Strbo Natasa, Yamazaki Koichi, Lee Kelvin, Rukavina Daniel, Podack Eckhard R
Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL, USA.
Am J Reprod Immunol. 2002 Oct;48(4):220-5. doi: 10.1034/j.1600-0897.2002.01118.x.
As shown previously, gp96-Ig peptide complexes secreted by an ovalbumin transfected tumor (EG7) mediate strong, specific tumor immunity through a CD4 T cell independent CD8+ CTL response. In this study, we set out to develop a system to quantitatively determine the CD8 CTL response to gp96-Ig and to evaluate the influence of an established wild type tumor.
Secreted heat shock protein gp96-Ig was constructed by replacement of the endoplasmic reticulum retention signal with the Fc portion of IgGI, transfected into EG7 (EG7-gp96-Ig) and used to induce CD8+ CTL expansion in vivo. Adoptively transferred, ovalbumin specific T-cell receptor (TCR) transgenic CD8+ cells (OT-1) responded with clonal expansion to the immunization with EG7-gp96-Ig. OT-1 expansion was quantitated with K(b-peptide)-tetramers by flow cytometry.
In response to primary immunization with EG7-gp96-Ig, OT-1 expand from an initial frequency of 0.5 to 25% of all CD8 cells, and to 50% of all CD8 cells after a booster immunization. Endogenous ovalbumin specific CD8 cells also expand strongly. Antigen specific effector function was measured by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) for interferon-gamma (IFN-gamma). While effector function was strongly induced by secreted gp96-Ig, not all expanded OT-1 produce IFN-gamma. EG7 does not cause OT-1 expansion, but rather induces anergy. If OT-1 are transferred into wild type EG7 tumor bearing mice to induce anergy of OT-1, immunization with EG7-gp96-Ig can partly overcome unresponsiveness.
We conclude that secreted gp96-Ig is a powerful mediator of specific CD8+ CTL responses in vivo. Secretory gp96 mimics release of gp96 by damaged or necrotic cells that is able to activate dendritic cells without CD4 help. Gp96-Ig associated peptides have not been selected by binding to major histocompatibility complex (MHC). Specific immunization by secreted gp96-Ig therefore is expected to occur also in allogeneic settings.
如先前所示，由卵清蛋白转染肿瘤（EG7）分泌的gp96-Ig肽复合物通过不依赖CD4 T细胞的CD8⁺CTL反应介导强大的特异性肿瘤免疫。在本研究中，我们着手开发一种系统来定量测定对gp96-Ig的CD8 CTL反应，并评估已建立的野生型肿瘤的影响。
通过用IgG1的Fc部分替换内质网保留信号构建分泌型热休克蛋白gp96-Ig，将其转染到EG7（EG7-gp96-Ig）中，并用于在体内诱导CD8⁺CTL扩增。过继转移的、卵清蛋白特异性T细胞受体（TCR）转基因CD8⁺细胞（OT-1）对EG7-gp96-Ig免疫产生克隆扩增反应。通过流式细胞术用K(b-肽)-四聚体定量OT-1扩增。
响应于EG7-gp96-Ig的初次免疫，OT-1从初始频率0.5%扩增至所有CD8细胞的25%，在加强免疫后扩增至所有CD8细胞的50%。内源性卵清蛋白特异性CD8细胞也强烈扩增。通过酶联免疫吸附斑点形成细胞测定法（ELISPOT）检测干扰素-γ（IFN-γ）来测量抗原特异性效应功能。虽然分泌的gp96-Ig强烈诱导效应功能，但并非所有扩增的OT-1都产生IFN-γ。EG7不会引起OT-1扩增，而是诱导无反应性。如果将OT-1转移到携带野生型EG7肿瘤的小鼠中以诱导OT-1无反应性，用EG7-gp96-Ig免疫可部分克服无反应性。
我们得出结论，分泌的gp96-Ig是体内特异性CD8⁺CTL反应的强大介质。分泌型gp96模拟受损或坏死细胞释放的gp96，其能够在没有CD4辅助的情况下激活树突状细胞。与gp96-Ig相关的肽尚未通过与主要组织相容性复合体（MHC）结合来选择。因此，预计分泌的gp96-Ig在同种异体环境中也会发生特异性免疫。
