Khandekar S S, Brauer P P, Naylor J W, Chang H C, Kern P, Newcomb J R, Leclair K P, Stump H S, Bettencourt B M, Kawasaki E, Banerji J, Profy A T, Jones B
Procept Inc., Cambridge, MA 02139, U.S.A.
Mol Immunol. 1997 Apr;34(6):493-503. doi: 10.1016/s0161-5890(97)00044-8.
Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR beta-chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted alphabeta TCR and its superantigen and MHC/peptide ligands. The extracellular domains of the murine D10 TCR (Valpha2, Vbeta8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble D10 TCR bound to Staphylococcus aureus enterotoxin C2 with an association rate of 1.69+/-0.12 x 10(4)M(-1) sec(-1) and a dissociation rate of 1.9+/-0.47 x 10(-2) sec(-1), giving a dissociation constant of 1.1 microM. Binding of the TCR to S. aureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-A(k) molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I A(k) chimera specifically stimulated proliferation of the D10 T-cell clone, and bound to immobilized soluble D10 TCR with an association rate of 1.07+/-0.19 x 10(4)M(-1)sec(-1) and a dissociation rate of 2.2+/-0.65 x 10(-2) sec(-1), giving a dissociation constant of 2.1 microM.
免疫激活是由T细胞受体（TCR）与结合到主要组织相容性复合体（MHC）的抗原肽之间的特异性相互作用介导的。超抗原也可刺激T细胞激活，超抗原可结合某些TCRβ链的种系编码可变域序列。我们使用表面等离子体共振生物传感器来表征II类限制性αβTCR与其超抗原以及MHC/肽配体之间的分子相互作用。小鼠D10 TCR（Valpha2，Vbeta8.2）的胞外结构域在昆虫细胞中表达并作为二硫键连接的异二聚体分泌。在没有II类MHC的情况下，纯化的可溶性D10 TCR与金黄色葡萄球菌肠毒素C2结合，结合速率为1.69±0.12×10⁴M⁻¹sec⁻¹，解离速率为1.9±0.47×10⁻²sec⁻¹，解离常数为1.1μM。TCR与金黄色葡萄球菌肠毒素B的结合几乎检测不到，并且由于解离速率过快而无法准确测量。可溶性D10 TCR也与含有融合的抗原性伴清蛋白肽和互补亮氨酸拉链序列的可溶性小鼠II类MHC I-A(k)分子结合，以促进有效的链配对。纯化的I A(k)嵌合体特异性刺激D10 T细胞克隆的增殖，并以1.07±0.19×10⁴M⁻¹sec⁻¹的结合速率和2.2±0.65×10⁻²sec⁻¹的解离速率与固定化的可溶性D10 TCR结合，解离常数为2.1μM。
