Biophysical analysis of T-cell discrimination among classes of self-ligands

描述(申请人提供)：在自然界中，细胞毒性T细胞负责识别显示外来抗原的细胞并杀死它们。然而，T细胞不仅对典型的外来抗原完全反应，而且在自身免疫或有效的抗肿瘤反应中，T细胞在外周也表现为共激动剂活性(其中某些宿主自身配体可以与激动剂配体协同触发T细胞受体复合体启动信号)或显性激动剂活性。一种潜在的策略是通过增加TCR-CD8-肽-MHC的结合亲和力来补偿无效的抗肿瘤反应。然而，目前还不清楚人们可以在多大程度上提高系统的效力，以便在不介导危险的对宿主抗原的自身反应的情况下，调节更大的T细胞活性。我们的长期目标是改变T细胞激活阈值，使这些免疫效应器对疾病相关的自身(肿瘤)抗原产生有效的免疫反应，同时保持对宿主的非肿瘤相关自身抗原的无反应。本申请中提出的实验的目的是通过确定T细胞如何区分不同类别的自我配体(定义为肿瘤(SELF)和宿主(SELF)配体)的生物物理机制，朝着这一目标迈出重要的一步。应用的中心假设是，具有共激动剂活性的自体配体和介导自毁反应的自体配体之间的功能区分是通过TCR和CD8共受体的联合作用介导的动力学阈值实现的。在强大的初步数据的指导下，这一假说将通过追求三个具体目标来检验：1)确定特定多肽-MHC配体的TCR-CD8结合亲和力与这些配体的共激动剂与效应器诱导活性之间的定量关系；2)确定TCR亲和力，该亲和力定义了维持效应器诱导配体特异性的阈值；3)确定CD8亲和力的变化将如何影响T细胞维持对自身配体类别的区分的能力。为了实现这些目标，我们将使用一个成熟的模型系统来识别“真正的”自身抗原，该系统涉及在小鼠CD8+T细胞上表达人类自身/肿瘤反应性的HLA-A2限制性受体。我们将使用新的分子荧光成像和生物物理技术来研究配体识别的生物物理和功能反应之间的关系。最后，我们将使用体外噬菌体展示来筛选出高亲和力的TCR和CD8分子，这将使我们能够获得比使用自然产生的TCR和CD8分子更大的动力学窗口。这项研究的基本原理是，在正常组织共激动剂和弱肿瘤激动剂之间存在足够大的亲和力窗口，从而增强后者的结合TCR-CD8亲和力可以改善效应器反应，而不会产生不良的抗组织反应。与人类健康相关：我们认为这个问题对于了解和提高癌症免疫力很重要，并可能导致对这种疾病的更有效的治疗。公共卫生相关性：我们预计，我们的研究将为我们提供洞察，了解如何通过增加TCR-CD8-肽-MHC相互作用的亲和力来操纵免疫反应，以中介更有效地识别肿瘤(自身)抗原，同时避免自身反应。所获得的知识对于产生高亲和力的TCR或CD8分子非常重要，当这些分子被转移到CD8+T细胞中时，可以用于有效的过继免疫治疗转移疗法，并且仍然保持特异性。或者，具有明确特异性的可溶性TCR也可以被修饰，以输送放射性核素、毒素或免疫调节分子，以更有效地杀死肿瘤细胞。

DESCRIPTION (provided by applicant): In nature, cytotoxic T-cells are responsible for identifying cells that display foreign antigens and killing them. However, T-cells do not only respond fully to typical foreign antigens but also show responses to self in the periphery in the form of co-agonist activity (wherein certain host self-ligands can synergize with agonist ligands in triggering the T-cell receptor complex to initiate signaling) or overt agonist activity in the case of autoimmunity or effective anti-tumor responses. One potential strategy is to compensate for ineffective anti- tumor responses by increasing TCR-CD8-peptide-MHC binding affinity. However, it is still not clear how much one can increase the potency of the system in order to mediate greater T-cell activity without mediating dangerous auto-reactivity to host antigens. Our long-term goal is to modify the T-cell activation threshold so that these immune effectors mediate efficient immune responses to disease-associated, self (tumor)-antigens while remaining unresponsive to the host's non-tumor associated self-antigens. The objective of the experiments proposed in this application is to make a major step towards this goal by determining the biophysical mechanism underlying how T-cells discriminate between different classes of self-ligands defined as tumor (self) and host (self) ligands. The central hypothesis of the application is that functional discrimination between self-ligands with co-agonist activity and those that mediate self-destructive responses is achieved by kinetic thresholding mediated by the conjoint action of the TCR and the CD8 co-receptor. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the quantitative relationship between TCR-CD8 binding avidity for specific peptide-MHC ligands and the co-agonist verses effector-inducing activity of these ligands; 2) Determine the TCR affinity that defines the threshold for maintaining specificity to effector-inducing ligands; 3) Determine how alterations in CD8 affinity will affect the ability of the T-cells to maintain discrimination among self-ligand classes. To achieve these aims we will use a well-established model system for recognition of "true" self-antigens involving expression of human self/tumor- reactive HLA-A2 restricted receptors on mouse CD8+ T-cells. We will use novel molecular fluorescent imaging and biophysical techniques to investigate the relationship between the biophysics of ligand recognition and functional responses. Finally, we will use in vitro phage display to select out high-affinity TCR and CD8 molecules which will allow us to access a larger kinetic window than available using naturally occurring TCR and CD8 molecules. The rationale for the proposed research is that a sufficiently large affinity window exists between normal tissue co-agonists and weak tumor agonists such that enhancing the conjoint TCR-CD8 avidity for the latter can improve effector responses without engendering undesirable anti-tissue responses. Relevance to human health: We believe this issue is important in understanding and enhancing cancer immunity and could lead to more effective therapies for this disease. Public Health Relevance: We expect that our studies will provide insight into how we can manipulate the immune response by increasing the affinity of the TCR-CD8-peptide-MHC interaction to mediate more effective recognition of tumor (self) antigens while avoiding self-reactivity. The knowledge obtained would be important in terms of generating high-affinity TCR or CD8 molecules that when transferred into CD8+ T-cells could be used for effective adoptive immunotherapy transfer therapies and still retain specificity. Alternatively, soluble TCRs with well-defined specificities could also be modified to deliver radionuclides, toxins or immunomodulatory molecules for more efficient killing of tumor cells.