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 ４ｕｇ／ｍＬ ＳＥＢ刺激静息的ＳＥＢ应答ＣＤ４＾＋Ｔ细胞１２ｈ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     

超抗原SEB体外诱导胸腺细胞凋亡的研究

目的：观察超抗原SEB能否在APC的辅助下体外诱导胸腺细胞凋亡，并研究其机制。方法：SEB作用于与APC混合培养的胸腺细胞，用DNA凝胶电泳、DNA片段测定等判定胸腺细胞凋亡并作定量分析；用间接免疫荧光法标记并以流式细胞仪检测Fas、FasL的表达。结果：SEB处理组胸腺细胞有凋亡表现且凋亡率明显高于对照组，Fas、FasL的表达明显高于对照组。结论：SEB可以于体外在APC辅助下诱导胸腺细胞凋亡，Fas、FasL的高表达可能在凋亡中起重要作用，这可能为体外研究胸腺细胞阴性选择过程提供一种方法。     

超抗原SEB对CD8+T细胞凋亡诱导配体表达的影响

为了探讨超抗原SEB对CD8 T细胞凋亡诱导配体在细胞胞内和膜表面表达的影响,以SEB及PHA刺激PBMC,用免疫荧光染色结合三色流式细胞术,分析比较刺激前后PBMC中CD8 T细胞三种主要的凋亡诱导配体TRAIL、FasL和TNF-α在细胞胞内和膜表面表达的变化.结果表明,SEB上调FasL在CD8 T细胞胞内(34.8%至42.5%)和TRAIL在CD8 T细胞膜表面(7%至20.7%)的表达,SEB还可同时上调TNF-α在CD8 T细胞膜表面(7%至45.7%)及胞内(23.9%至88.7%)的表达.说明SEB能上调不同凋亡诱导配体在CD8 T细胞膜表面或胞内的表达,这可能是SEB影响CD8 T细胞效应功能的方式之一.     

献血员CD8~+T细胞的增加对CD4~+T细胞的抑制作用

实验发现 6 5 %体检合格的献血员外周血CD8+T细胞数量明显增加 ,并伴随CD16 +淋巴细胞数量的增加。这些异常的淋巴细胞与葡萄球菌肠毒素B (SEB )共同培养 6d ,CD4+T细胞无增殖反应。预先用抗CD8抗体去除CD8+T细胞 (去除率 >90 % )再用SEB刺激淋巴细胞 ,其应答能力恢复正常。增殖的细胞是CD4+T细胞 ,它们由 34 0 4%增加到 99 34%。CD8+T细胞由最初的 9 5 7%降低到 0 12 %。这些结果显示过量的CD8+T细胞有可能是被外原性抗原活化的T细胞。它们直接抑制CD4+T细胞对SEB的免疫应答反应 ,但不破坏CD4+T细胞的TCRVβ结构。     

超抗原SEA对CD4~+T细胞TCR V_β链的取用格局

用超抗原葡萄球菌肠毒素A (SEA )在体内或体外刺激C57BL/ 6小鼠 2种不同的TCRVβ链T细胞亚群 (Vβ3+ CD4+ 、Vβ1 1 + CD4+ T细胞 ) ,观察二者对超抗原的免疫应答。结果显示 :体内初次刺激后 ,2种T细胞亚群在刺激后第 2天都发生增殖 ,随后均逐渐下降至对照水平。但第 2次用同样剂量刺激后 ,Vβ3+ CD4+ T细胞群对SEA的再次刺激表现出增殖 ,而Vβ1 1 + CD4+ T细胞群对SEA再次刺激则表现出免疫无能。体外初次、再次刺激也分别获得与体内实验相同的结果。表明超抗原SEA可选择性地取用Vβ3+ CD4+ T细胞群并对其发生增殖 ,即超抗原SEA对CD4+ T细胞TCRVβ链的取用格局各有不同。     

超抗原SEA诱导T细胞失能体外实验模型的建立

目的 建立超抗原诱导T细胞失能的体外实验模型。方法 SEA刺激人外周血淋巴细胞,再加入外源性的rIL-2维持,建立短期SEA反应性T细胞系;进行Ficoll-Hypaque密度梯度离心获取经SEA多次刺激过SEA反应性T细胞;加入处理过的PBMC作APCs,建立超抗原SEA诱导T细胞的化组和失能组。结果 SEA和rIL-2共同作用可得到静息SEA反应性T细胞系;SEA反复刺激使T细胞处于一低反应状态,与活化组相比,其DNA合成能力明显下降,IL－2分泌显著减少,静息一段时间后,其增殖能力慢慢恢复。结论 超抗原SEA多次刺激诱导SEA反应性T细胞处于失能状态。     

超抗原诱导T细胞凋亡或增殖的体外研究

研究抗原递呈细胞在超抗原诱导人外周血特异性Ｔ细胞反应中的作用。方法金黄色葡萄球菌Ａ肠毒素刺激短期人外周血ＳＥＡ反应Ｔ细胞系的作用中,通过去除或加入Ａｐｃ观察细胞形态,ＦＣＭ检测细胞亚ＧＯ／Ｇ１峰大小及１．８％ＤＮＡ琼脂糖凝胶电泳观察凋亡特征条带。     

超抗原SEA诱导T细胞无能的作用机制探讨

目的 探讨超抗原诱导T细胞无能的分子机制。方法 采用ELISA的FACS，分别检测超抗原对诱导T细胞无能的过程中，IL－2，IL－10的产生和IL－2Rα链（CD25）的表达，并以^3H-TdR掺入法，测定无能T细胞对rhIL-2,PMA及ionomycin的应答能力，而L－10的产生则逐渐升高；CD25的表达与活化组相比较无显著差异。rhIL-2的加入可恢复T细胞增殖。PMA单独作用能诱导无能T细胞的部分增殖能力，但PMA＋ionomycin则能更大程度地恢复T细胞的增殖。结论 超抗原SEA对T细胞无能的诱导，可能与降低IL－2的水平和升高IL－10的水平有关。超抗原SEA的反复刺激对T细胞无能的诱导，可能是干扰了TCR信号途径的近端事件，导致Ras/MAPK途径和Ca/calcineurin途径受阻，而使IL－2基因不能转录所致。     

APC辅助超抗原活化T细胞的机制

超抗原是ＭＨＣ非限制性及ＴＣＲＶβ特异性方式激活Ｔ细胞的一种新的蛋白质抗原分子。现已发现它所激活的Ｔ细胞可引发人体的多种急、慢性疾病。ＡＰＣ以与辅助普通抗原不同的方式辅助超抗原是超抗原活化Ｔ细胞的关键，了解ＡＰＣ辅助超抗原活化Ｔ细胞的机制有助于理解超抗原激活Ｔ细胞的机理及预防超抗原所引发的疾病。     

CTLA-4在超抗原诱导T细胞无能中的作用研究

目的 探讨CTLA—4对T细胞无能的诱导作用。方法 通过使用超抗原SEA作为一种无能诱导剂，建立体外无能模型，检测了无能T细胞在受到SEA的再次刺激时其膜分子CD28和CTLA—4的表达。结果 与活化T细胞相比，无能T细胞在免疫应答的后期阶段表面表达高水平的CTLA—4分子，而CD28的表达则只较活化组T细胞稍高一些。结论 CTLA—4表面表达水平的升高很可能与T细胞无能状态的诱导有关。     

SEB诱导的CD4+ T细胞无能、凋亡及MHC-I类分子表达下调

目的：探讨超抗原金黄色葡萄球菌肠毒素（SEB）体外诱导外周T细胞免疫耐受的作用机制。方法：采用SEB体外刺激C57BL/6J（B6）小鼠的脾细胞后，以MTT比色法检测脾细胞的增殖，并用PI染色后以流式细胞术（FCM）分析不同时间段处于S期，G0-G1期的细胞及无能T细胞的凋亡，测定T细胞亚群及MHC-I（H-2K^b)表达的变化。用琼脂糖凝胶电泳，观察不同时间段凋亡T细胞的DNA特征。结果：部分去除CD8^ T细胞后。SEB可刺激B6小鼠脾细胞中CD4^ T细胞大量增殖，在SEB刺激后第3天，CD4^ T细胞中处于S期的比率最大，此后开始下降；而处于G0-G1期的CD4^ T细胞变化则相反，在初次刺激后第3天，增殖的CD4^ T细胞出现无能，FCM检测及用琼脂糖凝胶电泳检查DNAladder证实，在第7天，无能CD4^ T细胞出现凋亡，且凋亡细胞的比率逐渐增多，不因加入抗CD3抗体或CoN A而逆转，在SEB刺激后，CD4^ T细胞表面MHC-I类分子（H-2K^b)的表达，随细胞无能的出现而明显下调。结论：SEB诱导的T细胞免疫耐受，可能与CD4^ T细胞的无能，凋亡及细胞表面分子MHC-I的表达下调有关。     

Regulation of apoptosis and T cell activation by Fas-specific mAb

Fas was initially described as a molecule expressed on the surfaceof certain cell lines that could mediate programmed cell death(apoptosis) subsequent to ligatlon by specific mAb. To determinewhether mAb to other epitopes on the Fas molecule might mediateother functions, we generated a panel of mAb to the extracellularportion of human Fas. Significant lysis of Fas-expressing targetcells was only observed when the new mAb were first bound toa solid-phase support and not when the mAb were added in solution.However, several of these mAb inhibited the killing of targetcells induced by the prototypic Fas-specific mAb, CH-11. ThosemAb that inhibited apoptosis of target cells mediated by theCH-11 mAb also blocked lysis of target cells mediated by cellsexpressing Fas ligand. Finally, some of the Fas-specific mAbwere found to co-stimulate proliferation of peripheral bloodT cells in the presence of immobilized CD3 mAb. Thus, the dataindicate the existence of a complex set of interactions mediatedby Fas in both normal and transformed lymphoid cells that mayhave important implications regarding the role(s) of this moleculein regulation of immune responses.     

Interaction between T cells and murine acquired immunodeficiency virus superantigen: effect of second signal on T cell reactivity to the MAIDS virus superantigen

A murine acquired Immunodeficiency (MAIDS) virus transformedB cell line, B6-1710, was shown to express superantigen activityand stimulate T cell hybridomas to produce IL-2. However, Tcell clones and lines from which B6-1710 reactive hybridomaswere established failed to proliferate upon stimulation withB6-1710, while B6-1710 cells were capable of presenting bacterialsuperantigen to stimulate both T cell hybridomas and clones.Proliferative response of T cells to MAIDS virus superantigencould be detected by the addition of the pharmacological agentphorbol myristate acetate (PMA). Thus, B6-1710 seems to lacka stimulatory activity necessary for the prollferative responseof T cells to the MAIDS viral superantigen. In the analysisof the response of naive splenic T cells to the MAIDS superantigen,T cells recovered from a culture stimulated with B6-1710 cellsin the presence of PMA showed no dominant TCR V_ß chainusage, while cells recovered from a culture stimulated withB6-1710 alone were dominated by T cells expressing V_ß5,11, and 12 TCR chains. These results suggest that T cells bearinga majority of TCR V_ß chains are capable of respondingto B6-1710 superantigen. The avidity of interaction betweenT cells and viral superantigen may differ significantly amongT cells and those T cells exhibiting weak interaction with MAIDSvirus superantigen may require additional signals, such as PMA,for an in vitro proliferative response to B6-1710 cells.     

Help signals provided by lymphokines modulate the activation and apoptotic programs induced by partially agonistic peptides in specific cytotoxic T lymphocytes

Inefficient recognition of altered peptide ligands (APL) by specific CTL is believed to contribute to the failure of immune control over tumors and progressive viral infections. A link between deficient help signals and the appearance of CTL epitope mutants has been suggested by recent studies. However, the regulation of APL activity by immunologic help is not well understood. We analyzed the capacity of exogenous IL-2 and IL-15, which are physiologically produced by cells of the adaptive and innate immune system, respectively, to modulate proliferation, responsiveness to repeated stimulation and apoptotic programs triggered in specific CTL by either fully or partially agonistic peptide ligands. We show that signals induced by the lymphokines synergize with weak TCR signaling induced by partially agonistic APL, converting many of these peptides from inhibitory to stimulatory ligands. Some APL partially suppress the responsiveness of specific CTL to secondary stimulation, while this inhibitory effect is diminished if APL-stimulated cells are cultured in the presence of either of the lymphokines. We also demonstrate that IL-2 and IL-15 suppress up-regulation of the Bcl-2 family member Bim and induction of a death receptor-independent apoptotic program triggered by partially agonistic APL. Our results suggest that under conditions of insufficient immunologic help, partially agonistic APL may actively suppress specific CTL responses and become especially advantageous for immune escape of tumors or viruses.     

Triggering via the alternative CD2 pathway induces apoptosis in activated human T lymphocytes

Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin-2 (IL-2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2-dependent activation pathway. In the presence of exogenous IL-2, a pair of mitogenic anti-CD2 mAb suppressed the IL-2-driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti-CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.     

Induction of apoptosis and modulation of activation and effector function in T cells by immunosuppressive drugs

Immunosuppressive drugs (ISD) are used for the prevention and treatment of graft rejection, graft-versus-host-disease (GVHD) and autoimmune disorders. The precise mechanisms by which ISD interfere with T cell activation and effector function or delete antigen-specific T cells are defined only partially. We analysed commonly used ISD such as dexamethasone (DEX), mycophenolic acid (MPA), FK506, cyclosporin A (CsA), rapamycin (RAP), methotrexate (MTX) and cyclophosphamide (CP) for apoptosis-induction and modulation of activation and effector function in human peripheral T cells, cytotoxic T cell lines (CTL) and Jurkat T cells. Of all drugs tested only CP and MTX prevented antigen-specific proliferation of T cells and decreased cytotoxicity of alloantigen specific CTL lines by direct induction of apoptosis. MTX and CP also slightly increased activation-induced cell death (AICD) and CD95-sensitivity. In contrast, all other drugs tested did not induce T cell apoptosis, increase CD95-sensitivity or AICD. CsA and FK506 even prevented AICD by down-modulation of CD95L. DEX, MPA, CsA, FK506 and RAP inhibited activation of naive T cells, but were not able to block proliferation of activated T cells nor decrease cytotoxic capacity of CTL lines. These results show that ISD can be classified according to their action on apoptosis-induction and inhibition of proliferation and would favour a rational combination therapy to delete existing reactive T cells and prevent further T cell activation.     

Enhanced murine CD4+ T cell responses induced by the CD2 ligand CD48

The physiological functions of murine CD2 and its ligand CD48 are uncertain. We have examined the role of the CD2-CD48 interaction in murine T cell activation using a series of Chinese hamster ovary (CHO) cell transfectants. CHO cells expressing I-A^d together with CD48 induced more potent activation of OVA-specific, I-A^d -restricted DO11.10-transgenic T cells than CHO cells expressing I-A^d alone. CD48 augmented proliferation and IL-2 production in response to antigen. The enhancing effect of CD48 was of the same magnitude as that seen for CD80 (B7-1). Conjugate assays revealed the ability of CD48 to increase adhesion between T cells and CHO transfectants. The enhancing effects of CD48 on T cell-antigen-presenting cell adhesion and T cell activation were inhibited by anti-CD2 monoclonal antibody. This report provides the first evidence that the CD2 ligand CD48 contributes to the interactions of murine CD4⁺ T cells with antigen-presenting cells.     

Virus-induced polyclonal T cell activation is followed by apoptosis: partitioning of CD8+ T cells based on {alpha}4 integrin expression

Systemic infection with lymphocytic choriomeningitis virus (LCMV)is accompanied by marked splenomegaly, primarily reflectingthe accumulation of CD8⁺ T cells with an activated phenotypee.g. VLA-4^hi). Analysis of DNA content using 7-aminoactinomycin-Drevealed that as many as 30% of CD8⁺ T cells are cycling aroundday 6 post-infection and that virtually all cycling cells expressa high level of VLA-4. In accord with the relatively stableCD4⁺ cell number, only few cycling CD4⁺ cells were observed.Following virus control, splenic lymphocyte numbers decreasedgradually and during this period many apoptotic cells were detectedin the white pulp using terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labeling. Flow cytometric analysis of DNAcontent revealed a high frequency of cells with subnormal levelsof DNA in the CD8⁺VLA-4^hi subset, whereas the frequency waslow for other lymphocyte subsets studied (CD4⁺, CD8⁺VLA-4¹⁰and B cells). In addition, numbers of CD8⁺VLA-4^hi cells constitute{small tilde}30% of splenocytes at the peak of the responseand undergo preferential decrease during normalization of splenocytenumbers. Together these findings indicate that LCMV-inducedactivation of T cells is followed by apoptosis of many of theactivated cells. Those CD8⁺VLA-4^hi cells which do persist inLCMV Immune mice are more sensitive to treatment with the cell-cycle-specificdrug hydroxyurea than are phenotypically naive T cells. Ourresults therefore indicate that LCMV infection induces polyclonalactivation of CD8 ⁺ cells which is followed by apoptosis ofmost of the triggered cells while a smaller subset persistsas a primed population which include cycling cells.