Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide gamma-chain-specific monoclonal antibody.
Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide gamma-chain-specific monoclonal antibody.
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通过抗肽 γ 链特异性单克隆抗体鉴定人和小鼠中的第二种 T 细胞抗原受体。
DOI：
10.1073/pnas.84.12.4244
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发表时间：
1987
影响因子：
11.1
通讯作者：
Platsoucas,CD
中科院分区：
文献类型：
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作者：
Ioannides,CG;Itoh,K;Fox,FE;Pahwa,R;Good,RA;Platsoucas,CD
我们从小鼠t细胞抗原受体(TCR) γ链恒定区（氨基酸118-130）推断的氨基酸序列中选择合成肽（P13K），开发了一种单克隆抗体（mAb） （9D7）。使用该单抗，我们鉴定了在严重联合免疫缺陷（SCID）患者外周血淋巴细胞上表达的推定第二TCR，这些淋巴细胞在重组白细胞介素2 （il -2）和Con a的培养中繁殖。该单抗在非还原条件下免疫沉淀两条40和58 kDa的多肽链，在还原条件下免疫沉淀两条40和56 kDa的多肽链，这些多肽链来自于SCID患者培养中扩增的T3+ WT31淋巴细胞的125i标记的变性裂解物。这些多肽链不是二硫连接的，也不存在于用植物血凝素培养5天或用il -2和多克隆激活剂培养2周的正常供体的人外周血淋巴细胞上，也不存在于Jurkat淋巴母细胞样人t细胞系上。对125i标记的细胞进行化学交联，然后用抗leu -4单抗在非还原或还原条件下进行免疫沉淀，结果显示40和56-kDa多肽链与T3分化抗原相关。这些结果通过抗leu -4单抗和9D7抗p13k单抗序贯免疫沉淀得到证实。9D7抗p13k单抗免疫沉淀了两条43 kDa和64 kDa的多肽链，这些多肽链来自于严重的常见可变免疫缺陷（CVI）患者的淋巴细胞的变性裂解物，这些细胞在rIL-2和Con a的培养中扩增。因此，第二个TCR可能由两条多肽链（γ γ′）组成，两者似乎都是γ链基因的产物。这些实验是在多克隆细胞群体中进行的。需要克隆T3+ WT31-细胞群来确定该TCR是否含有两条γ多肽链。相比之下，来自轻度CVI患者外周血淋巴细胞的9D7抗p13k单抗在rIL-2和多克隆激活剂培养中扩增，只有一条56 kDa的多肽链被免疫沉淀。使用相同的9D7抗p13k单抗和免疫印迹分析，我们鉴定了一个35 kDa的γ链多肽，在还原条件下在纯化的L3T4- Lyt2- BALB/c小鼠胸腺细胞上表达。该γ链TCR为二硫键，在非还原条件下分子量为80 kDa。
We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the 9D7 anti-P13K mAb from peripheral blood lymphocytes from a patient with mild CVI expanded in culture with rIL-2 and polyclonal activators. Using the same 9D7 anti-P13K mAb and immunoblotting analysis, we identified a 35 kDa gamma-chain polypeptide under reducing conditions expressed on purified L3T4- Lyt2- BALB/c mouse thymocytes. This gamma-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.