Theodore A C, Center D M, Cruikshank W W, Beer D J
Cell Immunol. 1986 Apr 1;98(2):411-21. doi: 10.1016/0008-8749(86)90300-x.
Human T-T hybridomas were developed as a strategy for obtaining lymphokines that alter T-lymphocyte motility. Mitogen-stimulated human T lymphocytes were fused with cells of the human CEM lymphoma line and the supernatants derived from these fusion products were assessed for chemoattractant activity in a modified Boyden chamber assay. Supernatants from hybridoma 41B2 enhanced lymphocyte migration to 198 +/- 13% (mean +/- SEM) of control. Characterization by Sephadex G-100 molecular sieve chromatography revealed a single peak of chemoattractant activity corresponding to a molecular weight (MW) of 56,000. This activity eluted from a Sephadex QAE anion-exchange column at 4-6 mS. Subsequent isoelectric focusing in sucrose revealed an isoelectric point of 9.0-9.2. Fractions with activity after sequential molecular sieve and anion-exchange chromatography were concentrated, radiolabeled with 125I, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography revealed a band which corresponded to a MW of 14,000 (representing four similar monomeric chains) and to the region from which chemoattractant activity could be detected in eluates from slices of unstained gels run in parallel. The biological activity of this hybridoma-derived lymphocyte chemoattractant was abolished by treatment with trypsin and neuraminidase but was unaffected by heating to 56 degrees C. We conclude that certain human T-T-cell hybridomas constitutively elaborate a lymphocyte chemoattractant that appears to be physicochemically identical to a previously described human lymphokine, lymphocyte chemoattractant factor.
人T-T杂交瘤作为一种获取可改变T淋巴细胞运动性的淋巴因子的策略而被开发出来。用丝裂原刺激的人T淋巴细胞与人CEM淋巴瘤细胞系的细胞融合，并在改良的Boyden小室试验中评估这些融合产物的上清液的趋化活性。杂交瘤41B2的上清液使淋巴细胞迁移增强至对照的198±13%（平均值±标准误）。通过Sephadex G-100分子筛层析进行表征，发现趋化活性的单一峰，其分子量（MW）为56,000。该活性在4-6 mS时从Sephadex QAE阴离子交换柱上洗脱下来。随后在蔗糖中进行等电聚焦，显示等电点为9.0-9.2。经过连续的分子筛和阴离子交换层析后具有活性的级分被浓缩，用125I进行放射性标记，并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。放射自显影显示一条带，其分子量为14,000（代表四条相似的单体链），并且与平行运行的未染色凝胶切片洗脱物中可检测到趋化活性的区域相对应。用胰蛋白酶和神经氨酸酶处理可消除这种杂交瘤来源的淋巴细胞趋化因子的生物学活性，但加热至56℃则无影响。我们得出结论，某些人T-T细胞杂交瘤组成性地分泌一种淋巴细胞趋化因子，其在物理化学性质上似乎与先前描述的人淋巴因子淋巴细胞趋化因子相同。
