Arimilli S, Mumm J B, Nag B
Anergen Inc., Redwood City, California, USA.
Immunol Cell Biol. 1996 Feb;74(1):96-104. doi: 10.1038/icb.1996.13.
The recognition of T cell receptors (TCR) by purified major histocompatibility complex (MHC) class II-peptide complexes in the absence of costimulatory signals leads to the induction of T cell non-responsiveness or anergy. In a recent study using human T cell clones, it was observed that prolonged incubation of resting T cells with soluble MHC II-peptide complexes appears to result in T cell apoptosis. The present study shows that the engagement of TCR by soluble MHC II-peptide complexes also results in antigen-specific apoptosis in immortalized T cells. Apoptosis was demonstrated in a herpes saimiri virus (HSV) transformed human T cell clone (SS8T) restricted for HLA-DR2 in association with an epitope from the myelin basic protein [MBP(84-102)]. A dose- and time-dependent T cell death was observed upon incubation of SS8T cloned T cells with purified complexes of native human HLA-DR2 and MBP(83-102)Y83 peptide. The specificity of T cell apoptosis was demonstrated by exposing SS8T cells with DR2 alone and DR2 bound to another high affinity epitope [MBP(124-143)] from the same MBP. Recently, we have shown that the complexes of HLA-DR2 and [MBP(83-102)Y83] can be reconstituted by refolding Escherichia coli expressed individual DR2 alpha and beta (B5*0101) polypeptide chains lacking the transmembrane region. When SS8T cloned T cells were exposed to purified reconstituted rDR2.MBP(83-102)Y83 complexes, similar apoptosis of T cells was observed. Agarose gel analysis of T cells incubated with complexes showed a degradation of celluar deoxyribonucleic acid (DNA) to oligonucleosomal bands, a characteristic of apoptosis. The quantitative detection of DNA strand breaks was performed by pulsing T cells with 5-bromo-2'-deoxyuridine (BrdU) followed by the detection of BrdU-labelled DNA fragments using an antibody sandwich enzyme-linked immuno assay (ELISA). The fragmentation of DNA was also measured by double fluorescence flow cytometry by 3' end labelling of fragmented DNA with biotinylated-deoxyuridine triphosphate (dUTP) in the presence of terminal deoxynucleotide transferase (TdT) enzyme. The expression of the bcl-2 protein in SS8T cells following TCR engagement by soluble MHC II-peptide complexes was monitored by chemiluminescence blot analysis using anti-bcl-2 monoclonal antibody. Finally, the nucleosomal condensation of T cells following complex treatment, characteristics of typical apoptosis, was demonstrated by transmission electron microscopy. These results suggest that the binding of soluble MHC class II-peptide complexes to TCR induces antigen-specific apoptosis in transformed CD4 positive T cells in vitro. Such induction of apoptosis by soluble MHC II-peptide complexes may provide a novel therapeutic strategy to delete autoreactive T cells in various autoimmune diseases.
在没有共刺激信号的情况下，纯化的主要组织相容性复合体（MHC）II类 - 肽复合物识别T细胞受体（TCR）会导致T细胞无反应性或无能的诱导。在最近一项使用人T细胞克隆的研究中，观察到静息T细胞与可溶性MHC II - 肽复合物长时间孵育似乎会导致T细胞凋亡。本研究表明，可溶性MHC II - 肽复合物与TCR的结合也会导致永生化T细胞中的抗原特异性凋亡。在一种限制于HLA - DR2且与髓鞘碱性蛋白[MBP(84 - 102)]的一个表位相关的疱疹病毒（HSV）转化的人T细胞克隆（SS8T）中证实了凋亡。将SS8T克隆的T细胞与天然人HLA - DR2和MBP(83 - 102)Y83肽的纯化复合物孵育后，观察到剂量和时间依赖性的T细胞死亡。通过仅用DR2以及与来自同一MBP的另一个高亲和力表位[MBP(124 - 143)]结合的DR2处理SS8T细胞，证明了T细胞凋亡的特异性。最近，我们已经表明，HLA - DR2和[MBP(83 - 102)Y83]的复合物可以通过重折叠缺乏跨膜区的大肠杆菌表达的单个DR2α和β（B5*0101）多肽链来重构。当SS8T克隆的T细胞暴露于纯化的重构rDR2.MBP(83 - 102)Y83复合物时，观察到类似的T细胞凋亡。用复合物孵育的T细胞的琼脂糖凝胶分析显示细胞脱氧核糖核酸（DNA）降解为寡核小体条带，这是凋亡的一个特征。通过用5 - 溴 - 2'-脱氧尿苷（BrdU）脉冲T细胞，然后使用抗体夹心酶联免疫测定（ELISA）检测BrdU标记的DNA片段来进行DNA链断裂的定量检测。DNA片段化也通过双荧光流式细胞术在末端脱氧核苷酸转移酶（TdT）酶存在下用生物素化 - 脱氧尿苷三磷酸（dUTP）对片段化DNA进行3'末端标记来测量。使用抗bcl - 2单克隆抗体通过化学发光印迹分析监测可溶性MHC II - 肽复合物与TCR结合后SS8T细胞中bcl - 2蛋白的表达。最后，通过透射电子显微镜证明了复合物处理后T细胞的核小体凝聚，这是典型凋亡的特征。这些结果表明，可溶性MHC II类 - 肽复合物与TCR的结合在体外诱导转化的CD4阳性T细胞中的抗原特异性凋亡。可溶性MHC II - 肽复合物诱导的这种凋亡可能为在各种自身免疫性疾病中清除自身反应性T细胞提供一种新的治疗策略。
