A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.
A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.
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DOI：
10.1016/j.vetimm.2012.08.012
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发表时间：
2012-12-15
影响因子：
1.8
通讯作者：
Hess PR
中科院分区：
文献类型：
--
作者：
Ross P;Holmes JC;Gojanovich GS;Hess PR
识别免疫优势CTL表位是研究人群中CD8+T细胞反应的关键，但仍然很困难，因为抗原中的多肽通常太多，无法合成和筛选。相反，为了便于发现，在对蛋白质进行电子扫描以寻找与限制性MHC I类等位基因的基序或结合偏好匹配的序列时，可以使用限制性MHC I类等位基因-免疫优势的最大决定因素-来预测可能的候选基因。这种分析的高假阳性率理想情况下需要结合确认，这是通过使用RMA-S等细胞系常规检测获得的，这些细胞系具有与抗原处理(TAP)机制相关的缺陷转运蛋白，因此很少有表面I类分子。通过添加真正的结合剂，多肽-MHC复合体在细胞表面的稳定性和由此产生的延长寿命证实了它们的身份。为了确定是否可以为狗开发类似的检测方法，我们将流行的I类等位基因Dla-88*50801导入RMA-S。在BARC3克隆中，重组重链与小鼠MHC 2-微球蛋白结合，重要的是可以通过表面β稳定来区分基序匹配和错配的多肽。这项工作证明了利用转导犬等位基因的S细胞作为发现该物种细胞毒性T淋巴细胞表位的工具的潜力。
Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele – the largest determinant of immunodominance – can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species.
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DOI：
10.1073/pnas.92.18.8166
发表时间：
1995-08-29
影响因子：
11.1
作者：
POGUE, RR;ERON, J;MATSUI, M
通讯作者：
MATSUI, M
影响因子：
3.7
作者：
Andersen, MLM;Ruhwald, M;Claesson, MH
通讯作者：
Claesson, MH
影响因子：
4.4
作者：
Tian, Shaomin;Maile, Robert;Frelinger, Jeffrey A.
通讯作者：
Frelinger, Jeffrey A.
DOI：
10.1084/jem.167.2.288
发表时间：
1988-02-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Seong RH;Clayberger CA;Krensky AM;Parnes JR
通讯作者：
Parnes JR
影响因子：
8
作者：
Morgan, CS;Holton, JM;Mayo, SL
通讯作者：
Mayo, SL