DNA－转染体细胞系产生单克隆抗体

本文研究淋巴细胞转染体的产生和转染体单克隆抗体的抗原结合特性．以小鼠宫颈癌细胞系Ｕ１４为抗原免疫小鼠。以制备的Ｕ１４和小鼠骨髓瘤细胞系Ｓｐ２／０ＤＮＡ分别转染经Ｕ１４免疫的小鼠原代脾细胞．将所形成的转染体细胞进行克隆，建成２株淋巴细胞转染体细胞系ＵＤ和ＳＤ．它们能连续生长于含血清和无血清培养液中，但不能在同基因小鼠体内生长．ＵＤ和ＳＤ细胞系所产生的单克隆抗体不仅能与小鼠宫颈癌细胞表面抗原及其可溶性抗原发生特异反应，且能识别人宫颈癌组织和宫颈癌病人血清中的肿瘤抗原决定簇．本实验结果进一步证明了我们提出的肿瘤＂进化抗原＂理论，并提示瘤细胞ＤＮＡ转染造成的淋巴细胞永生化，可能是产生人源性单克隆抗体具有前景的途径．     

DNA－转染体细胞系产生单克隆抗体

本文研究淋巴细胞转染体的产生和转染体单克隆抗体的抗原结合特性。以小鼠宫颈癌细胞系Ｕ１４为抗原免疫小鼠。将制备的Ｕ１４和小鼠骨髓瘤细胞系ＳＰ２／０ＤＮＡ分别转染经免疫的小鼠原代脾细胞。将所形成的转染体细胞进行克隆，建成２株淋巴细胞转染体细胞系ＵＤ和ＳＤ，它们能连续生长于含血清和无血清培养液中，但不能在同基因小鼠体内生长。ＵＤ和ＳＤ细胞系所产生的单克隆抗体不仅能与小鼠宫颈癌细胞表面抗原及其可溶性抗原发生特异反应，且能识别人宫颈癌组织和宫颈癌病人血清中的肿瘤抗原决定簇。本实验结果进一步证明了我们提出的肿瘤“进化抗原”理论，并提示，瘤细胞ＤＮＡ转染造成淋巴细胞永生化，可能是产生人源性单克隆抗体具有前景的途径。     

光敏生物素标记胃癌单克隆抗体MG_7的制备

本文介绍一种新的蛋白质标记技术，即用光源照射光敏生物素对本室制备的胃癌单克隆抗体ＭＧ_７进行标记。以亲和素－碱性磷酸酶检测，标记的单抗最低值达０．７５ｐｇ。免疫组化研究显示标记抗体对胃癌组织有很强的亲和力和特异性。上述结果表明这种标记技术具有操作简单，对蛋白质的生物活性影响小等特点。     

CD86对CD8+T细胞分化的影响

目的：探讨CD86(B7-2)对CD8^ T细胞分化的影响。方法：用限制性内切酶Xho Ⅰ酶切质粒pCDM8得到CD86基因，并将其插入pCDNA3，用BamH Ⅰ酶切鉴定。用脂质体法介导pCDNA3-CD86真核表达载体转染人肝癌细胞株HMCT／21，600μg/ml G418筛选，稳定且高表达CD86的抗性克隆用流式细胞仪进行鉴定。从健康志愿者血中分离外周血单个核细胞(PB-MC)，使PBMC与靶细胞之比为20：1，共同培养48h后，用流式细胞仪检测CD3^ T细胞内IL-4和IFN-γ的表达率。结果：成功构建pCDNA3-CD86真核表达载体；CD86在HMC7721-CD86细胞中的表达率为30．8％，而在HMC7721细胞中的表达率为0．98％；健康志愿者CD3^ T细胞内IL-4和IFN-γ的表达率分别为1．92％和24．4％；PBMC与靶细胞共同培养48h后，无论是否用IFN-α刺激，IL-4，IFN-γ的阳性比值在HMC7721-CD86转染组均大于1，而在HMC7721未转染组均小于1。结论：在细胞培养中，CD86可诱导CD8^ T细胞活化，并向Tc2表型转化。     

联合应用CD3/CD28单克隆抗体对小鼠T淋巴瘤细胞EL4活化的影响

目的:研究联合应用CD3/CD28单克隆抗体对小鼠T淋巴瘤细胞EL4活化的影响.方法:ELISA方法检测不同浓度CD3/CD28抗体在不同时间点体外刺激EL4细胞后培养上清中IL-2的分泌;CCK-8方法检测EL4细胞增殖能力;流式细胞术检测EL4细胞周期;RT-PCR检测IL-2、NF-κB和NF-AT转录因子的转录情况.结果:在48小时抗CD3抗体(25μg/ml)与抗CD28抗体(2μg/ml)共刺激EL4细胞明显增加IL-2的分泌和细胞增殖能力,不影响细胞周期.结论:EL4细胞可以作为研究小鼠T淋巴瘤细胞活化的细胞模型.     

恶性淋巴瘤单克隆抗体的制备和初步鉴定

采用人恶性淋巴瘤细胞株Raji细胞作为免疫原免疫BALB/C小鼠, 按常规方法融合并经间接ELISA法筛选, 成功地制备了1株抗Raji细胞单克隆抗体(MAb), 命名为SZ138.用琼脂糖双扩散鉴定其为IgG1类, ELISA法测定其腹水效价为1:3.2×104.MAb流式细胞仪检测表明, 该抗体与Raji和Daudi呈阳性反应;与外周红细胞、白细胞(包括淋巴细胞和中性粒细胞)、未活化血小板、内皮细胞株ECV呈阴性反应.免疫组化SP法显示, MAb SZ138识别的抗原在胃肠道的腺体和肝脏有所表达, 而在心、脑、扁桃体、脾、脂肪组织、动脉血管壁、神经节、横纹肌、平滑肌等组织不表达或表达很低.Western blotting分析显示, MAb SZ138可特异识别还原条件下相对分子量为170kD的蛋白多肽.结果提示: MA SZ138对恶性淋巴瘤的诊断和治疗可能具有一定的临床意义.     

识别CD1a不同表位单克隆抗体HI149的制备、鉴定和初步应用

单克隆抗体(单抗)HI149经免疫荧光和免疫组化检测证明是CD1a抗体。竞争抑制试验证实,HI149单抗识别的表位与一个标准CD1a单抗OKT6和两个OKT6样单抗HIT6-1和HIT6-2完全相同,但不同于另一个CD1a单抗T6。在白血病免疫分型诊断中,HI149是有用的试剂。并对CD1分子研究进展进行了讨论。     

抗CD3单抗对T细胞的作用

抗ＣＤ３单抗可作为一种免疫调节剂对Ｔ细胞的多种功能产生调节作用，它与ＩＬ－２具协同作用，诱导Ｔ细胞扩增，增强胞毒活性，体内转输ＣＤ３－ＡＫ细胞能降低肿瘤的转移和生长，高剂量的抗ＣＤ３单抗具有免疫抑制作用。该抗体对Ｔ细胞的作用机制目前尚不十分清晰，可能与磷酸肌醇，细胞内Ｋ＾＋、Ｃａ＾＋浓度的变化以及ＩＬ－１，ＩＬ－６和ＩＬ－２的级联相关作用有关。     

大鼠CD4+CD25-T细胞的Foxp3基因转染及其表达***◆

背景：调节性T细胞在维持机体免疫应答稳态和免疫耐受方面具有非常重要的作用,但外周血中CD4+CD25+调节性T细胞含量极低,且增殖能力较差。 目的：以携带Foxp3基因的慢病毒EGFP+载体转染大鼠CD4+CD25- T细胞,观察其在大鼠CD4+CD25- T细胞中的表达。 方法：以免疫磁珠两步法分选大鼠CD4+CD25- T细胞,用携带大鼠Foxp3基因的慢病毒载体体外转染分选的细胞,以转染Foxp3基因的CD4+CD25- T细胞为实验组,EGFP空白质粒组及CD4+CD25- T细胞为阴性对照组,CD4+CD25+ T细胞为阳性对照组。荧光显微镜和RT-PCR分别从蛋白和mRNA水平检测Foxp3基因的表达。 结果与结论：成功完成了免疫磁珠的分选,获得了纯度较高的CD4+CD25-T细胞和CD4+CD25+ T细胞,细胞存活率为(94±2)%,慢病毒转染的CD4+CD25- T细胞高表达Foxp3基因。表明以携带Foxp3基因的慢病毒载体系统可有效介导Foxp3基因在大鼠CD4+CD25- T细胞中高表达。     

Kail/CD82基因转染对结肠癌细胞株LoVo生物学特性的影响

目的　探讨Kail/CD82对LoVo细胞株生长、黏附、分离与侵袭能力的影响。方法　转染KailcDNA ,建立稳定Kail表达克隆株。采用体外实验的方法 ,绘制转染细胞与对照细胞的生长曲线 ,并检测其黏附、聚集、分离与侵袭能力 ,对比有无差别。结果　与对照细胞相比 ,转染对LoVo细胞的生长无明显影响 ;震荡 10、2 0、3 0、40min时 ,转染细胞与空白对照细胞的黏附细胞数 (× 10 5 )分别为0 0 8、0 63、0 83、0 91和 0 0 4、0 48、0 71、0 82 ,其中震荡 2 0、3 0与 40min时二者差异有显著性 (P <0 0 5) ;震荡 5、10、15min时 ,转染细胞与空白对照细胞的脱落率分别为 13 %、2 0 %、53 %和 11%、2 8%、60 % ,其中震荡 10与 15min时二者的差异有统计学意义 (P <0 0 5) ;5h的温和震荡后 ,转染细胞的聚集体形成率较空白对照细胞为高 (64 8%和 58 6% ,P <0 0 5) ;1d共培养后 ,转染细胞穿过内皮细胞的数目较空白对照细胞的少 (6 3 3 /视野和 17 67/视野 ,P <0 0 5) ;总之 ,转染可以增强细胞的黏附与聚集能力 ,降低其分离与侵袭能力。结论　Kail/CD82对结肠癌细胞的转移力具有抑制作用     

抗人CD33单克隆抗体(HIM3-4、WM53、M195)抗原识别表位的初步研究

目的：确定抗人CD33单克隆抗体（HIM3-4、WM53、M195）抗原识别表位所在区域，进而阐明抗体识别表位不同于其介导的生物学作用之间的关系。方法：将四个CD33胞外区不同长度的基因片段定向克隆入真核表达载体pDisplay，构建成真核表达载体pDisplay／EP1、2,3、4，分别将空载体和构建的质粒通过磷酸钙沉淀法转染入293T细胞，瞬时转染48小时后利用流式细胞仪通过抗血凝素A（HA）抗体标记以检测各质粒在细胞表面的表达，同时检测HIM3-4、WM53、M195和不同区段的结合活性。结果：抗人CD33单克隆抗体HIM3-4能识别pDisplay／EP1、2、3表达的蛋白，WM53能识别pOisplay／EP1表达的蛋白，M195则均能识别四个片段所表达的蛋白。结论：HIM3-4、WM53、M195识别的CD33抗原表位分别位于115—170、17—60、170—262氨基酸。     

Specific hyporesponsiveness of alloreactive peripheral T cells induced by CD4 antibodies

We investigated whether exposure of naive and in vitro pre-activated T cells to CD4 monoclonal antibodies (mAb) could induce specific hyporesponsiveness to a subsequent challenge in the absence of CD4 mAb. Unfractionated peripheral blood mononuclear cells were cultured with mitomycin-treated B cell lines as stimulator cells, in the presence or absence of CD4 mAb, then challenged with the same or unrelated stimulator cells. The kinetics of [³H] thymidine incorporation, blast transformation and CD25 expression were determined. Cells activated in primary or secondary culture in the presence of CD4 mAb demonstrated a markedly decreased response to subsequent challenge in the absence of antibody. This effect was reproduced with three different CD4 mAb of the IgG₁ and IgG_2a subclasses, which recognize two distinct epitopes of the CD4 molecule. Addition of recombinant interleukin-2 (rIL-2) during exposure to CD4 mAb failed to prevent the induction of specific hyporesponsiveness. Similarly, exogenous rIL-2, added together with stimulating cells, failed to restore the specific proliferative response, indicating that the mechanisms were different from those of classical anergy. The hyporesponsiveness was clonally restricted since CD4 mAb-pretreated cells developed a normal primary response to third-party stimulator cells. No increase in the percentage of apoptotic cells was observed in hyporesponsive cell populations, but selective clonal deletion cannot be excluded. The data demonstrate a delayed effect of CD4 ligation on T cell responses to a subsequent challenge.     

A Novel Anti-Human Syndecan-1（CD138） Monoclonal Antibody 4B3： Characterization and Application

Syndecan-1 （CD138）, a member of integral membrane heparin sulfate proteoglycans, is an essential matrix receptor for maintaining the normal morphological phenotypes. In this study, we generated a specific mouse anti-human syndecan-1 monoclonal antibody （mAb） 4B3 and identified it by competition assay with the available syndecan-1 mAb （BB4）. Stained by 4B3, the expression of syndecan-1 was detected on tumor cell lines, such as 8226, U266, XG-1, XG-2, Daudi and Jurkat. The expression was also found on neuron stem cells. It was established that 4B3 mAb could inhibit XG-1 and XG-2 proliferation. The data not only determined that 4B3 mAb was a functional anti-human syndecan-1 mAb, but also indicated that syndecan-1 might be a valuable surface antigen and play an important role in regulation of tumor pathology and differentiation of neural stem cells. This novel antibody 4B3 may be value of study of tumor proliferation/survival mechanism and contributes to diagnosis and treatment of diverse diseases. Cellular ＆ Molecular Immunology.     

CD_3McAb对PHA和TPA诱导人胚胸腺细胞增殖反应调节作用的研究

本文观察了CD3 McAb 对PHA 和TPA 诱导的20～36周龄人胚胸腺细胞增殖反应的调节作用。结果表明:CD3McAb 不能单独诱导人胚胸腺细胞增殖反应,但却能显著地促进TPA 诱导的人胚胸腺细胞增殖和抑制PHA 诱导的人胚胸腺细胞增殖,并呈剂量依赖关系;加入外源性rHulL—2能促进人胚胸腺细胞对CD3McAb 诱导的应答反应,而rHuIL—1则无此作用。此结果对于了解人胚胸腺表达CD_3抗原细胞的功能水平及CD_3分子的生物学作用有一定的意义.     

Mechanisms in CD4 antibody-mediated transplantation tolerance: kinetics of induction,antigen dependency and role of regulatory T cells

CBA/Ca mice may be made tolerant to minor histoincompatible B10.BR skin grafts by treatment with a short course of non-depleting anti-mouse CD4 and CD8 monoclonal antibodies (mAb), during the transplantation period. We wished to determine when, in relation to antibody therapy, the T cells became tolerant. This was investigated by a series of adoptive transfer experiments in which mAb-treated cells were removed from therapeutic antibody at defined times after skin grafting, and exposed to fresh antigen in the absence of further mAb treatment. We show here that T cells do not become fully tolerant until 5 weeks after skin grafting. If antibody therapy is continued for the full 5 weeks, T cell tolerance can still be established, suggesting that antibody therapy does not prevent lymphocytes from registering the presence of antigen. Once the tolerant state is established, it is difficult to break that tolerance by lymphocyte infusions from normal donors. This “resistance” is mediated by T cells of the tolerant host. We show that the maintenance of both tolerance and “resistance” requires a continuous supply of antigen. When tolerant cells were “parked” in T celldepleted mice, tolerance and “resistance” were eventually lost by 6 months. In contrast, “parked” cells exposed to fresh antigen at any time up to 4 months remained tolerant and “resistant” indefinitely. Finally, we wished to establish whether “resistance” was peculiar to this form of peripheral tolerance, or whether it might also be present in tolerance considered to be classically central. We observed resistance to be greater in the mAb-treated peripherally tolerant group, but noted that some of the centrally tolerant animals also exhibited a level of resistance above that of T cell-ablated controls. This suggests that a tolerance mechanism whose role is only minor in central tolerance may have a major role in antibody-mediated peripheral tolerance.     

无血清培养体系中CD3AK细胞诱导HL—60细胞凋亡的研究

目的：采用无血清培养体系,研究CD3单克隆抗体激活的杀伤（CD3AK）细胞诱导HL－60细胞凋亡情况。方法：用血清代用品代替人血清培养CD3AK细胞,将诱导生成的CD3AK细胞与HL－60细胞共同孵育后,通过观察细胞形态、琼脂糖凝胶电泳检测DNA条带及充式细胞仪（FCM）检测分析HL－60细胞凋亡情况。结果：与CD3AK细胞孵育7h后的HL－60细胞出现凋亡细胞形态特征,DNA凝胶电泳典型梯状电泳带,FCM检测凋亡细胞比例为24.59%。结论：无血清培养条件下CD3AK细胞可诱导HL－60细胞凋亡。     

抗人CD20单克隆抗体诱导Daudi细胞凋亡的功能研究

目的　观察由基因重组抗原制备的抗人CD2 0单克隆抗体 (CD2 0 McAb) 1- 2 8对B淋巴瘤细胞的促凋亡作用 ,并探讨其作用机理。方法　利用流式细胞术测定所获 1- 2 8单抗对标准CD2 0 FITC单抗的竞争抑制作用及对胞内游离钙的影响 ;通过电镜观察其诱导Daudi细胞凋亡后形态上的改变 ,免疫印迹实验显示Daudi细胞凋亡后功能上的变化。结果　 1- 2 8能明显竞争标准CD2 0 FITC单抗与Daudi细胞表面CD2 0分子的结合。电镜结果证实了 1- 2 8能够促进Daudi细胞发生凋亡的结论 ;实验结果显示Daudi细胞与 1- 2 8作用后胞内游离钙浓度明显升高 (P <0 .0 5 ) ,细胞内Bcl 2蛋白和caspase酶原的表达量下降 ,而Bax蛋白和caspase酶原降解的活性产物表达增加。结论　 1- 2 8能竞争结合Daudi细胞表面的CD2 0分子并诱导其发生凋亡     

Prevention of adjuvant arthritis by the W3/25 anti-CD4 monoclonal antibody is associated with a decrease of blood CD4(+)CD45RC(high) T cells

Imbalance between Th1 and Th2 functions is considered to play a key role in the induction and development of several autoimmune diseases, and the correction of that imbalance has led to effective therapies of some experimental pathologies. To examine whether CD4(+)CD45RC(high) (Th1-like) and CD4(+)CD45RC(low) (Th2-like) lymphocytes play a role in the pathogenesis of adjuvant arthritis (AA) and in its prevention by anti-CD4 antibody, CD45RC expression on CD4(+) T cells was determined in arthritic rats and in animals treated with an anti-CD4 MoAb (W3/25) during the latency period of AA. The phenotype of regional lymph node lymphocytes from arthritic rats in the active phase of the disease was determined by flow cytometry. Peripheral blood lymphocytes from rats treated with W3/25 MoAb were also analysed for 2 weeks after immunotherapy finished. IgG2a and IgG1 isotypes of sera antibodies against the AA-inducing mycobacteria, considered to be associated with Th1 and Th2 responses, respectively, were also determined by ELISA techniques. Fourteen days after arthritis induction, regional lymph nodes presented an increase in CD4+CD45RC(high) T cell proportion. Preventive immunotherapy with W3/25 MoAb inhibited the external signs of arthritis and produced a specific decrease in blood CD4(+)CD45RC(high) T cells and a diminution of antibodies against mycobacteria, more marked for IgG2a than for IgG1 isotype. These results indicate a possible role of CD4(+)CD45RC(high) T lymphocytes in the pathogenesis of AA, and suggest that the success of anti-CD4 treatment is due to a specific effect on CD4(+)CD45RC(high) T subset that could be associated with a decrease in Th1 activity.     

GPA33 is expressed on multiple human blood cell types and distinguishes CD4+ central memory T cells with and without effector function

The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4⁺ T cell compartment. Naïve and CXCR5⁺ regulatory T cells were GPA33^high, and naïve conventional CD4⁺ T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4⁺ central memory T cell (Tcm) population. GPA33⁺ CD4⁺ Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33^– Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4⁺ T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4⁺ T cell lineage.     

New polymorphic HLA-DR epitopes recognized by three monoclonal antibodies produced against DR103 transfected L cells

Production of monoclonal antibodies directed against polymorphic epitopes of HLA class II molecules using whole human cells as immunogen has often proved ineffective, because most of the antibodies produced are directed against non-MHC human cell surface molecules. One approach to overcome this problem is the use of transfected mouse L cells expressing a single HLA class II allele as immunogen. By immunizing C3H mice with DR103-transfected L cells, we obtained 3 mAb, OHA TM901, OHA TM902, and OHA TM903, that recognize different polymorphic epitopes of the HLA-DR molecule. The molecular specificities of the 3 mAb were determined on a large panel of B-lymphoblastoid cell lines (B-LCL), peripheral blood cells and HLA class II transfectants from the XIth International Histocompatibility Workshop. Interestingly, the 3 polymorphic mAb detect new HLA-DR epitopes shared by several specificities: OHA TM901 reacts with DR1 (DR101, DR103), DR9 (DR901) and DR10 (DR1001) molecules; OHA TM902 recognizes the same molecules but also DR8 (DR801, 802, 803); OHA TM903 reacts with all DR types except DR3 (DR301, 302), DR7 (DR701, 702) and DR52. Surprisingly, OHA TM901 reacts with DR9 transfectants and B-LCL but not with DR9 peripheral blood lymphocytes. Biochemical analyses indicate that the 3 mAb immunoprecipitate HLA-DR products and react in western blots with DR alpha/beta-dimer but not with free alpha- or beta-chains. This study shows that transfected L cells are very useful tools for the production and the fine characterization of mAb recognizing polymorphic epitopes of HLA class II molecules.