Mechanisms of impaired T-cell mechanosensing of melanoma antigens

项目摘要 本项目研究肿瘤微环境(TME)如何损害T细胞表面的原位相互作用 黑色素瘤细胞上带有抗分子的分子，以抑制抗肿瘤免疫。详细机械论 将通过综合方法获得理解，该方法将基于物理科学(PS)的工具与 黑色素瘤T细胞免疫的两个互补的临床前小鼠模型，将使用 来自黑色素瘤患者的生物标本。分子焦点是启动T细胞的T细胞受体(TCR) 结合多肽主要组织相容性复合体(PMHC)和辅受体CD8的抗原识别 这与pMHC共同连接。第一个PS工具是通过单分子作用力来量化TCR机械传感 细胞内信号同步成像分子相互作用的原位动力学分析 在一个单细胞上。第二个PS工具是基于DNA的数字张力探头，它报告细胞产生的拉力 在TCR和CD8上通过接通的pMHC。一种动物模型是使用黑色素瘤的公认标准 与OT-I TCR识别的鸡卵清蛋白抗原结合。另一种动物模型是 黑色素瘤自身抗原gp100与JR209人源化转基因小鼠联合。通过分析 TCR和/或CD8在T细胞膜上的机械调节二维(2D)配体结合 观察小鼠原发黑色素瘤CD8T细胞对pMHC的TCR亲和力 与非肿瘤相关组织(脾和血液)中的T细胞相比显著减少。是这样的 用pMHC四聚体的常规检测不能检测不同的亲和力，这证明了这种能力 我们分析TCR-pMHC相互作用的基于力学的方法。我们还发现黑色素瘤 显著改变力依赖的TCR-pMHC键的持久性：在无肿瘤动物中，TCR和pMHC 形成了一种接滑粘结，它的寿命随着力的增加而先增加后减少，我们已经 TCR-pMHC键寿命仅随力的增加而减小，即表现为滑移键，并且 与T细胞效应器功能降低有关。 结果，至少部分是由于TME内的抗原识别受损，如改变的TCR所示 PMHC的机械传感。 对T细胞抗原识别至关重要的相互作用受到TME的损害；2)定义功能 抑制T细胞抗原识别的后果；以及3)阐明TME的潜在机制 抑制T细胞抗原识别。完成这些目标有可能发现新的 提高晚期黑色素瘤患者预后的免疫治疗靶点 疾病。

Project Summary This project investigates how the tumor microenvironment (TME) impairs in situ interactions of T-cell surface molecules with counter-molecules on the melanoma cells to suppress anti-tumor immunity. Detailed mechanistic understanding will be obtained by an integrated approach that combines physical science (PS) based tools with two complementary pre-clinical mouse models of melanoma T cell immunity, which will be further tested using biospecimens from melanoma patients. The molecular focus is the T-cell receptor (TCR) that initiates the T-cell antigen recognition upon binding to peptide-major histocompatibility complex (pMHC), and the coreceptor CD8 that co-ligates with the pMHC. The first PS tool is quantifying TCR mechanosensing by single-molecule force probes through in situ kinetic analyses of molecular interactions with concurrent imaging of intracellular signals on a single cell. The second PS tool is DNA-based digital tension probes that report cell generated pulling forces on the TCR and CD8 via engaged pMHC. One animal model is a recognized standard that uses melanoma conjugated with a chicken ovalbumin antigen recognized by the OT-I TCR. The other animal model is a melanoma self-antigen gp100 in conjunction with JR209 humanized transgenic mice. By analyzing the mechanically regulated two-dimensional (2D) ligand binding of TCR and/or CD8 at the T-cell membrane, we observed that the TCR avidities for the pMHC of CD8 T cells infiltrating primary murine melanomas grown in vivo are significantly reduced relative to T cells within non-tumor associated tissues (spleen and blood). Such differential avidities were not detected by the conventional assay using pMHC tetramer, attesting to the power of our mechanics-based methods for analyzing TCR–pMHC interactions. We also found melanomas to substantially alter the force-dependent TCR–pMHC bond durability: in tumor-free animals, the TCR and pMHC formed a catch-slip bond whose lifetime first increased and then decreased with increasing force, which we have previously demonstrated to govern T cell signaling and effector function, whereas in melanoma-bearing animals, the TCR–pMHC bond lifetime only decreased with increasing force, i.e., behaved as a slip bond and were associated with reduced T cell effector functions. We hypothesize that deficient CD8 T cell immunity in melanoma results, at least in part, from impaired antigen recognition within the TME, as manifested by the altered TCR mechanosensing of pMHC. Three specific aims are proposed to test our hypothesis: 1) Determine the molecular interactions crucial to T cell antigen recognition that are impaired by the TME; 2) Define the functional consequences of suppressed T cell antigen recognition; and 3) Elucidate the mechanisms underlying the TME suppression of T cell antigen recognition. Completing these aims has the potential to identify new immunotherapeutic targets for the treatment of melanoma to improve the outcomes of patients with advanced disease.