Kaiser M, Hoskin D W
Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada.
Cell Immunol. 1992 Apr 15;141(1):84-98. doi: 10.1016/0008-8749(92)90129-d.
Coculture of purified murine T cells with anti-CD3 monoclonal antibody (145-2C11) results in the induction of nonspecific cytotoxic T lymphocytes (CTL) with MHC-unrestricted cytolytic activity against a range of tumor targets. Serine proteases associated with effector cell granules are among the molecules postulated to play a role in cell-mediated cytolysis. The present study examines the ability of exogenous serine protease substrates to inhibit anti-CD3-activated cytotoxic T (ACT) cell-mediated killing of P815 mastocytoma and YAC1.2 lymphoma target cells. The chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester (ATEE) was found to significantly inhibit ACT cell-mediated cytolysis. In contrast, the trypsin substrate N-benzoyl-L-arginine ethyl ester (BAEE) had little, if any, effect on ACT cell-mediated cytolysis. These effects were observed with both target cell populations. Conjugate inhibition studies performed with ATEE indicated that a chymotrypsin-like serine protease is involved in a postbinding event during cytolysis. Pretreatment of either target or effector cells with ATEE prior to cytolytic assay revealed that the chymotrypsin-like serine protease involved in cytotoxicity is of effector cell origin. Northern blot analysis of total RNA extracted from ACT cells revealed the presence of transcripts coding for CCP1 and CCP2 serine proteases known to be involved in antigen-specific CTL function, but little or no expression of the HF serine protease which has also been implicated in antigen-specific CTL killing. CCP2 exhibits chymotrypsin-like activity while HF displays trypsin-like activity. On the other hand, the CCP1 gene product has protease activity which resembles neither chymase nor tryptase activities. Thus, the level of mRNA expression for these serine proteases is consistent with our earlier observations, using the serine protease substrates, that a chymotrypsin-like serine protease but not a trypsin-like serine protease is involved in ACT cell-mediated cytolysis. "Lymphocyte panning" of ACT cells revealed abundant CCP1 and moderate CCP2 mRNA expression in CD4- and CD8+ anti-CD3-activated T cells with strong tumoricidal activity. CD8- anti-CD3-activated T cells with moderate cytolytic activity also expressed substantial levels of CCP1 and CCP2 mRNA, suggesting that both CD4- CD8- and CD4- CD8+ ACT cells participate in killing tumor targets. In contrast, CD4+ anti-CD3-activated T cells lacked both cytolytic activity and significant CCP1 and CCP2 mRNA expression. These findings are consistent with the involvement of chymotrypsin-like, as well as other, serine proteases in CTL-mediated lysis.
将纯化的小鼠T细胞与抗CD3单克隆抗体（145-2C11）共培养，可诱导产生非特异性细胞毒性T淋巴细胞（CTL），其具有针对一系列肿瘤靶标的MHC非限制性溶细胞活性。与效应细胞颗粒相关的丝氨酸蛋白酶被认为是在细胞介导的细胞溶解中发挥作用的分子之一。本研究检测了外源性丝氨酸蛋白酶底物抑制抗CD3激活的细胞毒性T（ACT）细胞介导的P815肥大细胞瘤和YAC1.2淋巴瘤靶细胞杀伤的能力。发现胰凝乳蛋白酶底物N-乙酰-L-酪氨酸乙酯（ATEE）能显著抑制ACT细胞介导的细胞溶解。相比之下，胰蛋白酶底物N-苯甲酰-L-精氨酸乙酯（BAEE）对ACT细胞介导的细胞溶解几乎没有影响。在两种靶细胞群体中均观察到了这些效应。用ATEE进行的共轭抑制研究表明，一种胰凝乳蛋白酶样丝氨酸蛋白酶参与了细胞溶解过程中的结合后事件。在溶细胞试验前用ATEE预处理靶细胞或效应细胞，结果显示参与细胞毒性的胰凝乳蛋白酶样丝氨酸蛋白酶起源于效应细胞。对从ACT细胞中提取的总RNA进行Northern印迹分析，发现存在编码已知参与抗原特异性CTL功能的CCP1和CCP2丝氨酸蛋白酶的转录本，但几乎没有或没有表达也与抗原特异性CTL杀伤有关的HF丝氨酸蛋白酶。CCP2表现出胰凝乳蛋白酶样活性，而HF表现出胰蛋白酶样活性。另一方面，CCP1基因产物的蛋白酶活性既不像糜酶也不像胰蛋白酶的活性。因此，这些丝氨酸蛋白酶的mRNA表达水平与我们早期使用丝氨酸蛋白酶底物的观察结果一致，即参与ACT细胞介导的细胞溶解的是胰凝乳蛋白酶样丝氨酸蛋白酶而非胰蛋白酶样丝氨酸蛋白酶。对ACT细胞进行“淋巴细胞淘选”发现，在具有强大杀瘤活性的CD4 - 和CD8 + 抗CD3激活的T细胞中，CCP1表达丰富，CCP2表达中等。具有中等溶细胞活性的CD8 - 抗CD3激活的T细胞也表达大量的CCP1和CCP2 mRNA，这表明CD4 - CD8 - 和CD4 - CD8 + 的ACT细胞都参与杀伤肿瘤靶标。相比之下，CD4 + 抗CD3激活的T细胞既缺乏溶细胞活性，也缺乏显著的CCP1和CCP2 mRNA表达。这些发现与胰凝乳蛋白酶样以及其他丝氨酸蛋白酶参与CTL介导的裂解作用一致。
