Understanding HIV escape using atomic force microscopy

描述(申请人提供)：使用T细胞受体(TCR)作为传感器，HIV特异性CD8+T细胞检测由人类白细胞抗原(Phla)提呈在抗原提呈细胞(APC)或靶细胞上的HIV多肽。HIV容易突变这些多肽的能力对设计基于T细胞的疫苗和免疫疗法构成了巨大的挑战，因为逃逸变异体不再有效地被针对原始肽的TCR识别。这些分子水平的突变如何改变它们与TCR的相互作用尚不完全清楚。以前的研究主要集中在溶液参数上，如亲和力、开启速率和关闭速率，这些参数是在没有外力作用的情况下测量的。然而，这些参数并不能描述T细胞和APC或靶细胞之间界面上Phla-TCR相互作用的真实行为。T细胞与APC或靶细胞以高度动态的方式相互作用，不断地膜脱离和重新附着。因此，Phla-TCR相互作用是在恒定的机械应力下进行的。我们认为，机械力作用下Phla-TCR的结合强度决定了Phla-TCR相互作用的特异性。只有具有足够强度的结合才能将细胞运动产生的机械力传递给TCR，而TCR的构象变化才是导致TCR信号和T细胞反应的原因。因此，我们假设HIV逃逸变异体通过降低其与特定TCR结合的机械强度来逃避T细胞攻击。在这里，利用原子力显微镜(AFM)的最新进展，我们将通过测量人类白细胞抗原-A2呈递HIV Gag多肽SL9(HLA-A2-SL9)或其逃逸变异体与SL9特异性868 TCR之间的结合机械强度来验证我们的假设，然后将其与诱导的T细胞反应相关联。此外，我们还将研究人类白细胞抗原-A2-SL9变异体与最近发现的识别SL9及其所有逃逸变异体的超生理学的a11b6 TCR之间的结合。我们预计逃逸变异体与868 TCR结合的机械强度低于野生型SL9，而11b6 TCR通过与逃逸变异体结合的机械强度高于野生型868 TCR来识别逃逸变异体。具体地说，在目标1中，我们将使用纯化的868 TCR、a11b6 TCR、HL A-A2-SL9及其变异体的蛋白，用原子力显微镜测量它们在机械力作用下的结合强度。打破结合所需的力(破裂力)将与它们诱导T细胞反应的能力相关。在目标2中，我们将使用经HLA-A2-SL9或其变体功能化的AFM TIPS直接探测表达868 TCR或a11b6 TCR的T细胞，并测量破裂力。荧光显微镜下的实时比值成像将同时监测由细胞内钙离子流量或PI3K激活所指示的T细胞信号。破裂力将与T细胞反应水平相关。通过首次利用原子力显微镜表征TCR和HIV逃逸变异体之间的相互作用，我们的研究将对HIV感染的疫苗和基于T细胞的免疫疗法的设计具有直接意义。 与公共卫生相关：艾滋病毒不断变异以逃避免疫系统。为了了解突变体是如何逃避免疫检测的，我们使用了一种尖端的生物物理工具--原子力显微镜，来剖析突变体如何改变它们与T细胞受体的相互作用。我们的数据将有助于设计有效的艾滋病毒疫苗或针对艾滋病毒感染的免疫疗法。

DESCRIPTION (provided by applicant): Using T cell receptors (TCRs) as sensors, HIV-specific CD8+ T cells detect HIV peptides presented by human leukocyte antigen (pHLA) on antigen presenting cells (APCs) or target cells. The ability of HIV to readily mutate these peptides poses a great challenge for designing T cell-based vaccines and immunotherapies, since the escape variants are no longer effectively recognized by TCRs specific for the original peptide. How these molecular level mutations change their interactions with TCRs is not fully understood. Previous studies have focused on solution parameters, such as affinity, on-rate and off-rate, which were measured without external forces exerted on the interactions. These parameters, however, do not describe the real behavior of the pHLA-TCR interaction at the interface between T cells and APCs or target cells. T cells interact with APCs or target cells in a highly dynamic fashion, with constant membrane detachment and re-attachment. Therefore, the pHLA-TCR interaction is under constant mechanical stress. We believe that pHLA-TCR binding strength under mechanical force determines the specificity of pHLA-TCR interaction. Only bindings with sufficient strength allow the transfer of mechanical force originating from cell locomotion to TCR, and it is force-induced TCR conformational change that leads to TCR signaling and T cell response. Therefore, we hypothesize that HIV escape variants evade T cell attack by reducing the mechanical strength of their binding to specific TCRs. Here, taking advantage of recent advances in atomic force microscopy (AFM), we will test our hypothesis by measuring the mechanical strength of the bindings between HLA-A2 presenting HIV Gag peptide SL9 (HLA- A2-SL9) or its escape variants and SL9-specific 868 TCR, and then correlating this with the induced T cell response. In addition, we will examine the binding between HLA-A2-SL9 variants and a recently discovered 'supraphysiological' a11b6 TCR that recognizes SL9 and all its escape variants. We expect that the escape variants bind to the 868 TCR with less mechanical strength than wild type SL9, and that a11b6 TCR recognizes the escape variants by binding them with higher mechanical strength than wild type 868 TCR. Specifically, in Aim 1, using purified proteins of 868 TCR, a11b6 TCR, HLA-A2-SL9 and its variants, we will measure their binding strength under mechanical force with AFM. The forces it takes to rupture the binding (rupture force) will be correlated with their ability to induce T cell responses. In Aim 2, we will use AFM tips functionalized with HLA-A2-SL9 or its variants to directly probe T cells expressing 868 TCR or a11b6 TCR and measure rupture force. T cell signaling as indicated by intracellular calcium flux or PI3K activation will be monitored simultaneously using real time ratio imaging under a fluorescence microscope. Rupture force will be correlated with the level of T cell responses. By characterizing the interactions between TCR and HIV escape variants using AFM for the first time, our study will have direct implications for the design of vaccines and T cell-based immunotherapies for HIV infection. PUBLIC HEALTH RELEVANCE: HIV constantly mutates to escape from the immune system. To understand how the mutants evade immune detection, we employ a cutting edge biophysical tool, the atomic force microscopy, to dissect how the mutants change their interactions with the T cell receptor. Our data will help design effective HIV vaccines or immunotherapies for HIV infection.