免疫性肝损伤动物模型建立及4-1BB、CD4+CD25+T淋巴细胞表达

4-1BB是一种主要表达在活化T淋巴细胞上共刺激信号,是神经生长因了/肿瘤坏死因子受体家族成员之一。CD4^＋CD25^＋T淋巴细胞是一种免疫耐受细胞,具有免疫无能性及免疫抑制性两方面功能。本实验对免疫性肝损伤中4 1BB及CD4^＋CD25^＋T淋巴细胞的变化进行研究。[第一段]     

隔药饼灸对环磷酰胺诱导免疫抑制兔共刺激分子CTLA-4、4-1BB、CD28的影响

目的 探讨隔药饼灸对环磷酰胺诱导免疫抑制兔共刺激分子细胞毒性T淋巴细胞相关抗原4(CTLA-4)、4-1BB、CD28的影响。方法 选择大耳白兔32只,随机分为空白组、模型组、艾条灸组和隔药饼灸组,每组8只。模型组、艾条灸组和隔药饼灸组予环磷酰胺诱导免疫抑制模型。模型制备成功次日,艾条灸组和隔药饼灸组分别予艾条灸和隔药饼灸,隔日1次,共10次。空白组和模型组不予艾灸。艾条灸组和隔药饼灸组末次干预次日,空白组和模型组同时间,采集腹腔静脉血,离心留取血清,采用ELISA法检测血清CTLA-4、4-1BB、CD28;腹腔静脉取血后,摘取脾脏和肝脏,免疫组化法检测脾脏和肝脏组织CTLA-4、4-1BB蛋白表达。结果 模型组血清CTLA-4、CD28水平均高于空白组,血清4-1BB水平低于空白组（P均<0.05）;与模型组比较,艾条灸组和隔药饼灸组血清CTLA-4、CD28水平均降低,血清4-1BB水平均升高（P均<0.05）;与艾条灸组比较,隔药饼灸组血清CTLA-4、CD28水平均降低,血清4-1BB水平升高（P均<0.05）。模型组脾脏和肝脏组织CTLA-4阳性表达均高于...     

淋巴细胞激活诱导的受体4-1BB在类风湿关节炎T淋巴细胞亚群上的表达

目的探讨类风湿关节炎(RA)T淋巴细胞上淋巴细胞激活诱导的受体4-1BB的表达及其作用机制。方法应用流式细胞术检测30例RA患者和20名正常对照者外周血T细胞活化前后4-1BB的表达。结果RA患者CD4~ T和CD8~ T细胞表达的4-1BB明显高于正常对照组[表达百分率分别为(18．56±4．08)％,(10．33±2．13)％,(1．24±0．12)％,(0．87±0．09)％,P＜0．01],经抗CD3单抗体外刺激后CD4~ T和CD8~ T细胞表达的4-1BB均显著高于活化前[表达百分率为(33±4)％和(21±8)％,P＜0．01]。RA患者CD4~ T/CD8~ T比值明显升高,而且与4-1BB~ CD4~ T细胞数呈正相关关系(r=0．84,P＜0.01),另外4-1BB~ CD4~ T细胞数与血沉、IgA呈正相关关系(r=0．476,P＜0．05；r=0．659,P＜0．05)。结论RA患者T细胞表达的4-IBB在RA的发生发展中具有重要意义,4-1BB可能通过对CD4~ T活化与增殖参与关节炎症和免疫反应。     

急性冠状动脉综合征患者B7∶CD28/CTLA4共刺激分子的表达

目的:探讨急性冠状动脉综合征(ACS)患者外周血中单核细胞共刺激分子CD28、细胞毒T淋巴细胞抗原(CTLA)4、CD80(B7-1)的变化,探讨这些分子在发病中的意义。方法:采用直接荧光标记流式细胞仪测定23例ACS患者(ACS组)和31例稳定型心绞痛(SA)患者(SA组)入院时外周血CD4 ,CD8 T淋巴细胞CD28、CTLA4、B7-1分子的表达,同时选健康人群15例作为对照(对照组)。结果:与对照组相比,SA组及ACS组发病时CD4 ,CD8 T淋巴细胞表面共刺激分子CD28均显著升高(均P<0.01);SA组与ACS组比较差异无统计学意义。与对照组相比,SA组CD4 ,CD8 T淋巴细胞表面CTLA4表达均显著升高(P<0.01);而ACS组CD4 ,CD8 T淋巴细胞表面CTLA4表达均显著下降(P<0.01)。各组B7-1的表达差异无统计学意义。结论:①共刺激分子B7-1:CD28/CTLA4途径参与了冠心病的发病过程;②ACS的强烈的炎症反应与CT-LA4的低表达有关。     

合并糖尿病的急性冠脉综合征患者共刺激分子与炎症指标的研究

目的探讨CD4^＋CD28^nullT淋巴细胞及炎症因子C反应蛋白（CRP）在合并糖尿病的冠心病患者发病机制中的作用。方法51例经冠状动脉造影确认为急性冠脉综合征（ACS）的患者,根据1999年WHO标准分为2型糖尿病合并ACS组21例,单纯ACS组30例,对所有研究对象均通过流式细胞术测量外周血中CD4^＋CD28^nullT淋巴细胞的数量,对比分析两组患者的临床资料和冠状动脉造影结果。结果与单纯ACS患者相比较,合并糖尿病的ACS患者外周血CD4^＋CD28^nullT淋巴细胞的数量显著增多[（4．66±4．24）％VS（8．89±6．45）％,JP〈0．05],CRP浓度无明显统计学差异[（1．97±1．05）VS（2．22±1．14）mg／L,P〉0．05],冠状动脉造影显示完全闭塞、弥漫病变比例较高（7w11,P〈0．05;6YS12,P〈0．01）。结论糖尿病合并ACS患者冠状动脉病变累及范围广且程度重,共刺激分子可能与合并糖尿病的ACS患者的免疫调节异常有关。     

4-1BB单克隆抗体对免疫性肝损伤治疗及CD4+CD25+T淋巴细胞影响研究

目的 探讨4-1BB单克隆抗体(4-1BBmAb)对免疫性肝损伤治疗及对CD4+CD25+T淋巴细胞影响.方法 建立小鼠刀豆蛋白A(ConA)肝损伤模型,检测4-1BB表达,ConA注射后2 h给予4-1BBmAb(100μg/只),观察肝功能及病理学变化,流式细胞仪检测CD4+CD25+T淋巴细胞.结果 丙氨酸转氨酶(ALT)模型组(139±22)U/L,对照组(32±12)U/L,天冬氨酸转氨酶(AST)分别(130±16)U/L及(29±11)U/L,两组差异有统计学意义(P<0.01);模型组4-1BB表达(8.1±2.6)比对照组(5.3±2.6)升高(P<0.01).4-1BBmAb治疗后ALT(98±14)U/L,AST(89±11)U/L降低(P<0.01).模型组CD4+CD25+T淋巴细胞(2.9±0.8)低于对照组(3.6±1.2)(P<0.05),4-1BBmAb组(8.3±3.0)高于生理盐水组(3.0±0.8)(P<0.01).结论 4-1BBmAb对免疫性肝损伤有治疗作用,影响CD4+CD25+T淋巴细胞达到治疗肝损伤目的 .     

T淋巴细胞与1型糖尿病

CD4^ 、CD8^ T淋巴细胞在1型糖尿病（DM）发病中起重要作用。近年研究发现T淋巴细胞在胰岛自身抗原刺激下发生增殖，该细胞在浸润胰岛过程中需要粘附分子参与，CD4^ T淋巴细胞通过分泌细胞因子参与1型DM的发病过程，CD8^ T淋巴细胞在1型DM发病早期有重要作用，且与CD4^ T淋巴细胞有协同作用。     

B7-1、CD40和CD54分子在Graves病患者甲状腺组织中的表达

目的探讨T细胞共刺激分子B7-1、CD40和CD54在Graves病(GD)患者甲状腺组织中的表达及其发病机制。方法采用免疫组织化学方法(HRP)观察51例GD患者和50例非毒性结节性甲状腺肿(NTG) 患者甲状腺组织T细胞共刺激分子B7-1、CD40和CD54的表达,应用Mias 99图像分析系统进行定量分析。结果 B7-1、CD40和CD54分子在所有GD患者的甲状腺组织中呈阳性表达,强阳性染色区域主要分布在甲状腺滤泡的表面和细胞质,在浸润的淋巴细胞区部分呈强阳性表达;而在NTG患者的甲状腺组织几乎不着色。B7-1、 CD40和CD54分子阳性表达例数、阳性百分率、阳性颗粒面积及平均吸光度值,在GD组明显高于NTG组(P< 0．01)。结论 T细胞共刺激分子B7-1、CD40和CD54的异常表达在GD的发病中起一定的作用。     

茶多糖对NOD小鼠1型糖尿病的预防作用

目的探讨茶多糖（TPS）对非肥胖糖尿病（NOD）小鼠1型糖尿病（DM）的预防作用。方法比较TPS预免疫组和生理盐水（NS）对照组NOD小鼠1型DM的发病率、血清C肽和谷氨酸脱羧酶抗体水平、胰岛组织病理学和免疫组化、脾脏T细胞亚群比例。结果TPS预免疫组与NS组比较，DM发病率显著降低，血清C肽水平显著增高，胰岛炎症程度减轻，CD8T细胞亚群比例显著增高，CD4／CD8比例显著降低。结论早期应用茶多糖预免疫可以预防或延缓NOD小鼠1型糖尿病的发生。     

食管癌切除术患者围手术期外周血淋巴细胞亚群的变化

采用流式细胞仪对20例食管癌患者术前、术毕及术后1d三个时间点的外周血CD4、CD8、CD3、CD19、自然杀伤细胞（NK）及CD28、CD80细胞进行检测。结果显示。术毕外周血CD3^＋、CD^＋、CD28^＋、CD28^＋CD4^＋、CD28^＋CD8^＋、CD19^＋细胞比率降低，CD4^＋／CD8^＋降低，NK细胞比率升高，术后1dCD3^＋细胞依然低于术前，CD4^＋、CD28^＋、CD28^＋CD4^＋细胞升高但尚未完全恢复至术前水平，CD8^＋细胞低于术前。CD28^＋＋CD8^＋细胞及CD4^＋／CD8^＋恢复至术前水平，CD19^＋细胞比率升高且高于术前，NK细胞恢复至术前水平。认为食管癌患者围手术期特异性免疫受抑制，而非特异性免疫处于相对优势；术后1d患者的免疫状态尚未完全恢复至术前水平。     

Role of 4-1BB (CD137) in the functional activation of cord blood CD28(-)CD8(+) T cells

The CD28(-) subset of CD8(+) T cells is associated with cytotoxic T lymphocyte (CTL) effector function. We investigated a potential role for 4-1BB, a costimulatory molecule structurally related to members of the tumor necrosis factor (TNF) receptor family, in the generation and functional activation of CD28(-) CTLs by using human cord blood (CB) cells composed exclusively of naive CD8(+) T cells with few or no CD28(-) CTLs. The 4-1BB was induced preferentially on the CB CD28(-)CD8(+) T cells when CD28 down-regulation was induced by interleukin 15 (IL-15) and IL-12 stimulation. Anti-4-1BB costimulation induced dramatic phenotypic changes in the CD28(-) CTLs, including restoration of CD28 expression as well as that of memory markers such as CD45RO and CC chemokine receptor 6 (CCR6). Anti-4-1BB costimulation also promoted long-term survival of CD28(-) CTLs, which were sensitive to activation-induced cell death upon anti-CD3 stimulation. The memory-type CD28(+) CTLs induced by anti-4-1BB costimulation acquired a greatly enhanced content of granzyme B, a cytolytic mediator, and enhanced cytotoxic activity as compared with CD28(-) CTLs. Strong cytotoxicity of memory-type CTLs to a 4-1BB ligand-expressing Epstein-Barr virus (EBV)-transformed B-cell line was almost completely abrogated by 4-1BB-Fc, a soluble form of 4-1BB, suggesting involvement of 4-1BB in cytolytic processes. Taken all together, our results suggest that 4-1BB plays a role in the differentiation of effector memory CTLs.     

Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering

To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo. In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo. Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type beta transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and prevention of autoimmune disease.     

Modulation of cord blood CD8+ T-cell effector differentiation by TGF-beta1 and 4-1BB costimulation

Transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, inhibits cytotoxic T cell (CTL) immune responses. In contrast, 4-1BB (CD137), a costimulatory molecule in the tumor necrosis factor (TNF) receptor family, amplifies CTL-mediated antitumor immune responses. We investigated whether TGF-beta1 responses could be reversed by 4-1BB costimulation during in vitro differentiation of naive CD8+ T cells into effector CTL cells. TGF-beta1 potently suppressed CTL differentiation of human cord blood naive CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. TGF-beta1-mediated suppression of CTL differentiation was abrogated by 4-1BB costimulation but not by CD28 or another member in the TNF receptor family, CD30. 4-1BB costimulation suppressed Smad2 phosphorylation induced by TGF-beta1, suggesting that 4-1BB effects were at the level of TGF-beta1 signaling. 4-1BB effects on the TGF-beta1-mediated suppression were enhanced by interleukin 12 (IL-12) but counteracted by IL-4; 4-1BB expression was up- or down-regulated, respectively, by IL-12 and IL-4. IL-4 was more dominant than IL-12 when both cytokines were present during 4-1BB costimulation in the presence of TGF-beta1. This indicates critical roles for IL-4 and IL-12 in regulating 4-1BB effects on TGF-beta1-mediated suppression.     

Immunotherapy Targeting 4-1BB and Its Ligand

T-cell activation in the absence of costimulation is futile because T-cells deprived of costimulatory signals enter a state of unresponsiveness or anergy. The interaction of 4-1BB and 4-1BB ligand (4-1BBL) activates an important costimulatory pathway with diverse and important roles in immune regulation. Signals relayed through 4-1BB generate strong CD8(+) T-cell responses rather than CD4(+) T-cell responses; this action results in cytokine induction and promotes T-cell survival. In recent years, 4-1BB-mediated immune regulation has gained great significance because of the seemingly contradictory dual roles of agonistic anti-4-1BB in vivo disease models. To date, agonistic 4-1BB monoclonal antibody has shown therapeutic potential against a variety of tumors, CD4(+) T-cell-mediated autoimmune diseases, and chronic graft-versus-host disease. In addition, blockade of 4-1BB/4-1BBL interaction has produced therapeutic effects against coxsackievirus-induced myocardial inflammation, herpetic stromal keratitis, and graft rejection. We propose that the dual roles of agonistic anti-4-1BB--an enhanced effector function and a suppressor function--are mediated by a novel CD11c(+)CD8(+) T-cell population.     

The expression and the regulatory role of OX40 and 4-1BB heterodimer in activated human T cells

OX40 and 4-1BB are members of the tumor necrosis factor (TNF) family of costimulatory receptors whose signaling is important for differential immune responses mediated by CD4+ or CD8+ T cells. Although activated T cells may acquire OX40/4-1BB double-positive phenotype and signaling from each receptor is expected to influence cell functions, the relevance between OX40 and 4-1BB has never been investigated before. While we were investigating the expression of OX40 and 4-1BB on activated human T cells, we found that they colocalize. The study of receptor gene-transfected cells showed that both receptors coendocytose and the complex of OX40 and 4-1BB was detected by specific ligands or antibodies (Abs). The heterodimer of OX40 and 4-1BB was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreduced conditions and was associated with the tumor receptor-associated factor (TRAF) family proteins in a unique manner. Furthermore, the stimulation of OX40/4-1BB rendered cells sensitive to apoptosis induced by TNF-alpha that accompanied reduced activation of nuclear factor-kappaB (NF-kappaB). Finally, the OX40/4-1BB stimulation repressed the mitogen response in activated CD25+CD4+ T cells and preactivated CD8+ T cells. Thus, the OX40/4-1BB heterodimer appears to represent a unique regulatory receptor in activated T cells.     

4-1BB regulates NKG2D costimulation in human cord blood CD8+ T cells

Ligation of NKG2D, a potent costimulatory receptor, can be either beneficial or detrimental to CD8(+) cytotoxic T cell (CTL) responses. Factors for these diverse NKG2D effects remain elusive. In this study, we demonstrate that 4-1BB, another costimulatory receptor, is an essential regulator of NKG2D in CD8(+) T cells. Costimulation of NKG2D caused down-modulation of NKG2D, but induced 4-1BB expression on the cell surface, even in the presence of TGF-beta1, which inhibits 4-1BB expression. Resulting NKG2D(-)4-1BB(+) cells were activated but still in an immature state with low cytotoxic activity. However, subsequent 4-1BB costimulation induced cytotoxic activity and restored down-modulated NKG2D. The cytotoxic activity and NKG2D expression induced by 4-1BB on NKG2D(+)4-1BB(+) cells were refractory to TGF-beta1 down-modulation. Such 4-1BB effects were enhanced by IL-12. In contrast, in the presence of IL-4, 4-1BB effects were abolished because IL-4 down-modulated NKG2D and 4-1BB expression in cooperation with TGF-beta1, generating another CD8(+) T-cell type lacking both NKG2D and 4-1BB. These NKG2D(-)4-1BB(-) cells were inert and unable to gain cytotoxic activity. Our results suggest that 4-1BB plays a critical role in protecting NKG2D from TGF-beta1-mediated down-modulation. Co-expression of NKG2D and 4-1BB may represent an important biomarker for defining competency of tumor infiltrating CD8(+) T cells.     

The B7/BB1 antigen is expressed by Reed-Sternberg cells of Hodgkin's disease and contributes to the stimulating capacity of Hodgkin's disease-derived cell lines

The B7/BB1 molecule has recently been found to be expressed on professional antigen-presenting cells and to be the natural ligand for CD28 and CTLA-4 on T cells. On binding of B7/BB1, CD28 transduces a signal that synergizes with triggering of the T-cell antigen receptor, resulting in enhanced cytokine secretion. In view of the data supporting an antigen-presenting function of Reed-Sternberg cells, we evaluated the expression of B7/BB1 in lymph nodes affected by Hodgkin's disease. B7/BB1 was found to be strongly expressed by the Reed- Sternberg cells in all 47 cases of Hodgkin's disease studied. Moreover, Reed-Sternberg cells were frequently surrounded by CD28-expressing T cells. Evidence for a functional role of B7/BB1 on Reed-Sternberg cells was obtained by our findings that T-cell proliferation and interleukin- 2 (IL-2) production in the primary allogenic mixed lymphocyte reaction (MLR), using the B7/BB1-expressing Hodgkin's disease-derived cell lines L428 and KM-H2 as stimulators, could be partially blocked by adding anti-B7 monoclonal antibody. B7/BB1 expression was also evaluated in a group of non-Hodgkin's lymphomas (n = 46). Whereas B7/BB1 was not expressed by the neoplastic cells of most non-Hodgkin's lymphomas, including T-cell-rich B-cell lymphoma (n = 11), it was present on the neoplastic cells of anaplastic large-cell lymphoma (Ki-1 lymphoma) (n = 5) and follicular lymphoma (n = 4). Our data provide further evidence for an accessory cell function of Reed-Sternberg cells. The accessory cell function of Reed-Sternberg cells might lead to pronounced T-cell activation in vivo, which might contribute to the Hodgkin's syndrome. In addition, our study indicates that B7/BB1 may be a useful marker for differentiating Hodgkin's disease from morphologically similar conditions such as T-cell-rich B-cell lymphoma.     

Treatment of acute lymphoblastic leukaemia with the second generation of CD19 CAR‐T containing either CD28 or 4‐1BB

T cells modified with anti‐CD19 chimeric antigen receptor (CAR) containing either CD28 or 4‐1BB (also termed TNFRSF9, CD137) costimulatory signalling have shown great potential in the treatment of acute lymphoblastic leukaemia (ALL). However, the difference between CD28 and 4‐1BB costimulatory signalling in CAR‐T treatment has not been well elucidated in clinical trials. In this study, we treated 10 relapsed or refractory ALL patients with the second generation CD19 CAR‐T. The first 5 patients were treated with CD28‐CAR and the other 5 patients were treated with 4‐1BB CAR‐T. All the 10 patients were response‐evaluable. Three patients achieved complete remission and 1 patient with extramedullary disease achieved partial response after CD28‐CAR‐T treatment. In the 4‐1BB CAR‐T treatment group, 3 patients achieved complete remission. Furthermore, FLT‐3 ligand (FLT3LG) was highly correlated with response time and may serve as a prognosis factor. No severe adverse events were observed in these 10 treated patients. Our study showed that both CD28 CAR‐T and 4‐1BB CAR‐T both worked for response but they differed in response pattern (peak reaction time, reaction lasting time and reaction degree), adverse events, cytokine secretion and immune‐suppressive factor level.     

Blockade of the 4-1BB pathway attenuates graft arterial disease in cardiac allografts

4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, binds the 4-1BB ligand (4-1BBL) and works as a costimulatory molecule and regulates T cell-mediated immune responses. Because T cell-mediated immunity is associated with graft arterial disease (GAD), we investigated the role of the 4-1BB pathway in the progression of GAD. Hearts from C57BL/6 mice were transplanted into Bm12 mice (class II mismatch). 4-1BB expression was induced on CD4(+) and CD8(+) splenocytes in allografts after cardiac transplantation. 4-1BBL was detected in the vessel wall of the rejecting cardiac allograft and in cultured smooth muscle cells (SMCs) stimulated with fetal calf serum. Recipients were injected intraperitoneally with 4-1BBIg every 7 days for 8 weeks. GAD was significantly attenuated by 4-1BBIg treatment (luminal occlusion, 15.4 +/- 3.1% versus control IgG treatment, 75.6 +/- 4.6%, P < 0.001). T-cell infiltration of cardiac allografts and expression of interferon-g , interleukin-6, and interleukin-15 in cardiac allografts were suppressed by 4-1BBIg treatment. Coculture of SMCs with sensitized splenocytes after transplantation induced SMC proliferation, and this was inhibited by addition of 4-1BBIg. The 4-1BB pathway regulates not only T-cell activation but also SMC proliferation. Blockade of the 4-1BB pathway is a promising strategy to prevent progression of GAD.