Induction of anergy in resting human T lymphocytes by immobilized anti-CD3 antibodies

How the T cell receptor (TcR)/CD3 complex mediates not only the induction of T cell activation but also suppressive effects like T cell anergy or apoptosis is not well understood. Here we describe a series of preincubation and restimulation experiments which demonstrate that primary stimulation of resting, unseparated human T cells with mitogenic doses of immobilized anti-CD3 antibodies induces hyporesponsiveness upon restimulation of the cells. Various costimuli can prevent this type of anergy to a variable degree if present during the preincubation period, phorbol 12-myristate 13-acetate (PMA) being the most and anti-CD4 antibody the least effective. If employed together with anti-CD3 antibody during the restimulation phase of the assay, interleukin (IL)-2, IL-4 and anti-CD28 antibody break anergy almost completely. Proliferation induced by a submitogenic dose of anti-CD3 antibody supplemented by costimulatory signals (anti-CD2, anti-CD4, anti-CD28, IL-2, IL-4 or PMA) does not result in hyporesponsiveness. Taken together, these results support a modified view of the two-signal model for T cell activation according to which anergy induction in resting T cells occurs if primary proliferation is induced by high density triggering of the TcR/CD3 complex in the absence of accessory signals. We discuss possible implications of these findings for the induction of peripheral tolerance.     

Activation of resting T lymphocytes by cross-linked anti-CD3 (T3)

In this study we have examined the role of small numbers of adherent cells in the stimulation of highly purified resting T lymphocytes by cross-linked monoclonal anti-CD3 antibodies (T3). T cells were obtained by 3 cycles of purification, using adherence to plastic surface, nylon wool column separation and rosetting with 2-aminoethyl isothiouronium bromide-treated sheep red blood cells. They were stimulated either with T3 antibodies or with solid-phase rabbit anti-mouse IgG-T3 complexes. In standard high density cultures (1 X 10(5)-2 X 10(5) cells), soluble T3, interleukin (IL) 1 and IL 2, used separately, did not induce proliferation of T cells. Soluble T3 together with IL 2 slightly activated T cells to proliferate. Rabbit anti-mouse IgG-T3 complexes attached to the plastic wells induced stimulation in some cultures. The response was markedly increased after addition of IL 2, but not IL 1. The irregular response of these cultures suggested the presence of another variable signal. When cell numbers were reduced (12 X 10(3) cells), neither cross-linking of the CD3-Ti complex nor addition of exogenous IL 1 or IL 2, alone or in combination, stimulated the T cells to increase DNA synthesis. A positive response, comparable to complete peripheral blood mononuclear cells, was restored with 0.5% supplemented adherent cells, provided that IL 2 was present.     

Generation of alloreactive cytolytic T lymphocytes by immobilized anti-CD3 monoclonal antibodies. Analysis of requirements for human cytolytic T-lymphocyte differentiation.

Requirements for the induction of human cytolytic T-lymphocyte (CTL) activity were studied in a monocyte-free T-cell activation system that uses immobilized anti-CD3 monoclonal antibodies (mAb) as a stimulus. Alloreactive CTL with specificity for HLA-A and -B locus antigens could be demonstrated within 2 days after the initiation of activation. CTL induction in purified T cells initiated by an optimal concentration of immobilized anti-CD3 mAb was not enhanced by the addition of monocytes or exogeneous cytokines, whereas addition of anti-CD25 mAb largely blocked the response. Upon suboptimal anti-CD3 mAb stimulation, addition of recombinant interleukin (rIL)-2, rIL-1 and rIL-4, but not recombinant interferon-gamma (IFN-gamma) or rIL-6, potentiated the development of CTL activity. Finally it was shown that immobilized anti-CD3 mAb induced significant levels of CTL activity in both purified CD4+ and CD8+ cells. This study indicates that the requirement for cytokines in the differentiation of CTL precursors depends on the strength of the activation signal delivered through the T-cell receptor.     

Two monoclonal anti-CD3 antibodies can induce different events in human T lymphocyte activation

Two monoclonal antibodies, WT32 and CLB-T3/4.2a, directed against the CD3 complex were used to study the mechanism of activation of human peripheral T lymphocytes. WT32, a mouse monoclonal IgG2a antibody with a low avidity (much less than OKT3) for the CD3 complex, effectively induces mitogenesis of purified T lymphocytes when used in the 1 ng-10 micrograms range in the presence of monocytes or recombinant interleukin 2 (IL2). In contrast, CLB-T3/4.2a, a mouse monoclonal antibody of the same isotype with a high avidity (much greater than OKT3) for the CD3 complex, induces IL2 receptor expression and IL2 responsiveness only at very low concentrations (less than 5 ng/ml), yet in the presence of monocytes this antibody induces proliferation within a similar dose range as WT32. Apparently, in the absence of accessory cells which can cross-link the antibody CD3 complexes, the binding properties (avidity) of an antibody and thereby the number of receptors that are occupied are important parameters for induction of IL2 responsiveness. Furthermore, we show that Ca2+ mobilization only occurs when the cells are stimulated by saturating amounts of antibody, so that, when the conditions are optimal for the induction of IL2 responsiveness, no Ca2+ mobilization will be detected.     

Immobilized anti-CD3 monoclonal antibodies induce accessory cell-independent lymphokine production, proliferation and helper activity in human T lymphocytes.

Monoclonal antibodies (mAb) directed against the human CD3 molecular complex are able, when immobilized on the plastic of microtitre wells, to induce accessory cell-independent T-cell proliferation. In this study, we show that the anti-CD3 mAb CLB-T3/3 induces strong T-cell stimulation that is proportional to the density of the immobilized antibody. T cells, optimally stimulated with plastic-immobilized CLB-T3/3, showed a five-fold higher proliferation compared to cells that were stimulated with soluble anti-CD3 in the presence of accessory cells. The difference in magnitude of proliferation was found to be correlated with the expression of the CD25 (TAC) antigen and the production of interleukin (IL)-2, but not with the number of high-avidity IL-2 receptors expressed on the surface of these differentially activated cells. In addition, immobilized CLB-T3/3 initiated the production of interferon-gamma (IFN-gamma), but not of IL-4, in purified T lymphocytes. Coated anti-CD3 mAb induced helper activity in T cells for IgM and IgG production by B lymphocytes. Whereas addition of IL-1 or IL-2 had only a moderate effect on T-cell proliferation induced by immobilized anti-CD3 mAb, helper activity was strongly enhanced in the presence of these factors. This T-cell activation system may prove useful for a standardized analysis of both activation requirements and immunoregulatory capacities of human T cells.     

The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells.

Antigen-specific T cell clones are useful reagents for studies of the fine specificity of antigen recognition and of potential therapeutic use in adoptive immunotherapy for human viral and malignant diseases. Culture methods which require antigen and APC for stimulation can be problematic for the generation and long-term growth of human virus and tumor-specific T cells. We have developed an alternative culture method using monoclonal antibodies to T cell activation molecules, CD3 and CD28, as stimulation to efficiently grow CD4+ and CD8+ antigen-specific T cells from single progenitors and expand T cell clones in long-term culture. This method alleviates the requirement for large amounts of viral or tumor antigens and MHC compatible APC to sustain the growth of virus and tumor-specific T cell clones, and, as demonstrated for CD8+ CMV-specific cytotoxic T cells, overcomes the difficulties cloning CD8+ T cells using virally infected cells as antigen-presenting cells. T cell clones generated and maintained with monoclonal antibody stimulation are rapidly expanded and retain antigen-specific responses after 3 months in culture, suggesting this approach may prove useful for growing large numbers of antigen-specific T cell clones for cellular immunotherapy.     

Comparison of the staining of peripheral blood T lymphocytes by various anti-CD8 and HLA-DR monoclonal antibodies

T cell subset analysis with monoclonal antibodies is performed in variety of clinical situations, including the monitoring of transplant recipients. We compared the percentages CD8+ cells in the peripheral blood of ten normal volunteers and 26 renal transplant patients using fluorescein isothiocyanate (FITC)- or phycoerytrin (PE)-conjugated Leu2a and Leu2b monoclonal antibodies (mAb). Furthermore, the percentage of HLA-DR+ cells within this population was determined using two anti-HLA-DR antibodies of differing isotype (designated 203 and 243) and either an FITC or a PE label. For both controls and transplant recipients, the PE-conjugated Leu2a and Leu2b mAbs gave significantly higher percentages of positive cells than the FITC-conjugated antibodies (P less than 0.01). Furthermore, the percentage of Leu2b+ cells was higher than the percentage of Leu2a+ cells, irrespective of the fluorochrome used (P less than 0.01); this was particularly true for samples with less than or equal to 10% Leu2a+ cells. Cell sorting experiments indicated that up to 30% of the Leu2a- population were able to bind OKT8 or Leu2b mAb in blood samples with a low percentage of Leu2a+ cells. The percentage of Leu2+ cells that were stained with anti-HLA-DR mAb 203 or 243 varied considerably, depending on the fluorochrome and the isotype of the antibodies. We conclude that the analysis of peripheral blood T cells with mAbs that apparently have the same specificity may give significantly different results, depending on the patient population, the fluorochrome and the isotype of the antibodies.     

Regulation of T cell proliferation by anti-CD49d and anti-CD29 monoclonal antibodies.

The beta 1 integrin VLA-4 (alpha 4 beta 1, CD49d/CD29), which is expressed on a large subpopulation of peripheral blood T lymphocytes, functions as a receptor for the endothelial adhesion protein VCAM-1 and the extracellular matrix protein fibronectin. Previous studies showed that immobilized fibronectin enhanced anti-CD3 monoclonal antibody (mAb)-induced T cell proliferation through binding to the integrins VLA-4 and VLA-5 (alpha 5 beta 1, CD49e/CD29). We studied the ability of the anti-CD49d mAb L25 to potentiate proliferation. T cell proliferation was induced by subthreshold concentrations of anti-CD3 mAb (mAb OKT3) coimmobilized with mAb L25 but not with coimmobilized anti-CD29 (beta 1) mAb. Soluble anti-CD29 mAb inhibited the proliferation induced by coimmobilized mAb OKT3 and L25 but not proliferation induced by mAb OKT3 with PMA or coimmobilized anti-CD26 mAb.     

Human T cell activation: differential response to anti-CD28 as compared to anti-CD3 monoclonal antibodies

Monoclonal antibodies (mAb) against CD3 or CD28 in conjunction with the tumor promoter phorbol 12-myristate 13-acetate (PMA) induce interleukin 2 receptor (IL2R) expression, IL2 production and proliferation in resting T cells. Recent studies indicate that these two pathways are biochemically distinct. In this study T cell activation induced by PMA and anti-CD28 mAb 9.3 is compared to the effects of PMA plus anti-CD3 mAb (T3-II and 235) in the presence or absence of cyclosporin A (CsA), dibutyryladenosine 3':5' cyclic monophosphate (db-cAMP) or cholera toxin (CT). Proliferation of T cells stimulated with PMA plus mAb 9.3 is resistant to the inhibitory effects of CsA, db-cAMP and CT. Only at the highest dose did CsA have any effect on PMA plus mAb 9.3-induced T cell proliferation. Conversely, CsA, db-cAMP and CT inhibit PMA plus T3-II-induced T cell proliferation. mRNA analysis further demonstrates the similarities and the differences between the CD28 and CD3 activation pathways. Recently, T3-II was reported to induce tumor necrosis factor (TNF) and lymphotoxin (LT) mRNA synthesis in PMA-treated T cells. In this study mAb 9.3 is shown to substitute for T3-II in the induction of TNF and mRNA. However, the production of TNF and LT mRNA in PMA plus mAb 9.3-treated T cells is greater than that seen in PMA plus T3-II-treated cells. mRNA synthesis included by PMA plus T3-II is blocked by CsA. mRNA production in T cells activated with PMA plus mAb 9.3 is resistant to CsA. Similar results are noted with IL2 and IL2R mRNA. Flow cytometric analysis of the IL2R confirms the mRNA data. CsA blocks the T3-II-induced potentiation of PMA-induced IL2R expression but not the mAb 9.3-induced potentiation. This differential inhibitory effect of CsA on IL2R expression is also seen with db-cAMP and CT. We examined the effects of these two pathways on the expression of the early activation antigen EA 1 and cytoplasmic free calcium. Recently, we have shown anti-CD3 mAb potentiate EA 1 expression induced by 1,2-sn-dioctanoylglycerol and this potentiation is calcium dependent. dp-cAMP blocks T3-II- and 235-induced potentiation of EA1 expression and inhibits the T3-II- and 235-mediated rise in intracellular free calcium [( Ca2+]i). Conversely, 9.3 does not potentiate EA 1 expression or induce a rise in [Ca2+]i. These results provide further evidence that the CD28 and CD3 activation pathways utilize distinct signal transduction pathways.     

Production of human allotype-specific anti-CD45 monoclonal antibodies.

The aim of this work was to raise allotype specific monoclonal antibodies to human CD45, with the long-term objective of producing a reagent which could be used to prolong graft survival in renal transplantation through removal of passenger leukocytes from the graft. At present there are no anti-CD45 monoclonal antibodies able to distinguish between host and donor leukocytes. An in vitro immunisation technique has been developed through which donated human leukocytes are sensitised to CD45 prior to fusion with a myeloma cell line. IgM was produced by all the anti-CD45-positive clones. Flow cytometric analysis using these antibodies showed their ability to differentiate between blood from individual donors, indicating the existence of allotypic forms of human CD45, in conformity with the findings in rats and pigs. Therefore, a reagent which could be used in renal transplantation is a technical possibility.     

Mechanisms of killing by anti-CD20 monoclonal antibodies

CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells, but not stem or plasma cells. It is an ideal target for monoclonal antibodies (mAb), such as rituximab and ofatumumab, as it is expressed at high levels on most B-cell malignancies, but does not become internalized or shed from the plasma membrane following mAb treatment. This allows mAb to persist on the cell surface for extended periods and deliver sustained immunological attack from complement and FcR-expressing innate effectors, particularly macrophages. CD20 can also generate transmembrane signals when engaged by certain mAb which, although unproven, might provide an important element of the therapeutic success of anti-CD20 mAb. These favourable characteristics have led to anti-CD20 mAb being developed and exploited for use in immunotherapy, where they have proven remarkably efficacious in both the treatment of malignant disease and autoimmune disorders by deleting malignant or normal B cells, respectively. In this review, we discuss how these mAb have driven research in the immunotherapy field over the last decade, detail their likely modes of action and their limitations in terms of effector exhaustion, and explore ways in which they might be enhanced and further exploited in the future.     

Mechanisms of killing by anti-CD20 monoclonal antibodies

CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells, but not stem or plasma cells. It is an ideal target for monoclonal antibodies (mAb), such as rituximab and ofatumumab, as it is expressed at high levels on most B-cell malignancies, but does not become internalized or shed from the plasma membrane following mAb treatment. This allows mAb to persist on the cell surface for extended periods and deliver sustained immunological attack from complement and FcR-expressing innate effectors, particularly macrophages. CD20 can also generate transmembrane signals when engaged by certain mAb which, although unproven, might provide an important element of the therapeutic success of anti-CD20 mAb. These favourable characteristics have led to anti-CD20 mAb being developed and exploited for use in immunotherapy, where they have proven remarkably efficacious in both the treatment of malignant disease and autoimmune disorders by deleting malignant or normal B cells, respectively. In this review, we discuss how these mAb have driven research in the immunotherapy field over the last decade, detail their likely modes of action and their limitations in terms of effector exhaustion, and explore ways in which they might be enhanced and further exploited in the future.     

抗CD71人-鼠嵌合抗体对活化淋巴细胞的效应

目的　通过体外实验探讨抗CD71人 鼠嵌合抗体对活化淋巴细胞效应的影响 ,并与其鼠源性单克隆抗体进行比较。方法　以丝裂原诱导的人外周血单个核细胞 (PBMC)为靶细胞 ,测定嵌合抗体和鼠源性单抗对其增殖抑制率 ;在新鲜补体存在下 ,测定补体依赖性嵌合抗体介导的细胞毒效应(CDC)。以EBV转化的B细胞为刺激细胞 ,测定两种抗体对其诱导的同种异体PBMC的增殖抑制率 ;以同种异体的PBMC为刺激细胞 ,测定两种抗体在单向、双向混合淋巴细胞培养 (MLC)中的增殖抑制率。结果　嵌合抗体和鼠源性单抗均可明显抑制丝裂原诱导的PBMC的增殖反应 ,且二者抑制率差异无显著性(P >0 .0 5 ) ,其抑制作用随抗体浓度增加而增强 ,PBMC和丝裂原共同孵育 12h后加入抗体的增殖抑制效应最明显 ;在新鲜补体存在下 ,嵌合抗体对丝裂原诱导增殖的PBMC具有CDC作用 ,而鼠源性单抗CDC作用较弱 ;两种抗体对混合淋巴细胞培养反应有明显的抑制作用 ,且嵌合抗体组抑制率明显高于鼠源性单抗组 (P <0 .0 5 )。结论　抗CD71人 鼠嵌合抗体在体外实验中可抑制淋巴细胞的活化及其效应 ,其作用明显强于抗CD71鼠源性单克隆抗体。     

Synergistic stimulation of human B lymphocytes by anti-CD40 monoclonal antibodies and synthetic lipopeptide analogues from Escherichia coli lipoprotein.

Human tonsillar B lymphocytes were stimulated with synthetic lipopeptide analogues of Escherichia coli lipoprotein alone or together with anti-CD40 and/or interleukin-4 (IL-4). While lipopeptides alone or lipopeptides plus IL-4 did not include proliferation of B lymphocytes, synergistic stimulation was observed when anti-CD40 antibodies were added. Proliferation was even more pronounced in the presence of Fc receptor type II (FcRII)-transfected L cells. Compared to the stimulus anti-CD40 plus IL-4 plus FcRII-transfected fibroblasts exerted, the addition of lipopeptides induced a more rapid maximal response which peaked on day 4. Antibody production could also be enhanced by lipopeptides. Our data provide evidence that lipopeptides, which act as mitogens toward murine B lymphocytes, also stimulate human B lymphocytes, provided that co-signals are added.     

Isolation and partial characterization of a novel subset of human T lymphocytes defined by monoclonal antibodies

Delayed-type hypersensitivity (DTH) responses to alloantigens were found to correlate with both skin and tumour allograft rejection in 224 reconstituted ATXBM-CBA mice. Furthermore, DTH responses and allograft rejection were observed only in mice that had received Ly-1 cells. Depletion of Thy-1+ or Ly-1+ cells led to indefinite graft survival and the absence of DTH responses, whereas depletion of Ly-2+ cells led to rapid graft rejection and strong DTH responses. The same result was obtained with CBA mice responding to grafts of either C57BL/6 skin, the B16 melanoma, or the EL4 lymphoma; and for (CBA X A)F1 mice responding to H-2K region alloantigens of AQR skin grafts. Thus, DTH and allograft rejection are both mediated by a Ly-1 T cell and it is considered that these are two different manifestations of the same transplantation response.     

Induction of T-cell proliferation by recombinant mouse and chimeric mouse/human anti-CD3 monoclonal antibodies.

The isotype of anti-CD3 mAb has a dramatic effect on anti-CD3 induced T-cell activation, as was previously reported for switch variants (IgG2b to IgA) of a high-avidity IgG1 anti-CD3 mAb (CLB-T3/4.1). In order to study and compare the isotype dependency of T-cell activation with anti-CD3 mAb of various mouse and human subclasses, we now prepared recombinant anti-CD3 mAb. The variable region of the anti-CD3 Ig heavy chain was cloned, joined with genes for the heavy chain constant region and expressed in a cell line only secreting autologous mouse chi light chains. Thus we obtained cell lines that produced mouse (m) IgM, mIgG3 and chimaeric mouse/human (h) IgM, hlgG1, hlgG2, hlgG3, hlgG4, hlgE and hlgA2 anti-CD3. The matched set of mouse and mouse/human chimaeric anti-CD3 isotypes switch variants was then used to study activation of T cells in an accessory cell-dependent system. hlgG1, hlgG4, hlgE, mlgG2a and mlgE induced T-cell proliferation in PBMC of all donors tested, whereas PBMC from a subset of donors were unresponsive to stimulation with hlgG2, hlgG3, hlgA2, mlgG1 and mlgG2b anti-CD3 mAb. hlgM, mlgM and mlgA were only able to induce T-cell mitogenesis in combination with PMA. Our panel of anti-CD3 mAb variants may prove a powerful tool to study mouse and human isotype-dependent effector functions and their influence on T-cell activation requirements in detail.     

CD3抗体诱导的新生儿脐带血淋巴细胞信号传导的研究

目的：从TCR／CD复合物信号传导的角度，研究CD3抗体、PMA和ionomycin对新生儿脐带血淋巴细胞增殖及胞内钙离子浓度的影响，以及与磷脂酶Cγ1（PLCγ1）表达的关系。方法：利用^3H-TdR渗入法检测淋巴细胞的DNA合成；利用荧光分光光度法检测淋巴细胞内钙离子浓度；利用蛋白印迹法检测PLCCγ1的表达。结果：应用CD3抗体或PMA＋ionomycin协同处理新生儿脐带血和成人外周血淋巴细胞，其DNA合成和胞内钙离子浓度均明显增高，并且新生儿脐带血淋巴细胞的DNA合成和胞内钙离子浓度明显低于成人外周血水平，但是，与CD抗体相比，PMA＋ionomycin协同所诱导的新生儿脐带血淋巴细胞的增殖接近成人外周血水平，用CD3抗体处理前后的新生儿脐带血淋巴细胞PLCγ1的表达均明显低于成人外周血水平，结论：新生儿脐带淋巴细胞信号传导发育的不完善可能主要存在于细胞活化的早期阶段，并且这种不完善的原因可能与PLCγ1表达降低以及不能有效地被激活有关。