特发性血小板减少性紫癜患儿外周血T细胞PKC活性变化的研究

为了探讨ITP患儿外周血T细胞蛋白激酶C(PKC)的活性变化及其与T细胞活化和血小板减少程度之间的关系,无菌采集35例ITP患儿及30例正常儿童外周血,采用T细胞分离富集柱法分离纯化T细胞,分别用非同位素标记法检测T细胞PKC的活性变化,用流式细胞仪检测T细胞活化标志FasL蛋白的表达,血细胞计数仪计数血小板的减少程度。结果ITP患儿T细胞PKC的总活性与正常儿童相比明显增强[(0.97±0.21)nmol/ml·min和(0.55±0.13)nmol/ml·min,x±s,P<0.05],T细胞活化标志FasL蛋白表达与正常儿童比较显著升高(CD4+TFasL:32.7%±3.4%和14.7%±4.2%;CD8+TFasL:17.3%±9.7%和11.6%±8.5%,x±s,P<0.05),并且T细胞PKC的活性变化与CD4+TFasL、CD8+TFasL的表达均为显著正相关(r1=0.68,r2=0.53,P<0.05),与血小板计数成显著负相关(r=-0.75,P<0.05)。上述研究结果表明ITP患儿PKC活性增强可能引起T细胞的活化,活性T细胞增多可导致患儿血小板大量损伤,提示PKC信号转导在ITP的免疫病理机制中发挥重要作用。     

Synergistic action of protein kinase C theta and calcineurin is sufficient for Fas ligand expression and induction of a crmA-sensitive apoptosis pathway in Jurkat T cells

Deletion of activated peripheral T cell clones by apoptosis requires the regulated expression of Fas ligand (FasL) and sensitization of these cells to CD95-mediated signaling. To investigate the signaling pathways responsible for FasL expression in T cells, we tested-besides subfamily-selective protein kinase C (PKC) inhibitors - the effect of constitutively active mutants of representatives of all PKC subfamilies, i.e. PKCalpha,epsilon,theta,iota, on FasL luciferase promoter reporter constructs. In synergy with a constitutively active form of protein phosphatase 2B calcineurin (CaN), only PKCtheta, but not PKCalpha,epsilon,iota, preferentially induced FasL promoter reporter activity and, consequently, FasL protein expression in Jurkat T cells. Activation of an inducible PKCtheta AE-estrogen receptor fusion mutant led to a CaN-dependent and rapid FasL reporter activity detected as early as 4 h after addition of 4-hydroxytamoxifen, incidating a direct effect of PKCtheta action on FasL expression. Consistently, in Jurkat T cells, expression of PKCtheta AE / CaN significantly enhanced FasL protein expression and apoptosis in a CD95-dependent manner since cell death was not observed in T cells co-expressing the caspase-8 inhibitor crmA. Taken together, our results support the notion that PKCtheta and CaN are sufficient to regulate apoptosis through FasL expression.     

Bmi-1 Regulates Autoreactive CD4+ T Cell Survival in Immune Thrombocytopenia Patients

Autoreactive T cells in immune thrombocytopenia(ITP) patients undergo a rapid clonal expansion and are resistant to apoptosis to maintain continuous effect in thrombocytopenia. As Bmi-1 is involved in memory CD4+ T cell survival and Th2 proliferation, we hypothesized that Bmi-1 may have a role in autoreactive CD4+ T cell clonal expansion and Th1/Th2 development in ITP patients. We found that CD4+ T cells from active ITP patients had a higher Bmi-1 expression in comparison with remission and healthy controls, and autoreactive CD4+ T cells had more capability to proliferate and resistance to apoptosis than that of healthy controls. We evaluated the part that Bmi-1 played in proliferation and Th1 bias condition of autoreactive CD4+ T cells in ITP. We used lentiviral transfer vectors containing Bmi-1 and shBmi-1 to infect CD4+ T cells from ITP patients and healthy controls during autologous platelets stimulation. Flow cytometry and ELISA were applied to detect various parameters. The results showed that suppression of Bmi-1 using short hairpin RNA inhibited the platelet-mediated proliferation and increased apoptosis of autoreactive CD4+ T cells from ITP patients.Increased Bmi-1 expression in CD4+ T cells from healthy controls promoted the proliferation and inhibited apoptosis of CD4+ T cells. Bmi-1 significantly promoted interleukin-4 secretion by CD4+ T cells. These findings suggest that Bmi-1 plays a part in autoreactive CD4+ T cell proliferative capability and apoptotic resistance in ITP patients.     

IFN-gamma inhibits the proliferation of allergen-activated T lymphocytes from atopic, asthmatic patients by inducing Fas/FasL-mediated apoptosis

The defect in interferon-gamma (IFN-gamma) production that results in a T helper cell type 2-dominated response may be responsible for a decrease in the apoptosis of allergen-activated T cells in asthma. We investigated the effect of recombinant IFN-gamma on proliferation, Fas/Fas ligand (FasL) expression, and apoptosis in allergen-stimulated peripheral blood mononuclear cells obtained from atopic, asthmatic patients and nonatopic, control subjects. The addition of IFN-gamma at the start of cultures markedly inhibited the proliferative response to a specific allergen in cells from all asthmatic patients, whereas no change was observed in cells from nonatopic, control subjects. IFN-gamma induced an increase in the expression of Fas and FasL by allergen-stimulated CD4+ T cells from asthmatic patients and caused the apoptosis of these cells. A Fas-blocking monoclonal antibody prevented the inhibitory effect of IFN-gamma on allergen-induced proliferation. These results suggest that IFN-gamma inhibits the proliferation of allergen-stimulated CD4+ T cells from atopic, asthmatic patients by inducing the surface expression of Fas and FasL, which in turn triggers their apoptotic program. The defect in IFN-gamma production involved in the allergic, immune response may therefore be responsible for a decrease in apoptosis of allergen-activated T lymphocytes in the airways of atopic, asthmatic patients.     

血必净对活化诱导T细胞凋亡的调节

目的 观察活化诱导对脾脏T淋巴细胞凋亡、凋亡相关基因mRNA表达及caspase3活性的影响,以及活血化瘀中药的调节作用.方法 提取BALB/c小鼠脾脏T淋巴细胞并培养,以Con A+IL-2诱导T细胞活化凋亡,MTT法检测细胞增殖活性,流式细胞仪检测细胞凋亡率,RT-PCR检测Fas、FasL、Bcl-2、Bax、IL-2 mRNA表达水平,分光光度法测定caspase3酶活性,并观察活血化瘀中药对上述各项指标的影响.结果 活化T淋巴细胞于诱导18h后凋亡率明显增加,于诱导6h时未见FasL、Bax mRNA表达,Fas、Bcl-2 mRNA表达无明显变化;于诱导18 h后Fas、FasL、Bax mRNA表达升高,Bel-2 mRNA表达下降,caspase3活性增高.活血化瘀中药可降低T细胞凋亡,并可分别降低Fas、FasL、Bax mRNA表达,提高Bcl-2 mRNA表达,减轻easpase3酶活性.在活化诱导早期(6 h)促进T淋巴细胞内IL-2 mRNA表达,在晚期(18 h)减少IL-2 mRNA表达.结论 过度活化是脾脏T淋巴细胞异常凋亡的诱发因素,而凋亡的发生与Fas、FasL、Bax、Bcl-2 mRNA表达的改变有关.活血化瘀中药可通过调节IL-2及凋亡相关基因mRNA表达而减轻脾脏T淋巴细胞凋亡,同时可以促进T淋巴细胞的增殖活性.     

B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice

The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell-mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune-prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas- targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas-mediated, cytotoxic effectors. The level of membrane-anchored FasL increases with the size of the B cell and cell-surface activation marker CD69 expression, indicating that the expression of FasL is up-regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan-1+ plasma cells.     

脐带间充质干细胞体外对脐带血CD4+ T 淋巴细胞免疫调节作用的研究

目的:研究脐带间充质干细胞(UCMSCs)在体外对异源脐血CD4+ T 淋巴细胞增殖、凋亡及向CD4+ CD25+ 调节性T 细胞(Treg)分化的免疫调控作用。方法:建立不同比例UCMSCs 与植物血凝素(PHA)活化的脐血CD4+ T 淋巴细胞共培养,或与Transwell 共培养体系,检测CD4+ T 淋巴细胞增殖情况、CD4+ CD25+ / CD4+ 比率的变化及调节性T 淋巴细胞标志基因Foxp3 相对表达量的变化。同时,建立UCMSCs 与地塞米松(DXM)刺激的脐血CD4+ T 淋巴细胞共培养,或与Transwell 共培养体系,检测CD4+ T 淋巴细胞凋亡情况。结果:PHA 活化的脐血CD4+ T 淋巴细胞在与UCMSCs 共培养3 d 后,较无UCMSCs对照组相比,细胞数量明显减少(P<0. 05)且CD4+ CD25+ / CD4+ T 淋巴细胞的比率及Foxp3 的相对表达量显著增加(P<0. 01),并随UCMSCs 数量增加,作用增强(P<0. 05)。DXM 诱导的脐血CD4+ T 淋巴细胞与UCMSCs 共培养7 d 后,细胞凋亡比率较无UCMSCs 对照组显著降低(P<0. 01)。上述效应在Transwell 共培养中部分减弱但无法消除。结论:UCMSCs 对脐血CD4+ T细胞介导的免疫反应具有负调节作用,其作用主要表现在对细胞增殖能力和分化的调控而不是促进凋亡。     

Polarization and apoptosis of T cell subsets in idiopathic thrombocytopenic purpura

It is well-known that idiopathic thrombocytopenic purpura （ITP） is an acquired organ-specific autoimmune hemorrhagic disease and dysfunctional cellular immunity is considered important in the pathophysiology of ITP. However, polarization patterns and apoptosis profiles of T lymphocytes remain unclear. In this study, we investigated the polarization of T cell subsets, the expressions of apoptotic proteins Fas/FasL on the subsets and the level of anti-apoptotic gene bcl-2 and bax mRNA. It was demonstrated that the ratios of Thl/Th2 and Tcl/Tc2 in ITP children were increased obviously and that the average percentages were increased clearly for Thl and Th2, but not for Tcl and Tc2. In ITP children, the enhancing expressions were detected for FasL on Thl and Tcl and for Fas on Th2 and Tc2. With increasing level of bcl-2 mRNA and decreasing expression of bax mRNA in ITP children, the ratio of bcl-2/bax mRNA was improved obviously, which was positive correlated with the ratio of Thl/Th2. Taken together, our findings indicate that ITP is a Thl predominant disease. This polarization pattern of T cell subsets might be related to the high ratio of bcl-2/bax mRNA and the abnormal expressions of Fas and FasL on T cell subsets.     

Soluble egg antigen-stimulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL(+) T and B cells during murine Schistosoma mansoni infection

Granuloma formation around schistosomal eggs is induced by soluble egg antigens (SEA) and mediated by the activity of CD4(+) Th lymphocytes and their cytokines. Regulation of the inflammatory Th cell response during infection is still insufficiently understood. The hypothesis of this study was that activation-induced cell death (AICD) of CD4(+) T cells is involved in the immune inflammatory response. This study investigated the dynamics of splenic and granuloma CD4(+) Th cell apoptosis and Fas ligand (FasL) expression during the acute and chronic stages of murine schistosomal infection. Enhanced apoptosis of freshly isolated CD4(+) Th lymphocytes commenced after egg deposition and persisted during the peak and modulated phases of granuloma formation. After oviposition, CD4(+), CD8(+), and CD19(+) splenocytes and granuloma cells expressed elevated levels of FasL but FasL expression declined during the downmodulated stage of infection. In culture, SEA induced splenic and granuloma CD4(+) T-cell apoptosis and stimulated expression of FasL on splenic but not granuloma CD4(+) T cells, CD8(+) T cells, and CD19(+) B cells. SEA-stimulated splenocytes and granuloma cells preferentially lysed a Fas-transfected target cell line. Depletion of B cells from SEA-stimulated splenic cultures decreased CD4(+) T cell apoptosis. Coculture of purified splenic B cells with CD4(+) T cells and adoptive transfer of purified B cells indicated that antigen-stimulated B cells can kill CD4(+) Th cells. However, CD4(+) T cells were the dominant mediators of apoptosis in the granuloma. This study indicates that AICD is involved in the apoptosis of CD4(+) T cells during schistosomal infection.     

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.     

Changes in sensitivity of peripheral lymphocytes of autoimmune gld mice to FasL-mediated apoptosis reveal a mechanism for the preferential deletion of CD4-CD8-B220+ T cells

During thymic selection 'mis-selected' CD8(+) T cells exit to the periphery where they are deleted by a Fas/FasL-mediated mechanism, presumably as a result of activation by self-antigens. In the absence of functional FasL, as is the case in autoimmune gld mice, these 'mis-selected' T cells develop into unique Thy1(+)CD4(-)CD8(-) TCRalphabeta(+)B220(+) lymphocytes [abnormal double negative T (DN T) cells]. Using bioactive FasL-bearing vesicles [FasL vesicle preparation (FasL VP)], we were able to induce acute apoptosis in freshly isolated lymphocytes and to demonstrate that peripheral lymphocytes of gld mice become more sensitive to the FasL-mediated apoptosis as they age. Furthermore, flow cytometric analyses indicated that within this peripheral lymphocyte population, the abnormal DN T cells were preferentially eliminated. The exquisite sensitivity of these abnormal DN T cells is attributed to their increased membrane Fas expression with a concomitant reduction of cytosolic FLIP(L). Our data support the hypothesis that specific components of the Fas-mediated apoptotic pathway are modulated in favor of the elimination of auto-reactive T cells as well as those CD8(+) T cells that are 'mis-selected' in the thymus and escape to the periphery.     

Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively

We show that an influenza hemagglutinin-specific CD4+ murine T cell hybridoma (IP-12-7) enters the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon T cell receptor (TCR) stimulation. These cells express Fas and FasL mRNA, cell surface Fas and intracellular FasL, but do not enter apoptosis upon Fas ligation prior to TCR stimulation. TCR stimulation additionally results in protein synthesis-dependent cell surface expression of the preformed FasL. Addition of phorbol dibutyrate (PBu2) alone was sufficient to induce susceptibility to Fas ligation induced apoptosis, while addition of both PBu2 and calcium ionophore A23187 were required to induce FasL cell surface expression. Addition of cyclosporin A completely inhibited TCR-mediated death and FasL cell surface up-regulation, but had no effect on apoptosis induced directly by Fas ligation following TCR stimulation. Inhibitors of protein kinase C (PKC) (G? 6976 and GF 109203X) completely inhibited TCR-induced susceptibility to Fas ligation, but only partially inhibited TCR-induced cell surface expression of FasL. PKC isoenzymes alpha, beta, delta and zeta were expressed by this cell line and only the alpha and betaI isoforms translocated to the membrane fraction upon TCR stimulation. Our data suggest that in activation-induced T cell apoptosis PKC is involved in pathways that mediate the acquisition of Fas susceptibility, while calcineurin is required for cell surface expression of the preformed FasL.     

Immunopathology of in situ seminoma

In this study of the seminomatous human testis the composition, activity and apoptosis of lymphocytes infiltrating the immune-privileged seminiferous tubules with in situ seminoma were studied by immunohistochemistry and DNA fragmentation detection. Likewise the lymphocytes infiltrating the invasive seminomas were studied. The study showed equal numbers of CD4+ and CD8+ T cells and B cells, about 30% of the cells. Very few T gamma/delta and NK cells were present. The activity in terms of IL-2-R, FasL and perforin expression was low. Apoptosis of the lymphocytic cells was limited. No differences were observed between the lymphocytes in seminiferous tubules with in situ seminoma and the lymphocytes in invasive tumours. The study suggests that either specifically committed lymphocytes are not present or, if present, immune-suppressing mechanisms in addition to FasL may be working.     

FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts

Fas-Fas ligand (FasL) interaction and apoptosis are important in the mechanism of allograft rejection. However, the interaction between donor and recipient cells, specifically focusing on antigen-presenting cells (APCs), under various conditions is poorly understood in human liver allografts. FasL expression on APCs, its association with apoptosis, and the origin of apoptotic lymphocytes in human liver allografts were assessed by immunohistochemistry and in situ hybridization. We found increased expression of FasL on Kupffer cells (KCs) and endothelium in acute cellular rejection (n = 20) and to lesser extent in chronic rejection (n = 6) and septic cholangitis (n = 5) compared with stable grafts and normal controls. In addition, the graft specificity of infiltrating T cells was confirmed by polymerase chain reaction examination of T-cell receptor-gamma loci. T-cell apoptosis occurred at a higher rate in acute cellular rejection than in chronic rejection or septic cholangitis. The number of apoptotic bodies derived from recipient lymphocytes correlated with the severity of rejection and was reversed by treatment. FasL(+) KCs phagocytosed CD4(+) interferon-gamma(+) T cells, rather than CD4(+) interleukin-4(+) T cells, suggesting a role of KCs in regulating CD4(+) T-cell subset differentiation. In conclusion, our data suggest that FasL expression on APCs and phagocytosis of apoptotic T cells by FasL(+) KCs are indicators of rejection activity in human liver allografts.     

1Alpha,25-dihydroxyvitamin D3 restores thymocyte apoptosis sensitivity in non-obese diabetic (NOD) mice through dendritic cells

AIMS/HYPOTHESIS: Resistance of NOD thymocytes to apoptosis-inducing signals is restored by 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3), a therapy preventing diabetes in NOD mice. We studied whether modulation of thymocyte apoptosis is due to direct effects on thymic T lymphocytes or indirect effects via thymic dendritic cells, since both cell types constitute known targets for 1alpha,25OH2D3. METHODS AND RESULTS: Female NOD mice were treated with 1alpha,25OH2D3 (5microg/kg/2d) from 21 to 70 days. Vehicle-treated NOD and NOR mice served as controls. Analysis of thymic T lymphocytes from 1alpha,25OH2D3)-treated mice revealed a decrease in number of apoptosis-resistant CD4+CD8+ and CD4+CD8-HSA(high) T lymphocyte subsets, higher pro-apoptotic IL-2 and FasL, and lower anti-apoptotic Bclx-L mRNA expression levels. Thymic dendritic cells from 1alpha,25OH2D3-treated NOD mice had increased CD8alpha+FasL+ and CD80+/86+ expression compared to control NOD mice. In a syngeneic co-culture system of thymocytes and thymic dendritic cells, apoptosis levels were 20% higher only in co-cultures where both T cell- and dendritic cell-compartments originated from 1alpha,25OH2D3-treated mice. Activation-induced cell death-sensitivity in peripheral T lymphocytes was comparable to levels present in NOR mice, confirming better thymic selection in 1alpha,25OH2D3-treated mice. CONCLUSION/INTERPRETATION: We conclude that 1alpha,25OH2D3 needs both thymic T cell- and dendritic cell-compartments to exert its apoptosis-restorative effects in NOD thymocytes.     

Apoptosis and apoptosis-associated perturbations of peripheral blood lymphocytes during HIV infection: comparison between AIDS patients and asymptomatic long-term non-progressors

This study was designed to compare the degree of lymphocyte apoptosis and Fas-Fas ligand (FasL) expression in AIDS patients and long-term non-progressors (LTNPs) and correlate these parameters with apoptosis-associated perturbations in lymphocyte function. LTNPs had a lower frequency of apoptotic CD4+ and CD8+ T cells compared with subjects with AIDS. This correlated with a lower frequency of cells expressing Fas and FasL. The frequency of selected lymphocyte populations exhibiting a disrupted mitochondrial transmembrane potential (DeltaPsim) and increased superoxide generation was lower in LTNPs than in patients with AIDS; these abnormalities were associated with lower levels of caspase-1 activation in LTNPs. The results indicate a significantly reduced level of apoptosis and apoptosis-associated parameters in LTNPs than in patients developing AIDS. Based on these findings, a crucial role for mitochondria can be predicted in the process of lymphocyte apoptosis during the evolution of AIDS.     

Taurine attenuates CD3/interleukin-2-induced T cell apoptosis in an in vitro model of activation-induced cell death (AICD)

Interleukin (IL)-2 immunotherapy is used for the treatment of metastatic melanoma and renal cell carcinoma and mediates its effects through the clonal expansion of lymphocytes. Although IL-2 remains the most effective form of therapy for these cancers, response rates are poor and dose escalation is hampered by side effects, which include vascular leak and lymphopenia. The mechanism underlying T cell loss is currently unidentified but could be the induction of activation-induced cell death (AICD) mediated by FasL. Our previous studies have shown that the amino acid taurine can attenuate apoptosis induced by a number of factors in different cell types. Here, we induced T cell AICD via CD3 and IL-2 stimulation and investigated the effect of taurine on lymphocyte apoptosis. Anti-CD3-activated Jurkat T cells treated with IL-2 significantly increased FasL expression, which was associated with increased apoptosis. Treatment with taurine prior to stimulation down-regulated FasL protein expression and partially inhibited apoptosis. Inhibition of FasL-signalling resulted in an identical reduction in apoptosis. As the kinetics of AICD are completely different in circulating T cells, we repeated these experiments in such cells to confirm our finding. Stimulation of CD4(+) circulating T cells induced apoptosis in sensitized, but not freshly isolated T cells, which was abrogated partially by taurine. In Jurkat cells it was determined that taurine-mediated down-regulation of FasL protein expression was associated with decreased FasL mRNA expression and reduced NFkappaB activation. These results reveal one possible mechanism underlying the lymphopenia observed with IL-2 immunotherapy, involving increased FasL expression leading to apoptosis. Taurine may be of use in reversing the lymphopenia associated with IL-2, thereby augmenting its immunotherapeutic potential.     

CD4+CD25-T细胞凋亡机制的研究

目的：探讨CD4^＋CD25^-T细胞AICD发生的机制.方法：磁性细胞分离器（MACS）分离CD4^＋CD25^- T细胞.以CD3/CD28单克隆抗体活化BALB/c小鼠CD4^＋CD25^-T细胞或以OVA323-339肽、抗原递呈细胞活化DO11.10小鼠CD4^＋CD25^-T细胞两种方式建立AICD模型.基因芯片检测CD4^＋CD25^-T细胞和CD4＋CD25＋T细胞凋亡相关基因的表达.流式细胞仪检测细胞的凋亡率.并观察FasL中和抗体、TRAIL中和抗体及zVAD-fmk对CD4^＋CD25^-T细胞凋亡的影响.结果：MACS成功分离CD4^＋CD25^-T细胞,纯度可达98%.建立了CD3/CD28抗体以及OVA特异性抗原活化的CD4^＋CD25^-T细胞AICD模型,CD4^＋CD25^-T细胞凋亡率达35%～40%.基因芯片分析发现CD4^＋CD25^-T细胞相对高表达TRAIL、FAS,而CD4^＋CD25^-T细胞相对高表达DR5、FasL.FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4＋CD25＋T细胞的凋亡.结论：FasL、TRAIL及其它凋亡相关分子可能参与了CD4^＋CD25^-T细胞的凋亡.