B7-H3单抗交联作用对Mo-DC体外生物学功能的影响

探讨B7-H3分子对人外周血单核细胞来源树突状细胞(Mo-DC)体外成熟和生物学功能的影响。采用常规方法从健康人外周血单核细胞诱导DC,在诱导过程中,加入B7-H3单抗21D4共培养,经流式细胞术检测Mo-DC上B7-H3分子和其他共刺激分子的表达,ELISA试剂盒检测培养上清中细胞因子IL-10和IFN-γ的分泌量,并采用3H-TdR掺入法测定T细胞的增殖。结果:B7-H3分子在未成熟和成熟Mo-DC上均有高水平表达,抗人B7-H3单抗21D4能上调Mo-DC表面CD80、CD86和CD83的表达,提高Mo-DC的共刺激能力,促进T细胞的体外增殖,并能显著促进T细胞分泌IL-10。由此表明,B7-H3单抗21D4交联作用可以促进Mo-DC体外成熟,上调其共刺激T细胞的能力。     

阻断CD40-CD40L共刺激途径对T细胞表型及细胞因子分泌的作用研究

目的探讨应用抗CD40L单克隆抗体阻断CD40-CD40L共刺激途径后对T细胞表型及其分泌的细胞因子的影响,为体外阻断该共刺激途径诱导T细胞对异体移植抗原的免疫耐受提供实验依据.方法供鼠(C57BL/6H-2b)脾T细胞作为反应细胞,受鼠(BALB/CH-2d)脾细胞作为刺激细胞,设单抗组(加抗CD40L单抗)和对照组(不加单抗),初次混合淋巴细胞培养(MLR)7天,在不同时间点采用3H-TdR掺入法检测细胞增殖率,以ELISA法测定培养上清液中IFN-γ、IL-2、IL-4、IL-10等的水平,第5天采用流式细胞仪检测CD4+T和CD8+T细胞上CD25、CD69、CD40L和CD45RA的表达.再次MLR 5天,第1、3、5天采用3H-TdR掺入法测定细胞的增殖情况和ELISA法测定培养上清液中的上述细胞因子的水平.结果初次和再次MLR结果均显示,单抗组细胞增殖反应率明显低于对照组.初次MLR单抗组中CD4+T和CD8+T细胞比例明显低于对照组(P＜0.05);单抗组中CD4+CD25+T、CD4+CD69+T、CD8+CD25+T、 CD4+CD40L+T和CD8+CD69+T细胞比例明显低于对照组(P＜0.05),而CD8+CD40L+T和CD4+CD45RA+T细胞的比例与对照组相比无明显差异(P＞0.05).初次MLR中单抗组和对照组培养上清中IL-4和IL-10几乎无法测出,而单抗组培养上清中IFN-γ和IL-2的水平均明显低于对照组(P＜0.01);再次MLR后培养上清中单抗组IFN-γ、IL-2和IL-4和IL-10的分泌水平明显低于对照组(P＜0.05),但处于低水平,仍明显低于对照组.结论在体外MLR体系中,应用抗CD40L单抗孵育供鼠脾T细胞,可同时作用于CD4+T和CD8+T细胞,使CD40L+,CD25+和CD69+表达下降,引起T细胞早期的活化和成熟障碍,T细胞增殖能力减低,抑制了Th1类细胞因子IFN-γ和IL-2及Th2类细胞因子IL-4和IL-10的分泌水平,可诱导供者T细胞免疫耐受.     

CD40配基化调节小鼠树突状细胞上B7-H3分子表达的研究

目的 探讨CD40配基化对小鼠骨髓来源树突状细胞上B7-H3分子表达的调节作用及其生物学意义。方法 采用GM-CSF和IL-4联合方案体外诱导小鼠髓系DC，并利用mCD40-CHO和TNF-α分别刺激凋亡肿瘤细胞负载的Dc制备成熟DC；采用间接免疫荧光标记法检测成熟Dc上B7-H3分子的表达；RT-PCR检测B7-H3 mRNA转录水平；混合淋巴细胞反应（MLR）和B7-H3单抗阻断实验分析CD40配基化的DC表面B7-H3分子在T细胞活化中的作用；^3H-TdR掺入试验检测DC对T淋巴细胞的促增殖效应；ELISA测定各组MLR反应和DC培养上清中IFN-γ分泌水平。结果 B7-H3分子在DC不同分化发育阶段均有表达，CD40配基化能显著上调凋亡肿瘤细胞负载的DC中B7-H3表达，TNF-α激发的DC弱表达（P〈0．05）；阻断CD40配基化的DC上B7-H3分子能抑制T细胞增殖和IFN-γ分泌；CD40配基化促进凋亡肿瘤细胞负载的DC分泌IFN-γ量也明显高于TNF-α组（P〈0．05）。结论 体外CD0配基化DC的B7-H3分子上调性表达有助于其刺激T细胞增殖和IFN-γ的产生。     

小鼠树突状细胞表面PD-L1分子对T淋巴细胞协同刺激效应的研究

目的：琛讨小鼠髓系DCs表面PD-L1分子在树突状细胞介导T细胞免疫应答中的作用。方法：采用流式细胞术分别检测未成熟DCs和凋亡肿瘤细胞负载并经CD40配基化的成熟DCs表面免疫分子的表达；混合淋巴细胞反应(MLR)和抗PD-L1单抗阻断试验分析未成熟DCs和成熟DCs表达的PD-L1分子对T淋巴细胞的协同刺激／抑制效应；^3H-TdR掺入试验检测未成熟DCs和成熟DCs对T淋巴细胞的促增殖效应；ELISA测定各组MLR反应上清中IL-10、IFN-γ的分泌水平；MTT比色法检测成熟DCs激发的肿瘤抗原特异性CTL对肿瘤细胞的杀伤效应。结果：未成熟DCs表面高表达PD-L1，负性调节未成熟DCs对自体T淋巴细胞的促增殖作用，抑制T细胞分泌IL-10、IFN-γ；凋亡肿瘤细胞负载并经CD40配基化的成熟DCs中等水平表达PD-L1，具有显著增强对自体T细胞的体外激发、扩增和细胞毒效应的作用，并可增加T细胞的IFN-γ分泌。结论：未成熟DCs高表达PD-L1抑制了对T细胞共刺激效应；CD40配基化成熟的DCs中度表达。PD-L1有助于激发T细胞介导免疫应答。     

CD40配基化的肿瘤特异性树突状细胞介导Th1细胞分化的研究

目的：探讨CD40配基化的肿瘤特异性DCs在介导Th1细胞分化中的作用。方法：采用GM-CSF和IL-4联合方案体外诱导小鼠髓系DCs，并利用mCD40L-CHO和TNF-α分别刺激凋亡肿瘤细胞负载的DCs制备DCs瘤莆；^3H-TdR掺入试验检测DCs对T淋巴细胞的促增殖效应；ELISA测定细胞培养上清中IL-10、IFN-1、IL-12的含量；胞内染色和流式细胞术检测经成熟DCs活化的T细胞中CD4^＋IFN-γ^＋T和CD4^＋IL-4^＋T的比例。结果：体外刺激T细胞增殖能力在CD40配基化DCs组最高（P〈0．05），CD40配基化DCs能更有效地促进活化T细胞分泌IFN-γ和介导CD4^＋IFN-γ^＋T细胞的分化（P〈0．05）。同时，CD40配基化DCs分泌IL-12的量也明显高于TNF-α组（P〈0．05）。结论：CD40配基化的肿瘤特异性DCs体外能有效介导Th1细胞的分化。     

结直肠癌患者外周血Vδ2 T细胞IL-17A的表达研究1

目的:比较结直肠癌患者与正常人外周血单个核细胞来源的Vδ2 T细胞CD27、IL-17A、IFN-γ和TNF-α的表达变化。方法:抽取试验对象外周血,分离单个核细胞,采用流式细胞术分析其中CD27+Vδ2 T细胞与CD27-Vδ2 T细胞的IL-17A、IFN-γ和TNF-α的分泌表达情况。结果:结直肠癌患者外周血Vδ2 T细胞的IL-17A表达明显高于正常人[(5.99±0.80)%比(3.48±0.57)%,P=0.01;]IFN-γ与TNF-α的分泌表达具有明显正相关性(P0.0001)。结直肠癌患者和正常人CD27+Vδ2 T细胞的IL-17A表达均较CD27-Vδ2 T细胞低,而CD27+Vδ2 T细胞的IFN-γ和TNF-α分泌表达则高于CD27-Vδ2 T细胞。结论:结直肠癌患者Vδ2 T细胞的IL-17A表达是升高的,而IFN-γ与TNF-α的分泌表达之间具有显著正相关性;共刺激受体CD27影响VδT细胞的功能表达。     

一个简单敏感的检测IL-2活性的生物学方法

本文介绍了一种简单一次定量的检测IL-2生物活性的方法。它以培养纯CD3~+T细胞为基础,CD3~+T 细胞是从血液的单个核细胞中,用微球(M450)结合CD3McAb 筛选的。培养时,经抗CD3活化的T 细胞将表达IL-2受体而不产生IL-2。加入IL-2后,可引起增殖反应,IL-2浓度在0.01～20U/ml时,可以得到一个线性剂量-反应曲线。细胞对IL-1、IL-4、TNF-α、IFN-α、INF-γ、PHA、ConA 不反应。IL-2的反应性可为TNF-α增强,被IFN-α抑制。试验用的促分裂素不影响增殖反应。抗IL-2和抗IL-2受体的抗体可以抑制IL-2反应,并有一定的剂量依赖关系。本法既简单又特异,不用长期培养实验用的细胞。     

Th2细胞因子和抗IL-12Rβ1mAb抑制由IL-23诱导PBMC IFN-γ的产生

目的：探讨重组人白介素23（IL-23）诱导正常人PBMC IFN-γ的产生，细胞亚群和调节因素。方法：正常人PBMC在不同条件下与IL-23进行培养，采用酶联免疫吸附试验（ELISA）检测细胞培养液中IFN-γ的水平。同时采用流式细胞仪，分析IL-23诱导PBMC IFN-γ表达的细胞亚群。结果：IL-23呈剂量依赖性诱导PBMC IFN-γ产生，并可与IL-2协同诱导IFN-γ的产生。细胞亚群分析的结果表明，IL-23诱导高表达CD56^＊NK细胞产生IFN-γ，对CD4^＊和CD8^＊T细胞无明显作用。Th2细胞因子和抗IL-12受体β1 mAb（IL-12Rβ1）抑制IL-23诱导IFN-γ产生。结论：IL-23可直接作用于CD3^＊CD56^＊NK细胞，诱导产生IFN-γ。Th2细胞因子和抗IL-12Rβ1 mAb抑制IL-23诱导IFN-γ产生，提示可以用于由IL-23引起自身免疫病的治疗。     

IL-4、IL-10和抗IL-12受体β1 mAb抑制IL-23诱导正常人记忆T细胞 IFN-γ产生

目的 探讨重组人白介素23（IL-23）是否能够诱导正常人T细胞IFN-γ的产生，作用的靶细胞亚群和调节因素。方法 正常人PBMC在抗CD3（anti-CD3）单克隆抗体或anti-CD3和抗CD28（anti-CD28）单克隆抗体刺激的条件下与IL-23进行培养，采用酶联免疫吸附试验（ELISA）检测细胞培养液中IFN-γ的水平；同时采用流式细胞仪，在单个细胞水平上分析IL-23诱导PBMC IFN-γ表达的T细胞亚群。结果 在未经任何刺激的情况下，PBMC产生很低或不产生IFN-γ。IL-23呈剂量依赖方式促进由anti-CD3活化的PBMC IFN-γ产生。细胞亚群分析的结果表明，IL-23诱导记忆CD4^＋和CD8^＋T细胞表达IFN-γ，对活化的CD4^＋T细胞作用较为明显。Th2细胞因子（IL-4、IL-10）和抗IL-12受体β1 mAb（IL-12Rβ1）抑制IL-23诱导T细胞IFN-γ产生。结论 IL-23促进活化的记忆CD4^＋和CD8^＋T细胞IFN-γ的产生。Th2细胞因子和抗IL-12Rβ1 mAb抑制由IL-23诱导IFN-γ产生，提示这些细胞因子和抗体对IL-23引起的自身免疫病具有拮抗作用。     

抗CD28单抗和IL-15对CIK增殖和杀肿瘤作用的研究

CIK是肿瘤过继性细胞免疫治疗中的免疫效应细胞。为使CIK在实验室里能被更有效地诱导增殖并赋予其更强的杀肿瘤效应,我们在CIK常规培养环境中加入抗CD28单抗和IL-15,探讨抗CD28单抗和IL-15对CIK增殖和杀肿瘤效应。取人外周血单个核细胞(PBMC),预先以常规方法诱导CIK,然后加入抗CD28单抗和IL-15与CIK共培养。用全自动五分类血液分析仪计数CIK增殖率;用流式细胞术测定CIK中粒酶B、穿孔素和CD107a等分子的表达率;用ELISA方法检测CIK分泌IL-10、IL-12、INF-γ和TNF-α水平;用乳酸脱氢酶释放法测定CIK对人肺癌细胞株(A549)、乳腺腺癌细胞株(MFC-7)和人黑素瘤细胞株(HME1)的杀伤活性。PBMC经常规CIK诱导培养以后再加入抗CD28单抗和IL-15与对照组比较,前者细胞增殖率明显增强(P<0.05);在CIK培养体系中加入抗CD28单抗和IL-15可促进颗粒酶B、穿孔素和CD107a等分子的表达率进一步增强(P<0.05);加入抗CD28单抗和IL-15,培养8d后CIK对A549、MFC-7和HME1细胞杀伤活性分别为82.2%、59.3%和70.6%,与对照组(分别为60.9%、49.6%和48.4%)相比差异有统计学意义(P<0.05);在培养体系中加入抗CD28单抗和IL-15,培养8d后其细胞因子IFN-γ、TNF-α分泌水平显著高于对照组(P<0.05),组间IL-10和IL-12的分泌量未见显著差异(P>0.05)。实验说明在CIK培养体系中加入抗CD28单抗和IL-15可增加CIK增殖率并提高其抗肿瘤效应。     

Treatment of old mice with IL-2 corrects dysregulated IL-2 and IL-4 production

Splenic T cells from old BALB/c mice, activated in vitro withantibody to CD3e, secrete more IL-4 but less IL-2 than splenicT cells from young mice. The age-associated increase in IL-4secretion is associated with a significantly increased concentrationof intracellular IL-4 and its mRNA, although there is no increasein the number of activated T cells with intracellular IL-4.In contrast, the age-associated decrease in IL-2 secretion isassociated with a significant decrease in the number of activatedT cells with intracellular IL-2. In vivo there is a similarage-associated change in the number of activated T cells withdetectable cytokine. The number of activated T cells with intracellularIL-4 is comparable in old and young mice, while the number ofactivated T cells with intracellular IL-2 is significantly decreasedin old compared with young mice. Of great interest is the factthat old mice continuously exposed to IL-2 In vivo followingthe transplantation of J558 cells expressing the transfectedIL-2 gene product have an increased number of splenic T cellswith intracellular IL-2 that equals the level of such cellsobserved in young mice. Most important, the effect of continuousIL-2 administration in vitro was stable as spleen cells fromold, IL-2-treated mice when stimulated in vitro with anti-CD3ehad a young-like pattern of both intracellular IL-2 and IL-4expression as well as IL-2 and IL-4 secretion following in vitroactivation. Thus, it appears that exposure of old mice to exogenousIL-2 can redress the age-associated imbalance in cytokine expressionin vivo and cytokine secretion in vitro.     

Role of alpha chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor

Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta chains in the dynamics of the formation of high-affinity IL-2 binding in T cells.     

A disturbance of the IL-2/IL-2 receptor system parallels the activity of multiple myeloma.

The IL-2/IL-2 receptor (IL-2R) system has been investigated in 64 patients with multiple myeloma (MM), 31 with monoclonal gammopathies of undetermined significance (MGUS) and 20 normal controls. The MM data were related to clinical status by comparing active disease, i.e. at diagnosis and at relapse, and stable disease, i.e. complete remission and off-treatment plateau phase. Serum and urinary values of the soluble IL-2R (sIL-2R) were significantly increased in MM patients compared with normal controls and this increase was related to activity. MM patients with active disease gave significantly higher values than those with stable disease. Compared with normal controls, enriched B cell (but not T cell) preparations from peripheral blood mononuclear cells (PBMC) showed significantly increased proportions of IL-2R+ cells in MM and MGUS. However, the highest proportions were detected in active MM compared with stable MM and MGUS. Also, 16% of all MM patients, as opposed to 9% of MGUS, had well-defined bone marrow IL-2R+ plasma cell populations. The lowest serum IL-2 values were found in active MM. Serial follow up of serum sIL-2R suggested that this peptide can be used as an additional marker of active malignancy. The data indicate that a disturbance of IL-2/IL-2R system is most pronounced in active MM. These findings may provide clues as to the T cell abnormalities in MM.     

Immunotherapy through the IL-2 receptor

The IL-2 receptor system is a suitable target for immunotherapy in conditions in which activated (T) cells play a pivotal role, such as transplantation reactions and auto-immune diseases. Antibodies directed at the IL-2R can be used as such or modified by conjugation to toxins or through chelators to radiolabels. These antibodies have, however, the disadvantage that the recipient may mount an immune response against it, thus limitng its therapeutic use.     

Ligand binding kinetics of IL-2 and IL-15 to heteromers formed by extracellular domains of the three IL-2 receptor subunits

Studies on the binding of IL-2 to its receptor (IL-2R) havegenerally been limited to receptors expressed on cell surfaces.This has hampered detailed kinetic and mechanistic studies atthe molecular level. We have prepared the soluble extracellulardomains of all three receptor subunits (called , ßand ) by recombinant techniques and have used these to performdetailed kinetic studies of their binding properties using thetechnique of surface plasmon resonance. We describe a novelapproach whereby the receptors are assembled on an antibodysurface, being held by an epitope engineered into the C-terminusof each of these domains. Thus the receptors are oriented naturallyleading to homogeneous ligand binding kinetics. We have characterizedthe interactions of the heteromeric complexes of these subunitswith mouse and human IL-2 and their analogs, as well as therecently discovered cytokine, IL-15. We have also studied theextracellular domains of the mouse receptor subunits for thefirst time and have used these as well as mouse-human hybridreceptors to probe the mechanism of assembly of these complexes.We show that no additional proteins are required to reproducethe properties of these complexes in vitro. In addition, kineticstudies with site-specific analogs of IL-2 and the mouse-humanreceptor hybrids clearly indicate that the extracellular domainsof and ) can together readily bind ligand with kinetic propertiesdistinct from those of the constituent subunits. In contrast,a complex containing ligand and the extracellular domains of and ß was comparatively difficult to assemble andrequired prolonged exposure to IL-2. Our method enabled us tocalculate the stoichiometry of these complexes and to determinethat anchoring these subunits is necessary to efficiently drivecomplex formation. The kinetic and equilibrium differences betweenthe mouse and human receptor complexes, and between IL-2 andIL-15 binding to these receptors clarify the roles of the andß subunits in the differential response of cells todifferent cytokines that may be present simultaneously in theenvironment.     

Demonstration of a cross-talk between IL-2 and IL-5 in Phosphorylation of IL-2 and IL-5 receptor {beta} chains

Malaria infection induces the production of serum antibodiesto a variety of malaria antigens but the prevalence of antibodiesto any particular antigen ins typically mucb less than 100%.It has been assumed that non-responsiveness to defined antigensin malaria immune subjects is due to HLA mediated restricutionof the Immune response. In this study we have investigated therole of HLA and non-HLA genes in the antibody response to twomerozoite surface antigens (MSP1 and MSP2) and a sexual stageantigen (Pfs260/230) opf P{lasmodium falcpartum, and concludethat host genotype is not a major determinant of responsiveness.Although antibody levels vary in accordance with seasonal variationsin malaria transmission in semi-immune children, antibiody levelsremain stable in clncall immine adults.     

IL-2 regulation of soluble IL-2 receptor levels following thermal injury.

In the immunosuppressed burn patient serum levels of both IL-2 and a soluble form of IL-2 receptor alpha (sIL-2R alpha) are significantly elevated. Strikingly, the production of these markers by the in vitro activated patients' cells is decreased. This study examines the role of IL-2 in the decreased production of the sIL-2R alpha in vitro in patients with major burns (n = 18, 30 to greater than 70% total body surface area). Peripheral blood mononuclear cell (PBMC) cultures from patients with highly elevated serum sIL-2R alpha, and from healthy controls (n = 12) were activated with concanavalin A (Con A) at initiation. In patients' cultures mitogen-induced increments of sIL-2R alpha levels were significantly lower. There was a significant negative correlation (r = 0.64, P less than 0.001) between a high serum sIL-2R alpha level and a decreased lectin-induced sIL-2R alpha release in vitro. Low levels of sIL-2R alpha in patients' samples were not normalized by increasing the number of T lymphocytes. Also exogenous rIL-1 was without effect, whereas rIL-3 increased sIL-2R alpha release in some cultures. However, sIL-2R alpha levels were significantly increased in patients' cultures by (i) addition of exogenous IL-2; (ii) removal of adherent cells; (iii) addition of cyclooxygenase inhibitor, indomethacin; (iv) bypassing cell surface activation by the combination of the calcium ionophore A23187 and the phorbol ester 12-o-tetradecanoyl acetate. The cyclic AMP-elevating drug, forskolin, abrogated the ability of exogenous IL-2 to increase sIL-2R alpha production. Thus, in the burn patient, the reduced in vitro sIL-2R alpha release appears to relate to abnormalities in IL-2 production and action mediated through its functional surface receptor. Elevated levels of sIL-2R alpha in vivo may, therefore, reflect systemic activation of T lymphocytes in response to biologically active IL-2.