用抗人CD3,CD4,CD8单抗识别精液中的T淋巴细胞

用鼠抗人ＣＤ３，ＣＤ４，ＣＤ８单克隆抗体（ＭＣＡｂ）识别精液中的淋巴细胞，用羊抗鼠荧光抗体（ＦＩＴＣ）作为第２抗体染色体镜检，细胞膜呈绿色荧光为阳性，结果表明，生育男性与精索静脉曲张及附睾结核的男性精液中Ｔ淋巴细胞检出率差异显著（Ｐ〈０．０１）。     

脓毒症患者外周血CD3+、CD4+、CD8+ T淋巴细胞的凋亡及Bcl-2蛋白表达

目的探讨脓毒症患者外周血淋巴细胞凋亡及其与Bcl-2蛋白表达的关系。方法采集44例脓毒症患者（脓毒症组，年龄19～86岁）和25例健康体检者（正常对照组，年龄29～80岁）的外周静脉血5ml，采用流式细胞仪分别检测CD3^＋、CD4^＋、CD8^＋T淋巴细胞凋亡率和Bcl-2蛋白表达。分析细胞凋亡率与Bcl-2蛋白表达的相关性。脓毒症诊断标准参考1991年美国胸科医师协会和美国危重病医学会联席会议制定的诊断标准。结果与正常对照组比较，脓毒症组患者CD3^＋、CD4^＋T淋巴细胞凋亡率升高，Bcl-2蛋白表达下降（P〈0．05），CD8^＋T淋巴细胞凋亡率及其Bcl-2蛋白表达差异均无统计学意义（P〉0．05），CD3^＋、CD4^＋T淋巴细胞凋亡率分别与其Bcl-2蛋白表达呈负相关（r=-0．389，P〈0．05；r=-0．689，P〈0．05）。结论脓毒症患者细胞免疫功能异常可能与Bcl-2蛋白表达下调导致淋巴细胞凋亡增加有关。     

肝癌肿瘤浸润淋巴细胞的培养及抗CD3单抗对其增殖及细胞毒的 …

目的 研究人原发性肝癌肿瘤浸润淋巴细胞（ＴＩＬ）的分离，培养方法和增殖及体外细胞毒变化特点以及抗ＣＤ３单克隆抗体（Ａｎｔｉ－ＣＤ３ＭｃＡｂ）对ＴＩＬ增殖和细胞毒的影响。方法 采用Ｉ型和Ⅳ型胶原酶，Ｖ型透明质酸酶和Ｉ型ＤＮＡ酶四联酶消化，应用不连续密度梯度的离心分离，ＴＩＬ，采用ＭＴＴ比色法检测ＴＩＬ对自体癌细胞及肝癌细胞株的细胞毒。结果 ＴＩＬ对自体肝癌细胞的杀伤具有特异性（Ｐ〈０．０１）。Ａｎｔ     

CD4+CD25-T淋巴细胞转化为CD4-CD8-调节T细胞的体外实验研究

目的 探讨体外诱导和纯化CD4+ CD25-T淋巴细胞(effector T cell,Teff)转化为CD4-CD8-双阴性调节T细胞(double negative regulatory T cell,DN Treg)的最适条件.方法 采用免疫磁珠分选方法提取C57BL/6小鼠的CD4+ CD25-T淋巴细胞、DBA/2小鼠的成熟树突状细胞共培养,加入不同剂量的IL-2,通过流式细胞检测CD4-CD8-T细胞的转化比例并确定最适条件,免疫磁珠阴性选择分选提纯转化的CD3+ CI4-CD8-T细胞,流式细胞仪检测转化的CD4-CD8-调节T细胞对CD4+ CD25-效应T细胞增殖抑制情况.结果 CD4+ CD25-T淋巴细胞与DBA/2小鼠的树突状细胞共培养6d后检测CD4-CD8-调节T细胞的转化比率为6.21％ ±2.03％,实验组加入不同浓度IL-2的转化率:A组(25 ng/ml)为14.77％±2.15％,B组(50 ng/ml)为21.29％±2.68％,C组(75 ng/ml)为43.45％ ±4.45％,D组(100 ng/ml)为28.59％±3.05％,IL-2浓度在75 ng/ml时,转化获得率最高(C组与对照组、实验A、B、D组比较分别t=10.700,8.288,6.158,3.932,均P＜0.05);分离提纯CD4-CD8-双阴性调节细胞纯度达到98.10％,CD4-CD8-双阴性调节细胞与CFSE染色的CD4+ CD25-T淋巴细胞、小鼠树突状细胞共培养6d,实验组增殖指数为1.15明显低于对照组2.07.结论 小鼠CD4+ CW25-T淋巴细胞在体外,成熟树突状细胞刺激下可转化为CD4-CD8-双阴性调节T细胞,IL-2可显著提高其转化率.     

结合半乳糖基抗CD3单抗的肿瘤浸润淋巴细胞对自体肝癌细胞杀伤的形态学研究

目的　获取与半乳糖基抗CD3 单抗结合的肿瘤浸润淋巴细胞 (McAb TIL)杀伤自体肝癌细胞的形态学证据 ,并进行杀伤机制探讨。方法　制备McAb TIL ,将其与H2 2 小鼠肝癌细胞共同孵育 ,于不同时间在倒置显微镜和透射电镜下观察。结果　McAb TIL呈增殖活化状态 ,与靶细胞共同孵育 0 .5h即可导致靶细胞严重损伤。杀伤靶细胞后的McAb TIL结构清晰、完整。凋亡与坏死形态学改变可共存于同一个被杀伤靶细胞。结论　偶联了大量半乳糖基的抗CD3 单抗仍具有很强的肿瘤浸润淋巴细胞 (TIL)活化作用。McAb TIL通过多种杀伤机制杀伤自体肝癌细胞 ,杀伤力极强 ,具有连续杀伤靶细胞的能力。     

肿瘤浸润性淋巴细胞结合偶联半乳糖抗CD3单抗趋肝性初探

目的 探讨结合偶联半乳糖（Ｇａｌ）的大鼠抗小鼠ＣＤ３单克隆抗体（Ｍｏｕｓｅ－ａｎｔｉ－ｒａｔ－ＣＤ３ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｙ Ａｎｔｉ－ＣＤ３－ＭｃＡｂ）肿瘤浸润性淋巴细胞的（ＴＩＬ）趋肝性，方法 本实验把小鼠抗小鼠ＣＤ３单克隆缺本和半乳糖（Ｇａｌ）偶联在一起，与＾３Ｈ－ＴｄＲ标记ＴＩＬ结合后，从尾静脉注入小鼠体内，分别在注射后不同时间抠眼取血０．５ｍｌ，然后处死，切除肝、脾、肺组织     

抗T淋巴细胞单克隆抗体与丝裂霉素偶联的方法学研究

我们采用抗Ｔ细胞单克隆抗体Ｔ３、Ｔ４，与具有抑制细胞ＤＮＡ合成的药物丝裂霉素交联，形成免疫偶联物，以期在临床应用中获得特异性的、对机体无副作用的、能选择性地作用于免疫活性细胞、延长移植物存活的效果。交联后的免疫偶联物，经过洗脱曲线，紫外扫描以及抗体效价测定等，实验结果表明单克隆抗体Ｔ３、Ｔ４和交联物葡聚糖Ｔ－４０与丝裂霉素Ｍｉｔｏｍｙｃｉｎ交联后的免疫复合物，抗体效价和药物活性均无明显影响，体外淋巴细胞转化试验表明交联后的免疫偶朕物具有很强的免疫抑制作用。     

结合半乳糖基抗CD3单抗的肿瘤浸润淋巴细胞对自体肝癌 …

目的 获取与半乳糖基抗ＣＤ３单抗结合的肿瘤浸润淋巴细胞（ＭｃＡｂ－ＴＩＬ）杀伤自体肝癌细胞的形态学证据，并进行杀伤机制探讨。方法 制备ＭｃＡｂ－ＴＩＬ，将其与Ｈ２２小鼠肝癌细胞共同孵育，于不同时间在倒置显微镜和透射电镜下观察。结果 ＭｃＡｂ－ＴＩＬ呈增殖活化状态，与靶细胞共同孵育０．５ｈ即可导致靶细胞严重损伤。杀伤靶继细胞后的ＭｃＡｂ－ＴＩＬ结构清晰、完整。凋亡与坏死形态学改变可共存于同一个被杀伤     

人CD8+CD28-抑制性T淋巴细胞的体外扩增及其抗原特异性调节作用

目的 探讨在体外大量扩增CD8+CD28-抑制性T淋巴细胞(Ts细胞)的方法,并检验其免疫调节作用.方法 分离健康志愿者全血中CD8+T淋巴细胞,在含不同细胞因子和异体抗原提呈细胞(APC)的培养条件下进行体外扩增.应用流式细胞术对扩增过程中CD28 -细胞亚群的比例进行监测.分为3组进行混合淋巴细胞培养,反应细胞均为CD4+T淋巴细胞:B-APc组以来源于原致敏供者的APC作为刺激细胞,I-APC组以HLA-A、B、DR全错配的无关供者的APC作为刺激细胞,Dynabeads组以包被有抗CD3和CD28单克隆抗体的免疫微球Dynabeads作为刺激细胞;扩增后的Ts细胞作为第三方调节细胞加入混合淋巴细胞培养中,测定其对CD4+T淋巴细胞增殖的抑制作用.结果 在含白细胞介素2(IL-2)+ IL-7+ IL-15的培养条件下,CD8+T淋巴细胞培养后CD8+CD28-T淋巴细胞亚群的比例最高(P＜0.05).B-APC组不加入Ts细胞时,增殖的CD4+T淋巴细胞比例为66.7％,当加入的Ts细胞与反应细胞的比例分别为0.5∶1、0.1∶1和0.02∶1时,增殖的CD4+T淋巴细胞比例分别为16.5％、34.1％和62.6％.Ts细胞对CD4+T淋巴细胞的增殖有明显抑制作用,而在I-APC组和Dynabeads组中,此抑制作用不明显.结论 应用含IL-2+ IL-7+ IL-15的培养条件联合异体APC刺激可以在体外大量扩增Ts细胞,扩增所得Ts细胞在体外对供者CD4+T淋巴细胞的增殖有明显抗原特异性抑制作用.     

Initial experience with the human anti-human CD154 monoclonal antibody, ABI793, in pig-to-baboon xenotransplantation

BACKGROUND: ABI793 (ABI) is a human monoclonal antibody (mAb) specific for human CD154. To assess the suitability of ABI for baboon transplantation studies, we carried out in vitro studies to determine ABI's reactivity with baboon cells expressing CD154, performed in vivo pharmacokinetic studies in two baboons, and tested the effect of ABI administration on elicited antibody production in two baboons undergoing either pig hematopoietic progenitor cell (PBPC) or heterotopic heart transplantation. METHODS: In vitro: Baboon peripheral blood mononuclear cells were activated in vitro to upregulate CD154, and binding of ABI to CD154 was measured by flow cytometry. In vivo: Serum levels of ABI were measured immediately before and 15 min after the intravenous administration of ABI (20 mg/kg) to two baboons over 28 days. Subsequently, ABI (25 mg/kg on days 0, 1, 4 and 7, and then 20 mg/kg every 5 days) was included in the immunosuppressive regimen in two pig-to-baboon transplants (PBPC or heart transplantation). RESULTS: In vitro: ABI was almost non-reactive to baboon T cells before stimulation, but bound to activated T cells. In vivo: In the pharmacokinetic study, trough levels of ABI (before the next dose) ranged between 190 and 580 microg/ml, and the estimated half-life was 10-15 days. There was no apparent toxicity. Following pig PBPC or heart transplantation, no elicited antibody was detected while ABI was being administered or during several weeks of follow-up. CONCLUSIONS: ABI functions in baboons, is well-tolerated, and prevents an elicited antibody response to pig antigens.     

Long-term survival of hamster islet xenografts in mice under short-course treatment with nondepleting versus depleting anti-CD4 monoclonal antibodies

ABSTRACT: Xenogeneic grafts provide a potential alternative to the current shortage of human organs for transplantation. However, the prevention of rejection and tolerance induction of xenografts still remain to be further explored. Islet xenografts appear more promising than vascularized whole organ xenografts and additionally also more resistant to the recurrence of autoimmune disease than allografts. Recently, the nondepleting monoclonal antibody (mAb), which blocks the CD4 molecule on lymphocytes, was reported to be able to induce tolerance in allotransplantation and CD4 positive cells were further confirmed to be a major factor responsible for cellular xenograft rejection. Therefore, we hypothesize that anti-CD4 nondepleting mAb could also be effective in protecting cellular xenografts and inducing unresponsiveness of recipients. We studied the effect of the nondepleting anti-CD4 mAb YTS177.9 on islet xenograft survival by using the hamster-to-mouse islet transplantation model. Results were compared with that of the depleting anti-CD4 mAb GK1.5 that was shown to have similar binding sites on the CD4 molecule to mAb YTS 177.9. Our data show that mAb YTS 177.9 did effectively prolong the survival of islet xenografts and, in addition, also successfully did induce long-term acceptance of 40% grafts after only three penoperative injections of 0.5 mg mAb per mouse. The average survival of the graft was markedly prolonged to >66.8±37.1 days compared with controls (8.3±1.4 days) or with the depleting anti-CD4 mAb GK1.5 (25.7±5.5 days). However, the latter displayed a more profound inhibition in in vitro and ex vivo mixed lymphocyte xenoreaction than mAb YTS 177.9. Moreover, the activity of this nondepleting mAb was found to be dose-dependent and 80% of grafts survived permanently when the dose was increased to six injections of 0.5 mg mAb. Like mAb GK1.5, mAb YTS 177.9 also prevented rejection when given after a delay of two days posttransplant. In addition, we found that neither depleting nor nondepleting anti-CD8 mAb was effective in this model. Our results strongly suggest that an anti-CD4 nondepleting or blocking mAb alone is able to induce long-term acceptance of islet xenografts and that blocking the CD4 molecule is significantly superior to depleting CD4 positive cells for the protection of islet xenografts. This may indicate that CD4 cells play a major role in xenograft tolerance induction.     

Prolonged survival of baboon renal allografts using idarubicin‐conjugated anti‐CD4 monoclonal antibodies

Abstract Tolerance to organ allografts in rodents and pigs can be easily achieved. However, tolerance induction in a large primate model has been more elusive. In this study, we have used an anti‐CD4, murine monoclonal antibody as a carrier for the cytotoxic drug idarubicin (IDA) to delete or inactivate alloreactive T‐cells responding to a renal allograft in a baboon transplant model. Fourteen Chacma baboons weighing between 15‐25 kg received heterotopic renal allografts. Recipient and donor pairs were selected on the basis of ABO compatibility. Seven animals were given no immunosuppression and served as the control group. The remaining 7 animals received anti‐CD4 IDA. The first 2 animals in this group received 2 mg IVI intraoperatively and three doses at 48‐h intervals thereafter. The last 5 animals received a larger dose of 1 mg/kg, starting 24 h pre‐operatively and again on postoperative days 2 and 5. The untreated animals promptly rejected their allografts with a mean survival of 10 days. The survival of the 2 animals treated with 2 mg anti‐CD4 IDA was 7 days each. However, the animals treated with 1 mg/kg anti‐CD4 IDA survived 7, 18, 20, 40 and > 40 days. Peritransplant administration of anti‐CD4 IDA prolonged renal allograft survival in a large primate model. This unique immunoconjugate has the potential of tolerance induction.     

Development of new prostate specific monoclonal antibodies

BACKGROUND: Despite the need for new prostate-specific diagnostic and therapeutic targets, very few unique prostate (cancer) specific antigens have been characterized. Monoclonal antibody (mAb) technology is a powerful tool to identify specific antigenic markers, which could be potential targets for cancer diagnostics or therapy. METHODS: Splenocytes from mice immunized with prostate cancer (PCa) homogenates of different origin were fused using standard techniques. Employing a differential high-throughput screening method followed by immediate screening in immunohistochemistry (IHC) a large number of hybridomas were screened for prostate (cancer) specificity. RESULTS: From 25 successful fusions approximately 300 clones were identified excreting PCa-reactive antibodies. Subsequent immunohistochemical fine-specificity analysis reduced this number to 26. Eventually, after extensive fine-specificity analysis, the number of mAbs appearing to define prostate-specific antigenic structures that might serve as new diagnostic or therapeutic targets was reduced to three. CONCLUSIONS: Using mAb technology combined with a high throughput screening method we have developed three mAbs (1.8, 2.26, and 3.10) directed against prostate associated antigens that might identify potential new therapeutic targets.     

Prediction of graft prolongation by mixed lymphocyte culture following anti-CD4 monoclonal antibody treatment among different donor-recipient combinations

+ cells, were examined to determine which was the most suitable for predicting graft survival, the MLC results showed that the responses of LN cells correlated most closely with graft outcome. In conclusion, MLC using LN cells as the responder is a useful tool for predicting allograft survival induced by anti-CD4 MAb therapy. (Received for publication on Feb. 10, 1998; accepted on Jan. 7, 1999)     

Characterization of a monoclonal anti‐porcine CD3 antibody

Huang CA, Lorf T, Arn JS, Koo GC, Blake T, Sachs DH. Characterization of a monoclonal anti-porcine CD3 antibody. Xenotransplantation 1999; 6: 201-212. © Munksgaard, Copenhagen Abstract: Partially inbred miniature swine have been developed in this laboratory as a large animal model for studies related to transplantation tolerance and as a source of hematopoietic cells and organs for xenotransplantation. The identification of swine CD3 specific mAbs capable of activating or depleting T cells in vitro and inducing an immunosuppressive state in vivo greatly facilitates studies of the swine immune system, transplantation tolerance and xenotransplantation research. Flow cytometry was used to determine the phenotypic profile of the swine specific mAb 898H2-6-15 (2-6-15). The specificity of 2-6-15 was further defined biochemically by surface labeling and immunoprecipitation. The ability of this mAb to activate pig T cells in vitro was examined by several criteria including proliferation assays, calcium flux analysis and detection of surface CD25 upregulation by fluorescence activated cell sorter (FACS) analysis. Monoclonal antibody 898H2-6-15 is specific for swine CD3 and is capable of inducing proliferation and CD25 upregulation in cultured swine peripheral blood lymphocytes. In addition, it induces calcium flux in purified pig T cells. Surprisingly, in contrast to described antibodies to CD3 in swine and other species, the binding of this antibody to porcine CD3 is dependent on the presence of extracellular calcium. Thus calcium was required in order to immunoprecipitate labeled surface molecules for biochemical analysis and to stain cell surfaces for FACS analysis of swine lymphocytes. In this paper, we describe a new swine CD3 specific mAb, 898H2-6-15 (2-6-15) the characteristics of which make it an extremely useful tool for in vitro and in vivo studies of the swine immune system and xenotransplantation. The availability of swine T cell specific reagents should facilitate the monitoring of swine T cell engraftment and/or release amongst xenogeneic mixed chimeras and thymic transplant recipients as well as provide a means to treat potential GvHD across xenogeneic barriers. We are currently evaluating the in vivo effects of 2-6-15 in the pig.     

Human glioma associated and related antigens,identified by monoclonal antibodies

Summary Tumour specific and various tumour-associated antigens expressed on human glioma cells were reported from many laboratories. These have been identified by xeno-, allo-, or auto-sera and more recently by utilizing monoclonal antibodies produced by hybridoma technique.In this article, the results of reports are summarized comparing the studies with conventional antisera and monoclonal antibodies. The present and future clinical applications of these monoclonal antibodies are described.