Bottino C, Tambussi G, Ferrini S, Ciccone E, Varese P, Mingari M C, Moretta L, Moretta A
Ludwig Institute for Cancer Research, Epalinges, Switzerland.
J Exp Med. 1988 Aug 1;168(2):491-505. doi: 10.1084/jem.168.2.491.
Two mAbs directed to the TCR-gamma/delta were analyzed for their pattern of reactivity with CD3+WT31- cell populations or clones. In normal individuals, the BB3 mAb reacted with approximately 2/3 of peripheral blood CD3+WT31- lymphocytes, whereas delta-TCS-1 stained approximately 1/3 of such cells. In addition, the sum of the percentages of BB3+ and delta-TCS-1+ cells approximated the percentages of peripheral blood CD3+WT31- lymphocytes in seven normal donors tested. Also, in peripheral blood-derived polyclonal CD3+WT31- populations, cultured in IL-2, cells reacting with one or another mAb accounted for the whole cell population. On the other hand, only delta-TCS-1-reactive cells, but not BB3+ cells, could be detected in unfractionated as well as in CD4-8-thymocyte populations. Analysis of peripheral blood-derived CD3+WT31- clones showed that 70% of 72 clones analyzed reacted with BB3 mAb, but not with delta-TCS-1 mAb. On the other hand, delta-TCS-1 mAb stained the remaining BB3- clones. Five clones expressing medium-low amounts of CD8 antigen were BB3- delta-TCS-1+. Both types of clones lysed the Fc gamma receptor-bearing P815 target cell in the presence of anti-CD3 mAb (but not of mAb directed against HLA-DR, CD7 molecules, or TCR-alpha/beta). In this cytolytic assay, BB3 mAb induced target cell lysis only by BB3+ clones, whereas delta-TCS-1 mAb was effective only with delta-TCS-1+ clones. The CD3-associated surface molecules expressed by BB3+ or delta-TCS-1+ clones were analyzed after cell surface iodination and immunoprecipitation with the corresponding anti-TCR mAb or with anti-CD3 mAb (in digitonin-containing buffer). In SDS-PAGE, molecules immunoprecipitated from 13 BB3+ clones displayed, under nonreducing conditions, a molecular weight of 80 kD (in some cases, a minor 38-kD band could be detected). Under reducing conditions, two major components of 44 and 41 kD (and a minor component of 38 kD) were detected. On the other hand, TCR molecules immunoprecipitated from 11 different delta-TCS-1+ clones appeared as a diffuse band of 41-44 kD, both under reducing and nonreducing conditions (under non-reducing condition, an additional 38-kD band was present). Therefore, BB3+ cells express a disulphide-linked form of TCR-gamma/delta whereas delta-TCS-1+ cells express a non-disulphide-linked form.(ABSTRACT TRUNCATED AT 400 WORDS)
分析了两种针对TCR-γ/δ的单克隆抗体(mAb)与CD3+WT31-细胞群体或克隆的反应模式。在正常个体中,BB3单克隆抗体与约2/3的外周血CD3+WT31-淋巴细胞发生反应,而delta-TCS-1则使约1/3的此类细胞染色。此外,在测试的7名正常供体中,BB3+和delta-TCS-1+细胞百分比之和接近外周血CD3+WT31-淋巴细胞的百分比。同样,在IL-2中培养的外周血来源的多克隆CD3+WT31-群体中,与一种或另一种单克隆抗体发生反应的细胞占整个细胞群体。另一方面,在未分离的以及CD4-CD8-胸腺细胞群体中,只能检测到delta-TCS-1反应性细胞,而不能检测到BB3+细胞。对外周血来源的CD3+WT31-克隆的分析表明,在分析的72个克隆中,70%与BB3单克隆抗体反应,但不与delta-TCS-1单克隆抗体反应。另一方面,delta-TCS-1单克隆抗体使其余的BB3-克隆染色。五个表达中低量CD8抗原的克隆为BB3-delta-TCS-1+。在抗CD3单克隆抗体(而非针对HLA-DR、CD7分子或TCR-α/β的单克隆抗体)存在的情况下,两种类型的克隆均能裂解携带Fcγ受体的P815靶细胞。在这种细胞溶解试验中,BB3单克隆抗体仅通过BB3+克隆诱导靶细胞裂解,而delta-TCS-1单克隆抗体仅对delta-TCS-1+克隆有效。在用相应的抗TCR单克隆抗体或抗CD3单克隆抗体(在含洋地黄皂苷的缓冲液中)进行细胞表面碘化和免疫沉淀后,分析了BB3+或delta-TCS-1+克隆表达的与CD3相关的表面分子。在SDS-PAGE中,从13个BB3+克隆免疫沉淀的分子在非还原条件下显示分子量为80 kD(在某些情况下,可检测到一条较小的38-kD条带)。在还原条件下,检测到44 kD和41 kD的两个主要成分(以及38 kD的一个次要成分)。另一方面,从11个不同的delta-TCS-1+克隆免疫沉淀的TCR分子在还原和非还原条件下均表现为41-44 kD的弥散条带(在非还原条件下,还存在一条38-kD条带)。因此,BB3+细胞表达二硫键连接形式的TCR-γ/δ,而delta-TCS-1+细胞表达非二硫键连接形式。(摘要截断于400字)