酪氨酸激酶抑制剂对T淋巴细胞生物学特性影响的研究

目的探讨酪氨酸激酶抑制剂（TKI）在体外对T淋巴细胞生物学特性的影响。方法应用尼龙毛柱法分选人外周血T淋巴细胞,加入植物血凝素和抗人CD3单克隆抗体体外培养;运用MTT法检测不同浓度的伊马替尼（imatinjb）、尼罗替尼（nilotib）对T细胞增殖的影响;酶联免疫吸附试验（ELISA）法检测其分泌的细胞因子浓度;通过流式细胞仪检测T细胞表面免疫活性标志;实时荧光定量PCR法检测各组T细胞信号分子P56^lck。mRNA的表达。结果伊马替尼、尼罗替尼对T淋巴细胞增殖活性有抑制作用（P=0）,且呈浓度和时间依赖性;各处理组细胞因子IFN-γ、TNF-α、IL-4、IL-2浓度较对照组低（P=0．03）,IL—10浓度在处理组与对照组无差异（P=0．53）,且表面活性分子CD25和CD69明显减少（P=0．03,P=0．01）,尼戮替尼处理组CD25、CD69表达更低（P=0．05,P=0）;各处理组T细胞信号分子P56^lck。表达水平降低（P=0．03）,而尼罗替尼处理组P56^lck。表达更低（P=0．04）。结论有效治疗浓度的伊马替尼、尼罗替尼在体外可能通过抑制T淋巴细胞受体P56^lck的表达进而抑制T细胞的增殖和活性。     

SiRNA干扰靶向抑制酪氨酸蛋白激酶Lck对哮喘小鼠T细胞功能的影响

目的通过siRNA技术靶向抑制哮喘小鼠T细胞非受体酪氨酸蛋白激酶Lck的基因表达,研究Lck特异性siRNA对哮喘小鼠T细胞功能的影响。方法化学法合成小鼠T细胞Lck基因21—23bp的RNA片段,以INTERFERinTMsiRNA Transfection Reagent作为转染试剂,将合成的siRNA片段转染哮喘小鼠脾脏来源的T细胞,作用48h后再与哮喘小鼠骨髓来源的树突状细胞（DC）混合反应48h,收集细胞上清液,ELISA法检测细胞因子IL4、IL-13、IL-2、INF-1;Western Blot法检测T细胞Lck蛋白的含量,判断其表达是否被沉默。结果siRNA干扰组T细胞中几乎检测不到Lck蛋白的表达,且其细胞上清液中IL-4、IL-13的水平（10．19±1．66、12．34±0．79）较非siRNA干扰组（28．06±2．88、27．87±1．61）及对照组（22．07±2．51、20．47±2．37）明显下降,差异有统计学意义（P〈0．01）。结论特定的21—23bp的RNA片段能够以siRNA干扰的方式有效的抑制特定基因的表达,Lck特异性siRNA可以阻断哮喘小鼠T细胞的激活和分化,减少哮喘炎症因子的释放。     

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR β locus is incomplete but thymocyte development is not restored by TcR β or TcR αβ transgenes

The protein tyrosine kinase, p56^lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56^lck expression (p56^lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4⁺CD8⁺ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56^lck-/- mice and found an increase in the proportion of the CD44^?CD25⁺ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) β expression, may be delayed in the absence of p56^lck expression. In addition, the expression of a transgenic TcR β chain or TcR αβ pair did not restore thymic development in p56^lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56^lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56^lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56^lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one Vβ chain in TcR transgenic p56^lck-/- mice, we found that inhibition of endogenous TcR β gene rearrangement was almost complete in thymocytes of Vβ transgenic p56^lck-/- mice and we could not detect any peripheral T cells that expressed more than one Vβ chain in non-transgenic p56^lck-/- mice.     

HIV-1 glycoprotein gp120 disrupts CD4-p56lck/CD3-T cell receptor interactions and inhibits CD3 signaling

Using the CD4⁺ human T cell clone P28, we demonstrated that the HIV-1 glycoprotein gp120 inhibited CD3-induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56^lck from CD4 in CEM cells, with a concommittant inhibition of CD4-linked kinase activity. We have addressed the question whether disruption of CD4/p56^lck or CD4/CD3-T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti-CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4-associated p56^lck and CD4-linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti-CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3-induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide-specific phospholipase C-γ-1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3-induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4-p56^lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down-modulation of CD4-p56^lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3-linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.     

Accumulation of p60lck in HTLV-I-transformed T Cell Lines Detected by an Anti-Lck Monoclonal Antibody, MOL 171

The lck gene encodes a protein tyrosine kinase of nonreceptor type, p56^lck, whose expression occurs almost exclusively in T lymphocytes. MOL 171, an anti-p56^lck monoclonal antibody, was produced by using a 25-amino-acid synthetic polypeptide as the antigen, its structure corresponding to the N-terminal region deduced from the lck cDNA sequence. Immunoblot analysis with MOL 171 showed the accumulation of 60 kD form of Lck protein, p60 ^lck , and the decrease of p56 ^lck in human T cell leukemia virus type I (HTLV-I)-transformed T cell lines. Another anti-Lck monoclonal antibody, MOL 294, raised by using a synthetic peptide corresponding to the C-terminal region deduced from the lck cDNA sequence, also detected the accumulation of p60 ^lck in those HTLV-1-transformed T cell lines. The appearance of p60 ^lck with the decrease of p56 ^lck in normal T lymphocytes after stimulation suggested the origin of p60 ^lck in HTLV-I-transformed T cells.     

The C terminus of T cell-specific adapter protein (TSAd) is necessary for TSAd-mediated inhibition of Lck activity

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, is expressed in activated T cells. The function of TSAd is as yet unknown. We previously showed that TSAd may modulate T cell receptor-triggered signaling events. TSAd contains a Src homology (SH)2 domain, ten tyrosines and a C-terminal proline-rich region. Here, we show that human TSAd interacts with Lck through the Lck SH2 and SH3 domains and is a substrate for Lck. The TSAd C terminus, including the proline-rich region and five tyrosines, is both necessary and sufficient for TSAd interaction with and phosphorylation by Lck. Expression of TSAd in Jurkat TAg cells results in hyperphosphorylation of endogenous Lck on Y394 and to an even larger extent on Y505, resulting in a reduced Y394/Y505 phosphorylation ratio in these cells. Furthermore, full-length TSAd, but not TSAd lacking the C terminus, inhibits the hyperactive Lck Y505F mutant when both are expressed in Jurkat T cells. In contrast, expression of the TSAd C terminus alone is sufficient to inhibit Lck Y505F in phosphorylating its substrates in Jurkat T cells. Our results indicate that the TSAd C terminus is essential for inhibition of Lck activity by TSAd, and suggest a mechanism for how TSAd may inhibit early T cell activation events.     

T cell receptor and CD8-dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein

Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56^lck kinase have now been examined in this two-step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56^lck. When CD8 bound to class I, phosphorylation of p56^lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56^lck activity.