Blanchard D, Aubry J P, de Vries J E, Spits H
UNICET, Laboratories for Immunological Research, Dardilly, France.
J Immunol. 1989 Apr 1;142(7):2173-80.
Human T cell clones contain enzymes that can cleave the substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). All CTL clones tested in this study secreted BLT-serine esterase activity, whereas only one of three tested non-cytolytic T cell clones secreted this enzymatic activity upon Ag-specific activation. BLT-serine esterase secretion could also be induced by the Fc gamma+ target cell Daudi in the presence of mAb specific for the TCR/CD3 complex, CD2, or the T cell activation Ag Tp 103. In addition, anti-CD3 and a mitogenic combination of anti-CD2 mAb, induced secretion of BLT-serine esterase in the absence of target cells, whereas anti-Tp 103 failed to do so. The secreted BLT-serine esterase activity induced by the various ligands was inhibited by the serine esterase inhibitors PMSF and m-ABA, but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone. Significant BLT-serine esterase activity was induced by target cells or soluble anti-CD3 in the absence of extracellular Ca2+ ions, provided that extracellular Mg2+ ions were present. The cytotoxic activities by the human CTL clones were completely blocked under these conditions. All ligands that induced BLT-serine esterase secretion in the absence of extracellular Ca2+, induced a transient rise of intracellular Ca2+. Soluble anti-CD3 mAb did not induce a transient rise in intracellular Ca2+ or secretion of BLT serine esterase in CTL preincubated for 2 h with 5 mM EGTA. These findings indicate that mobilization of intracellular Ca2+ in human CTL clones is required for induction of secretion of BLT-serine esterase.
人T细胞克隆含有能够切割底物N-α-苄氧羰基-L-赖氨酸硫代苄酯（BLT）的酶。在本研究中测试的所有细胞毒性T淋巴细胞（CTL）克隆均分泌BLT-丝氨酸酯酶活性，而在三个测试的非细胞溶解性T细胞克隆中，只有一个在抗原特异性激活后分泌这种酶活性。在存在针对T细胞受体/CD3复合物、CD2或T细胞激活抗原Tp 103的单克隆抗体的情况下，Fcγ⁺靶细胞Daudi也可诱导BLT-丝氨酸酯酶的分泌。此外，抗CD3以及抗CD2单克隆抗体的促有丝分裂组合在无靶细胞的情况下可诱导BLT-丝氨酸酯酶的分泌，而抗Tp 103则不能。各种配体诱导分泌的BLT-丝氨酸酯酶活性受到丝氨酸酯酶抑制剂苯甲基磺酰氟（PMSF）和间氨基苯甲酸（m-ABA）的抑制，但不受N-α-对甲苯磺酰-L-赖氨酸氯甲基酮的抑制。在存在细胞外镁离子（Mg²⁺）的情况下，即使没有细胞外钙离子（Ca²⁺），靶细胞或可溶性抗CD3也可诱导显著的BLT-丝氨酸酯酶活性。在这些条件下，人CTL克隆的细胞毒性活性被完全阻断。在没有细胞外Ca²⁺的情况下，所有诱导BLT-丝氨酸酯酶分泌的配体都会引起细胞内Ca²⁺的短暂升高。在与终浓度5 mM乙二醇双（2-氨基乙基醚）四乙酸（EGTA）预孵育2小时的CTL中，可溶性抗CD3单克隆抗体不会诱导细胞内Ca²⁺的短暂升高或BLT丝氨酸酯酶的分泌。这些发现表明，人CTL克隆中细胞内Ca²⁺的动员是诱导BLT-丝氨酸酯酶分泌所必需的。
