BTLA信号在T细胞活化启动和早期阶段的调节作用

发布时间：2018-04-05 14:31

本文选题：BTLA　切入点：抑制　出处：《苏州大学》2012年硕士论文

【摘要】：BTLA是2003年发现的Ig家族成员，是与CTLA-4及PD-1类似的抑制性受体。人的BTLA基因定位于染色体3q13.2，与小鼠具有48%的同源性。BTLA mRNA在脾脏及淋巴结组织中高表达，在肾脏、肝脏、肺、脑、心脏及肌肉等组织中表达量都很低，甚至不表达。BTLA广泛表达于T细胞、B细胞、巨噬细胞、树突状细胞和NK细胞表面。 BTLA含有一个信号序列、一个免疫球蛋白可变区（IgV）样的胞外段、跨膜区和大约100个氨基酸的胞浆区。BTLA胞浆区存在3个重要的含酪氨酸残基的基序:潜在的Grb2结合位点，免疫受体酪氨酸抑制基序和免疫受体酪氨酸转换基序。研究表明，，BTLA与其配体HVEM结合可以抑制T细胞的增殖和细胞因子的分泌，推迟细胞周期的进程，从而抑制或终止免疫应答。在持续的抗原刺激下，BTLA高度选择性表达在无能CD4+T细胞上，表明BTLA信号在诱导与维持T细胞无能状态的过程中有重要作用。另外，BTLA不仅在在维持外周免疫耐受中具有重要作用，还可以预防自身免疫疾病的发生，终止炎症反应，抑制移植免疫应答。 BTLA在初始和活化T细胞上都有表达，因此，Murphy提出BTLA可能在T细胞活化的各个阶段尤其是早期阶段发挥作用。本研究首先分析了BTLA-HVEM顺式复合物的存在对细胞表面BTLA分子表达水平检测的影响，进而研究了BTLA在T细胞活化起始和早期阶段的调节作用。一、BTLA-HVEM顺式复合物对细胞表面BTLA分子检测的影响【目的】T细胞和U937细胞表面存在BTLA和HVEM共表达的现象。探讨BTLA-HVEM顺式复合物存在对细胞表面BTLA分子检测的影响。【方法】分离人外周血单个核细胞，经阴性选择磁珠分离纯化获得T淋巴细胞。采用流式细胞术的方法，用抗不同BTLA位点的商品化单抗MIH26和本室研制的单抗8H9分别检测U937和T细胞等细胞表面BTLA分子的表达水平。293T/BTLA基因转染细胞上BTLA分子的检测采用8H9单抗。【结果】商品化单抗MIH26(0.5μg)检测T细胞和U937细胞上BTLA分子的表达量分别为90%以上，而8H9单抗（1μg）检测到二者的表达水平分别为42.2%、30.8%。采用0.02μg和0.05μg的8H9单抗检测293T/BTLA转基因细胞株，显示BTLA分子的表达量分别为80%和91.4%。【结论】T细胞和U937细胞上共表达的BTLA和HVEM形成顺式复合物，影响8H9对BTLA的检测，而不影响MIH26的检测。二、BTLA对T细胞活化启动和早期阶段的调节作用【目的】观察BTLA在T细胞活化过程中的表达变化，探讨其在T细胞活化各个阶段尤其是早期阶段的抑制作用。【方法】分离人外周血单个核细胞，经磁珠分选获得纯化的T细胞。用激发型CD3抗体和CD28抗体刺激T细胞活化，采用流式细胞术，检测BTLA、CTLA-4和PD-1在T细胞活化过程中的动态表达。激发型CD3抗体和CD28抗体在加入BTLA单抗8H9的情况下刺激T细胞活化，流式细胞术检测T细胞活化早期标志分子CD25和CD69的表达。激发型CD3抗体和CD28抗体联合HVEM-Fc或者BTLA单抗8H9，观察BTLA信号在活化后的不同时间对T细胞增殖的作用。同时，采用ELISA方法检测8H9对CD3单抗联合CD28单抗刺激的T细胞分泌IL-2和IFN-γ的影响。预先用CD3和CD28抗体刺激T细胞活化后，在24h，48h，72h分别加入8H9抗体并交联，观察缺失BTLA信号对T细胞活化抑制的减弱程度。【结果】BTLA在T细胞上组成性高表达，活化后表达有所降低，然后迅速回升；CTLA-4在静止T细胞上不表达，PD-1的表达水平很低，二者都是活化后被诱导表达。激发型CD3抗体和CD28抗体联合8H9刺激T细胞后，CD25和CD69的表达均受到抑制。激发型CD3抗体和CD28抗体联合8H9或者HVEM-Fc刺激后，在刺激后的1d，2d，3d，4d检测，均对T细胞有抑制作用。同时，8H9对CD3单抗联合CD28单抗刺激的T细胞分泌的IL-2和IFN-γ具有抑制效应。CD3抗体和CD28抗体预先活化T细胞24h，48h，72h后，再加入8H9抗体仍然具有抑制功能，但不如在T细胞活化之初加入8H9的效应。【结论】BTLA可以在T细胞活化的启动和早期阶段发挥重要的负性调节作用，从而抬高T细胞活化的阈值。
[Abstract]:BTLA is found in 2003 the members of the Ig family, is similar to the CTLA-4 and PD-1 inhibitory receptors. The BTLA gene is located on chromosome 3q13.2 with 48% homology to.BTLA mRNA high expression in spleen and lymph node tissues in the kidney, liver, lung, brain and heart in mice, expression and muscle tissue volume is very low, or no expression of.BTLA was widely expressed in T cells, B cells, macrophages, dendritic cells and NK cell surface.
BTLA contains a signal sequence, a variable region of immunoglobulin (IgV) extracellular sample, there are 3 important motifs containing tyrosine residues in transmembrane and about 100 amino acid cytoplasmic.BTLA cytoplasmic region: potential Grb2 binding sites, immunoreceptor tyrosine inhibitory motifs and the immune receptor tyrosine conversion motif. The study shows that the combination of BTLA and its ligand HVEM can inhibit the proliferation and cytokine secretion of T cells, delay the progression of the cell cycle and inhibit or terminate the immune response. In the continuous stimulation of antigen, expression of BTLA highly selective in anergic CD4+T cells, suggesting that BTLA signal is an important role in the process of induction and maintenance of T cell anergy. In addition, BTLA not only play an important role in sustaining peripheral tolerance, but also can prevent the occurrence of autoimmune diseases, termination of inflammatory reaction, inhibition of graft immune response.
In the initial BTLA and activated T cells have the expression, therefore, Murphy put forward the various stages of the BTLA activation in T cells especially the early stage play a role. This study first analyzes the existing BTLA-HVEM CIS complex expression level detection of cell surface BTLA molecules, and study the regulation of initiation and activation of BTLA in the early stage of T cells.
The effect of BTLA-HVEM CIS complex on the detection of BTLA molecules on the surface of cells
[Objective] the co expression of BTLA and HVEM on the surface of T cells and U937 cells was investigated. The effect of BTLA-HVEM CIS complex on the detection of BTLA molecules on the cell surface was investigated.
[method] isolated from human peripheral blood mononuclear cells were isolated and purified by negative selection using T cells. By using the method of flow cytometry, developed by different anti BTLA site commercial monoclonal antibody MIH26 and monoclonal antibody 8H9 were detected in the laboratory detection of BTLA molecular level.293T/BTLA gene transfected cells on the expression of U937 using 8H9 monoclonal antibody and T cell BTLA cell surface molecules.
[results] the commercialization of monoclonal antibody MIH26 (0.5 g) to detect the expression of BTLA molecule on T cells and U937 cells were more than 90%, while the 8H9 monoclonal antibody (1 g) to detect the expression level of two were 42.2%, 30.8%. by 0.02 g and 0.05 g 8H9 monoclonal antibody 293T/BTLA transgenic fine cell lines showed that the expression level of BTLA molecules were 80% and 91.4%.
[Conclusion] the co expression of BTLA and HVEM on T and U937 cells forms CIS complex, which affects the detection of BTLA by 8H9, but does not affect the detection of MIH26.
Two, BTLA regulates the activation and early stages of T cell activation
[Objective] to observe the expression of BTLA in the activation of T cells, and to explore the inhibition of the activation of T cells in various stages, especially in the early stage.
[method] isolated from human peripheral blood mononuclear cells, purified T cells by Macs. Agonist CD3 and CD28 antibody stimulated the activation of T cells, flow cytometry was used to detect BTLA, CTLA-4 and PD-1 in T cell activation in the dynamic process. The expression of agonist CD3 and CD28 antibody in join the BTLA mAb 8H9 under the condition of stimulated T cell activation, activation of T cells was detected by flow cytometry the expression of early marker CD25 and CD69. Agonist CD3 and CD28 antibody combined with HVEM-Fc or BTLA mAb 8H9, to observe the effect of BTLA signal on T cell proliferation in different time after activation. At the same time, the influence of ELISA method for detecting 8H9 of CD3 monoclonal antibody combined with CD28 mAb stimulated T cells secreting IL-2 and IFN- gamma. Advance with CD3 and CD28 antibodies to stimulate T cell activation, in 24h, 48h, 8H9 and 72h were added to the antibody cross-linking, observe the deletion of BTLA signal to T cells The degree of attenuation of activation inhibition.
[results] BTLA of high expression in T cells after activation, expression decreased, and then quickly rebounded; CTLA-4 was not expressed in resting T cells, the expression level of PD-1 is very low, the two are activated are induced. Combined with 8H9 agonist CD3 and CD28 antibody stimulated T cells. The expression of CD25 and CD69 were suppressed. Agonistic CD3 antibody and CD28 antibody combined with 8H9 or HVEM-Fc stimulation, after stimulation of 1D, 2D, 3D, 4D detection, all have inhibitory effect on T cells. At the same time, the secretion of 8H9 CD3 monoclonal antibody and CD28 monoclonal antibody of T cells stimulated by IL-2 and IFN- with gamma the inhibitory effect of.CD3 antibody and CD28 antibody pre activated T cells 24h, 48h, 72h after adding 8H9 antibody has inhibiting function, but not in T cell activation at the beginning of accession to the 8H9 effect.
[Conclusion] BTLA can play an important negative regulatory role in the initiation and early stages of T cell activation, thus raising the threshold of activation of T cells.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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