激发型CD28单抗诱导多发性骨髓瘤细胞凋亡的作用研究

为了探讨激发型CD2 8单抗诱导多发性骨髓瘤细胞凋亡的作用及其机制 ,在表达CD2 8分子的多发性骨髓瘤细胞U2 6 6和XG 1的培养体系中加入终浓度为 10 μg/ml的激发型CD2 8单抗 ,逐日观察和分析细胞的生长与增殖状况。结果显示 ,在加入激发型CD2 8单抗后 2 4h即可见细胞聚集且逐渐加剧 ,细胞的立体感和折光性逐渐减弱 ,细胞的生长与增殖抑制 ,台盼蓝着色阳性率增加 ;凝胶电泳的结果表明 ,多发性骨髓瘤细胞出现降解的DNA条带 ;透射电镜分析的结果显示 ,加入激发型CD2 8单抗培养 2 4～ 4 8h ,30 %以上的细胞出现核质边聚的凋亡早期的病理性改变 ,培养 72h后 ,5 0 %以上的细胞出现空泡及凋亡小体。提示激发型CD2 8单抗具有诱导表达CD2 8分子的多发性骨髓瘤细胞凋亡的作用。     

5株鼠抗人CD28单克隆抗体的研制及生物学特性的研究

目的：制备鼠抗人CD28分子功能性单克隆抗体，研究其对T细胞活化、增殖及信号转导等方面的生物学效应。方法：以天然高表达CD28分子的人多发性骨髓瘤细胞株U266和小鼠淋巴瘤细胞转人CD28基因细胞株CD28-T分别作为免疫原和检测细胞株，采用B淋巴细胞杂交瘤技术进行单抗的研制；以快速定性试纸法鉴定单抗亚类；腹水诱生法和免疫亲和层析法进行单抗的制备和纯化；经间接免疫荧光法分析单抗对不同细胞膜表面CD28分子的识别；采用竞争抑制法分析单抗识别的抗原位点；利用^3H-TdR掺入法分析单抗对PBTC的刺激效应和免疫荧光法分析PBTC活化前后的表型变化。结果：成功获得5株鼠抗人CD28功能性单克隆抗体，分别命名为2D5、2F5、3136、3F8和8G8，其中2D5为IgG2a亚类，其余均为IgG1亚类；流式细胞仪分析结果显示，5株单抗均能良好识别CD28-T、U266、XGI和Jurkat细胞表面的CD28分子；竞争抑制试验表明，2D5和8G8能完全阻断标准抗人CD28单抗与U266膜CD28分子的结合，其余3株为部分阻断；^3H-TdR掺入法实验结果表明，单抗8G8联合激发型CD3单抗能明显促进PBTC的增殖，刺激指数为7．4，活化细胞CD4、CD25、ICOS、4IBB及OX40分子的表达上调。结论：5株单抗均为抗人CD28单克隆抗体，具有重要的基础研究及潜在的临床应用价值。     

急性冠脉综合征患者CD4~+CD28~-T细胞表面OX40和4-1BB表达上调

目的探讨急性冠脉综合征(ACS)患者CD4+CD28-T细胞表面OX40和4-1BB的表达及其临床意义。方法采用流式细胞术检测57例ACS患者、46例稳定性心绞痛(SA)患者和60例健康体检者(HC)外周血CD4+CD28-T细胞表面OX40和4-1BB的表达。结果 HC组、SA组和ACS组,三组相比CD4+CD28-T细胞表面OX40的表达水平依次升高;HC组、SA组和ACS组,三组相比CD4+CD28-T细胞表面4-1BB的表达水平依次升高。结论 ACS患者外周血CD4+CD28-T细胞表面OX40和4-1BB表达上调。     

再生障碍性贫血患者CD4~+T细胞中共刺激分子和免疫抑制受体的表达特点和意义

目的:分析再生障碍性贫血患者外周血CD4+T细胞中共刺激分子和免疫抑制受体情况,为全面了解再生障碍性贫血异常T细胞免疫机制提供研究资料。方法:收集12例健康人和16例再生障碍性贫血患者外周血,利用流式细胞术分别分析CD4+T细胞中共刺激分子OX40、ICOS、4-1BB、GITR和HVEM,以及免疫抑制受体TIM-3、LAG-3和CTLA-4的分布情况。结果:健康人CD4+T细胞中OX40、ICOS、4-1BB、GITR和HVEM的比例依次为3. 26%、2. 40%、1. 39%、25. 95%和49. 60%,而再生障碍性贫血患者上述信号分子的比例依次为8. 01%、2. 44%、1. 16%、11. 55%和82. 20%。再生障碍性贫血患者CD4+OX40+T细胞比例显著高于与健康人(P0. 01),而CD4+GITR+T细胞的比例显著低于健康人(P0. 01)。不同病情程度再生障碍性贫血患者CD4+GITR+T细胞比例均显著低于健康人(P0. 05)。重型再生障碍性贫血患者和极重型再生障碍性贫血患者CD4+OX40+T细胞比例均显著高于健康人(P0. 05)。极重型再生障碍性贫血患者CD4+HVEM+T细胞比例显著高于健康人(P0. 05)。再生障碍性贫血患者CD4+T细胞中免疫抑制受体TIM-3、LAG-3和CTLA-4分布比例与健康人之间无显著差异。结论:再生障碍性贫血患者异常CD4+T细胞活化可能与OX40、HVEM和GITR等密切相关,不同病情程度再生障碍性贫血患者CD4+T细胞中共刺激分子OX40和HVEM存在差异变化特点。     

激发型4-1BB单抗联合凋亡肿瘤细胞负载的树突状细胞在恶性肿瘤免疫治疗中的作用

目的：研究激发型4-1BB单抗（2A）联合凋亡肿瘤细胞负载的DC（AP-DC）疫苗在小鼠B细胞淋巴瘤免疫治疗中的作用.方法：凋亡小鼠B细胞淋巴瘤细胞A20负载的DC用来制备AP-DC.A20荷瘤小鼠分别被注射AP-DC、2A单抗或二者联合.观察肿瘤生长情况及小鼠生存期.流式细胞术检测免疫治疗后荷瘤鼠脾脏T细胞的表型和胞浆内细胞因子;3H-TdR掺入试验检测T细胞体外增殖能力;ELISA法检测荷瘤鼠血清和脾脏T细胞培养上清中IL-2、IFN-γ和IL-10含量.结果：应用激发型4-1BB单抗2A或AP-DC疫苗对荷瘤小鼠开展免疫治疗,可抑制肿瘤生长,延长荷瘤鼠生存期,获得12.5%或25%的肿瘤完全缓解率.但二者联合应用,治疗效果更好,可获得62.5%的肿瘤完全缓解率,并且荷瘤鼠可长期生存.联合治疗后荷瘤鼠脾脏T细胞体外增殖更为显著,CD4^＋IFN-γ^＋细胞的比例也明显增加.IL-2和IFN-γ的分泌水平在联合治疗荷瘤鼠的血清和脾脏T细胞的培养上清中升高更明显,而IL-10的分泌则降低.结论：激发型4-1BB单抗和AP-DC在肿瘤免疫治疗中有协同作用.     

慢性肾炎患者外周血T细胞亚群和共刺激分子的表达及其意义

为研究慢性肾炎患者外周血T细胞亚群和共刺激分子的表达特点及其在慢性肾炎免疫病理机制中的作用 ,本文采用免疫荧光标记和流式细胞仪分析 ,对 35例慢性肾炎患者外周血T淋巴细胞亚群和共刺激分子CD2 8、 4 1BB等的表达进行研究。结果表明 :(1)慢性肾炎患者T细胞亚群明显失衡 ,表现为CD4减少 ,CD8增加 ,CD4/CD8比值显著降低 ;(2 )共刺激分子CD2 8表达显著低于正常对照组 (CD2 8表达百分率分别为 45 95± 5 6 7和 6 6 42± 4 5 8,P <0 0 0 1) ,且CD4+ CD2 8+ T细胞和CD8+ CD2 8+ T细胞均显著减少。治疗后缓解期患者T细胞亚群失衡明显纠正 ,CD2 8+ T细胞 ,尤其是CD4+ CD2 8+ T细胞显著增多 ,而且CD4+ CD2 8+ T细胞数与患者的 2 4h尿蛋白定量呈负相关 (r= 0 47,P <0 0 1) ;(3)慢性肾炎患者共刺激分子 4 1BB在T细胞中的表达显著高于正常对照组 (表达百分率分别为 30 5 7± 8 12和 0 74± 0 2 8,P <0 0 0 1) ,治疗后的 4 1BB表达水平显著降低 ,而且 4 1BB异常高表达与CD8+ T细胞数呈正相关 (r=0 6 3,P <0 0 5 )。从而表明慢性肾炎外周血T细胞亚群失衡和T细胞活化所必需的共刺激分子CD2 8、 4 1BB异常表达 ,可能在慢性肾炎发生和病变进展中起着重要作用。     

4-1BB在人外周血γδT细胞活化中的协同刺激作用

采用结核杆菌(Mtb)低分子多肽刺激人外周血单个核细胞,流式细胞术(FCM)检测不同活化时相γδT细胞膜表面4-1BB分子的表达;用阻断型4-1BB配体(4-1BBL)单抗阻断4-1BB/4-1BBL信号,FCM检测γδT细胞的增殖比率和细胞内产生IFN-γ的情况,同时与阻断CD28/B7-1信号相比较。结果显示,静止的γδT细胞膜表面不表达4-1BB分子,Mtb抗原刺激后6 h,4-1BB即有明显表达(29.71%),48 h达到高峰(49.79%);与未阻断组相比,阻断4-1BB/4-1BBL信号,γδT细胞的增殖效应和细胞内IFN-γ的产生均明显下降(P<0.01),与CD28/B7-1信号阻断组相比,差异无显著性(P>0.05)。提示4-1BB/4-1BBL信号同CD28/B7-1信号一样,可为γδT细胞活化提供协同刺激作用。     

(Pyr~1)apelin-13对Jarkat T淋巴细胞增殖和活化的影响

目的探讨(Pyr~1)apelin-13对Jurkat T淋巴细胞增殖和活化的影响。方法用(0、10、50)nmol/L(Pyr~1)apelin-13处理Jurkat T淋巴细胞24 h后,用CCK-8法检测细胞增殖能力的变化;用抗CD3抗体和抗CD28抗体包被并活化Jurkat T淋巴细胞,观察(Pyr~1)apelin-13对Jurkat T淋巴细胞活化的影响;实时定量PCR检测CD69、CD25、细胞毒性T淋巴细胞相关蛋白4(CTLA-4)及程序性死亡蛋白1(PD-1)的mRNA水平。结果 (Pyr~1)apelin-13对Jurkat T淋巴细胞增殖无影响,但通过增加CD69和CD25的表达促进Jurkat T淋巴细胞活化,但对CTLA-4和PD-1表达影响不明显。结论 (Pyr~1)apelin-13促进Jurkat T淋巴细胞活化。     

BTLA信号对T细胞活化的起始和早期阶段的调节作用

目的:观察BTLA分子在T细胞上的表达并探讨其在各个阶段不同时相对T细胞活化的抑制。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。检测T细胞上BTLA、CTLA-4和PD-1的表达;用CD3抗体刺激T细胞活化,比较BTLA、CTLA-4和PD-1在T细胞活化过程中的动态表达。CD3抗体联合CD28抗体活化T细胞,在不同的活化时间,MTT法检测BTLA单抗8H9对T细胞增殖的影响。GM-CSF和IL-4体外诱导单核细胞分化成未成熟DC,CD40抗体刺激DC成熟,流式检测HVEM在DC上的表达。用DC诱导T细胞活化,加入游离8H9或抗HVEM抗体,阻断HVEM和BTLA结合,MTT法检测T细胞增殖。结果:静止T细胞组成性高表达BTLA,不表达CTLA-4和PD-1分子。T细胞活化后,BTLA分子表达有所降低,然后迅速回升至高水平。CTLA-4、PD-1分子在活化后两天几乎不表达,第三天开始表达并逐渐上升。8H9可以抑制CD3和CD28抗体活化的T细胞增殖。CD3和CD28抗体预先活化T细胞24小时或48小时后,再加入8H9仍然具有抑制效应,但不如在T细胞活化之初加入8H9的抑制效应。单核细胞诱导的不成熟DC上高表达HVEM,当DC成熟后,HVEM表达降低。用游离8H9或HVEM抗体阻断DC表面HVEM与T细胞表面BTLA结合,48小时之内均明显增强了DC诱导的T细胞增殖。结论:BTLA信号可以提高T细胞的活化阈值,在T细胞活化的起始和早期阶段发挥重要的负性调控作用。     

类风关滑膜浸润T细胞特性与致病机制研究

为研究类风湿关节炎时关节滑膜浸润性T细胞生物学特性与致病机制 ,对 10例RA患者滑膜液中淋巴细胞的免疫表型、细胞因子分泌格局与趋化因子受体表达进行了分析。用双色荧光标记法分别测定滑膜液中和外周血淋巴细胞表型与趋化因子受体表达。用ELISA方法检测滑膜液与外周血中IFN γ、IL 10、IL 4与IL 12的含量。结果是滑膜液中的CD4 + T淋巴细胞为 4 0 0 %± 11% ,CD8+ T细胞为 34 0 %± 6 % ,CD4 + 与CD8+ T细胞的比值为 1 2 ,显著低于外周血中CD4 /CD8的比值。滑膜液中CD3和CD2 5双阳性的活化T细胞占 16 %± 6 0 %。趋化因子受体CCR5表达较低 ,与外周血无明显差异。但CX CR3表达水平较高 ,为 16 %± 4 0 % ,远远高于外周血 (仅为 0 5 %± 0 3% )。IFN γ在滑膜液中含量很高 ,达 (36 6 7± 4 3 2 )pg/ml,而外周血中含量仅为 (2 0 1± 3 2 )pg/ml。IL 4含量未能测得 (<15pg/ml ) ,与外周血相似。IL 12含量为 (4 19 9±89 2 )pg/ml,远高于外周血中的含量 (6 5 32± 34 2 )pg/ml。IL 10含量为 (187 7± 34 5 )pg/ml,高于外周血中的含量 (85±12 7)pg/ml。在所测细胞因子中 ,关节滑膜液中IFN γ和IL 12的含量与外周血相比具有显著的统计学差异。表明RA关节滑膜液中有相当数量的T细胞浸润。这些T细胞     

Costimulation through OX40 is crucial for induction of an alloreactive human T-cell response

The alloreactive immune response is a series of events initiated by the interaction of T cells with allogeneic dendritic cells (DCs), involving alloantigen recognition and costimulatory signals. In this study, we investigated the role of OX40 in alloreactivity in vitro. We first demonstrated that anti-OX40 ligand (anti-OX40L) monoclonal antibody (mAb) could markedly suppress the mixed leucocyte reaction (MLR) of peripheral blood mononuclear cells (PBMC). To further define the contribution of the OX40/OX40L system to the MLR, we set up a co-culture system of CD4+ T cells and allogeneic monocyte-derived dendritic cells (DCs). After 2 days, OX40 expression was induced on CD4+ T cells and this induction was strongly inhibited by the addition of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein, suggesting that the expression of OX40 during alloreaction is dependent on CD28 signalling. Next we examined the effects of anti-OX40L mAb, CTLA-4-Fc fusion protein and anti-human leucocyte antigen (HLA)-DR mAb on the proliferative response of CD4+ T cells to allogeneic DCs. The proliferation of T cells was almost completely suppressed by anti-OX40L mAb, which was comparable with that of CTLA-4-Fc. Measurement of interleukin-2 (IL-2) production in the culture supernatants showed that suppression of a proliferative response was at least in part ascribed to reduced IL-2 production. Furthermore, purified OX40L- allogeneic DCs could induce considerable proliferation of CD4+ T cells, which was suppressed by anti-OX40L mAb. These results suggest that the OX40/OX40L system is crucial for induction of the allogeneic T-cell response and the OX40/OX40L system is subsequent to and dependent on CD28 signalling, but is crucial for the end outcome of the human alloreactive T-cell response.     

Differential expression of costimulatory molecules in chronic inflammatory periodontal disease tissue

Although B cell activation and subsequent immunoglobulin production are the immunopathological features of chronic inflammatory periodontal disease, in situ expression of costimulatory molecules in humoral immunity has not been investigated. In the present study we examined the expression of CD40, CD40 ligand (CD40L), CD80, CD86, CD28 and cytolytic T lymphocyte-associated antigen-4 (CTLA-4) on lymphocytes immunohistochemically. Cryostat sections were prepared from the gingival tissue samples of 14 patients with moderate to advanced adult periodontitis. In vitro kinetics of the expression of CD40L and CTLA-4 by peripheral blood T cells and that of CD80 and CD86 by peripheral blood B cells were also investigated by flow cytometry. Positive percentage expression of CD40L, CD28 and CTLA-4, and CD40, CD80 and CD86 was calculated for the number of CD3⁺ and CD19⁺ cells, respectively. Flow cytometric analysis demonstrated that the expression of CD40L and CTLA-4 on T cells, and CD80 and CD86 on B cells of peripheral blood was up-regulated upon activation. While most T cells and B cells expressed CD28, and CD80 and CD86, respectively, in gingival tissues, the expression of CD40L and CTLA-4 was lower but highly variable between specimens. Furthermore, these two molecules seemed to be expressed reciprocally in the lesion. As both CD40L and CTLA-4 expression are induced transiently by stimulation, variability in the expression of the molecules may reflect immunological activities and participation in the regulation of B cell activation of the lesion.     

T淋巴细胞清除促进脐血造血细胞的体外扩增

采用抗CD3或抗CD8单克隆抗体的补体细胞毒方法清除脐血单个核细胞 (MNC )中T淋巴细胞 ,CD34免疫亲和柱纯化MNC中CD34 +细胞 ,流式细胞技术 (FACS )分析细胞表面标志。将CD34 +细胞中加入含多种造血生长因子的培养基进行体外液态扩增 ,并观察粒 巨噬集落 (CFU GM )和多向祖细胞集落 (CFU GEMM )形成能力。结果CD3+或CD8+细胞清除组和MNC经CD34免疫亲和柱纯化后 ,CD34 +细胞分别为 5 9 5 2 %、 5 6 70 %和 5 0 72 % ,比未纯化组 (1 0 7% )纯度大幅度提高。抗CD3单抗清除 +CD34纯化组和单纯CD34纯化组经造血生长因子刺激培养第 14天 ,细胞总数分别扩增 110 40倍和 87 0 0倍。抗CD3单抗清除 +CD34纯化组、抗CD8单抗清除 +CD34纯化组和单纯CD34纯化组的CFU GM产率分别为 2 86 5 0± 12 0 2、2 88 5 0± 17 68和 2 19 5 0± 5 3 0 3,前者虽高于后者 ,但差异无显著性。CFU GEMM产率分别为 376 67± 43 2 4、 438 33± 36 73和 311 0 0± 40 11,抗CD3单抗清除 +CD34纯化组无显著差异 ,抗CD8单抗清除 +CD34纯化组显示出显著性作用 (P <0 0 5 )。     

Blocking CD40 - CD154 and CD80/CD86 - CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production.

Although CD28 triggering provides an important co-stimulatory signal to T cells, blocking the CD80/CD86 - CD28 interaction with CTLA-4lg fusion protein is not sufficient for tolerance induction in vivo or in vitro. According to more recent data, interruption of the CD40 - CD154 interaction might complement the effect of CTLA-4lg and induce graft acceptance. We studied the effects of a blocking anti-CD40 monoclonal antibody (mAb) and/or blocking anti-CD80/anti-CD86 mAb in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with allogeneic PBMC. T cells activated by alloantigens in the presence of anti-CD80, anti-CD86 and anti-CD40 entered a state of alloantigen-specific non-responsiveness as evidenced upon restimulation by lack of proliferation, cytotoxic activity, and IL-2, IL-5 and IL-13 production. IFN-gamma production during restimulation was less than in the control cultures, while the production of IL-10 was enhanced. Addition of recombinant IL-2 during the restimulation rescued alloantigen-specific activity. We conclude that the simultaneous blocking of the CD40 - CD154 and CD80/CD86 - CD28 interaction during allogeneic T cell activation induces T cell anergy. Since anergic cells induced by this treatment still produce high levels of IL-10, the latter could contribute to modulation of antigen-presenting cell activity and to bystander suppression of residual reactive T cells.     

抗CD28单抗对T细胞体外抗瘤作用的影响

采用健康人外周血T淋巴细胞分别与BEL 740 2人肝癌细胞和抗CD2 8混合培养 ,检测淋巴细胞活化增殖能力、CTL细胞的杀伤活性、培养上清IFN γ和TNF α的水平。结果发现 ,在BEL 740 2刺激细胞存在时 ,抗CD2 8促进增殖的最佳浓度为 6 μg/ml,而单一抗CD2 8不能刺激T细胞增殖。BEL 740 2细胞和抗CD2 8共刺激后IFN γ和TNF α分泌均比单纯BEL 740 2高 ,同时其诱生的CTL细胞杀伤BEL 740 2的能力也强于对照组 ,其差异均具有显著性 (P <0 0 1)。上述结果提示 ,抗CD2 8单抗共刺激诱导CD4+ 和CD8+ T细胞参与抗肿瘤免疫 ,使IFN γ、TNF α的分泌增多 ,增强了体外T细胞抗瘤作用。     

Non-responsiveness of antigen-experienced CD4 T cells reflects more stringent co-stimulatory requirements.

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.     

慢性乙肝病人外周血T细胞表面HLA-DR抗原的表达

本文采用单克隆抗体间接免疫荧光法,对15个慢性活动性肝炎病人以及1对照者外周血中T淋巴细胞膜表面的HLA-DR表达进行了测定。结果是慢性乙型活动性人外周血中的TDR~+细胞的百分率明显高于正常对照(P<0.001),并且增高的T细胞是以性细胞为主。     

Superantigen responses and co-stimulation: CD28 and CTLA-4 have opposing effects on T cell expansion in vitro and in vivo

Co-stimulation via the CD28/CTLA-4 system appears critical forT cell proliferation to peptide antigens presented in associationwith MHC. In this study, we examine the roles of CD28 and CTLA-4in the response of murine T cells to the superantigen staphylococcalenterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2or Fab fragments of anti-CD28 antibodies significantly inhibitthe response of splenocytes to SEB. Conversely, Fab fragmentsof anti-CTLA-4 antibodies augment the proliferative response.Further, addition of blocking antibodies directed against B7-1/B7-2augment proliferation co-stimulated by intact anti-CD28 antibodies.These data support the hypothesis that CD28 and CTLA-4 exertopposing effects upon early T cell activation. In vivo, Intactanti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28appear to have similar inhibitory effects upon the expansionof V_ß8⁺ T cells. In contrast, both intact and Fabfragments of anti-CTLA-4 appear to amplify this expansion. Weconclude that the SEB response is significantly augmented byCD28-derived signaling and this in turn may be attenuated bysignals through CTLA-4.     

SLE患者Th1/Th2平衡状态及与外周血淋巴细胞CD28及CTLA-4分子表达的关系

目的:了解SLE患者Th1/Th2平衡状态以及共刺激分子CD28/CTLA-4与Th1/Th2平衡状态的关系。方法:研究对象为18例SLE患者(活跃期12例、缓解期6例)。对照组14例,为健康体检者。外周血单个核细胞(PBMCs)经梯度密度离心法分离后置于含PMA(5μg/L)及ionomycin(500μg/L)培养液中培养72 h。采用ELISA方法检测培养的PBMCs上清液中IFN-γ及IL-10的含量。应用流式细胞技术检测培养的淋巴细胞CD28及CTLA-4分子的表达。结果:活跃期SLE患者培养的PBMCs分泌IL-10的量(351.29 ng/L±153.31 ng/L)较对照组(254.48 ng/L±120.69 ng/L)有一定程度的升高,但差异无显著(P0.05),IFN-γ的分泌量(25.76 ng/L±16.09 ng/L)明显低于对照组(50.71 ng/L±27.92 ng/L,P0.05),IL-10/IFN-γ比值(18.74±13.77)明显高于对照组(6.66±4.95,P0.05)。培养前、后SLE患者CD3+及CD8+T细胞CD28分子表达量与对照组比较均无显著差异。培养前活跃期SLE患者CD3+T细胞CTLA-4分子表达量(0.79%+0.37%)较对照组(1.31%+0.61%)明显降低(P0.05)。培养后SLE患者CD3+T细胞及CD8+T细胞CTLA-4分子表达量仍低于对照组,但差异无显著(P0.05)。活跃期SLE患者培养的PBMCs中CD3+T细胞CTLA-4分子的表达量与上清液中IFN-γ含量呈明显的直线正相关关系(r=0.681,P0.05)、与上清液中IL-10及IL-10/IFN-γ比值呈明显的直线负相关关系(r=-0.624,P0.05;r=-0.738,P0.01)。结论:SLE患者存在Th1/Th2平衡向Th2方向偏移,即Th2优势状态。CTLA-4分子可能通过抑制CD28的信号转导参与Th2优势状态的形成。