多发性骨髓瘤患者树突状细胞介导的特异性抗瘤活性的体外诱导

目的 :观察U2 6 6 细胞及其可溶性抗原激发的多发性骨髓瘤 (MM)患者树突状细胞 (DC)体外诱导U2 6 6 特异性CTL的作用。方法 :将MM患者外周血来源的单核细胞在rhGM CSF 80 0U ml与IFNα 6 0 0U ml条件下利用无血清技术培养生成DC ,应用丝裂霉素C处理的U2 6 6 细胞及用U2 6 6 细胞制备的可溶性抗原预刺激DC ,然后与自体淋巴细胞共同孵育 5～ 7d以诱生特异性CTL ,采用MTT法检测对U2 6 6 细胞的特异性杀伤效果。结果 :MM患者外周血单核细胞在GM CSF IFNα条件下培养 8d后生成具有典型特征的DC ,高度表达CD86、CD5 4及MHCII类分子HLA DR。应用MTT法检测U2 6 6 细胞及其可溶性抗原激发的DC诱导特异性CTL对靶细胞U2 6 6 的杀伤率分别为 2 1 2 %± 5 4 %和 2 8 0 %± 7 6 % ,对照组未用抗原刺激组都为 11 7%±4 3%。而以抗原直接刺激自体淋巴细胞组为 15 6 %± 4 8%和 13 1%± 5 5 % (P <0 0 1)。结论 :U2 6 6 细胞及其可溶性抗原激发的DC与自体淋巴细胞孵育能诱导抗U2 6 6 特异性CTL。     

催乳素对葡萄球菌肠毒素A诱导的人外周血T细胞凋亡的影响

目的探讨催乳素（PRL）在T细胞的活化诱导凋亡（activation induced cell death，AICD）中的作用。方法用葡萄球菌肠毒素（SEA）体外刺激人外周血T细胞作为T细胞AICD的模型，在第2次向模型中加入SEA诱导T细胞凋亡的同时，分别加入3种不同浓度的hPRL（20、300和1000ng/ml）进行干预，同时设不含hPRL的对照组。0～24h内，以MTT法检测T细胞的增殖情况。PI染色后用流式技术检测细胞凋亡情况，并通过琼脂糖凝胶电泳检测T细胞凋亡DNA。流式检测T细胞的Fas和FasL的表达水平变化。Westem blot法检测T细胞内凋亡相关蛋白Bcl-2和Bax。结果在T细胞的AICD模型中高浓度PRL组（300、1000ng／ml）其T细胞增殖得到显著维持（P〈0．05），各PRL处理组T细胞凋亡率比对照组降低了32．9％～78．2％（P〈0．05）。PRL组（300、1000ng/ml）Bax／Bcl-2比值比对照组下降了44．4％～46．0％（P〈0．05）。PRL可明显抑制细胞表面Fas和FasL的表达，其中各PRL处理组Fas的细胞阳性率比对照组下降了51．1％～75．0％（P〈0．01），FasL的细胞阳性率比对照组下降了28．2％～39．1％（P〈0．01）。结论在SEA诱导T细胞的AICD过程中，PRL可通过抑制Fas、FasL和Bax的表达，并提高Bcl-2的表达，来抑制T细胞凋亡，维持T细胞的增殖。     

MHC-I类分子在诱导异体肾癌特异性 CTL中的作用

目的探讨MHC-I类分子匹配的异体人肾癌细胞诱导肾癌细胞特异性CTL的作用。方法用经照射的HLA-I类分子匹配的异体人肾癌细胞作为刺激细胞,诱导外周血CTL,并分析其表型及杀伤效应。结果11例肾癌患者外周血淋巴细胞经HLA匹配的肾癌细胞刺激后,诱导的CTL细胞主要以TCRαβ＋、CD8＋为主,具有明显的杀伤效应。结论用MHC-I类分子匹配的异体肾癌细胞可诱导肿瘤特异性、并具有明显杀伤活性的的CTL。     

Th1 and Th2 subsets equally undergo Fas-dependent and -independent activation-induced cell death

Stimulation of previously activated T cells results in apoptosis, termed activation-induced cell death (AICD). Recent analysis revealed that the Fas/Fas ligand (FasL) interaction is predominantly involved in AICD of T cells. Furthermore, based on the analysis of various T cell clones and lines, it has been reported that FasL is expressed mainly in Th1 but not in Th2 cells. However, the exact expression pattern of FasL and its function in normal activated T cells has not been determined. In the present study, by utilizing completely differentiated Th1 and Th2 cell populations obtained from ovalbumin-specific T cell receptor (TCR)-transgenic mice, the FasL expression on Th1 and Th2 was determined. Furthermore, involvement of Fas-FasL interaction in AICD of Th1 and Th2 cells was analyzed by two approaches: one was the inhibition of AICD by anti-FasL monoclonal antibodies, and the other AICD of Th1/Th2 subsets from TCR-transgenic mice backcrossed to lpr mice. We demonstrated that Th2 cells express FasL on the cell surface at a level similar to that expressed by Th1 cells, and that both subsets were equally susceptible to the Fas-mediated AICD. These observations suggest not only that the expression of FasL is not always correlated with Th subsets as defined by the cytokine-producing profile, but also that the responses of both Th1 and Th2 subsets are regulated by Fas-mediated AICD. Finally, analysis of the kinetics of AICD revealed a novel Fas/FasL-independent pathway in its initial stage. These findings revealed the precise function of Fas/FasL-mediated as well as Fas/FasL-independent AICD in the regulation of helper T cell responses.     

Activation-induced NK cell death triggered by CD2 stimulation

Both T cells and natural killer (NK) cells express CD2, the target of an alternative activation pathway that induces the proliferation of both cell types. The mitogenic response to CD2 ligation requires the co-expression of CD3 : TCR in T cells and FcγRIII in NK cells, suggesting that these receptors are involved in transducing the response initiated by CD2. The ability of FcγRIII to trigger the activation-induced death of IL-2-primed NK cells led us to investigate the potential for CD2 to trigger activation-induced NK cell death. Our results reveal that the same anti-CD2 monoclonal antibodies (mAb) that activate freshly isolated NK cells induce apoptosis in IL-2-primed NK cells. CD2-induced apoptosis results in chromatin condensation, DNA fragmentation and cleavage of caspase-3. Activation-induced NK cell death triggered by CD2 ligation is extremely rapid (DNA fragmentation is first observed at 90 min) and it is not inhibited by neutralizing antibodies reactive with TNF-α or Fas ligand. Whereas mAb reactive with distinct CD2 epitopes (i.e. T11.1, T11.2, and T11.3) are required for activation-induced T cell death, mAb reactive with a single CD2 epitope are sufficient for activation-induced NK cell death. The ability of CD2, CD16, and CD94 to induce apoptosis in IL-2-primed lymphocytes suggests that cytokine priming changes the response to a signaling cascade that is common to each of these activation receptors.     

Distinct temporal programming of naive CD4+ T cells for cell division versus TCR-dependent death susceptibility by antigen-presenting macrophages

Naive T cells become programmed for clonal expansion and contraction during the early hours of antigenic signaling. Recent studies support an 'autopilot' model, wherein the commitment to proliferate and the magnitude of the proliferative response are simultaneously determined during a single, brief period of antigen exposure. Here, we have examined whether the proliferation of naive CD4+ T cells must occur on 'autopilot', or whether extended periods of antigenic signaling can impact primary proliferative responses to antigen-presenting macrophages (macrophage APC). We found that a single exposure to antigen (18 h) simultaneously committed T cells to (1) up-regulate surface TCR above the level expressed on naive T cells, (2) undergo minimal cell division, and (3) acquire susceptibility to TCR-dependent activation-induced cell death. However, continued antigenic signaling between 18 and 72 h was required to amplify the number of daughter cells derived from the already committed T cells. Thus, a discrete commitment time was followed by a 'tuning' period, where extended antigenic signaling determined the volume of the proliferative response. We conclude that T cell commitment to full clonal expansion versus TCR-dependent death susceptibility represent two separate programming events whose timing can be segregated by macrophage APC.     

Activation-induced peripheral blood T cell apoptosis is Fas independent in HIV-infected individuals

T cell apoptosis has been proposed as an Important contributorto the functional defects and depletion of T cells in HIV-lnfectedIndividuals. However, the mechanisms Involved in this apoptosishave not been elucidated. We recently showed that peripheralblood T cells from HIV-infected individuals are especially susceptibleto Fas antigen-induced apoptosis. In this study we examine therole of Fas, CTLA-4, tumor necrosis factor (TNF) receptors (TNFR)and CD30, receptors known to be involved In T cell activation-inducedcell death (AICD), in the spontaneous and activation (anti-CD3)-lnducedapoptosis of peripheral blood T cells from asymptomatic HIV-infectedindividuals. We report here that spontaneous and activation-InducedT cell apoptosis cannot be inhibited by reagents that blockinteractions of Fas, CTLA-4, p55 and p75 TNFR and CD30 withtheir respective ligands. We also show that IL-12, IFN-, IL-4and IL-10 cannot modify spontaneous, activation- and anti-Fas-inducedapoptosis. Anti-Fas preferentially Induced CD4⁺ T cell apoptosiswhereas AICD induced apoptosis equally In CD4⁺ and CD8⁺ T cells.We conclude that T cell AICD In HIV infection Is not mediatedby Fas, thus Indicating that Fas-Induced and activation-InducedT cell apoptosis are independent mechanisms of apoptosis whichmay play different roles in the pathogenesls of HIV infection.     

The ratio between dendritic cells and T cells determines the outcome of their encounter: proliferation versus deletion

Dendritic cells (DC) either induce T cell tolerance or contribute to the initiation and modulation of T and B cell responses. Since many of the variables determining the thresholds of naive T cell priming were defined in vitro using a homogeneously matured DC population, we here focused on partially mature DC which might reflect the occurrence of tumor-infiltrating and thymic DC. To predict how those DC regulate the induction of antigen-specific T cell proliferation and T cell tolerance, we co-cultured ovalbumin-pulsed murine DC at different ratios with antigen-specific DO11.10 transgenic T cells. Whereas partially mature DC at a DC/T cell ratio of 1:10 supported proliferation, a DC/T cell ratio of 1:2 induced proliferation arrest in naive CD4+ T cells. The acquisition of the NK cell inhibitory markers NK1.1 and KLRG on T cells exposed to high numbers of DC suggests a role for these molecules in the protection of antigen-responsive T cells from exhaustion by overstimulation. Mechanistically, abortive T cell proliferation upon encounter of high numbers of partially mature DC is caused by an apoptosis-related pathway, suggesting that excessive antigen density without sufficient costimulation results in activation-induced cell death.     

Selection of Epstein-Barr virus specific cytotoxic T lymphocytes can be performed with B lymphoblastoid cell lines created in serum-free media

Epstein-Barr Virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) are currently used for numerous applications in cellular immunology. Where protocols destined for clinical application are concerned, the final choice of assay is made according to a risk/benefit ratio analysis. In this balance the use of xenogenic or allogenic serum has always been a major concern, as it carries both an infectious and an immunological risk. So far, it is unknown whether serum can be omitted from the entire BLCL selection procedure. In addition, as BLCL have been described as heterogeneous, serum deprivation may affect their antigen-presenting capacity. In the present study, BLCL were generated in the absence or presence of fetal calf serum (referred to as BLCL0 or BLCL(FCS), respectively). Next, in order to assess the antigen-presenting capacity of these cells, we compared the ability of BLCL0 and BLCL(FCS) cells to stimulate the EBV-specific repertoire of the corresponding donor's peripheral blood mononuclear cells in vitro. Our results showed that addition of serum was not essential for BLCL infection and culture, and that as far as we could determine, BLCL0 cells were as effective as BLCL(FCS) in reactivating the EBV-specific T-cell repertoire in vitro. Notably, FCS-specific T-lymphocytes can be detected among the BLCL(FCS)-specific CD4+-CTL. Not only was this latter observation unexpected for an EBV-seropositive donor, but it implied that the BLCL had captured and processed the corresponding FCS-derived solubles antigens; taken together our results emphasized the interest of the possibility to generate BLCL0, both for research and for clinical applications.     

Activation of human alloreactive cytotoxic precursor T lymphocytes

The requirements for allogeneic T-cell activation have been studied in experiments with T and/or B cells as stimulator. Although target determinants (TDs, defined by CTL effectors in CML) are present on B and T cells used as target cells, this study indicates that TDs are functionally different when expressed on B and T cells used as stimulator cells, as only B cells can activate CTL precursors. Further, the study confirms that inducing TDs and strong lymphocyte-activating determinants (LADs, defined by proliferation in MLC) can be distinct structures found on two different stimulator B cells. The study suggests that binding of cytotoxic precursor T cells to TDs per se does not allow any detectable activation or start of proliferation and differentiation but requires another function of the stimulator cells in the non-T-cell compartment. The nature of this function is unknown, but it is the background for the first signal received by the TD-specific clones of CTL precursors, resulting in the expression of growth receptors for T-cell growth factor or interleukin 2 which is the second signal necessary for clonal expansion and differentiation.     

Requirement of the T cell antigen receptor occupancy for the target cell lysis by cytolytic T lymphocytes

We have constructed a bivalent bifunctional F(ab)2 fragment with binding specificity for a V beta 8 T cell antigen receptor and human tumor-associated antigen. Using the bifunctional antibody to focus cytolytic activity of mouse CTL to a human carcinoma cell line and anti-V beta 8 TCR Fab' as a competitor, we demonstrate that only a small percentage (-0.5%) of the TCR engaging on the target molecule is sufficient to deliver a lytic signal to the target cells.     

Loss of core fucosylation enhances the anticancer activity of cytotoxic T lymphocytes by increasing PD-1 degradation

As an immune checkpoint, programmed cell death 1 (PD-1) and its ligand (PD-L1) pathway plays a crucial role in CD8⁺ cytotoxic T lymphocytes (CTL) activation and provides antitumor responses. The N-glycans of PD-1 and PD-L1 are highly core fucosylated, which are solely catalyzed by the core fucosyltransferase (Fut8). However, the precise biological mechanisms underlying effects of core fucosylation of PD-1 and PD-L1 on CTL activation have not been fully understood. In this study, we found that core fucosylation was significantly upregulated in lung adenocarcinoma. Compared to those of Fut8^+/+OT-I mice, the lung adenocarcinoma formation induced by urethane was markedly reduced in Fut8^−/−OT-I mice. De-core fucosylation of PD-1 compromised its expression on Fut8^−/− CTL, resulted in enhanced Fut8^−/− CTL activation and cytotoxicity, leading to more efficient tumor eradication. Indeed, loss of core fucosylation significantly enhanced the PD-1 ubiquitination and in turn led to the degradation of PD-1 in the proteasome. Our current work indicates that inhibition of core fucosylation is a unique strategy to reduce PD-1 expression for the antilung adenocarcinoma immune therapy in the future.     

超抗原SEB激活诱导CD4~+T细胞凋亡模型的建立

目的为了探讨超抗原活化诱导 CD4+ T淋巴细胞凋亡分子机理及其信号传导途径 ,有必要建立超抗原活化诱导 CD4+ T细胞凋亡模型。方法采用电镜观察细胞凋亡的形态学特征 ,借助流式细胞仪 PI染色观察细胞凋亡的光散射特征及亚二倍体核型峰特征 ,琼脂糖凝胶电泳分析细胞凋亡的 DNA片段化图谱 ,最后采用改进的二苯胺 (DPA)法定量分析细胞凋亡 DNA片段化百分率。结果 4μg/ m L SEB刺激静息的 SEB应答 CD4+ T细胞 12 h后 ,电镜下观察呈现典型的凋亡形态学特征 ;流式细胞仪 PI染色分析表明 ,在二倍体峰的左侧出现亚二倍体核型峰典型凋亡特征 ,在光散射图谱上呈现低于正常细胞的前向散射和高于正常细胞的侧向散射 ;琼脂糖凝胶电泳分析呈现典型的凋亡 DNA梯状图谱 ;DNA片段化百分率分析表明 ,超抗原 SEB活化诱导 CD4+ T细胞凋亡率的升高具有时间依赖性 ,添加 Fas- Ig融合蛋白能够特异性抑制凋亡 DNA片段化百分率的升高。结论超抗原 SEB活化诱导 CD4+ T细胞凋亡模型的建立 ,为进一步深入探讨超抗原活化诱导 T细胞死亡的分子机理和信号通路奠定了坚实基础     

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H

Although the importance of CD4+ T cell responses to human cytomegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4+ CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HCMV isolates revealed that the gB and gH epitope sequences recognized by human CD4+ T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4+ CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse peptide-sensitized Patr-DR7+ cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class II lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.     

Soluble TNF‐α but not transmembrane TNF‐α sensitizes T cells for enhanced activation‐induced cell death

In addition to its proinflammatory effects, TNF‐α exhibits immunosuppression. Here, we compared the capacities of transmembrane TNF‐α (tmTNF) and soluble TNF‐α (sTNF) in regulating expansion of activated T cells by apoptosis. Splenic CD4⁺ T cells from wtTNF, TNF‐α‐deficient (TNF^?/?) and TNF^?/? mice expressing a non‐cleavable mutant tmTNF showed comparable proliferation rates upon TCR‐mediated stimulation. Activation‐induced cell death (AICD), however, was significantly attenuated in tmTNF and TNF^?/?, compared with wtTNF CD4⁺ T cells. Addition of sTNF during initial priming was sufficient to enhance susceptibility to AICD in tmTNF and TNF^?/? CD4⁺ T cells to levels seen in wtTNF CD4⁺ T cells, whereas addition of sTNF only during restimulation failed to enhance AICD. sTNF‐induced, enhanced susceptibility to AICD was dependent on both TNF receptors. The reduced susceptibility of tmTNF CD4⁺ T cells for AICD was also evident in an in vivo model of adoptively transferred CD4⁺ T‐cell‐mediated colonic inflammation. Hence, the presence of sTNF during T‐cell priming may represent an important mechanism to sensitize activated T cells for apoptosis, thereby attenuating the extent and duration of T‐cell reactivities and subsequent T‐cell‐mediated, excessive inflammation.     

Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the migration of a melanoma patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic melanoma culture. In this model, CTL migrated toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by the CC chemokine receptor (CCR)4 expressed by the CTL and the CC chemokine ligand (CCL)2 secreted by the tumor cells, as evidenced by blockage of CTL migration by CCL2 or antibodies to CCL2 or CCR4. These results were confirmed in a Transwell migration assay in which the CTL actively migrated toward isolated CCL2 and migration was inhibited by anti-CCR4 antibody. These studies, together with previous studies in mice indicating regression of CCL2-transduced tumor cells, suggest that CCL2 may be useful as an immunotherapeutic agent for cancer patients.     

Linomide inhibits programmed cell death of peripheral T cells in vivo

Programmed cell death (PCD) is involved in the physiological regulation of lymphocyte turnover, as well in the antigen-driven selection of T and B cells. Here it is shown that the immunomodulator linomide (quinoline-3-carboxamide) inhibits the apoptotic decay of peripheral T lymphocytes in response to three different stimuli. First, linomide reduces the superantigen-mediated apoptosis and deletion of specific T lymphocytes of both the CD4⁺ and the CD8⁺ subsets without affecting other superantigen-triggered phenomena such as T cell expansion and anergy. Second, linomide abolishes the T lymphopenia and inhibits PCD of splenic CD4⁺ and CD8⁺ T cells induced by exogenous glucocorticoids. This effect is restricted to peripheral T lymphocytes and does not concern thymocytes. Finally, linomide abolishes the development of lymphopenia that follows infection with vaccinia virus, while reducing PCD of CD4⁺ and CD8⁺ peripheral T cells. The anti-apoptotic effect of linomide could account for its immunostimulatory properties and might be relevant to the treatment of immunodeficiencies associated with an increased apoptotic decay of T lymphocytes.     

Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-alpha.

Human primary effector T cells were analyzed for their susceptibility to anti-CD3-induced activation-induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95-mediated apoptosis, although cell-surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti-CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF-alpha/TNFR interactions because although type 1 cells secreted more TNF-alpha than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z-AAD-CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.