一组抗胸腺细胞单克隆抗体的制备与鉴定——Ⅱ.生物学特性和临床的初步应用

本文主要报道一组抗胸腺细胞单克隆抗体的生物学特性及其在临床上的应用。HIT_4和 HIT_1 在补体存在下有明显的细胞毒作用。其它单抗结合补体能力很弱。HIT_(1-3)抗原出现在激活T细胞表面,不出现在休止外周T细胞上。HIT_4和HIT_(6-1.2)与休止和激活T细胞都不发生反应。HIT_2和 HIT_3单抗共同识别一个 43KD的抗原决定簇,而 HIT_4则独立识别一个 35KD的抗原。脐血单个核细胞中 HIT_(1-3)和OKT_(10)阳性率明显高于正常外周血,但Tac 阳性率较低,提示脐血中存在大量不成熟T细胞。通过研究这一种单抗的生物学特征,我们认为人类T细胞分化过程可能比 Rein-herz提出的模式更为复杂。我们还联合应用 HIT_(1-3)和 HIT_4及其它标准单抗对白血病细胞表现型进行分析和研究。     

单克隆抗体Leu8诱导的CD4抑制性T细胞

以前的资料表明,CD4T细胞的功能是多样性的。CD4T细胞用PWM激活后,在无CD8T细胞的条件下,能抑制免疫球蛋白(Ig)的合成。为了进一步研究CD4T细胞介导的抑制,作者检查了单克隆抗体Leu8鉴别出的CD4T细胞亚群的功能。结果发现,在无CD8T细胞的条件下,CD4,leu 8 ~+T细胞能直接抑制体外PWM诱导的多克隆Ig的合成。作者用E玫瑰花法从外周血单个核细胞中分离出T细胞,用单抗CD4(leu 3a+3b)和CD8(leu2b)及荧光激活细胞     

单克隆抗体(抗单)的荧光标记及其在竞争抑制试验中的应用

本文报告了一种用于直接免疫荧光试验、改进的单克隆抗体(单抗)荧光标记条件与方法 及其在单抗之间的竞争抑制试验中的应用,该法在标记条件上与用于间接免疫荧光的多克隆抗体(第二抗体)的标记有较大不同。最主要的是要求较高的F/P值。作者按此法标记了六种单克隆抗体Wu T_3,Wu T_4,Wu T_6,WuT_8,Wu T_(10)和Wu T(11)。并且将它们与已知的单抗进行了竞争抑制试验。结果表期:Wu T_3与OKT3,Wu T_4与OKT_4,Wu T_8与OKT_8,Wu T_6与H1T_(6-1),H1T(6-2)以及Wu T_(11)与抗CCT_3具有相互抑制结合抗原的再用。证明它们均能相应识别CD_3,CD_4,CD_8,CD_(1a)和CD_2靶抗原分子。此法对检定、比较抗细胞性抗原的单抗,具有很大价值。     

识别HIV-1 P_(24)蛋白的单克隆抗体杂交瘤细胞株的建立

以细胞培养的HIV-1全病毒抗原免疫,通过鼠-鼠杂交瘤技术获得3株分泌单克隆抗体(以下简称单抗)的杂交瘤细胞系(21A、51G、47B)。免疫印迹结果显示3株单抗均能识别病毒的核心元原-P_(24)蛋白。ELISA阻断抑制试验提示,3株单抗分别识别两个不同的抗原决定簇。进而为HIV-1病毒抗原的研究和制备以双抗体夹心法检测P_(24)抗原的试剂打下基础。     

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。     

武汉(Wu)系列抗入T细胞及其亚群单抗

本文报告了Wu系列抗人T及其亚群单抗按国际第三次人白细胞分化抗原专题讨论会要求进行检定的结果。这是国内第一套较全的OKT样系列单抗,即WuT1(OKT1,CD5);WuT3(OKT3,CD3);WuT4(OKT4,CD4);WuT6(OKT6,CD1a):WuT8(OKT8,CD8);WuT9(OKT9,抗转铁蛋白受体,抗TfR);WuT10(OKT10,CD38);WuT11(OKT11,CD2)及Wu-Tac(Tac,CD25)。     

人T细胞对猪白细胞抗原的直接识别

猪已被认为是异种移植的最理想供体。有关人 猪器官间超急性排斥研究已有所突破 ,而细胞性排斥报道尚少。本文用混合淋巴细胞反应体系研究异种移植细胞性排斥机制 ,发现异种反应明显弱于同种反应 ,而且以直接识别为主 ,这和同种移植细胞性排斥机制一致。抗人CD4单抗和抗猪白细胞抗原SLA DR、DQ单抗都能明显地抑制 (XMLR) ,而抗人CD8单抗无抑制功能 ,表明CD4+T细胞对SLA的识别在异种排斥应答中起重要作用     

利用基因重组抗原研制人CD20单克隆抗体及其功能的研究

目的　利用基因重组抗原免疫小鼠 ,制备人CD2 0单克隆抗体 ,研究其特异性和功能。方法　用表达重组人CD2 0基因的细胞NIH 3T3免疫BALB c小鼠 ,取其脾细胞与Sp2 0细胞融合 ,以间接免疫荧光法筛选杂交瘤上清。免疫沉淀法及流式细胞术鉴定其识别抗原的相对分子质量 (Mr)和特异性 ;以MTT法、流式细胞术及形态学方法检测诱导细胞凋亡和抑制细胞增殖的功能。结果　利用杂交瘤技术获得了 1株抗人CD2 0的单克隆抗体 1 2 8,它具有CD2 0抗体特异的细胞反应谱 ,其识别的抗原Mr 为 33× 10 3 。MTT实验证实 1 2 8能显著抑制Daudi和Raji细胞生长。结论　利用基因重组抗原制备了 1株能够稳定分泌抗人CD2 0的单克隆抗体的杂交瘤细胞株 1 2 8,此单抗具有抑制CD2 0阳性细胞增殖和诱导其凋亡的功能。     

CD107a/b分子用于评价SARS-CoV/S抗原特异性免疫应答

目的 观察抗原刺激以后CD4+和CD8+T细胞表面CD107a/b分子的表达与效应性细胞因子的产生之间的关系.方法 正常Balb/c小鼠脾细胞经多克隆刺激剂刺激或者SARS-CoV/S DNA疫苗免疫小鼠脾细胞经S抗原多肽刺激以后,使用流式细胞仪检测CD4+和CD8+T细胞表面CD107a/b分子的表达与IFN-γ、INF-α、IL-2等细胞因子的表达之间的关系.结果 经抗原多肽刺激后,抗原特异性CD4+ 和CD8+T细胞表面均表达CD107a/b.约80%的CD8+IFN-γ+细胞同时表达CD107a/b;约25%～40%的CD8+TNF-α+细胞表达CD107a/b;而大部分分泌IL-2的抗原特异性T细胞均不表达CD107a/b.结论 可以通过对抗原特异性T细胞表面CD107a/b分子的检测来鉴定抗原特异性T细胞.同时检测效应性细胞因子,可以提高检测的准确性和灵敏度.     

一组CD8单抗的研制(研究简报)

用胎儿胸腺细胞,T细胞系HPB-ALL和正常人外周血单个核细胞序贯免疫BALB/c小鼠,取脾细胞与NS_1小鼠骨髓瘤细胞融合,经间接免疫荧光法筛选,获得3个CD8单抗,分别命名为HI208、HI209和HI212。 经琼脂双向免疫扩散方法测定,HI208为IgG2b,HI209和HI212均为IgG1。组织分布结果表明,三个单抗的反应谱十分相似,它们均与33.3％、35.7％的外周血T细胞反应,与82.0％、84.6％的胎儿胸腺细     

Serum soluble CD4 and CD8 levels in Kawasaki disease.

The levels of soluble CD4 (sCD4) and sCD8 in serum correlate with the T cell subset activation and may be important in monitoring and characterizing disease processes during immunological diseases. We compared acute Kawasaki disease (KD) with anaphylactoid purpura (AP) and acute febrile viral infections, such as measles and infectious mononucleosis (IM), in terms of serum sCD4 and sCD8 levels. The levels of serum sCD4 and sCD8 were measured by a sandwich enzyme immunoassay. In addition, peripheral blood mononuclear cell subsets were analysed by single and two-colour flow-cytometric analyses in KD and IM patients. The levels of serum sCD4 and sCD8 were significantly elevated in patients during acute stages of KD, measles and IM, but not AP. Peripheral blood CD4+, CD8+ and also HLA-DR+ T cells count did not increase during the acute stage of KD; however, peripheral blood CD8+ and HLA-DR+ T cell counts were increased during the acute stage of IM. Our results suggest that there is a low level of activation of peripheral blood T cells during acute KD, or that infiltrated T cells in some local tissues of KD patients contribute to the elevated levels of serum sCD4 and sCD8.     

免疫组化技术分析人淋巴组织的多种分化抗原

用间接免疫过氧化物酶和PAP技术检测本室制备的31种抗人分化抗原单抗与淋巴组织的反应性.结果表明,CD3、CD5单抗与扁桃腺和淋巴结的T细胞、大部分成熟髓质胸腺细胞和脾白髓的中央动脉周围淋巴鞘呈非常强的反应.CD4~+细胞在扁桃腺的分布与CD3~+细胞类似,但数量稍少.只有少部扁桃腺和淋巴结T细胞与CD8单抗反应,CD8单抗主要染大部分胸腺皮质细胞,但抗CD8单抗与脾窦的内皮细胞呈强阳性交叉反应.Wu59单抗同时与扁桃腺、淋巴结和脾脏的T、B细胞呈非常强的膜染色,并与胸腺皮质和髓质细胞呈阳性反应,该单抗可能识别白细胞共同抗原或LFA-1.Wu 26.145单抗除与扁桃腺生发中心呈弱阳性反应外,还与脾红髓窦状结构内的血小板呈强阳性反应.此外,抗B细胞及其亚群单抗与扁桃腺、淋巴结、脾白髓生发中心呈强阳性反应.抗IL-2受体单抗与上述组织基本上呈阴性反应。     

Characterization of CD4+ T helper cells in patients with Kawasaki disease (KD): preferential production of tumour necrosis factor-alpha (TNF-alpha) by V beta 2- or V beta 8- CD4+ T helper cells.

KD is an acute febrile illness in children characterized by coronary arteritis accompanied by aneurysm and thrombotic occlusion. The etiology of KD is unknown. It has been recently reported that KD is associated with the selective expansion of V beta 2+ and V beta 8.1+ T cells in peripheral blood lymphocytes (PBL), by studying the T cell receptor (TCR) repertoire of in vitro activated T cells. KD may therefore be caused by a superantigen [1-3]. To understand better the immunopathology of KD, we investigated TCR V beta 2 and V beta 8.1 expression on both the T cells of freshly isolated PBL and T cell clones (TCC) from patients with KD. Cytokine production by TCC was also studied. Blood samples were obtained from patients with acute (n = 20) and convalescent (n = 20) KD, age-matched children with non-infectious diseases (n = 18), and healthy adults (n = 20). Among these four groups, there were no significant differences in the percentages of either V beta 2+ or V beta 8.1+ T cells of freshly isolated PBL. The same was true for the CD4+ or CD8+ T cell subsets. One hundred and five TCC (98 CD3+ CD4+ CD8- and seven CD3+ CD4- CD8+) established from the affected skin, lymph node or PBL of six patients with KD were also negative for either V beta 2 or V beta 8.1 TCR. Sixty-eight of 105 TCC (65%) produced detectable levels (> 5 pg/ml) of TNF-alpha (6-1016 pg/ml), in the absence of any stimuli. In contrast, only 11 (10%) of 105 TCC or 7 (7%) of 97 TCC produced detectable levels of IL-2 or IL-6, respectively, in the absence of any stimuli. Stimulation with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) induced most TCC to produce higher amounts of TNF-alpha, IL-2 and IL-6. These results suggest that CD4+ T helper cells expressing TCR-beta other than V beta 2 or V beta 8 receptor, primarily through TNF-alpha production, are involved in the immunopathology of KD.     

Immunopathology of skin lesions in Kawasaki disease]

Histopathological findings of skin lesions in Kawasaki disease (KD) have been characterized by extensive edema associated with the dilatation of small vessels in the papillary dermis. Although the inflammation in KD skin lesions was exudative in nature, neutrophil emigration was slight and most of the infiltrates were mononuclear cells. Immunofluorescent studies using skin biopsy specimens from 10 patients with KD aged from six months to eight years and seven months showed that the infiltrating cells in the dermis and epidermis were mainly composed of CD 3+ T cells and Leu M3+ macrophages, but not B cells. In double immunofluorescence staining with combinations of anti HLA-DR, CD4 and CD8 monoclonal antibodies, the infiltrating T cells were mainly CD4+ HLA-DR+ T cells. Leu 23% cell were also positive on these cells, thereby suggesting those to be activated. Studies of skin specimens obtained from the site of BCG vaccinations in patients with KD showed basically similar but more extensive lesions. As a control, the infiltrating cells in the dermis from patients with measles were examined. In contrast to KD, these cells were mainly CD8+ T cells. Together with the findings that the keratinocytes in the epidermis were positive for HLA-DR, the skin lesions in KD appear to be similar to those found in delayed type hypersensitivity. Thus, macrophages and helper T cells may play a crucial role in the pathogenesis of KD.     

人外周血T细胞主要功能亚群的分离及某些性质的研究

用本实验室自制CD_4类抗体T_(4a)T_(4b)和CD_3类抗体T_(3b)及流式细胞荧光激活分类仪(FACS440)将正常人外周血分离成T_(4a)(T_(4b))~+、T_(4a)(T_(4b))~-、T_(8b)~+、T_(8b)~-细胞。对所分离的细胞纯度分析表明.其纯度达95%以上.用CD_4、CD_(?)类对所分离细胞进行双染分析,发现T_(4a)(T_(4b)~+、T_(4a)(T_(4b))~-,T_(8b)~+中不含T_(4a)(T_(ab))~-、T_(8b)细胞.同样在阴性细胞中,阳性细胞的含量不超过2.2%.用此高度纯化的T_(4a)(T_(4b))~+、T_(8b)细胞,观察PHA诱导分泌IL-2的情况.发现仅T_(4a)(T_(4b)_~+细胞分泌IL-2.在本实验条件下,T_(8b)细胞不分泌IL-2.     

抗淋球菌PIA单克隆抗体的制备和应用分析

用淋球菌全菌体免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与Ｓｐ２／０骨髓瘤细胞融合，以纯化ＰＩＡ做ＥＬＩＳＡ间接法筛选，获得５株稳定分泌抗ＰＩＡ的ＭｃＡｂ的杂交瘤细胞株。５株ＭｃＡｂ中３株为ＩｇＭ类（２Ｈ１１，４Ｈ８，４Ｅ１０），另２株分别为ＩｇＧ１（１Ｃ２）和ＩｇＧ２ｂ（５Ａ５）亚类。２Ｈ１１和１Ｃ２为高亲和力抗体，１Ｃ２与５Ａ５识别的抗原表位相同。Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ试验表明，５株ＭｃＡｂ均能从复杂的淋球菌菌体崩解物中特异地识别分子量为３５ＫＤａ的ＰＩＡ抗原，与淋球菌ＰＩＢ无交叉反应。     

Immunohistological relation between DR antigen expression on melanoma cells and infiltration by CD8+ T cells

Previous studies have suggested that class II major histocompatibility (MHC) antigen expression on melanoma cells may influence immune responses against melanoma and the nature of lymphocytic infiltrates against the tumor. This question was examined further by immunoperoxidase studies on sections from 29 primary and 30 metastatic melanoma with monoclonal antibodies (MAbs) against different lymphocyte subsets. The results indicated that expression of MHC class II DR* antigens on melanoma cells was associated with increased overall lymphocytic infiltration and that this applied particularly to the CD8+ subset of T cells. The CR3 receptor (CD 11b) was expressed predominantly on T cells and not macrophages but infiltration by CD11b+ cells did not correlate strongly with DR expression on melanoma cells. Dual staining with MAbs to CD8 and CD11b revealed that, whereas most of the CD8+ cells in DR- primary melanoma and DR+ metastatic melanoma were CD11b+, only approximately 50% of the CD8+ cells in DR+ primary melanoma were also CD11b+. Expression of CD11b on T cells was reported to define a suppressor subset of T cells. If the latter is correct the present results suggest that DR expression on primary melanoma is associated with infiltration by the cytotoxic T cell subset, whereas in the absence of DR antigens and in metastases this subset is absent and the predominant subset appears to be CD8+ CD11b+ T cells with suppressor activity. The biologic and prognostic significance of these findings remains to be determined.     

Expression of CD11b (Leu15) antigen on CD3+, CD4+, CD8+, CD16+ peripheral lymphocytes. Estimation of CD3+8+11b+ and CD3+4-8-11b+ T-cell subsets using a single laser flow cytometer.

CD11b (Leu15) epitope is expressed on 20-30% of peripheral blood lymphocytes, including CD16+ large granular lymphocytes and CD8+ cells. This study confirms that 30% of CD8+ lymphocytes and virtually all CD16+ NK cells from healthy subjects express this determinant. In parallel, our data show that various proportions of CD3+4-8-, TCR-delta cytotoxic T lymphocytes and occasionally CD4+ lymphocytes subsets could also express this epitope. The CD8+11b+ phenotype is associated with suppression of T-cell proliferative response and has been extensively used to characterize suppressor T lymphocytes. Since about 25% of CD8 lymphocytes are non-T (CD3-) and express the CD16 NK antigen (CD8+16+3-), the expression of CD11b was also studied on CD8+3+ T-cell and CD8+16+ NK-cell subsets. To this end, we developed three methods using a flow cytometer equipped with a single laser and two fluorescence detectors. Results showed that T CD8+3+11b+ and NK CD8+16+11b+ lymphocytes account for 30% and 70% of CD8+11b+ cells respectively. Consequently, the CD8+3+11b+ phenotype would be more specific for suppressor T lymphocytes than the total CD8+11b+ phenotype which includes high proportions of CD16+ NK cells.     

CD8类McAb—SM4的研制与鉴定

本文报导一株抗人T细胞亚群单克隆抗体SM_4的研制与鉴定。SM_4仅与T细胞中的一个亚群反应,在淋巴组织冰冻切片上,SM_4只与胸腺依赖区的部分细胞反应,其组织分布特征与CD_8单抗相类似。叠加试验结果表明SM_4与CD_8类单抗识别同一细胞亚群——Ts/Tc亚群。竞争抑制试验进一步证明SM_4识别CD_8分子上某一特定表位。本文还就CD_8亚群异质性问题及SM_4的应用潜力进行了讨论。