Steele J K, Stammers A T, Levy J G
J Immunol. 1985 Apr;134(4):2767-78.
In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.
在我们实验室中，我们描述了一种单克隆抗体B16G，它已被证明可与DBA/2小鼠中的抑制性T细胞因子（TsF）结合。因此，B16G被用作探针来鉴定分泌假定TsF的T细胞杂交瘤。杂交瘤是通过用P815肿瘤膜提取物在体内刺激的DBA/2胸腺细胞与胸腺瘤BW5147融合而获得的。选择了一种这样的杂交瘤A10并用于进一步研究。从该杂交瘤的上清液和腹水中都可以获得一种因子，该因子可以特异性地结合B16G和P815抗原免疫吸附柱，并且在洗脱后在ELISA中与B16G呈阳性反应。从在无关柱上吸附过的A10上清液或腹水中无法获得这种反应性，从在原始筛选中对B16G无反应的其他T细胞杂交瘤的上清液或腹水中也无法获得这种反应性。体内研究表明，将亲和纯化的A10物质注射到DBA/2J小鼠中可显著增强P815肿瘤细胞的生长，但不会增强其他DBA/2同基因肿瘤系（如L1210或M-I）的生长。此外，这种物质不会抑制DBA/2脾细胞与同种异体B10.BR靶细胞之间的体外混合淋巴细胞反应（MLR）（与从整个DBA/2脾脏中纯化的B16G物质不同，后者已被证明在这种类型的MLR中具有抑制作用）。对来自A10的这种肿瘤特异性TsF进行了生化分析；发现天然分子量在80,000和90,000之间。在还原条件下，亲和纯化的A10 TsF在SDS-PAGE中解析为似乎是45,000和43,000的异二聚体。在大多数制剂中，观察到一种约25,000处解析的相关分子。讨论了这些观察结果的意义。
