摘要
晚近,固有免疫中的细胞成分—γδT细胞在抗感染免疫、肿瘤免疫、免疫调节以及自身免疫等方面的重要作用广受关注。然而,与承担特异性细胞免疫功能、MHC—抗原肽—TCRαβ三元体结构解析较为清楚的αβT细胞形成鲜明对比的是,免疫学术界对γδT细胞受体(TCRγδ)识别抗原的结构基础所知甚少,同时TCRγδ所识别的抗原鉴定工作也进展缓慢。
研究表明,TCRγδ链,尤其是TCRδ链上的互补决定区(CDR3)是与抗原表位结合的关键部位。近年来,我们实验室选择TCRδ2链的CDR3(CDR3δ)为研究对象,在CDR3合成多肽、CDR3移植性Ig与肿瘤靶细胞、靶组织以及肿瘤细胞提取蛋白的相互作用机制方面做了大量的工作,验证了CDR3δ是TCRδ2链与抗原结合的关键部位。为了更真实地反应TCRγδ的结合活性,本研究中,我们通过TCRγδ重建技术,构建了一个利用细胞膜表达不同CDR3δ移植型TCRγδ异质二聚体的技术体系,来研究CDR3δ与抗原特异性结合的分子结构基础。另一方面,该技术体系也为寻找γδT细胞的抗原表位提供新的方法。
本文的主要工作及学术成果如下:
一、CDR3δ与抗原结合的分子结构基?
1.构建了CDR3区移植型TCRγδ转染细胞系
将嵌合了特异性CDR3区序列的全长γ9和δ2链共转染J.RT3-T3.5细胞,使其细胞膜表面表达TCRγδ,构建了CDR3区移植型TCRγδ转染细胞系。这种TCRγδ转染细胞系能够模拟γδT细胞特异性识别抗原。
2.TCRγδ识别抗原的关键部位是CDR3δ
我们构建了卵巢癌TIL中δ2的特异性CDR3序列OT3移植型TCRγδ转染细胞系,命名为OT3γδTCR细胞。OT3γδTCR细胞在多种肿瘤细胞的刺激作用后,活化分泌IL-2。同时,这种活化作用能被抗TCRγδ的单克隆抗体所阻断。OT3γδTCR细胞对多种肿瘤细胞也具有细胞毒作用,杀伤活性也能被抗TCRγδ的单克隆抗体所阻断。另外,γ9链和OT3δ2链共转染及单独的OT3δ2链转染的细胞在肿瘤抗原刺激后IL-2的表达没有差异,提示TCRγδ与肿瘤抗原的结合主要依赖TCRδ2链。OT3γδTCR细胞与肿瘤抗原的反应性高于无关CDR3序列对照转染细胞(即两种细胞表达的TCR具有相同的γ9链和携带不同CDR3区序列的δ2链)。这些结果进一步证实,OT3γδTCR细胞能够模拟γδT细胞特异性识别肿瘤细胞抗原,并且TCRγδ识别抗原的过程中CDR3δ起着关键的作用。
3.CDR3δ的侧翼序列是抗原结合特异性的关键决定簇
为了进一步探讨CDR3δ与配体作用的分子结构基础,我们将OT3的V、D和J区突变体相对应的核甘酸序列嵌入到完整的δ2链中,与相同的γ9链共转染,构建各个突变体转染细胞,分别命名为VmOT3γδTCR、DmOT3γδTCR和JmOT3γδTCR细胞。在γδTCR表达率相同的前提下,比较各个转染细胞与肿瘤细胞的反应性。结果显示,VmOT3γδTCR细胞和JmOT3γδTCR细胞在HO8910、Hela和SKOV3细胞总蛋白及HSP70、hMSH2 N端蛋白(MNS)和C端蛋白(MCS)刺激作用下,细胞活化表达IL-2的量要远远低于OT3γδTCR细胞的,而DmOT3γδTCR细胞表达IL-2的量降低不明显。同时,在不同的效靶比时,VmOT3γδTCR细胞和JmOT3γδTCR细胞对SKOV3细胞的杀伤作用远远低于OT3γδTCR细胞的,而DmOT3γδTCR细胞的杀伤活性降低不明显。这一结果与我们实验室前期用OT3突变肽检测与肿瘤细胞的结合特性相吻合,进一步提示,正是侧翼序列(V和J)决定了TCRδ2-CDR3与靶细胞的结合。
4.CDR3δ97位的疏水性氨基酸在识别非肽抗原和蛋白抗原方面的差异
我们用相同的方法也构建了OT10γδTCR转染细胞,OT10是在δ97位具有疏水性氨基酸的CDR3δ序列。非肽抗原刺激OT10γδTCR细胞能分泌IL-2,而当用点突变的方法将897的异亮氨酸(I)突变成同样疏水性氨基酸亮氨酸(L)以及酸性氨基酸天冬氨酸(D)和羟基类氨基酸丝氨酸(S)后,这些突变体转染细胞在非肽抗原刺激作用后IL-2的表达量有所差异。突变成亮氨酸后,IL-2的表达量下降不明显,而突变成另外两种氨基酸后,其IL-2的表达量降低明显或几乎没有。实验结果进一步说明δ97位置的疏水性氨基酸对于非肽抗原的识别至关重要。但是,δ97位的氨基酸突变却不能改变转染细胞对SKOV3细胞总蛋白的识别。提示CDR3δ97位的疏水性氨基酸在识别非肽抗原和蛋白抗原方面存在差异。
二、以OT3γδTCR细胞系为探针,利用差异筛选的方法在噬菌体随机展示肽库中进行抗原表位筛选
以OT3γδTCR细胞作为探针,用差异筛选的方法,在噬菌体十二肽库中,筛选TCRγδ特异结合的十二肽,得到了两条优势序列。人工合成的优势十二肽的结合特性分析与体外功能实验,证明这两条十二肽都可为OT3γδTCR所识别,提示可能是其抗原表位。这一实验结果证明我们的策略是可行的。
本研究通过结构生物学手段进一步验证了CDR3δ在抗原结合中所起的关键作用,即CDR3δ决定抗原结合的特异性。我们所取得重要创新性科学发现为,首次发现CDR3δ侧翼序列的V和J片段决定CDR3δ与配体的结合特异性。由于CDR3δ侧翼序列在结构上是呈现的保守特点,故在理论上推测其CDR3δ侧翼序列只能与结构同样保守的蛋白多肽序列结合。在此意义上说,TCRγδ所识别的配体是结构保守和数量有限的。因此,这一发现为TCRγδ识别抗原的有限多样性提供了强有力的结构生物学证据。
我们还首次创立了OT3γδTCR细胞作为探针,以差异筛选的方法,在噬菌体十二肽库中筛选TCRγδ特异结合的十二肽的新策略,并且通过实验验证了这一策略是可行的。利用这一策略将为γδT细胞所识别的肿瘤抗原表位或肿瘤相关抗原表位的发现提供新思路和技术支持,进而为肿瘤的诊断、免疫治疗提供更多更的生物标记物或治疗靶点,为围绕γδT细胞所进行的肿瘤治疗的应用开发奠定坚实的基础。
此外,实验中我们还发现了新的人NKG2D的功能性配体,即ULBP4的三个剪切变体(ULBP4-Ⅰ、ULBP4-Ⅱ和ULBP4-Ⅲ)和RAET1G2的一个剪切变体(RAET1G3)。
Recently,γδT cells,which had been considered as a member of natural immunity, play an important role in the field of anti-infection immunity,anti-tumor immunity, immuneregulation and autoimmunity.But until now,little was known about the structural basis withinγδTCR antigen recognition and little progress had been made on the verification ofγδTCR antigen.
The study suggested that CDR3 ofδchain was the important position for antigen recognition.In recent years,our groups selected the CDR3 ofδ2 chain(CDR3δ) as a research target and did some investigation about the interaction machnism of CDR3 peptide and CDR3 grafted Ig with tumor cell,tumor tissue and tumor cell protein extract. Our results suggested that CDR3δwas the crucial position for antigen recognition.In this paper,through the reconstruction ofγδTCR transfectant cell,we set up a technique system using CDRδgrafed TCRγδexpressed on cell membrane.The article focuses on elucidating the key structural basis for specificity ofγδTCR,especially the molecular mechanism betweenγδTCR CDR3 and its ligands and providing new method for searching antigen epitope ofγδT cell.
We conducted the experiments and got some results shown as follows:
1.The molecular structural foundation of antigen recognition of CDR3δ
1) The construction oftransfectant cells expressingγδTCR with specific CDR3 sequence
The full-lengthγ9 andδ2 chain containing specific CDR3 sequence were transfected into J.RT3-T3.5 cell.The transfectant cells expressingγδTCR with specific CDR3 sequence were successfully constructed and they could mimicγδTCR to recognize specific antigen.
2)γδT cells recognize tumor cells via CDR3δregion
The transfectant cells expressingγδTCR with OT3(OT3γδTCR cells) have the cytotoxicity effect on many tumor cells and the cytotoxicity effect could be blocked by functional grade purified anti-humanγδTCR.In addition,when the transfectant cells were stimulated by tumor cell protein extract,the production of IL-2 for OT3γδTCR cells was higher than that of the irrelative control and mock control.Meanwhile,the production of IL-2 between cells transfected byγ9 and OT3δ2 chains and single OT3δ2 chain had no significant difference.These data strongly indicated that the OT3γδTCR cells could specifically bind the molecules expressed in tumors andγδT cells recognize tumor cells mainly via CDR3δ2 region.
3) Specific binding of CDR3δto antigens depends upon its flanking sequences
In order to further explore the important recognition region ofδ2CDR3 to tumor cells, we construct transfectant cells with different domain variants of OT3,respectively.That is VmOT3γδTCR,DmOT3γδTCR and JmOT3γδTCR cells.After the transfectant cells were stimulated by tumor cells or tumor cell protein extracts,the production of IL-2 was detected to evalute the reactivity of various transfectant cells to tumor cell.In contrast to production of IL-2 by OT3γδTCR transfectant cells,VmOT3γδTCR and JmOT3γδTCR transfectant cells secret lower level of IL-2 after the stimulation by the protein extract of HO8910,Hela and SKOV3 cells and HSP70,N teminal(MNS) and C terminal(MCS) of hMSH2.DmOT3γδTCR transfectant cells secret equivalent level of IL-2.For cytotoxicity assay,under the condition of different ratio of effector cells and target cells,the cytotoxicity of the VmOT3γδTCR and JmOT3γδTCR transfectant cells to SKOV3 cells were lower than that of OT3γδTCR transfectant cells,while the cytotoxicity of DmOT3γδTCR transfectant cells decreased indistinctively.The results demonstrated that specific binding of OT3γδTCR transfectant cells to tumor cells and cell protein extracts depends upon its flanking sequences.
4) Isoleucine residue at position 97 in CDR3 of the TCRδ2 chain was not sufficient to account for the recognition mechanisms ofγδT cell to protein antigen
We construct OT10γδTCR transfectant cells,whose 97 position ofδ2 chain was a hydrophobic amino acid,isoleucine.In order to investigate the role of isoleucine at theδ97 position in tumor antigen recognition,we mutated it to leucine(δ97L),serine(δ97S) and glutamic acid(δ97D),respectively.In contrast to the higher level of IL-2 by OT10γδTCR transfectant cells,δ97LγδTCR transfectant cells secret lower level of IL-2,δ97S andδ97DγδTCR transfectant cells secrete little of IL-2 when these transfectant cells were stimuated by non-peptide antigen.Meanwhile,the amino acid substitution of isoleucine at positionδ97 did not change the response ofγδTCR transfectant cell to tumor cells.The data suggested that isoleucine residue at position 97 in CDR3 of the TCRδ2 chain was not sufficient to account for recognition mechanisms ofγδT cell to protein antigen,which was different from non-peptide antigen reactiveγδT cell clone.
2.A new strategy for the identification of tumor antigenic epitopes
We develop a new strategy to identify the epitopes specific forγδT cells using OT3γδTCR transfectant cells based on subtractive screening in vitro with a phage display 12 peptide library.Two preponderant peptides were selected.The results from binding analysis and functional test suggested that the antigenic epitopes,which was fished by screening phage display peptide libraries using OT3γδTCR transfectant cells,could be recognized by humanγδT cells.This new strategy had been confirmed reasonable by our experiments.
In conclusion,this study had demonstrated that CDR3δplayed a central part within the antigen binding,which was to determine the specificity of antigen binding.Besides, our results had also suggested for the first time that the V and J fragments,which lied in the flanking sequence of CDR3δdomain,determined the specific binding between CDR3δand epitope,which might contribute to explaining the limited specificity repertoire inγδT cells.Furthermore,by using the OT3γδTCR transfectant cells as probes and based on the subtractive screening method,for the first time,we had developed a new strategy to identify the epitopes bound specifically withγδT cells within a 12 peptides phage display library.And this new strategy had been confirmed reasonable by our experiments.With this new strategy,new way could be found on screening of tumor associate antigen(TAA) or tumor specific antigen(TSA),which could be identified byγδT cells.Thus more and more biomarkers or therapeutic target could be provided for the diagnosis and immunotherapy towards tumor and more stable theory basis could be set within the field of application and exploration ofγδT cells on anti-tumor treatments.
In addition,we identified ULBP4 and RAET1G2 splice variants including ULBP4-Ⅰ, ULBP4-Ⅱ,ULBP4-Ⅲand RAET1G3,which could be recognized by human NKG2D.
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