Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability, and allows identification of antigen-specific CD4+ T cells in peripheral blood mononuclear cells
Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability, and allows identification of antigen-specific CD4+ T cells in peripheral blood mononuclear cells
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DOI：
10.1002/1521-4141(200212)32:12
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发表时间：
2002-12-01
影响因子：
5.4
通讯作者：
Kelleher, AD
中科院分区：
文献类型：
--
作者：
Cunliffe, SL;Wyer, JR;Kelleher, AD
用于产生可溶性HLA-DR分子、适合于多聚化和用作染色试剂的可靠、有效的系统已被证明是难以实现的。我们发现在肽和DRB 1 *0101-链的N末端之间添加柔性接头（Crawford，F.，Kozono，H.，白色，J.，Marrack，R和Kappler，J.，1998年豁免。8：675-682.），导致大肠杆菌表达的α链和β链的体外折叠效率更高，并增加了DRA 1 *0101/DRB 1 *0101/肽复合物的产率和稳定性。尽管10个氨基酸的接头对于来自HIV p24的20聚体表位有效地起作用，但是需要更长的接头来产生具有流感血凝素表位（HA（306-318））的DR 1 MHC 11类四聚体。DR 1-HA四聚体能够仅用短暂的30分钟孵育对超过98%的特异性克隆（HA 1.7）进行阳性染色。四聚体复合物检测稀释到PBMC中的克隆细胞，具有高灵敏度，加上CD 4（+）细胞中的低背景染色。在用HA肽刺激的PBMC群体中检测抗原特异性CD 4（+）T细胞是可能的。这证明了在许多临床情况下监测外周血中CD 4（+）T细胞应答的潜力。
Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, R and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class 11 tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.