Understanding HIV escape using atomic force microscopy

描述（由申请人提供）：使用T细胞受体（TCR）作为传感器，HIV特异性CD 8 + T细胞检测抗原呈递细胞（APC）或靶细胞上人类白细胞抗原（pHLA）呈递的HIV肽。HIV容易使这些肽突变的能力对设计基于T细胞的疫苗和免疫疗法提出了巨大挑战，因为逃逸变体不再被对原始肽特异性的TCR有效识别。这些分子水平的突变如何改变它们与TCR的相互作用尚未完全了解。以前的研究主要集中在溶液参数，如亲和力，结合速率和解离速率，这是在没有外力作用的情况下测量的相互作用。然而，这些参数不能描述pHLA-TCR在T细胞和APC或靶细胞之间的界面处相互作用的真实的行为。T细胞与APC或靶细胞以高度动态的方式相互作用，具有恒定的膜脱离和重新附着。因此，pHLA-TCR相互作用处于恒定的机械应力下。我们认为，在机械力作用下pHLA-TCR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允许将源自细胞运动的机械力转移到TCR，并且力诱导的TCR构象变化导致TCR信号传导和T细胞应答。因此，我们假设HIV逃逸变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HLA-A2呈递HIV Gag肽SL 9（HLA-A2-SL 9）或其逃逸变体与SL 9特异性868 TCR之间的结合的机械强度，然后将其与诱导的T细胞应答相关联来测试我们的假设。此外，我们将研究HLA-A2-SL 9变体与最近发现的“超生理”a11 b6 TCR之间的结合，该TCR识别SL 9及其所有逃逸变体。我们预期逃逸变体以比野生型SL 9更低的机械强度结合868 TCR，并且a11 b6 TCR通过以比野生型868 TCR更高的机械强度结合它们来识别逃逸变体。具体地，在目的1中，使用纯化的868 TCR、a11 b6 TCR、HLA-A2-SL 9及其变体的蛋白，我们将用AFM测量它们在机械力下的结合强度。破坏结合所需的力（断裂力）将与它们诱导T细胞应答的能力相关。在目标2中，我们将使用HLA-A2-SL 9或其变体功能化的AFM针尖直接探测表达868 TCR或a11 b6 TCR的T细胞并测量断裂力。在荧光显微镜下使用真实的时间比率成像同时监测由细胞内钙通量或PI 3 K活化指示的T细胞信号传导。断裂力将与T细胞应答水平相关。通过首次使用AFM表征TCR和HIV逃逸变体之间的相互作用，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产生直接影响。 公共卫生相关性：艾滋病毒不断变异以逃避免疫系统。为了了解突变体如何逃避免疫检测，我们采用了尖端的生物物理工具，原子力显微镜，解剖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.