Activation of human T lymphocytes by crosslinking of anti-CD3 monoclonal antibodies

The proliferation of human T lymphocytes induced by anti-CD3 monoclonal antibodies (mAb) is used as a model for antigen-induced activation via the T cell receptor-CD3 complex. Since both systems are accessory cell (AC)-dependent, an understanding of the role of AC in anti-CD3-induced proliferation may provide an understanding of physiological activation via the T cell receptor. Previous work has implicated receptor crosslinking as an important AC function. To determine its necessity in anti-CD3-induced lymphocyte proliferation, we prepared highly purified T lymphocytes and found that these cells did not respond to the anti-CD3 mAb UCHT1, either alone or with interleukin 1 (IL1), interleukin 2 (IL2), or tetradecanoyl phorbol acetate (TPA). However, the response, as measured by appearance of IL2 receptors and proliferation, was restored by crosslinking with immobilized goat anti-mouse antibodies (GAM) and did not require the addition of IL1, IL2, or TPA. Thus, crosslinking of CD3 receptors was a sufficient signal for proliferation of these cells. Cyclosporine A (CsA) inhibited the activation induced by immobilized UCHT1. Since macrophages are the principle targets of CsA-mediated suppression of mitogen-induced proliferation, but macrophages do not participate in the response to immobilized anti-CD3, this may indicate that CsA was inhibiting crosslinking or a signal generated by it.     

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.     

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.     

Perturbation of naive TCR transgenic T cell functional responses and upstream activation events by anti-CD4 monoclonal antibodies.

It is well established that non-depleting anti-CD4 monoclonal antibodies (mAb) have potent immunomodulatory properties in vivo and as such can induce a profound state of tolerance. Receptor blockade, CD4 modulation, or the transmission of a negative signal have all been proposed to explain their effects, however their precise mechanism of action, particularly at the level of cellular signaling, remains obscure. Experiments were thus carried out to examine the underlying mechanisms of action of two non-depleting anti-mouse CD4 mAb, YTS177 and KT6, which differ in their ability to modulate CD4 expression. The effects of the mAb were examined on CD4(+) T cells derived from D0.11.10 TCR transgenic mice. Functional studies revealed that both mAb could effectively block antigen-driven proliferation and IL-2 production but had only modest effects at higher peptide doses. Importantly, mAb pre-treatment of T cells stimulated by sub-optimal peptide seemed to induce an anergy-like state making them unresponsive to subsequent re-stimulation. Analysis of intracellular signaling demonstrated that certain key upstream events such as the phosphorylation of Zap-70 and LAT were also blocked by mAb pretreatment which may be due to interference with stable T cell-APC conjugate formation.     

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.     

Signals involved in T cell activation. T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies

Monoclonal antibodies (mAb) directed against the T cell differentiation antigen CD28 (Tp44) induce proliferation of resting T lymphocytes in the presence of phorbol esters. Moreover, it has been reported that such antibodies augment and sustain T cell proliferation induced by soluble antigens, phytohemagglutinin and anti-CD3 mAb. Recently, we have shown that in monocyte-depleted T cell suspensions, anti-CD28 mAb 9.3 and Kolt-2 were able to circumvent the requirement for interleukin 2(IL2) in T cell proliferation induced by soluble anti-CD3 antibodies. Apart from the synergy of anti-CD28 antibodies with phorbol myristate acetate and anti-CD3 antibodies, we found that anti-CD28 mAb were able to induce T cell mitogenesis in combination with an E rosette-blocking anti-CD2 antibody. In this report, we show that antibodies directed against different epitopes on the CD2 antigen can synergize with anti-CD28 mAb. Furthermore, we demonstrate that proliferation induced through the synergistic action of anti-CD28 mAb with anti-CD2 antibodies can be induced in the absence of accessory cells and is accompanied by the production of IL2 and the expression of IL2 receptors. We were unable to induce detectable Ca2+ mobilization through the simultaneous binding of anti-CD28 and anti-CD2 mAb. Taken together, these data show that IL2-dependent proliferation can be induced through the simultaneous binding of anti-CD28 and anti-CD2 antibodies, possibly through phosphatidyl inositol-independent pathways. The observations may provide further insight into the activation mechanisms of human T cells.     

Isologous monoclonal antibodies can induce anti-GBM glomerulonephritis in rats.

Injection of isologous monoclonal antibodies (SR2, SR3) caused anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY/NCrj rats. The antibodies were obtained from hybridoma cells derived from fusion of the spleen of a nephritic WKY/NCrj rat injected with rat solubilized renal basement membranes with adjuvant, and mouse SP2-myeloma cells. They belonged to the rat IgG2a subclass and bound to rat kidney in a linear pattern along the glomerular and tubular basement membranes. Histological changes in glomeruli were detected at day 1 after the injection; proteinuria with haematuria appeared on day 2; and proteinuria became severe and reached a plateau by day 5. These results demonstrate that anti-GBM nephritis can even be induced by an isologous monoclonal antibody and that the rat IgG2a subclass is at least nephritogenic. The experimental model of anti-GBM nephritis with isologous monoclonal antibodies makes it possible and easier to analyse further the mechanism of anti-GBM nephritis.     

CD3-mediated T cell activation is inhibited by anti-CD44 monoclonal antibodies directed to the hyaluronan-binding region.

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1-->S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

T cell activation by monoclonal antibodies directed to different epitopes on the human T cell receptor/CD3 complex: evidence for two different modes of activation

The mouse monoclonal antibody (mAb) BMA031 (IgG2b) has recently been described to be directed against a monomorphic part of the human T cell receptor (TcR) alpha/beta. In vitro analysis of its stimulatory potential for mononuclear cells revealed two patterns of responsiveness. Out of 35 tested individuals only 2 generated a proliferative response to low antibody concentrations (15 ng/ml; "high responders"), the others ("low responders") responded only to high antibody concentrations (1.5 micrograms/ml); the anti-CD3 mAb UCHT1 (IgG2b) stimulated only the two high responders. This response pattern to BMA031 was determined by the accessory cell compartment in the culture. Stimulation by BMA031 in low responders demonstrated some unusual features: (a) high antibody concentrations were required, (b) addition of autologous serum had no inhibitory effect and (c) vigorous depletion of macrophages reduced but did not abolish the proliferative response. These characteristics were shared by two other mAb, BMA032 and BW239/347, presumably directed against the TcR alpha/beta but not by several other antibodies to the TcR/CD3 complex. Thus, the results demonstrate unusual stimulatory properties of three anti-TcR alpha/beta mAb, inducing a proliferative response without antibody cross-linking. This suggests that the stimulatory effect of anti-TcR/CD3 complex mAb is not only determined by their isotype, but also strongly depends on their epitope specificity.     

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.     

Signals delivered via the Qa-2 molecule can synergize with limiting anti-CD3-induced signals to cause T lymphocyte activation.

Qa-2 is a glycolipid anchored, MHC encoded class I molecule expressed at high levels on all murine peripheral T lymphocytes. Anti-Qa-2 antibodies have previously been found to stimulate T cells to proliferate in the presence of crosslinking antibody and PMA. We have examined the effect of anti-Qa-2 antibodies on T cells stimulated with a suboptimal concentration of immobilized anti-CD3. When anti-Qa-2 antibodies were co-immobilized with limiting anti-CD3, in the absence of PMA, a clear augmentation of T cell proliferation was seen. Interestingly, the co-stimulatory anti-Qa-2 antibodies could be directed against epitopes mapped to either the alpha 3 or the alpha 1/alpha 2 Qa-2 domains. As was the case with activation induced by soluble/crosslinked anti-Qa-2 antibodies plus PMA, CD8+ T cells were less able to be costimulated with anti-Qa-2 antibodies than CD4+ cells. Surprisingly, Ca2+ mobilization was only seen when two anti-Qa-2 antibodies reactive to separate structural domains were co-crosslinked on the surface of Indo-1 loaded T cells with a suboptimal concentration of anti-CD3. Collectively these results raise questions regarding the mechanism of Qa-2 mediated signaling and its potential role in T cell activation.     

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.     

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.     

Requirements for activation of human peripheral blood T cells by mouse monoclonal antibodies to CD3

The present study was undertaken in an attempt to reconcile the conflicting results concerning the signals required for the activation of human resting T cells by antibodies to the T-cell receptor/CD3 complex (Ti/CD3). For this purpose we have used highly purified peripheral blood T cells, depleted of monocytes and of preactivated Ia + T cells, to the extent that they were unable to proliferate to interleukin 2 (IL-2) alone or to optimal doses of phytohemagglutinin (PHA). To further minimize the contribution of contaminating monocytes, we used the anti-CD3 mAb, Leu-4, and cells from Leu-4 nonresponder subjects, whose monocytes we show completely fail to bind the Leu-4 mAb. The parameters of T-cell activation which we measured were rises in intracellular free calcium ion [Ca2+]i, IL-2 receptor expression IL-2 production, and cell proliferation. Our results indicate that induction of proliferation of resting T cells requires at least two signals. Signal one is best delivered by multivalent anti-CD3 mAb, such as Leu-4 mAb covalently linked to Sepharose 4B (Seph-Leu-4), or with Leu-4 mAb and anti-mouse IgG. These reagents crosslink the CD3 receptor complex on the T cell, and result in a rise in intracellular [Ca2+]i, in expression of receptors for IL-2, and in proliferation upon addition of IL-2. In contrast, purified T cells exposed to soluble Leu-4 mAb do not exhibit a rise in intracellular [Ca2+]i, do not express receptors for IL-2, and do not proliferate upon addition of IL-2, indicating that the valency of anti-CD3 mAb is critical for the delivery of the first activation signal to the T cell. The essential step of crosslinking of CD3 antigens on T cells by anti-CD3 mAb is normally mediated by monocytes which have bound anti-CD3 mAbs via their Fc receptors. Monocytes from Leu-4 nonresponder subjects, which we show fail to bind Leu-4 mAb, fail to crosslink CD3 antigens on T cells, resulting in failure of T-cell activation. The second signal needed for the proliferation of T cells whose Ti/CD3 complexes are crosslinked is IL-2. IL-2 production by such T cells required a monocyte delivered signal, which must be delivered to these T cells simultaneously with the crosslinking of their Ti/CD3 antigens. This IL-2-inductive signal can be delivered by both Leu-4 nonresponder and Leu-4 responder monocytes, indicating that delivery of this IL-2 inductive signal is independent of anti-CD3 mAb binding by monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than OKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-β. Both humanized antibodies induced significantly lower production of IFN-γ and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-γ production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with OKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity.     

Clonal activation of cytotoxic lymphocyte precursors by monoclonal anti-CD3 antibody: analysis of feeder cell requirements

Activation of cytotoxic lymphocyte precursors (CLP) by the mitogenic monoclonal anti-CD3 antibody OKT3 was studied under limiting dilution (LD) culture conditions. One out of 2-6 E-rosette-purified T cells gave rise to a cytotoxic T cell (CTL) clone when cultured in the presence of OKT3 (0.2-2 ng/ml), recombinant IL-2 (100 U/ml), and irradiated feeder cells. Clonal CLP activation was optimally supported by a combination of E-rosette-depleted non-T feeder cells with small numbers of T cells added back. Among the cell lines tested, Fc-receptor-bearing monocytic cell lines U937 and HL-60 were efficient feeder cells whereas T cell lines (Jurkat, Molt-4, Ke37) did not support clonal CLP activation. These data indicate that clonal activation of CLP and differentiation into cytotoxic effector cells under LD culture conditions are critically influenced by the type and number of feeder cells used.     

Identification of anti-CD3 antibodies that do not modulate antigen but induce mitogenesis and block the mixed lymphocyte reaction

Using a panel of anti-CD3-TCR monoclonal antibodies (OKT3 A-E), it appears possible to separate the ability to cause surface antigen modulation from inhibition of MLR or induction of mitosis. OKT3D, an antibody that recognizes the CD3 antigen at a site that can be differentiated from the epitopes recognized by other members of this panel by competition binding, does not cause antigen modulation when incubated with human T cells for up to 3 days. Despite this, OKT3D is mitogenic and is capable of blocking MLR. Two different isotypes were produced from the OKT3D clone, IgG1 and IgG2b. The IgG2b isotype of OKT3D blocked MLR even in individuals unable to respond mitogenically to this antibody. Use of members of this panel may now permit dissection of the types of signals delivered by the CD3-TCR complex inducing mitosis, receptor modulation, and other T-cell responses.     

T lymphocyte subpopulations in progressive systemic sclerosis defined by monoclonal antibodies

Twenty two patients with progressive systemic sclerosis were studied by monoclonal antibodies to detect OKT4 and OKT8 positive cells. The absolute number of OKT4 and OKT8 cells was not altered as compared to control values. The ratio of T4/T8 cells slightly increased without statistical significance. The number of E-rosette forming T-cells was reduced in patients with progressive systemic sclerosis. Six months later 11 patients were reinvestigated, and similar results were obtained for the T-cell subsets and the ratio of T4/T8 positive cells. Individual data showed marked variability in the two periods of investigation without however, any correlation to the clinical findings. The sclerodermic patients had a long disease duration and showed no immunoregulatory T-cell imbalance.     

Rapid production of monoclonal antibodies to T lymphocyte functional antigens

Brief immunization of rats with mouse lymphoid cells was combined with the rat/mouse hybridoma technology and functional hybridoma screening to yield a rapid method for the production of monoclonal antibodies (MAb) against functionally important T lymphocyte cell surface antigens. Two protocols were used. In one, rats were immunized once with mouse thymocytes followed by fusion and screening of the hybridomas for interference with the thymocyte co-stimulator (interleukin 1) assay. The resultant hybridomas included producers of MAbs against the L3T4-antigen (inhibitory), the Ly-1-antigen (stimulatory), and the Thy-1-antigen (inhibitory?). In the second protocol, rats were immunized twice with a T cell hybridoma. The resultant hybridomas were screened for inhibition of polyclonal T cell activation, induced by an anti-Thy-1 (MAb G7). A panel of MAbs against the Thy-1 antigen with different reactivity profiles was generated by this procedure. Most of the MAbs were of the IgM class. Short-term immunization may lead to less selection of response to highly immunogenic determinants than a protocol involving several boosters. Thus, this alternative may be useful for producing MAbs against rare or weakly immunogenic cell surface molecules, as suggested by the ease with which we were able to make MAbs against the L3T4-molecule.     

Phagocyte activation with anti-ICAM-3 monoclonal antibodies

Monoclonal antibodies ICO-60 clustered as CDw50 (anti-ICAM-3) drastically boosted the chemiluminescence of human neutrophils, depending on the dose of antibodies, initial chemiluminescence, and individual human reactivity. The contact-adhesion interactions of neutrophils treated with ICO-60 and of intact neutrophils were associated with intensification of chemiluminescence. ICO-60 sharply increased the adhesion and aggregation of human neuroblasts and suppressed these phenomena induced by phorbol ester. The expression of ICAM-3 on neuroblasts is proposed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 4, pp. 447–449, April, 1996 Presented by A. A. Vorob'ev, Member of the Russian Academy of Medical Sciences