B7-1和B7-2基因转染神经母细胞瘤细胞免疫原性的研究

目的比较免疫共刺激信号B7-1和B7-2以腺病毒为载体转染神经母细胞瘤（NB）SH-SY5Y细胞株,观察其对肿瘤细胞免疫原性的影响。方法以携带绿色荧光蛋白的腺病毒质粒（Ad-GFP）转染SH—SYSY细胞,通过流式细胞仪（FCM）判断转染效率,确定合适的感染倍数（MOI）。分别以空病毒质粒（Ad）及携带B7-1或B7-2的腺病毒质粒转染SH-SYSY细胞,即对照组和B7-1或B7-2组;并以未转染的肿瘤细胞为空白对照（空白组）。采用流式细胞仪（FCM）检测B7-1分子和B7-2蛋白表达,Ficoll密度梯度离心法直接分离和纯化外周血单个核细胞,分别以效靶比5∶1和10∶1进行混合淋巴细胞培养后,测定放射性核素闪烁计数（cpm）/min,同时检测上清液中细胞因子IFN-γ及IL-2的分泌水平。结果腺病毒对SH-SY5Y细胞有较高的转染效率,转染后目的基因B7-1和B7-2得到了有效表达。B7-1组、B7-2组cpm值及上清液IFN-γ、IL-2水平明显高于Ad组、SH组（P〈0.01）;B7-1组与B7-2组比较,cpm值及细胞因子水平增高无明显差别（P〉0.05）。效靶比5∶1时,与10∶1相比,cpm值及IFN-γ、IL-2含量的增加更明显（P〈0.05）。结论 B7-1和B7-2分子可促进T淋巴细胞的增殖及活化T细胞分泌IFN-γ、IL-2,而B7-1与B7-2两组间cpm值及细胞因子水平无明显差别。     

4株鼠抗人B7-1分子单抗的制备及其生物学活性的鉴定

目的：制备鼠抗人B7—1分子(mAb)单抗及研究其生物学活性。方法：以人多发性骨髓瘤细胞转人B7-1基因细胞株XG7-B7为免疫原，采用B淋巴细胞杂交瘤技术制备鼠抗人B7—1分子单抗；以Western blot及间接免疫荧光标记法鉴定其特异性和亲和力；采用^3H—TdR掺入实验和Annexin-Ⅴ染色分析测定该单抗的生物学功能。结果：获得了4株持续分泌抗人B7—1分子的特异性单抗的杂交瘤细胞株，分别命名为1F11、3H8、6H2和7B10，其分泌的单抗类别分属于小鼠IgG1和IgM；4株单抗均具有良好的特异性和亲和力；1F11、3H8和6H2能部分组断B7—1分子基因转导细胞介导的共刺激信号，从而抑制T细胞的增殖效应(抑制率21％—52％)；且能诱导天然表达B7—1分子的人B系淋巴瘤细胞Raji的凋亡。结论：成功研制4株功能性抗人B7—1分子抗体，这些抗体在抗异体组织器官移植排斥反应及在B系淋巴瘤的治疗中具有潜在的应用价值。     

B7-1/B7-H1相对比例变化对Hsp70-肽复合物抗肿瘤免疫效应的影响

目的：探讨Hsp7肽复合物对B7-1／B7-H1相对比例的影响及真核表达可溶性PD-1(sPD-1)对Hsp70-肽复合物抗肿瘤作用的影响。方法：通过RT-PCR和半定量PCR技术检测Hsp70-肽复合物体外刺激和体内免疫对小鼠脾细胞正调控共刺激分子B7-1和抑制性共刺激分子B7-H1及其受体PD-1表达的影响；体内转染表达sPD-1后，观察Hsp70-肽复合物免疫小鼠的肿瘤生长以及脾淋巴细胞毒性的变化。结果：基因表达检测表明．Hsp70-肽复合物体外刺激的小鼠脾细胞B7-1 mRNA和B7-H1 mRNA的水平随时问而变化，B7-1／B7-H1比值随刺激时间而增高；Hsp70-肽复合物体内免疫小鼠后期脾细胞B7-1表达下降，B7-H1及其受体PD-1表达上调，B7-1／B7-H1比值逆转；体内表达sPD-1可显著增强和延长Hsp70-肽复合物的抑瘤效果；体内表达sPD-1可提高Hsp70-肽复合物免疫的荷瘤小鼠脾细胞的杀伤率。结论：Hsp70-肽复合物的刺激引起共刺激分子B7-1和B7-H1表达的变化，B7-1／B7-H1的比例与激活效应相关，sPD-1通过阻抑B7-H1／PD-1途径、上调B7-1／B7-H1比例，可增强免疫应答，提高Hsp70-肽复合物特异性免疫治疗肿瘤的效应。     

鼠抗人B7-1分子功能性单克隆抗体的制备及生物学特性

目的：制备鼠抗人Ｂ７－１分子的功能性单克隆抗体,研究其对高表达相应配基分子细胞的生物学效应。方法：用两株转入Ｂ７－１基因细胞株ＸＧ７－Ｂ７和Ｌ－Ｂ７分别作为免疫原及检测细胞株,利用Ｂ淋巴细胞杂交瘤技术制备单克隆抗体。以快速定性试纸分析法鉴定单抗所属的小鼠ＩｇＧ亚类,采用竞争抑制及间接免疫荧光法分析单抗的特异性和亲和力。以高表达Ｂ７－１分子的恶性淋巴瘤细胞Ｒａｊｉ和Ｄａｕｄｉ为靶细胞,分析单抗对其生     

B7-1抗体阻断B7/CD28信号通路对Pristane肾病的治疗作用

目的:探讨B7-1抗体阻断B7-1/CD28信号通路对小鼠Pristane肾病的治疗作用。方法:将8周龄雌性C57BL/J6小鼠随机分为3组,正常对照组予生理盐水0.5ml/只,造模组与治疗组予Pristane0.5ml/只后,分别予鼠同型IgG抗体(造模组)或鼠抗人B7-1抗体(治疗组)200μg/只,第10天分别检测局部淋巴结和脾脏中吞噬细胞、树突状细胞的活化及脾脏B细胞表面活化分子的变化;每月定期检测血清中抗核抗体(ANA)及尿蛋白,七个月后处死动物HE染色观测肾脏病理变化和冰冻切片直接免疫荧光法观察免疫复合物(IC)的沉积情况。结果:第10天,相对于正常对照组、治疗组和造模组小鼠局部淋巴结和脾脏中吞噬细胞、树突状细胞均有不同程度的活化,治疗组活化程度明显低于造模组(P0.05),脾脏B细胞表面CD21、CD40、CD86等分子上调程度低于造模组(P0.05);4个月后治疗组和造模组小鼠血清均检测到ANA,但治疗组的血清抗体滴度较造模组低(P0.05);治疗组7个月后,尿蛋白含量低于造模组,肾脏HE结果显示治疗组小鼠肾脏病理改变轻于造模组,直接免疫荧光检测可见治疗组免疫复合物荧光强度弱于造模组,正常对照组小鼠肾脏没有明显的病理改变。结论:鼠抗人B7-1抗体对Pristane肾病具有一定的治疗效果。     

共刺激分子4-1BBL和B7-1对人T淋巴细胞的协同激发作用

目的 :探讨 4 1BBL和B7 1这 2种共刺激分子在人外周血T淋巴细胞活化、增殖中的作用和机制。方法 :采用免疫磁珠阴性选择方法分离纯化人外周血T淋巴细胞 ;单向混合淋巴细胞反应 (MLR)分析共刺激分子对T细胞的激发作用 ;3H TdR掺入法测定细胞增殖 ;免疫荧光标记和流式细胞仪表型分析 ;ELISA检测细胞因子。结果 :经免疫磁珠阴性选择分离获外周血单个核细胞中的T细胞纯度 >90 %;人多发性骨髓瘤细胞株 (XG细胞 )转染 4 1BBL和B7 1cDNA后 ,细胞膜表面能稳定高表达这 2个分子 ,4 1BBL和B7 1转基因细胞也均能使同种异体T细胞发生活化、增殖、存活时间延长和介导细胞因子IL 2的分泌 ,且 2种共刺激分子具有一定的协同效应。结论 :4 1BBL和B7 1分子具有赋予XG细胞对T细胞的体外激发、促增殖和分泌IL 2的作用 ,4 1BBL和B7 1分子能产生协同效应。     

B7-H1/PD-1共刺激途径与病毒感染

B7-H1及其受体PD-1是共刺激分子B7-CD28家族的重要成员.B7-H1在淋巴组织及外周非淋巴组织广泛诱导性表达,PD-1则主要表达在活化的T细胞、B细胞及髓系细胞表面.B7-H1/PD-1共刺激途径主要作用是负性调节T、B细胞的免疫反应,参与维持外周组织的免疫耐受.病毒感染可以上调B7-H1及PD-1的表达,抑制病毒特异性T细胞的免疫功能,B7-H1/PD-1途径是病毒逃避免疫监视,引发慢性感染的重要通路,因而阻断B7-H1/PD-1共刺激途径能够恢复病毒特异性T细胞的功能,清除病毒感染,这对于病毒感染的免疫治疗,尤其是病毒慢性感染的免疫治疗具有重要意义.     

B7-H1/PD-1共刺激途径对慢性乙型肝炎患者HBV特异性T淋巴细胞功能的影响

目的研究B7-H1／PD-1共刺激信号途径对慢性乙型肝炎（chronic hepatitis B，CHB）患者HBV特异性T淋巴细胞免疫功能的影响。方法流式细胞术检测CHB患者和健康人外周血髓样树突细胞（myeloid dendritic cells，mDC）上B7-H1及T淋巴细胞上PD-1的表达水平，并分析患者B7-H1和PD-1的表达水平与ALT水平的相关性；体外培养CHB患者单核细胞来源的树突细胞（monocyte-derived cells，MoDC），HBcAg负载后用“细胞因子鸡尾酒”（TNF-α、IL-6、IL-1β和PGE2）促成熟。MoDC和自体T淋巴细胞混合培养，并对B7-H1／PD-1途径进行阻断处理，^3H-TdR掺入法检测HBV特异性T淋巴细胞增殖的能力；ELISA法检测混合淋巴细胞培养上清中细胞因子的浓度；ELISPOT法检测分泌IFN-γ的T淋巴细胞频数。结果B7-H1及受体PD-1在CHB患者外周血mDC和T淋巴细胞上的表达水平明显升高，且B7-H1和PD-1的表达水平与患者的ALT水平呈明显的正相关；阻断B7-H1／PD-1共刺激途径，不但能够促进HBV特异性T淋巴细胞的增殖和Ⅰ型细胞因子分泌，同时可以提高分泌IFN-γ的抗原特异性T淋巴细胞的频数。结论CHB患者B7-H1和PD-1表达水平的升高，降低了HBV特异性T淋巴细胞免疫功能。     

T细胞活化的新共刺激途径ICOS—B7h

近年来发现的共刺激途径ICOS-B7h在免疫应答中的作用正越来越受到重视。本文拟就ICOS-B7h的结构、表达等生物学特性及功能意义作一综述。     

小鼠抗人B7-1单克隆抗体抑制肿瘤细胞增殖的蛋白质组学分析

目的：研究鼠抗人单克隆抗体B7-1对恶性肿瘤细胞株Daudi（天然表达B7-1分子）体外生长、蛋白表达谱及信号通路的影响。方法：(1)采用诱生腹水法制备小鼠抗人B7-1单克隆抗体（4E5 mAb）,应用Protein A免疫层析法进行亲和纯化,流式细胞术对其纯化后产物的活性进行识别鉴定;(2)利用MTT法检测不同浓度的4E5 mAb(5、10、20、40μg/ml)对Daudi细胞体外增殖的影响;(3)应用Label-free蛋白定量技术分析比较4E5 mAb(20μg/ml)处理Daudi细胞48 h后与其对照组（同型对照IgG）蛋白质的差异表达情况,采用平行反应监测（PRM）对差异表达蛋白进行定量验证;(4)根据蛋白质的GO注释和生物信息学工具KEGG数据库将鉴定出来的差异蛋白质进行生物信息学富集分析。结果：(1)获得单克隆抗体的量为3.83 mg/ml,该抗体与Daudi肿瘤细胞的阳性结合率为93.7%;(2)当抗体终浓度为20μg/ml时,4E5 mAb显著抑制Daudi细胞的增殖;(3)与IgG对照组相比,4E5实验组鉴别出169个蛋白质的定量水平差异有统计学意义,其中131...     

Human B7-1 is more efficient than B7-2 in providing co-stimulation for alloantigen-specific T cells

Besides a signal via the T cell receptor/CD3 complex, an additional co-stimulatory signal is required for optimal T cell activation. This signal can be delivered by interaction of either B7-1 or B7-2 expressed by antigen-presenting cells with CD28 on the T cells. Comparison of the function of B7-1 and B7-2 in different experimental animal systems generated conflicting data on the roles for the co-stimulatory molecules. We therefore investigated whether there are differences between B7-1 and B7-2-mediated co-stimulation in an alloantigen-specific primary T cell response induced by B7-transfected human cell lines of epithelial origin. Both transfected keratinocyte cell lines efficiently induce T cell proliferation and the ratios of stimulator versus responder cells are similar. The kinetics of proliferation and interleukin (IL)-2, IL-4 and interferon-γ production are also comparable between both transfectant lines. However, despite equal B7 expression levels, it is consistently found that the magnitude of the B7-1-induced T cell proliferation was higher than that of B7-2. Comparison of precursor frequencies of helper T lymphocytes responsive with either B7-1 or B7-2 revealed that the frequency of B7-1-responsive T cells was higher than that of B7-2, and that the frequency of cells activated by a combination of B7-1 and B7-2 did not differ significantly from that of B7-1 alone. We therefore conclude that the B7-2-responsive T cells are part of the B7-1-responsive population, and that B7-1 on keratinocytes is more efficient in providing co-stimulation for alloantigen-specific T cells.     

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAb) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAb against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogeneic, as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence on B7-1 expression.     

Ultrastructural Analysis of Apoptosis Induced by the Monoclonal Antibody Anti-APO-1 on a Lymphoblastoid B Cell Line

Apoptosis (programmed cell death) is the most common form of death in eukaryotic cells. We recently described a monoclonal antibody, anti-APO-1, which induces apoptosis of cells from the human B-lymphoblastoid line SKW 6.4 and of cells from a variety of other human lymphoid cell lines. This model of apoptosis was now studied ultrastructurally.

SKW 6.4 cells undergoing apoptosis showed the following morphological changes: condensation of the cytoplasm and karyoplasm, formation of large electron-opaque aggregates of the chromatin lining the nuclear membrane, “blebbing” of the cell membrane at an early stage of apoptosis, and dilatation of the mitochondria. Two hours after adding anti-APO-1, the nuclear membrane was ruptured. Occasionally, large vesicular enlargement of endoplasmic reticulum or Golgi appeared in the cytoplasm. Finally, total breakdown of all cell membranes and cellular disintegration was observed.     

Ultrastructural Analysis of Apoptosis Induced by the Monoclonal Antibody Anti-APO-1 on a Lymphoblastoid B Cell Line

Apoptosis (programmed cell death) is the most common form of death in eukaryotic cells. We recently described a monoclonal antibody, anti-APO-1, which induces apoptosis of cells from the human B-lymphoblastoid line SKW 6.4 and of cells from a variety of other human lymphoid cell lines. This model of apoptosis was now studied ultrastructurally.

SKW 6.4 cells undergoing apoptosis showed the following morphological changes: condensation of the cytoplasm and karyoplasm, formation of large electron-opaque aggregates of the chromatin lining the nuclear membrane, “blebbing” of the cell membrane at an early stage of apoptosis, and dilatation of the mitochondria. Two hours after adding anti-APO-1, the nuclear membrane was ruptured. Occasionally, large vesicular enlargement of endoplasmic reticulum or Golgi appeared in the cytoplasm. Finally, total breakdown of all cell membranes and cellular disintegration was observed.     

B7-1分子诱导体外抗肝癌免疫反应

目的：了解Ｂ７分子在体外抗肝癌免疫中的作用。方法：健康人外周血单个核细胞（ＰＢＭＣ）与ＨｅｐＧ２／ｈＢ７－１，ＨｅｐＧ２／ｎｅｏ及亲代ＨｅｐＧ２瘤苗混合培养（ＭＬＴＣ），检测淋巴细胞活化增殖能力，淋巴细胞ＨＬＡ－Ｉ类抗原的表达，培养上清ＩＦＮ－γ水平，ＴＮＦ活性及ＬＡＫ，ＣＴＬ细胞活性。结果：ＨｅｐＧ２／ｈＢ７－１瘤苗促淋巴细胞增殖最高达１４．６倍，明显高于ＨｅｐＧ２／ｎｅｏ和ＨｅｐＧ２瘤苗的作用     

Expression and function of B7-1 and B7-2 in hapten-induced contact sensitivity

We analyzed the expression and function of the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) during contact sensitivity reactions induced by the hapten 2,4-dinitrofluorobenzene (DNFB). In the normal skin, only a few epidermal Langerhans cells or dermal dendritic cells express B7-2. In contrast, following challenge with DNFB, expression of B7-2 is up-regulated in both epidermis and dermis. Importantly, B7-1 is induced later and at lower levels compared to B7-2. Intravenous injections of anti-B7-2 mAb, but not anti-B7-1 mAb partially inhibit the hapten-induced contact sensitivity reaction. Experiments in which mice are injected differentially with anti-B7-2 mAb, either before the afferent or before the efferent phase of the contact sensitivity response, suggest that B7-2 is important for successful antigen priming.     

Both CD28 ligands CD80 (B7-1) and CD86 (B7-2) activate phosphatidylinositol 3-kinase, and wortmannin reveals heterogeneity in the regulation of T cell IL-2 secretion

In this report, the co-stimulatory signals provided by CD80(B7-1) or CD86 (B7-2) were compared to CD28 ligation by mAb.We demonstrate that while both anti-CD3 and anti-CD28 antibodiesinduced activation of phospholnositide (PI) 3-kinase, the kineticsof activation differed. Anti-CD28 produced a sustained activationof PI 3-kinase while anti-CD3 induced activation was transient.Both B7-1 and B7-2 could induce prolonged activation of PI 3-kinase.The co-stimulatory effects of B7-1 and B7-2 were dependent onCD28 cross-linking, based on complete inhibition of PI 3-kinaseactivation by CD28 antibody Fab fragments. While Jurkat T cellsco-stimulated with anti-CD3 and B7-1 or B7-2 secreted high levelsof IL-2, there were distinct effects of anti-CD28 mAb and B7-1or B7-2 on IL-2 secretion in conjunction with protein kinaseC activation. To assess functional effects of CD28 ligation,pharmacologic inhibitors of PI 3-kinase were evaluated. In Jurkatcells, efficient inhibition of PI 3-kinase activation afterB7-2 stimulation was achieved using wortmannin; however, weobserved a surprising increase in IL-2 secretion after B7 oranti-CD28 stimulation. The effect of wortmannin was concentrationdependent. Moreover, the effect was specific for receptor-mediatedactivation as wortmannin did not enhance phorbol ester pluslonomycin-induced IL-2 secretion. Another inhibitor of PI 3-kinase,LY294002, also resulted in augmentation of anti-CD28-inducedIL-2 secretion by Jurkat cells. The effects of wortmannin onIL-2 secretion were also examined in primary T cells. In markedcontrast, wortmannin resulted in a potent inhibition of anti-CD3plus B7-1 or anti-CD28-induced IL-2 secretion while phorbolester plus lonomycin-induced IL-2 secretion was wortmannin resistant.Together these observations demonstrate that signal transductionby both B7-1 and B7-2 involves PI 3-kinase, and that PI 3-kinaseor other wortmannin-sensitive targets are important for IL-2secretion. Finally, treatment of Jurkat cells with PI 3-kinaseinhibitors alone was sufficient to induce low levels of IL-2secretion. This is consistent with the notion that a wortmannin-sensitivetarget such as PI 3-kinase may down-regulate IL-2 secretionin Jurkat cells.     

抗B7-1和抗CD40L单抗延长小鼠皮肤移植物存活实验研究

将C57BL/6小鼠腹部全层皮肤移植于BALB/c小鼠中段背部,实验分组(每组7只小鼠):1.B7-1功能性单抗(4E5)治疗组;2.抗CD40L单抗(4F1)治疗组;3.4E5+4F1治疗组,各单抗以20㎎/㎏的剂量注入腹腔内;4.空白对照组,只注射同等量的生理盐水。注射时间为移植后0﹑1﹑3﹑5d,观察移植皮肤排斥情况。于术后第6天分别杀死各组受体和供体鼠,取受体脾细胞与供体作混合淋巴细胞反应(MLR)。收集培养6d的初次反应细胞,检测再次MLR。结果发现,与对照组相比,各单抗治疗组皮肤移植物存活时间延长(P<0.05﹚;与各单独应用4F1和4E5相比,联合使用4F1和4E5产生一定的协同作用,但未能进一步延长移植物的寿命(P>0.05)。初次单向MLR:4F1﹑4E5和4F1+4E5治疗组受体T淋巴细胞在MLR中表现对供体淋巴细胞特异性低反应性,能有效抑制T细胞对同种异体抗原的初次应答。再次单向MLR:4F1﹑4E5﹑4F1+4E5对供体淋巴细胞在再次反应中仍保持着对同种抗原的反应性,与对照组无显著差异,未能诱导特异性免疫耐受。综上结果证实,anti-CDB7-1mAb(4E5)和anti-CD40LmAb(4F1)作为新型免疫抑制剂,在一定程度上抑制细胞免疫应答,干预排斥反应。