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 在黑色素瘤细胞上具有反分子的分子以抑制抗肿瘤免疫。详细的机理 理解将通过一种综合方法获得，该方法将基于物理科学（PS）的工具与 两种互补的黑色素瘤T细胞免疫的临床前小鼠模型，将使用 黑色素瘤患者的生物样本分子焦点是T细胞受体（TCR），其启动T细胞 结合肽-主要组织相容性复合物（pMHC）和辅助受体CD 8后的抗原识别 与pMHC共连接。第一个PS工具是通过单分子力定量TCR机械传感 探针通过分子相互作用的原位动力学分析与细胞内信号的同时成像 在一个细胞上。第二个PS工具是基于DNA的数字张力探针，其报告细胞产生的拉力 通过接合的pMHC对TCR和CD 8的作用。一种动物模型是公认的标准， 与OT-ITCR识别的鸡卵清蛋白抗原缀合。另一种动物模型是 黑色素瘤自身抗原gp 100与JR 209人源化转基因小鼠联合。通过分析 机械调节TCR和/或CD 8在T细胞膜上的二维（2D）配体结合，我们 观察到TCR对体内生长的浸润原发性鼠黑色素瘤的CD 8 T细胞的pMHC的亲和力 相对于非肿瘤相关组织（脾和血液）内的T细胞显著降低。等 使用pMHC四聚体的常规测定法未检测到差异亲合力，证明了 我们的基于力学的方法来分析TCR-pMHC相互作用。我们还发现黑色素瘤 显著改变力依赖性TCR-pMHC结合的持久性：在无肿瘤动物中，TCR和pMHC 形成了一个捕捉滑移键，其寿命首先增加，然后随着力的增加而减少，我们已经 先前证明控制T细胞信号传导和效应器功能，而在携带黑素瘤的动物中， TCR-pMHC键的寿命仅随力的增加而降低，即，表现为滑动粘结， 与T细胞效应功能降低有关。我们假设黑色素瘤中CD 8 T细胞免疫缺陷 至少部分地由TME内受损的抗原识别引起，如通过改变的TCR所表现的 pMHC的机械传感。提出了三个具体目标来验证我们的假设：1）确定分子 T细胞抗原识别关键的相互作用，这些相互作用被TME损害; 2）定义功能性的 抑制T细胞抗原识别的后果;和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 治疗黑色素瘤的免疫靶点，以改善晚期黑色素瘤患者的结局 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.