T CELL RESPONSE TO GENETICALLY ENGINEERED AND MATURED DC

T 细胞对基因工程和成熟 DC 的反应

• 批准号: 6633514
• 负责人: BIJAY MUKHERJI
• 金额: $ 24.87万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633514
• 关键词: MHC class I antigen; MHC class II antigen; T lymphocyte; antigen presentation; apoptosis; biological signal transduction; cell population study; chimeric proteins; colony stimulating factor; cytokine receptors; cytotoxic T lymphocyte; dendritic cells; genetic manipulation; helper T lymphocyte; human tissue; immunogenetics; interleukin 2; intracellular transport; melanoma; neoplastic cell; receptor expression; tissue /cell culture; transfection /expression vector; tumor antigens

The major goal of this proposal is to test the hypothesis that "a more efficient anti-tumor T cell response can be generated through antigen presentation by specialized antigen presenting cells such as dendritic cells (DC) grown to immunogenic maturity in an environment of danger and/or tissue damage and made to present the relevant tumor associated antigen through the MHC class I and class II pathways". Using the human melanoma antigen MART-1 system as a prototype, the specific aims are: 1) to undertake a comprehensive analysis of both CD4+ and CD8+ T cell responses to MART-1 presented in vitro by engineered and immunogenically matured DC; 2) to define the role of and the mechanism by which CD4+ T cells facilitate and amplify CTL response; 3) to examine the molecular basis of "help" (IL-2 message/IL2R expression, signaling through cytokine common receptor gammac, and anti-apoptotic vs pro-apoptotic mechanism (Bcl/Bax); and 4) to examine the in vitro immunogenicity of compartmentalized epitope presentation by DC genetically engineered with VSV pseudotyped retrovector expressing EGFP-TAA fusion protein plus intracellular trafficking signal sequences and bacterial immuno-stimulatory sequences (ISS). Myeloid DC grown in GM-CSF and IL-4 will be transduced with an adenovector to express the MART-1 antigen and matured to "immunogenic competence" through CD40 signaling, with a variety of bacterial stimulants, or by making the DC capture MART-1 antigen from apoptotic cells. The conditioned DC will be used to generate CTL and helper T cell in vitro. T cell responses will be monitored in CTL assay, Fastimmune assay, and tetrameter binding assay. The role of the CD4+ T cells on the DC as well as on the CTL will be examined in appropriate co-cultures and the robustness of the CTL response will be determined in CTL assay, Fastimmune assay and in tetramer binding assay to obtain a quantitative assessment of CTL expansion. We shall also test a number of avenues to enhance antigen presentation by compartmentalized class I and/or class II loading of antigen via engineered DC. These are: a) endosomal localization with chimeric polypeptide expressing the TAA and intracellular trafficking signals: b) trafficking of TAA and heat shock -TAA fusions; c) TAA trafficking under a polarized Th1 condition engineered internally by expressing chimeric TAA and bacterial ISS sequences; and d) nuclear localization of EGFP:TAA chimeras. These studies will provide a much needed understanding of the rules of engagement of DC and CD4+ T cells with translational implications.

该提议的主要目标是检验以下假设：“更有效的抗肿瘤T细胞应答可以通过在危险和/或组织损伤的环境中生长至免疫原性成熟的特化抗原呈递细胞（例如树突细胞（DC））的抗原呈递来产生，并使其通过MHC I类和II类途径呈递相关的肿瘤相关抗原”。 以人黑色素瘤抗原MART-1系统为原型，具体目的是：1）对由工程化的和免疫原性成熟的DC在体外呈递的对MART-1的CD 4+和CD 8 + T细胞应答进行全面分析; 2）确定CD 4 + T细胞促进和放大CTL应答的作用和机制; 3）检查“帮助”的分子基础（IL-2信息/IL 2 R表达，通过细胞因子共同受体γ的信号传导，以及抗凋亡与促凋亡机制（Bcl/Bax）;和4）检测由用表达EGFP的VSV假型逆转录载体基因工程化的DC进行的区室化表位呈递的体外免疫原性。TAA融合蛋白加上细胞内运输信号序列和细菌免疫刺激序列（ISS）。 在GM-CSF和IL-4中生长的髓样DC将用腺病毒载体转导以表达MART-1抗原，并通过CD 40信号传导、用各种细菌刺激剂或通过使DC从凋亡细胞捕获MART-1抗原而成熟为“免疫原性能力”。条件DC将用于体外产生CTL和辅助性T细胞。 将在CTL试验、Fastimmune试验和四元结合试验中监测T细胞应答。将在适当的共培养物中检查CD 4 + T细胞对DC以及对CTL的作用，并将在CTL测定、Fastimmune测定和四聚体结合测定中确定CTL应答的稳健性，以获得CTL扩增的定量评估。 我们还将测试通过经工程化DC的抗原的区室化I类和/或II类负载来增强抗原呈递的许多途径。 这些是：a）用表达TAA和细胞内运输信号的嵌合多肽的内体定位; B）TAA和热休克-TAA融合物的运输; c）在通过表达嵌合TAA和细菌ISS序列内部工程化的极化Th 1条件下的TAA运输;和d）EGFP：TAA嵌合体的核定位。 这些研究将提供DC和CD 4 + T细胞参与翻译影响的规则的迫切需要的理解。

项目成果

Knockdown of T-bet expression in Mart-127-35 -specific T-cell-receptor-engineered human CD4(+) CD25(-) and CD8(+) T cells attenuates effector function.

Mart-127-35 特异性 T 细胞受体工程化人类 CD4( )、CD25(-) 和 CD8( ) T 细胞中 T-bet 表达的敲低会减弱效应子功能。

• DOI: 10.1111/imm.12431
• 发表时间: 2015
• 期刊: Immunology
• 影响因子: 6.4
• 作者: Jha,SidharthS;Chakraborty,NityaG;Singh,Prashant;Mukherji,Bijay;Dorsky,DavidI
• 通讯作者: Dorsky,DavidI

Inhibition of superoxide generation upon T-cell receptor engagement rescues Mart-1(27-35)-reactive T cells from activation-induced cell death.

• DOI: 10.1158/0008-5472.can-09-1176
• 发表时间: 2009-08-01
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Norell H;Martins da Palma T;Lesher A;Kaur N;Mehrotra M;Naga OS;Spivey N;Olafimihan S;Chakraborty NG;Voelkel-Johnson C;Nishimura MI;Mukherji B;Mehrotra S
• 通讯作者: Mehrotra S

Analyses of T cell-mediated immune response to a human melanoma-associated antigen by the young and the elderly.

分析年轻人和老年人对人类黑色素瘤相关抗原的 T 细胞介导的免疫反应。

• DOI: 10.1016/j.humimm.2013.01.015
• 发表时间: 2013
• 期刊: Human immunology
• 影响因子: 2.7
• 作者: Chakraborty,NityaG;Yadav,Meeta;Dadras,SoheilS;Singh,Prashant;Chhabra,Arvind;Feinn,Richard;Kerr,PhillipE;Grant-Kels,JaneM;Mukherji,Bijay;Hegde,UpendraP
• 通讯作者: Hegde,UpendraP