data suggest that dNKT cells have antigen specificities and functional capabilities that are distinct from those of iNKT cells. However, it is not known whether dNKT cells recognize microbial lipid antigens, whether they have the ability to generate memory responses, and what their function is during infection. Progress in understanding these critical questions is in large part hindered b the lack of markers, antigens, and tools to specifically identify and study dNKT cells. Our long-term goal is to understand the antigen specificity of dNKT cells, their function in response to microbial stimulation, and their role during Mtb infection. The objective here, which is the next step in pursuit of that goal, is to generate and characterize a TCR-retrogenic mouse model for the in vivo study of dNKT cells. Based on the previously unrecognized ability of dNKT cells to recognize diverse mycobacterial lipid antigens and to be stimulated by Mtb-infected APCs, our central hypothesis is that dNKT cells have a protective function during Mtb infection. Our strategy is to generate and characterize retrogenic (Rg) mice expressing the TCRs used by Mtb-specific dNKT cells. Generation of Rg mice is a novel method for rapid expression of defined TCR¿ and ¿ proteins that uses retroviral and stem cell-mediated gene transfer. We have used this method successfully to generate four Rg mouse lines that express MHC class I- restricted TCRs from Mtb-specific CD8+ T cells. We plan to test our central hypothesis and, thereby, accomplish the objective of this application by generating and characterizing TCR-retrogenic mice expressing CD1d-restricted Mtb-specific dNKT cell TCRs (Aim 1). We will then characterize Mtb-specific dNKT cell responses in those retrogenic mice following antigen stimulation and Mtb infection in vitro and in vivo (Aim 2). With respect to expected outcomes, the proposed studies will generate a murine in vivo system to enable studies of dNKT cell function in response to Mtb antigens and during Mtb infection. These studies are expected to have a significant impact because they will develop the first tractable small animal model for the study of dNKT cells in vivo, a model that can be used to study the contribution of dNKT cells to a variety of disease processes. Second, we expect to define the role of dNKT cells during mycobacterial infection, which may result in the identification of new antigen targets that could be incorporated into vaccines against Mtb. This work will fundamentally advance the field of CD1d/dNKT cell biology.