Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen

CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.     

Production of a monoclonal antibody against SARS-CoV spike protein with single intrasplenic immunization of plasmid DNA

Severe acute respiratory syndrome (SARS) is a highly infectious disease caused by a novel coronavirus (SARS-CoV). Specific monoclonal antibodies (mAbs) against the SARS-CoV are vital for early diagnosis and pathological studies of SARS. Direct intrasplenic inoculation of plasmid DNA encoding antigen is an effective and fast approach to generate specific mAb when the protein antigen is difficult to prepare or dangerous in use. In this study, we selected one fragment of SARS-CoV spike protein (S1-₃) as antigenic determinant by immunoinformatics. Single intrasplenic immunization of plasmid DNA encoding S1-₃ induced anti-spike protein antibodies. We established one hybridoma cell line secreting specific mAb and evaluated this mAb with murine leukemia virus pseudotyped with SARS-CoV spike protein (MLV/SARS-CoV). The mAb could recognize the spike protein on the MLV/SARS-CoV-infected Vero E6 cells albeit with no neutralizing effect on the infectivity of the pseudotype virus. Our results show that a single-shot intrasplenic DNA immunization is efficient for the production of specific mAb against SARS spike protein, and a linear epitope of the spike protein is recognized in this study.     

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。     

Inhibition of PHA-induced cell proliferation by polyclonal CD4 antibodies generated by DNA immunization.

Although the role of CD4 molecule as associative binding element to MHC class II is well documented, their role in T cell activation is unclear. In the present report we used DNA immunization, which is currently shown to induce potent immune responses, to produce the polyclonal antibodies specific for the CD4 molecule and used the generated antibodies to characterize the CD4 function. A rabbit was pre-treated with bupivacaine hydrochloride for 24 h which was followed by intramuscular injection of DNA encoding CD4 protein (CD4-DNA) at weekly interval. By this procedure, CD4 antibodies were detected in the immunized serum after two DNA inoculations. The CD4 antibodies titer was up to 1:800 after five DNA inoculations. The rabbit polyclonal CD4 antibodies recognized both recombinant CD4 protein expressed on CD4-DNA transfected COS cells and native CD4 protein presented on peripheral lymphocytes and CD4+ cell lines. These generated CD4 antibodies could block the binding of standard CD4 mAb, Leu3a and 13B8.2, to the CD4 molecule. To characterize the function of CD4 molecule, PBMC were cultured in the presence of sub-optimal dose of PHA and the produced polyclonal CD4 antibodies. We found that the polyclonal CD4 antibodies strongly suppressed PHA induced cell proliferation. The inhibitory effect of CD4 antibodies may be due to their steric inhibition of the CD4-TCR/CD3 association or may interfere with the binding of CD4 to its ligand IL-16, resulting in the reduction of signal transduction and subsequent cellular responses. Our results indicate the possibility of utilizing DNA immunization to produce polyclonal antibodies against cell surface molecule.     

CD45 molecule cross-linking inhibits natural killer cell-mediated lysis independently of lytic triggering.

The fact that certain CD45 [anti-leucocyte common antigen (LCA)] monoclonal antibodies (mAb) inhibit natural killer (NK) cell non-major histocompatibility complex (MHC)-restricted cytolysis led to the suggestion that these mAb block a 'trigger' for NK cell lytic activity. However, the discovery that the intracytoplasmic portion of the leucocyte common molecule has protein tyrosine phosphatase activity raises the possibility that the mAb initiate a direct inhibitory signal, independent of the triggering apparatus. To clarify this, we have tested the ability of CD45 antibodies to trigger NK cells and redirect cytotoxicity against mAb-producing hybridoma cells and autologous monocytes, an approach which has identified other cytotoxic trigger molecules. Peripheral blood NK cells failed to kill the CD45 antibody-producing hybridomas, although a CD3 antibody expressing hybridoma was susceptible to cytotoxic T-cell lysis. Furthermore, the CD45 mAb CMRF-12 + 26, 13.3 and HuLyM4 did not redirect lysis of autologous monocytes by NK cells, whereas the isotype-matched CD16 mAb did so. Bivalent CD45 antibody was necessary to block NK lysis of K562, as F(ab')2 but not F(ab') fragments of CMRF-12 + 26 antibody inhibited killing. Capping of the LCA appeared to correlate with the ability of the CD45 mAb to block killing, suggesting that cross-linking of LCA molecular isoforms on the NK cell surface is required for CD45 mAb to inhibit non-MHC-restricted cytolysis.     

A rapid and simple approach to preparation of monoclonal antibody based on DNA immunization

Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb).But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace theprotein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectorspcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNAimmunization.Female BALB/c mice developed a well antibody response to the target antigen after muscleinjection with corresponding plasmids.The mice with effective antibodies induced were used for preparation ofmAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted byintrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positivehybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that geneimmunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology.2004;1(4):295-299.     

CD45-mediated regulation of LFA1 function in human natural killer cells. Anti-CD45 monoclonal antibodies inhibit the calcium mobilization induced via LFA1 molecules

The TA218 and T205 monoclonal antibodies (mAb) were selected on the basis of their ability to inhibit the non-major histocompatibility complex-restricted lysis of the murine mastocytoma P815 cell line mediated by CD3^?CD16⁺ natural killer (NK) cells. Both mAb were found to react with CD45 molecules, as demonstrated by immunoprecipitation after surface iodination and western blot analysis. A panel of tumor target cells susceptible to lysis by polyclonal or clonal CD3^?CD16⁺ NK cells was used to study the mAb-mediated inhibitory effect. The inhibition of cytolysis mediated by TA218 and T205 mAb was found to consistently parralel the inhibition mediated (with the same tumor target cells) by the anti-LFAlα mAb TS.1.22 or by the anti-LFA1β mAb TS.1.18. However, different from the anti-LFAl mAb, T205 or TA218 mAb did not inhibit the binding of activated CD3^?CD16⁺ effector NK cells to the same tumor target cells. This finding supported the concept that the anti-CD45 mAb-mediated inhibition could occur at a post-binding stage. In polyclonal or clonal CD3-CD16⁺ NK cellsT205 orTA218 mAb were found to reduce by 50–70 % the intracellular Ca⁺⁺ ([Ca⁺⁺]i) mobilization induced by anti-LFAlα or anti-LFA1β mAb. On the other hand, TA218 and T205 mAb did not inhibit the Ca⁺⁺ mobilization induced by anti-CD 16 mAb or phytohemagglutinin, thus suggesting that, in NK cells, CD45 molecules may exert a selective inhibitory effect on the signal transduction mediated by LFA1 molecules. In line with this hypothesis, the cytolytic activity of human NK clones was triggered in the presence of the hybridoma cells secreting either anti-CD16 or anti-LFAla mAb (as “triggering targets”). This effect of anti-LFAlα, but not of anti-CD16 hybridoma was susceptible to inhibition by the anti-CD45 mAb T205 or TA218. Further, experiments on cloned NK cells indicated that T205 or TA218 mAb induced a strong decrease in the constitutive phosphorylation of the LFAlα chain (but not of HLA class I antigens). Taken together, these studies suggest that in human NK lymphocytes, CD45 molecule may regulate both the activation state and the function of the LFA1 molecule.     

A positive signal is transduced via surface CD4 molecules

We have previously reported on the identification of a monoclonal antibody (mAb) directed against the human CD4 antigen which is capable of activating CD4+ peripheral blood T cells in the absence of other stimuli. In the present study, we extended these findings by demonstrating that the mAb, termed B66, was able to induce the production of interleukin-2 in murine T-cell hybridoma transfectants expressing the human CD4 glycoprotein. Moreover, we found that incubation of Jurkat cells with mAb B66 resulted in the nearly complete disappearance of both CD4 and CD3 from the cell surface, whereas modulation of CD4, but not CD3, was observed after incubation with a non-stimulatory anti-CD4 mAb. Similar results were obtained in modulation experiments using human CD4-expressing murine transfectants. It is thus conceivable that the stimulatory activity of anti-CD4 mAb B66 may be associated with an effect on the CD3 molecular complex. While the biochemical basis for the unique stimulatory activity of mAb B66 has yet to be defined, these findings provide direct evidence that cross-linking of CD4 alone may cause T-cell activation, thus supporting the notion that this glycoprotein can transduce independent positive signals upon binding to class II major histocompatibility complex molecules.     

抗人CD38单克隆抗体的制备、鉴定及其功能的初步研究

目的：研制抗人CD38抗原分子的单克隆抗体，进一步研究其生物学功能。方法：采用高表达CD38抗原的Daudi细胞免疫Balb/c小鼠；取其脾细胞与SP2/0细胞融合；用间接免疫荧光法进行杂交瘤筛选；流式细胞术、免疫沉淀法与CD38分子原核表达产物的Western-blot分析鉴定单克隆抗体的特异性。以MTT（四甲偶氨唑盐）法检测单克隆抗体抑制细胞增殖以及介导补体杀伤靶细胞的功能。结果：获得了一株抗人CD38分子的单克隆抗体1C6，流式细胞术显示它具有CD38抗原特异的细胞反应谱，其识别的抗原分子质量为45000u。与CD38分子原核表达产物的Western-blot分析表明，其可特异识别CD38分子胞外结构域。MTT法显示其可介导补体杀伤靶细胞。结论：成功制备了抗人CD38分子的单克隆抗体，并进行了初步的功能研究。     

Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4⁺ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4⁺ cells were induced to proliferate and to synthesize interleukin 2 (IL2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL2-producing clones bearing either CD4 or CD8 on their surface. IL2 production was induced by mAb B66 in CD4+, but not CD8⁺, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4⁺ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4⁺ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.     

Monoclonal antibodies that identify the CD3 molecules expressed specifically at the surface of porcine gammadelta-T cells

The CD3 antigen is a surface structure associated with the T-cell receptor (TCR) to form a complex involved in antigen recognition and signal transduction. Reports on the structures of the CD3 molecules associated with alphabeta- and gammadelta-TCR have been contradictory. To investigate this issue, we raised a panel of monoclonal antibodies (mAb) against purified porcine CD3 molecules. Unlike the conventional anti-CD3, these mAb reacted specifically with peripheral gammadelta-T cells, but not with alphabeta-T cells. Immunoprecipitation showed that the antibody recognized a subset of CD3 molecules that were associated with gammadelta-TCR. Also unlike the conventional anti-CD3, these mAb, though directed at two different epitope groups, failed to induce antigenic modulation, T-cell proliferation and CD3-redirected cytotoxicity. Taken together, these results suggest that there are differences in the antigenicity, signal transduction potentials and probably structural differences between the CD3 molecules expressed at the surface of alphabeta- and gammadelta-T cells.     

Positive signal transduction via surface CD4 molecules does not need coexpression of the CD3/TcR complex.

We previously reported that the human CD4 molecule is capable of transducing a positive signal when activated by an anti-CD4 mAb B66. This antibody, in contrast to many other anti-CD4 mAb, induced IL2 production and proliferation of resting CD4+ peripheral blood T lymphocytes in the absence of any other signal. We further reported that anti-CD4 mAb B66 was able to induce IL2 production in murine T-cell hybridoma cells transfected with full-length human CD4 cDNA. In the present study, we extend these findings by demonstrating that anti-CD4 mAb B66 was able to induce Ca2+ mobilization and IL2 production in a CD3/TcR- variant 31-13, of the CD3/TcR+ Jurkat cell line. We further showed that anti-CD4 mAb B66 was able to activate CD4+ cells from the promonocytic cell line U937. In these cells, mAb B66 induced Ca2+ mobilization when cross-linked with a second antibody and, in addition, the production of large quantities of IL1 beta was measured. In essence, our findings provide direct evidence that cross-linking of CD4 may cause T-cell activation in the absence of the coexpression of the CD3/TcR molecular complex and that, in addition, CD4 might transduce a positive signal in CD4+ cells of the myeloid lineage.     

Helper effector function of human T cells stimulated by anti-CD3 mAb can be enhanced by co-stimulatory signals and is partially dependent on CD40-CD40 ligand interaction

In this study we have investigated whether anti-CD3-induced human T cell help for immunoglobulin production could be enhanced by co-stimulation of the T cells via other T cell surface molecules, and the contribution of CD40-CD40 ligand interaction to the execution of T helper effector function induced by these different stimulatory signals. In a system in which irradiated tonsillar T cells were stimulated with immobilized anti-CD3 monoclonal antibody (mAb), it was found that ligation of CD2 with a mitogenic pair of mAb considerably enhanced anti-CD3-induced T cell help for immunoglobulin production. Likewise, ligation of CD28 with mAb enhanced T helper activity, although to a lesser extent. Upon addition of anti-CD28 and anti-CD2 mAb together, an even higher immunoglobulin production was observed. This combination resulted in a four- to fivefold increase in immunoglobulin production as compared to cultures in which T cells were stimulated with anti-CD3 mAb alone. The effect of ligation with B7, the natural ligand of CD28, was studied in a system which utilizes the presentation of anti-CD3 mAb on human FcγRII-expressing mouse fibroblasts which were co-transfected with human B7. It appeared that B7 could stimulate help for immunoglobulin production much more efficiently than ligation of CD28 with mAb did. Physical separation of B cells from T cells led to complete abrogation of immunoglobulin production. Blocking of CD40 with specific mAb, which have no intrinsic B cell stimulatory properties, or the CD40 ligand with a soluble CD40-human IgM fusion protein, resulted in dose-dependent, but only partial, inhibition of T cell-dependent immunoglobulin production with all modes of T cell activation tested. A clear correlation was found between the induction of CD40 ligand expression on the T cells by the different modes of co-stimulation and subsequent immunoglobulin production by the B cells. It is concluded that ligation of CD28 and/or CTLA-4, and of CD2 can generate co-stimulatory signals for T cell help for immunoglobulin production, which was found to be only partially dependent on the CD40-CD40 ligand interaction.     

Single-shot plasmid DNA intrasplenic immunization for the production of monoclonal antibodies. Persistent expression of DNA

Monoclonal antibodies (Mc. Abs.) were generated against a 18-kDa protein from Brucella abortus 48 h and 25 days after a single intrasplenic injection of a DNA plasmid containing the expression vector for the protein. Hybridomas were also obtained from spleens injected 3, 5, and 10 days before fusion. Somatic cell fusion of spleen cells from mice, injected with the plasmid DNA, in saline, with the NS-0 myeloma cell line resulted in Mc. Abs of the IgG and IgM Isotypes. IgG antibodies were of the IgG2b and IgG1 subtype. Hybridoma tissue culture supernatants were strongly positive by ELISA at dilutions of up to 1/1200 and produced intense specific bands in immunoblotting. All these antibodies recognized the native recombinant protein (the screening antigen) and some of them also recognized the heat-denatured recombinant 18-kDa protein. When compared to standard procedures of immunization, as well as to intramuscular or gene gun DNA immunizations, this technique results in very early, time saving, strong Mc Abs. It is common knowledge that in order to generate specific hybridomas; spleen cells from immunized animals have to be fused no later than 5 days after the last boost. The fact that through single-shot intrasplenic immunization (SSI) specific hybridomas are generated 25 days after one single injection indicates that the gene coding the p18 protein is being expressed in the spleen for at least 20 days. We propose that plasmid DNA intrasplenic immunization can be a helpful tool for the production of specific hybridomas. This route of immunization could also be helpful in the further understanding of early events of the immune response to genetic immunization by naked DNA injection.     

CD27 synergizes with CD40 to induce IgM, IgG, and IgA antibody responses of peripheral blood B cells in the presence of IL-2 and IL-10

CD40, a member of the tumor necrosis factor receptor (TNFR) family, promotes IgM, IgG, and IgA antibody (Ab) synthesis in combination with a variety of cytokines. Another TNFR family member, CD27, causes B cells to differentiate into antibody-forming cells, with marginal effects on proliferation. In the present study, we examined whether anti-CD27 monoclonal antibody (mAb) modulates the antibody production induced by anti-CD40 mAb immobilized on L cells expressing FcgammaRII (CDw32) in the presence of IL-2 and/or IL-10. The anti-CD40 mAb substantially enhanced IgM, IgG, and IgA production in combination with IL-2 and IL-10, whereas anti-CD27 mAb augmented it only marginally, as assessed by enzyme-linked immunosorbent assay. The addition of anti-CD27 mAb enhanced the anti-CD40-mediated IgM, IgG, and IgA antibody production only when both IL-2 and IL-10 were present in the culture. The CD27-positive B cell compartment generated synergistic antibody responses in response to four different stimulants, anti-CD27/anti-CD40 mAb and cytokines IL-2/IL-10, whereas the CD27-negative B cell compartment failed to do so. Kinetic analysis showed that anti-CD40 might function in the early phase of B cell activation, while anti-CD27 mAb functioned in the late stage. The addition of CD27(-) to CD27(+) B cells in various ratios did not have any effect on the antibody production, suggesting that CD27(+) to CD27(-) B cell interaction does not occur in this system. Our findings suggest that a member of the TNFR family, CD27, cooperates with CD40 to induce efficient antibody production in combination with cytokines IL-2 and IL-10.     

CD23/CD21 interaction is required for presentation of soluble protein antigen by lymphoblastoid B cell lines to specific CD4+ T cell clones

Previous studies have documented a role for membrane-bound CD23 (the low affinity FcεRII) in presentation of alloantigens by B cells. The aim of the present study was to examine the involvement of cell surface CD23 in presentation of more conventional soluble protein antigens to T cells. We show that antibodies to CD23 and to its lymphocyte-associated second ligand, CD21, inhibit presentation of the cow's milk allergen casein, by autologous CD23⁺CD21⁺ B-EBV cell lines to casein-specific HLA-DP-restricted CD4⁺ T cell clones obtained from patients with either reaginic or enterophatic forms of cow's milk protein intolerance. Maximal inhibition was achieved when the antibodies were added at the initiation of the culture. The absence of specific inhibition by an anti-DRα monoclonal antibody (mAb) argues against a steric hindrance phenomenon impeding access of the T cell receptor to major histocompatibility complex class II molecules. Rather, anti-CD23 and anti-CD21 mAb-induced inhibition of antigen presentation seems to affect at least partly, heterotypic conjugate formation through CD23/CD21 interaction. Double immunofluorescence labeling of the T cell clones and antibody inhibition of T/B conjugate formation shows that functional CD23 and CD21 molecules are induced on T cells following contact with B-EBV cell lines. Taken together, these data indicate that CD23/CD21 interactions between T and B cells are required for presentation of soluble protein antigens by B-EBV cell lines to specific CD4⁺ T cells. The potential implications of these findings for allergen-specific T cell activation are discussed.     

一株识别CD40分子新位点的单克隆抗体研制及生物学特性鉴定

目的：研制识别CD40分子新位点的单克隆抗体（mAb）。方法：以转人CD40转基因细胞L929-CD40为免疫原，常规免疫6—8周龄的雌性BALB／c小鼠；采用B淋巴细胞融合技术，将免疫小鼠脾脏细胞与Sp2／0融合，以L929-CD40转基因细胞为抗体筛选阳性细胞，经免疫荧光标记分析对杂交瘤进行反复筛选和多次的克隆化培养；采用快速定性试纸法及竞争抑制结合试验分析了该mAb的亚类及抗原识别位点；采用MTT法分析mAb在体外对肿瘤细胞的促增殖效应、ELISA法测定细胞因子分泌以及PI染色分析细胞周期。结果：获得1株持续、稳定分泌鼠抗人CIM0mAb的杂交瘤细胞株（命名为286），该mAb能特异性地识别人CD40分子并较好的识别肿瘤细胞株H08910表达的CD40分子并能够体外促进肿瘤细胞增殖。结论：成功地研制1株识别CD40新位点mAb，该mAb能够很好地识别肿瘤细胞表达的CD40分子并具有体外促进肿瘤细胞生长的作用。     

Role of the adhesion molecule ICAM-1 (CD54) in staphylococcal enterotoxin-mediated cytotoxicity.

Staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules on target cells and directs human cytotoxic T lymphocytes (CTL) of irrelevant nominal specificity to mediate strong cytotoxicity against target cells. In this report we describe the importance of ICAM-1 (CD54) expression on the target cell in SEA-dependent cell-mediated cytotoxicity (SDCC), utilizing murine L cells co-transfected with HLA-DR and ICAM-1. Human CTL mediated a low but significant cytotoxicity against HLA-DR2- and HLA-DR7-transfected cells after preincubation with SEA, but no reactivity towards uncoated HLA-DR2 and HLA-DR7 cells or SEA-coated ICAM-1-transfected and untransfected L cells. In contrast, a strong cytotoxic response was mediated by CTL against L cells co-transfected with HLA-DR2/ICAM-1 and HLA-DR7/ICAM-1. Similar cytotoxic activity of the CTL was seen at a 30-fold lower effector-to-target cell ratio when comparing the HLA-DR2/ICAM-1-expressing cells with the HLA-DR2-expressing cells. SEA dose-response analysis demonstrated that the HLA-DR2/ICAM-1-expressing target cells enabled the CTL to respond to a 1000-fold lower concentration of SEA in comparison to the HLA-DR2-expressing cells. CD3+CD4+ and CD3+CD8+ cytotoxic T cell lines were equally dependent on the expression of ICAM-1 on the target cell. The strong CTL activity against HLA-DR2/ICAM-1-transfected cells could be blocked by anti-CD11a or anti-CD18 monoclonal antibodies (mAb), but not by anti-CD11b, anti-CD11c, anti-CD2 or unrelated control mAb. The great sensitivity of HLA-DR2/ICAM-1 expressing target cells to SDCC was strongly reduced by preincubation with various anti-ICAM-1 mAb but not by mAb against monomorphic HLA-DR or murine MHC class I determinants. The result in this study clearly demonstrates that efficient re-targeting of human CTL by SE is dependent on a proper interaction with the heterodimer CD11a/CD18 (Leu-CAMa, LFA-1) on the CTL and its target cell ligand ICAM-1.     

Activation of human B lymphocytes through CD40 and interleukin 4

We have produced and characterized a new CD40 monoclonal antibody, mAb 89, which in the presence of anti-IgM antibodies co-stimulates to induce B cell proliferation. mAb 89 activates resting B cells as shown by an increase in cell volume and an enhanced subsequent proliferation of B cells in response to anti-IgM antibody. However, mAb 89 does not prepare B cells to respond to the growth-promoting activity of interleukin (IL) 2 or IL 4. Unlike IL 2 and IL 4, mAb 89 only weakly stimulates the proliferation of anti-IgM pre-activated B cells. Thus, the activating properties of anti-CD40 are likely to explain its co-stimulatory effect on B cells. Interestingly, the anti-CD40 mAb 89 was found to act in synergy with IL 4, but not with IL 2, in co-stimulation and restimulation assays. In this respect, anti-CD40 does not induce a significant increase of B cell surface IL 4 receptors while IL 4, but not IL 2, induces a twofold increase of the CD40 antigen expression. Thus the synergistic interaction between IL 4 and anti-CD40 may be related to the IL 4-dependent increase of CD40 antigen expression.     

Generation of anti-human CD8 beta-specific antibodies using transfectants expressing mixed-species CD8 heterodimers

The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. The majority of monoclonal antibodies which recognize the human CD8 antigen react with the CD8 alpha chain, while a single mAb, referred to as T8/2T8-5H7 (or 2ST8-5H7), has been identified which binds to the CD8 alpha/beta heterodimer. In order to generate antibodies specific for CD8 beta, murine fibroblast transfectants were constructed which express the human CD8 beta chain in combination with either the human CD8 alpha chain or the murine CD8 alpha homologue, the Lyt-2 molecule. These transfectants were used to raise polyclonal heteroantisera in mice. Transfectants expressing human CD8 alpha/beta heterodimers induced moderate anti-CD8 alpha titers, but were weakly effective in generating anti-CD8 beta titers, despite high level cell surface expression of this protein. In contrast, transfectants expressing mixed-species CD8 heterodimers (murine CD8 alpha and human CD8 beta) induced high anti-CD8 beta titers in immunized mice. Following fusion of splenocytes from mice immunized with mixed-species CD8 transfectants, the mAb 5F2 was isolated which specifically recognizes the human CD8 beta chain. Unlike T8/2T8-5H7, the mAb 5F2 can bind the CD8 beta chain irrespective of its pairing partner, and can immunoprecipitate the CD8 beta protein from cells transfected with the CD8 beta gene in the absence of the human or mouse CD8 alpha gene product. Anti-CD8 beta antibodies should help elucidate the extent of biochemical heterogeneity of the CD8 beta protein, and will also be useful in defining the role of the CD8 beta protein in thymocyte and lymphocyte development, recognition and activation.