Mechanism of Human T-Cell Activation

人类 T 细胞激活机制

• 批准号: 7291791
• 负责人: JEFFREY SCHLOM
• 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/7291791
• 关键词: Mechanism; Human; Cell; Activation

ANALYSES OF RECOMBINANT VACCINIA AND FOWLPOX VACCINE VECTORS EXPRESSING TRANSGENES FOR TWO HUMAN TUMOR ANTIGENS AND THREE HUMAN COSTIMULATORY MOLECULES. The poor immunogenicity of tumor antigens and the antigenic heterogeneity of tumors call for vaccine strategies to enhance T-cell responses to multiple antigens. Two antigens expressed noncoordinately on most human carcinomas are carcinoembryonic antigen (CEA) and MUC-1. We have reported the construction and characterization of two viral vector vaccines to address these issues. The two viral vectors analyzed are the replication-competent recombinant vaccinia virus (rV-) and the avipox vector, fowlpox (rF-), which is replication incompetent in mammalian cells. Each vector encodes the transgenes for three human costimulatory molecules (B7-1, ICAM-1, and LFA-3, designated TRICOM) and the CEA and MUC-1 transgenes (which also contain agonist epitopes). The vectors are designated rV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM. Each of the vectors is shown to be capable of faithfully expressing all five transgenes in human dendritic cells (DC). DCs infected with either vector are shown to activate both CEA- and MUC-1-specific T-cell lines to the same level as DCs infected with CEA-TRICOM or MUC-1-TRICOM vectors. Thus, no evidence of antigenic competition between CEA and MUC-1 was observed. Human DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/ TRICOM are also shown to be capable of generating both MUC-1- and CEA-specific T-cell lines; these T-cell lines are in turn shown to be capable of lysing targets pulsed with MUC-1 or CEA peptides as well as human tumor cells endogenously expressing MUC-1 and/or CEA. These studies provided the rationale for the clinical evaluation of these multigene vectors in patients with a range of carcinomas expressing MUC-1 and/or CEA. INDUCTION OF HIGHER-AVIDITY HUMAN CTLS BY VECTOR-MEDIATED ENHANCED COSTIMULATION OF ANTIGEN-PRESENTING CELLS. The efficacy of antigen-specific CD8(+) CTLs depends not only on the quantity of CTLs generated but also perhaps, more importantly, on the avidity of the CTLs. To date, however, no strategy has been shown to preferentially induce higher-avidity human CTLs. In these studies, antigen-presenting cells (APC) generated from human peripheral blood mononuclear cells were infected with a recombinant avipox vector (rF-) containing the transgenes for a triad of costimulatory molecules (human B7.1, intercellular adhesion molecule-1, and LFA-3, designated as rF-TRICOM) and then used to elicit peptide-specific CTLs from autologous T cells. Compared with peptide-pulsed noninfected APCs or peptide-pulsed APCs infected with wild-type vector, peptide-pulsed APCs infected with rF-TRICOM induced not only more CTLs but also higher-avidity CTLs; this was shown by tetramer staining, tetramer dissociation, IFN-gamma production, and cytolytic assays. Peptide-pulsed rF-TRICOM-infected dendritic cells were also shown to induce CTLs with a >10-fold higher avidity than CTLs induced using CD40L-matured dendritic cells; the use of peptide-pulsed CD40L-matured dendritic cells infected with rF-TRICOM as APCs induced CTLs of even greater avidity. To our knowledge, these studies are the first to show a methodology to induce higher-avidity human CTLs and have implications for the development of more efficient vaccines for a range of human cancers. HUMAN B CELLS THAT HYPEREXPRESS A TRIAD OF COSTIMULATORY MOLECULES VIA AVIPOX-VECTOR INFECTION: AN ALTERNATIVE SOURCE OF EFFICIENT ANTIGEN-PRESENTING CELLS. Dendritic cells (DCs) are the most potent of the antigen-presenting cells (APCs). Preparation of sufficient numbers of mature DCs, however, is both costly and time-consuming. We have examined the possibility of using an alternative source of APCs that would be easier to obtain, would not require extensive culture, and thus would be more applicable to human immunotherapy protocols.

对两种人肿瘤抗原和三个人类共刺激分子的转基因的重组疫苗和Fowlpox疫苗向量的分析。肿瘤抗原的免疫原性和肿瘤的抗原异质性要求采用疫苗策略，以增强对多种抗原的T细胞反应。两种在大多数人癌上表达的两种抗原是癌胚抗原（CEA）和MUC-1。我们报告了两种病毒载体疫苗的结构和表征，以解决这些问题。分析的两个病毒载体是复制能力的重组疫苗病毒（RV-）和武毒载体Fowlpox（RF-），这在哺乳动物细胞中是无能的复制。每个载体均编码三个人类共刺激分子（B7-1，ICAM-1和LFA-3）和CEA和MUC-1转基因（还包含激动剂表位）的转基因。向量被指定为RV-CEA/MUC/Tricom和RF-CEA/MUC/Tricom。每个载体都显示能够忠实地表达人树突状细胞中的所有五个转基因（DC）。被感染的载体感染的DC被证明可将CEA-和MUC-1特异性T细胞线激活与感染CEA-TRICOM或MUC-1-TICOM载体的DC相同的水平。因此，未观察到CEA和MUC-1之间抗原竞争的证据。感染RV-CEA/MUC/Tricom或RF-CEA/MUC/Tricom的人DC也能够同时产生MUC-1和CEA特异性T细胞系。这些T细胞系又显示出能够用MUC-1或CEA肽脉冲的裂解靶标，以及内源表达MUC-1和/或CEA的人类肿瘤细胞。这些研究提供了对表达MUC-1和/或CEA的一系列癌患者的这些多基因载体临床评估的理由。通过载体介导的增强抗原呈递细胞的共刺激，诱导更高的持续性人CTL。抗原特异性CD8（+）CTL的功效不仅取决于产生的CTL的数量，而且更重要的是，也许还取决于CTL的亲和力。然而，迄今为止，尚未证明尚无优先引起更高避免人类CTL的策略。在这些研究中，用重组的抗原血液单核细胞产生的抗原呈递细胞（APC）被含有重组的丝维毒载体（RF-）感染，这些抗原载体载体（RF-）含有三合一的转基因的cost型分子的转基因（人B7.1，人类B7.1，人类b7.1，人体间粘附分子1，然后将其定义为lfa-1，然后将其指定为RFA-3-来自自体T细胞的CTL。与感染了野生型载体的肽脉冲未感染的APC或肽脉冲的APC相比，感染了RF-TICOM感染的肽脉冲APC，不仅引起更多的CTL，而且还引起了更高的持续性CTL；四聚体染色，四聚体解离，IFN-gamma产生和细胞溶性测定显示。与使用CD40L成熟的树突状细胞诱导的CTL相比，肽脉冲的RF-RF-TRICOM感染的树突状细胞具有高度> 10倍的CTL诱导CTL。用RF-TICOM感染的肽脉冲的CD40L成熟的树突状细胞作为APC诱发了更大的亲和力。据我们所知，这些研究是第一个展示诱导更高避免人类CTL的方法的方法，并对一系列人类癌症开发更有效的疫苗具有影响。通过抗原矢量感染过度刺激的costimulation分子三合会的人类B细胞：有效的抗原呈递细胞的替代来源。树突状细胞（DC）是抗原呈递细胞（APC）中最有效的。但是，准备足够数量的成熟DC既昂贵又耗时。我们已经检查了使用更容易获得的APC替代来源的可能性，不需要广泛的文化，因此更适用于人类免疫疗法方案。