T-Cell Antigen Recognition and Tumor Antigens

T 细胞抗原识别和肿瘤抗原

• 批准号: 7038111
• 负责人: ETTORE APPELLA
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7038111
• 关键词: T cell receptor; antigen presentation; cellular immunity; confocal scanning microscopy; cytotoxic T lymphocyte; dendritic cells; flow cytometry; gene expression; gene mutation; genetically modified animals; helper T lymphocyte; laboratory mouse; major histocompatibility complex; neoplasm /cancer immunology; neoplasm /cancer vaccine; p53 gene /protein; protein binding; surface antigens; synthetic peptide; synthetic tumor antigen; tumor antigens; vaccine development

For years, a major goal of tumor immunology has been the molecular characterization of human tumor antigens. Missense mutations in the p53 tumor suppressor gene are the most common genetic events associated with human cancer. Our work has focused on an immunological characterization of p53 missense mutations occurring in cytotoxic T-cell defined p53 epitopes in HLA-A0201+ squamous cell carcinomas of the head and neck. An analysis of p53 expressed in these tumors identified six missense mutations that occurred within the three HLA-A2-restricted, CTL-defined p53149-157, p53217-225 and p53264-272 epitopes. 3/6 mutations yielded mutant peptides with reduced or similar binding affinities for HLA-A2 molecules compared to the parental peptides, which failed to yield anti-peptide CTL following in vitro stimulation (IVS) of peripheral blood mononuclear cells (PBMC) with peptide-pulsed autologous dendritic cells. Two p53 mutations, which occur within the p53217-225 epitope, Y220C and Y220H, generated mutant peptides with slightly increased affinities to the HLA-A 0201 molecule relative to the weakly binding parental peptide, that were immunogenic. The anti-p53 Y220C effectors were reactive against an HLA-A0201+ SCCHN cell line, UD-SCC 6, which expresses the p53 Y220C mutation, indicating that this mutation can be naturally presented. IVS of the UD-SCC 6 patients' PBMC generated an anti-p53 Y220C CTL that recognized the autologous tumor cell line. Most importantly, the Y220C mutation was detected in 4/40 (10%) SCCHN in HLA-A2+ patients compared to a reported occurrence frequency of 1% of all human cancers. The observed increased representation of mutations at p53 codon 220 in HLA-A2.1+ SCCHN tumors, which also might be prevalent in other HLA-A2.1+ carcinomas, suggests that development of vaccines targeting this mutation would be more widely applicable than previously envisioned for any given p53 missense mutation. T cell activation is a critical step in mounting an appropriate immune defense against infectious agents such as viruses and bacteria. Central to understanding how antigen presenting cells mediate the full activation of T cells is knowledge of the interaction of MHC molecules with the T-cell receptor (TCR). We have previously described a novel experimental system in order to address how engagement of TCR leads to full T cell activation. We have isolated the mouse AHIII TCR and its MHC ligand, H2-Db-p1058, as well as a human pMHC complex with which it interacts, HLA-A2-p1049. Having demonstrated that we have in possession a functional AHIII cytotoxic T cell line and can produce large quantities of active protein for use in functional and biochemical analysis, we are currently performing experiments to distinguish between two models of T cell activation, the affinity-based model and the kinetic-rate model. Using our co-crystal structure of AHIII and HLA-A2-p1049, we have selected 11 mutations in the A2-p1049 molecule that will alter the affinity and/or kinetic off rate for the AHIII TCR. In the past year we have finished making sufficient quantities of the mutant pMHC complexes to determine its affinity and kinetic binding rates to the AHIII TCR by surface plasmon resonance. To relate these mathematical constants to T cell activity, we previously performed cytotoxic lysis assays. Realizing that cellular proliferation and cytokine secretion also make up the biological outcome of full T cell activation, we sought to establish the tools necessary to measure these responses. Thus, in the past year, we have finished making the cell lines expressing our mutant pMHC complexes needed to elicit T cell proliferation and cytokine secretion. Additionally, we have now obtained co-crystals of the AHIII TCR and some mutant pMHC complexes that will aid us in understanding how these mutations have affected the kinetic rate constants. Our current data supports the affinity-based model. The current study focuses on altered stimuli on the T cell and examines distal functional consequences, names cytotoxic lysis activity, proliferative response, and cytokine secretion. Upon the completion of this study in the near future, it will be essential to examine more immediate responses, such as changes in gene expression, activation of signaling intermediates, and spatial rearrangement of signaling components, in order to understand how the distal functional consequences are achieved. The establishment of an AHIII transgenic mouse will be necessary to compare our results with true primary T cells. Additionally, an AHIII transgenic mouse will allow this same study to be performed on naive and memory T cells, as opposed to cytotoxic T cells, in order to examine the differences in the requirements for activation.

多年来，肿瘤免疫学的一个主要目标一直是人类肿瘤抗原的分子表征。p53肿瘤抑制基因中的错义突变是与人类癌症相关的最常见的遗传事件。我们的工作集中在HLA-A0201+头颈部鳞状细胞癌中细胞毒性T细胞定义的p53表位中发生的p53错义突变的免疫学特征。对这些肿瘤中表达的p53的分析鉴定出在三个HLA-A2限制性、CTL定义的p53149-157、p53217-225和p53264-272表位内发生的六个错义突变。与亲本肽相比，3/6突变产生对HLA-A2分子具有降低或相似的结合亲和力的突变肽，其在用肽脉冲的自体树突细胞体外刺激（IVS）外周血单核细胞（PBMC）后不能产生抗肽CTL。发生在p53217-225表位内的两个p53突变，Y220 C和Y220 H，产生了相对于弱结合亲本肽对HLA-A 0201分子具有略微增加的亲和力的突变肽，其具有免疫原性。抗p53 Y220 C效应子对表达p53 Y220 C突变的HLA-A0201+ SCCHN细胞系UD-SCC 6具有反应性，表明该突变可以天然存在。UD-SCC 6患者PBMC的IVS产生识别自体肿瘤细胞系的抗p53 Y220 C CTL。最重要的是，在HLA-A2+患者的4/40（10%）SCCHN中检测到Y220 C突变，而报告的所有人类癌症的发生频率为1%。在HLA-A2.1+ SCCHN肿瘤中观察到的p53密码子220处突变的增加的代表性（其也可能在其他HLA-A2.1+癌中普遍存在）表明，靶向该突变的疫苗的开发将比先前针对任何给定的p53错义突变所设想的更广泛地适用。T细胞活化是对感染因子（如病毒和细菌）进行适当免疫防御的关键步骤。了解抗原递呈细胞如何介导T细胞的完全激活的核心是了解MHC分子与T细胞受体（TCR）的相互作用。我们之前已经描述了一种新的实验系统，以解决TCR的参与如何导致完全T细胞活化。我们已经分离出小鼠AHIII TCR及其MHC配体H2-Db-p1058，以及与其相互作用的人pMHC复合物HLA-A2-p1049。已经证明，我们拥有一个功能性AHIII细胞毒性T细胞系，并可以产生大量的活性蛋白用于功能和生化分析，我们目前正在进行实验，以区分两种模型的T细胞活化，亲和力为基础的模型和动力学速率模型。使用我们的AHIII和HLA-A2-p1049的共晶体结构，我们选择了A2-p1049分子中的11个突变，这些突变将改变AHIII TCR的亲和力和/或动力学解离速率。在过去的一年中，我们已经完成了足够数量的突变pMHC复合物，以确定其亲和力和动力学结合率的AHIII TCR的表面等离子体共振。为了将这些数学常数与T细胞活性相关联，我们先前进行了细胞毒性裂解测定。意识到细胞增殖和细胞因子分泌也构成了完全T细胞活化的生物学结果，我们试图建立测量这些反应所必需的工具。因此，在过去的一年中，我们已经完成了表达我们的突变型pMHC复合物的细胞系，这些复合物是引发T细胞增殖和细胞因子分泌所需的。此外，我们现在已经获得了AHIII TCR和一些突变pMHC复合物的共晶体，这将有助于我们理解这些突变如何影响动力学速率常数。我们目前的数据支持基于亲和力的模型。目前的研究重点是改变刺激的T细胞，并检查远端功能的后果，名称细胞毒性裂解活性，增殖反应和细胞因子分泌。在不久的将来完成这项研究后，将有必要检查更直接的反应，如基因表达的变化，信号传导中间体的激活和信号传导组分的空间重排，以了解如何实现远端功能性后果。建立AHIII转基因小鼠将是必要的，以比较我们的结果与真正的原代T细胞。此外，AHIII转基因小鼠将允许对初始和记忆T细胞（而不是细胞毒性T细胞）进行相同的研究，以检查活化要求的差异。