alpha beta T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization
alpha beta T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization
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DOI：
10.1073/pnas.94.25.13838
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发表时间：
1997-12-09
影响因子：
11.1
通讯作者：
Teyton, L
中科院分区：
文献类型：
--
作者：
Garcia, KC;Tallquist, MD;Teyton, L
细胞免疫是通过α-βT细胞受体(TCR)与主要组织相容性复合体(MHC)分子背景下的多肽相互作用而介导的。同种异体反应性T细胞具有识别自身和外源多肽-MHC(PMHC)复合体的α-βTCR，这意味着TCR与不同的pMHC具有显著的互补性。为了表征同种异体反应性TCR识别pMHC的分子基础，我们在果蝇黑腹细胞中生产了一种可溶的、重组形式的同种异体反应性α-βT细胞受体。这个重组的TCR，2C，被表达为正确配对的α-β异二聚体，链通过C-末端的二硫键共价连接。利用构象特异性的单抗以及同基因和同种异体的pMHC配体，通过表面等离子体共振(SPR)分析来探讨2C TCR的天然构象。2C与H-2K(B)-dEV8、H-2K(BM3)-dEV8、H-2K(B)-SiYR和H-2L(D)-p2Ca的相互作用跨越了同基因(H-2K(B))和异基因(H-2K(BM3)，H-2L(D))配体的亲和力K-d=10(-4)到10(-6)M的范围。一般来说，同基因配体与同种异体配体结合的亲和力比同基因配体弱，这与目前胸腺选择和T细胞激活的阈值模型一致。2C TCR的结晶需要对α链和β链的C末端残基进行蛋白质水解性修剪。生长了2C与H-2K(B)-dEV8、H-2K(BM3)-dEV8和H-2K(B)-SiYR的配合物晶体。
Cellular immunity is mediated by the interaction of an alpha beta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alpha beta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alpha beta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alpha beta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2K(b)-dEV8, H-2K(bm3)-dEV8, H-2K(b)-SIYR, and H-2L(d)-p2Ca spans a range of affinities from K-d = 10(-4) to 10(-6) M for the syngeneic (H-2K(b)) and allogeneic (H-2K(bm3), H-2L(d)) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2K(b)-dEV8, H-2K(bm3)-dEV8 and H-2K(b)-SIYR have been grown.