A monoclonal antibody specific for immunoglobulin A receptor triggers polymorphonuclear neutrophil superoxide release.

An immunoglobulin M (IgM) monoclonal antibody, My43, specific for IgA Fc receptor (Fc alpha R) on human monocytes, bound to human polymorphonuclear neutrophils (PMNs) and inhibited their ability to bind IgA but not IgG. It was observed that the PMN oxidative burst was induced by both polymeric IgA and aggregated IgG, whereas IgM was without effect. The IgG-mediated oxidative burst was inhibited by anti-Fc gamma RII Fab and anti-Fc gamma RIII F(ab')2 but not by My43. Conversely, the IgA-mediated oxidative burst was inhibited by My43 but not by anti-Fc gamma RII or anti-Fc gamma RIII. When anti-Fc receptor monoclonal antibodies (mAbs) were used directly as ligands, it was observed that both anti-Fc gamma RII Fab and anti-Fc gamma RII F(ab')2 promoted the oxidative burst when cross-linked. Moreover, My43, when cross-linked with F(ab')2 antimouse IgM, also triggered the oxidative burst, whereas an IgM anti-CD15 mAb, PM81, did not stimulate function. This demonstrates that IgA receptors on PMNs are function-triggering molecules and that an anti-IgA receptor mAb may be substituted as a ligand.     

A polymorphic Fc receptor for mouse IgG2b on human B cells and monocytes.

With respect to murine (m)IgG1 anti-CD3 monoclonal antibody (mAb), a polymorphic mitogenic response of peripheral blood mononuclear cells (PBMC) has been described which is caused by polymorphism of monocyte Fc gamma RII, and which defines high responders to mIgG1 (mIgG1-HR, approximately 70% of normal individuals) and low responders (mIgG1-LR). PBMC also exhibit a polymorphic mitogenic response to mIgG2b anti-CD3 mAb. In the present study 18 out of 550 individuals (3%) were mIgG2b-HR. Purified monocytes from mIgG2b-HR were able to support the mitogenic response to mIgG2b anti-CD3 mAb of purified T cells from mIgG2b-LR. Surprisingly, a significant mitogenic response to mIgG2b anti-CD3 mAb remained after vigorous depletion of monocytes from mIgG2b-HR PBMC. Apparently B cells are responsible for this accessory function since Epstein-Barr virus (EBV)-transformed B cells from mIgG2b-HR (but not from mIgG2b-LR) were able to support T-cell proliferation induced by mIgG2b anti-CD3 mAb. Only EBV B cells from mIgG2b-HR were able to form rosettes with human red blood cells (RBC) that had been sensitized with mIgG2b anti-glycophorin A mAb (EA-mIgG2b). These EBV B cells did not express Fc gamma RI or Fc gamma RIII, and could bind some but not all anti-Fc gamma RII mAb. The mitogenic response to mIgG2b anti-CD3 was not inhibited by any of the anti-Fc gamma RII mAb. From these studies we conclude that a polymorphic Fc receptor is expressed on human B cells and monocytes, which cross-reacts with mIgG2b. This receptor is different from Fc gamma RI and Fc gamma RIII, and apparently also from Fc gamma RII.     

Direct demonstration of binding of aggregated mouse IgG1 to the 40-kDa Fc receptor for IgG (Fc gamma RII) in both high and low responders.

Several directly fluorochrome-conjugated murine monoclonal antibodies (mAb) of the IgG1 subclass and directed against B or T cell antigens were found to bind to monocytes via the 40-kDa Fc receptor for IgG (Fc gamma RII). As expected from the established polymorphism of Fc gamma RII, strong staining was observed in about 75% of individuals. In the remaining 25% staining was clearly weaker, but could be definitely demonstrated with a mAb against the B cell-specific CD19 differentiation antigen. Specificity of binding to Fc gamma RII was confirmed by the ability to block the binding of the CD19 mAb by pre-incubation with aggregated IgG1 or with mAb against Fc gamma RII. The extent of T cell proliferation induced with a CD3 mAb of the IgG1 isotype (a-Leu 4), which is dependent on the interaction of monocyte Fc gamma RII with the Fc portion of the CD3 mAb, exactly correlated with the amount of binding to Fc gamma RII in all individuals. Proliferation was dose-dependent for both high and low responders; cells of low responders did not proliferate at concentrations below 16 ng/ml of mAb, whereas there was a small but unequivocal proliferation at higher concentrations. These results confirm that monocytes from previously characterized "non-responders" are able to bind aggregated murine IgG1 via Fc gamma RII. They also demonstrate that directly labeled mAb can cause extensive nonspecific staining which may not be excluded by the use of control antibodies of the same isotype.     

Studies on the monocyte dependence of T-cell proliferation induced by monoclonal antibodies directed against regions I and II of the CD2 antigen.

We produced three murine monoclonal antibodies (mAb) directed against the human T-cell differentiation antigen CD2 (formerly T11) and analysed their epitope specificity by E-rosette inhibition, cross-blocking and proliferation-induction experiments. When added together, two mAb (both IgG1 kappa) were able to induce proliferation in peripheral blood T cells. This proliferation was found to be strictly dependent on the presence of monocytes. The polymorphism of Fc receptors (FcR) expressed on human monocytes for murine IgG1 antibodies, which is readily demonstrable in anti-CD3-induced T-cell activation, was not found in the anti-CD2-driven system. Moreover, mAb directed against the 40,000 MW FcRII were unable to inhibit proliferation induced by the mitogenic anti-CD2 combination. Still, F(ab')2 fragments of the mitogenic anti-CD2 antibody combination could not initiate T-cell mitogenesis, despite their ability to induce a rise in the free intracellular [Ca2+]. The contributions of monocytes and of antibody Fc moieties to T-cell proliferation, induced by combinations of anti-CD2 mAb, will be discussed.     

Removal of monocytes from cell suspensions with anti-CD14 antibody and carbonyl-iron, using Fc gamma R-dependent accessory function as a sensitive measure of monocyte presence.

Human (Fc gamma RI-positive) monocytes are required as accessory cells when T cell proliferation is induced by murine IgG2a anti-CD3 monoclonal antibodies (mAbs). This T cell proliferation assay provides a sensitive method for detecting the presence of monocytes (less than 1% of monocytes can be detected), and we have used it to monitor the effectiveness of different procedures for the removal of monocytes from peripheral blood mononuclear cells. Counterflow centrifugation, phagocytosis of carbonyl-iron, adherence to plastic, monocyte depletion with magnetic beads (Dynabeads M450), and panning with anti-CD14 antibodies each strongly reduced the number of monocytes. However, none of these methods, when used on their own, were capable of completely abolishing the mitogenic response to murine IgG2a anti-CD3 mAb. A virtually complete depletion of monocytes was obtained when the panning procedure using anti-CD14 antibodies was combined with phagocytosis of carbonyl-iron. Importantly, this method could also be used with cryopreserved cells. We have applied this improved method for the removal of monocytes, to study T cell proliferation induced by murine IgG2b anti-CD3 mAb. We were able to demonstrate with this model that cells other than monocytes were able to provide accessory function.     

The heterogeneity of bovine IgG2. II. The identification of IgG2b

Goat and rabbit polyclonal reagents can be raised which recognize a new isotype of bovine antibodies. The polyclonal goat reagent was raised against a preparation enriched in the major IgG2 isotype (IgG2a) which contained the new isotype as a contaminant. The polyclonal rabbit reagent was prepared against a trypsin-derived Fc fraction of bovine IgG1 which contained the Fc of the new isotype as a contaminant. This new isotype is present in the sera of the cattle of all breeds tested regardless of their IgG2a allotype and is antigenically distinct from IgG2a, IgG1, IgA, IgM and IgE. The new isotype coelutes from DEAE anion exchangers with IgG1 and the more acidic populations of IgG2a. The isotype is tentatively designated IgG2b. The distribution of IgG2b antibody activity to E. coli K99 and phosphorylcholine among 15 cattle of different A allotypes is not correlated with the IgG2a or IgG1 antibody activity in these animals.     

Interferon-gamma restores T lymphocyte proliferation of nonresponders to IgG1 anti-CD3 via the induction of Fc gamma 1 receptors on monocytes

Human peripheral blood T lymphocytes are stimulated to grow and divide by some mouse anti-CD3 monoclonal antibodies. This polyclonal mitogenesis is dependent on both their immunoglobulin subclass and the presence of monocytes. The unresponsiveness of T lymphocytes from certain individuals to mouse IgG1 (or IgG2a) antibodies is due to a failure of their monocytes to bind these IgG isotypes. In this study, we have selected such nonresponder subjects to IgG1 anti-CD3 (UCHT 1) in order to study their monocytes. Two assays were used: IgG1 and IgG2 EA rosettes to evaluate their Fc receptor-binding capacity, and IgG-mediated monocyte chemiluminescence to test their receptor-related activation since mouse anti-T cell antibodies binding to lymphocytes trigger monocyte chemiluminescence via their Fc receptor. We have observed that in all nonresponder subjects the absence of IgG1 anti-CD3 monocyte chemiluminescence strictly correlates with the absence of IgG1 EA rosettes. Thus, the failure to respond to UCHT 1, in all nonresponders tested to date, is due to the absence of Fc gamma 1 receptors on their monocytes. Treatment of nonresponder monocytes by recombinant interferon-gamma was shown to restore T cell proliferation and monocyte chemiluminescence in nonresponders. This effect of interferon-gamma correlates with the appearance of Fc gamma 1 receptors on monocytes from these individuals. This work strongly suggests that nonresponder monocytes possess functional genes for Fc gamma 1 receptors which are not expressed normally at a detectable level but can be induced by interferon-gamma.     

Isolation of rat IgM to IgG hybridoma isotype switch variants and analysis of the efficiency of rat Ig in complement activation

Sequential sublining was used in combination with enzyme-linked immunosorbent assays to isolate mu----gamma isotype switch variants of the rat IgM secreting mouse-rat B cell hybridoma line BA1.8. Switch variants to all four subclasses of IgG were obtained. The variant antibodies retained the antigen specificity of the parental IgM for the O18 (lipopolysaccharide) antigen of Escherichia coli. In sodium dodecyl sulfate-polyacrylamide gels the apparent molecular mass of the gamma heavy chains decreased in the order gamma 2b greater than gamma 1 greater than gamma 2c greater than gamma 2a. IgM, IgG1, IgG2a, IgG2b and IgG2c of the BA1.8 variant family and IgG2b, IgE and IgA of the previously described BA1.2 family were used for a comparative analysis of the capacity of rat Ig to activate complement. Efficient lysis of sheep erythrocytes coated with the O18 antigen was observed with IgM and all IgG subclasses, but no lysis was triggered by IgE or IgA. One hundred to 1000 IgG molecules were required to mediate the same hemolytic activity as one IgM molecule. The four IgG subclasses were equally efficient at mediating lysis by rat or human complement, while IgG2a was less efficient with guinea pig complement than the other three IgG subclasses. Antibody-triggered binding of C3 to pathogenic O18:K1 E. coli bacteria was measured using serum containing 125I-labeled C3. K1-encapsulated strains did not fix C3 efficiently in the absence of specific antibodies while acapsular mutants fixed C3 via the alternative pathway. IgM and all IgG subclasses triggered C3 binding to the K1 encapsulated bacteria. The capacity of IgM to mediate C3 fixation was not greater than that observed with IgG.     

Mitogenicity of anti-Thy-1 monoclonal antibodies attributable to an Fc-dependent mechanism

We analyzed the mechanism by which certain anti-Thy-1 monoclonal antibodies (mAb) activate T cells directly without additional stimuli. Using a panel of rat anti-Thy-1 antibodies which included more than 30 IgG2c mAb, we found that only the IgG2c isotype was able to induce a strong proliferative response in both resting T cells and a T cell lymphoma, suggesting that this form of T cell activation is isotype restricted and might be a consequence of a unique physico-chemical property of the IgG2c heavy chain. Results from surface distribution studies of Thy-1 molecules, following specific interactions with anti-Thy-1 antibodies of different isotypes, again showed that only IgG2c mAb formed Thy-1 aggregates of high valence on the surface of a T cell lymphoma, and such clustering always evoked a biological response. This led us to propose that IgG2c mAb have the inherent tendency to self-associate, probably through homophilic Fc-Fc contacts, and that this feature renders anti-Thy-1 mAb mitogenic. To prove this, we set up cross-inhibition studies with randomly selected mitogenic (IgG2c) and non-mitogenic (IgG2b) anti-Thy-1 mAb. The results clearly demonstrated that IgG2c antibodies enhance their own binding, analogous to the new form of antibody binding that was recently demonstrated between murine IgG3 mAb and a multivalent antigen. Confirmation of this was also provided by IgG2c-derived F(ab')₂ fragments, which were unable to cause proliferation. Furthermore, masking the Fc part of cell-bound IgG2c mAb with a monomeric and thus non-aggregating IgG-binding protein A-derived fragment cancelled their mitogenic ability. Finally, induction of T cell proliferation appeared to be independent of cross-linking via FcγR. The results support a model in which noncovalent intermolecular homophilic contacts of the Fc regions of the IgG2c isotype bring about effective aggregation of Thy-1 molecules, thereby stimulating the mitotic apparatus of the cell.     

Induction of interleukin 2 receptiveness and proliferation in resting peripheral T cells by monoclonal anti-CD3 (T3) antibodies does not require the presence of macrophages

In this study, we sought to elucidate the sequence of events by which mitogenic monoclonal anti-CD3 antibodies (anti-CD3-MoAb) initiate T cell activation. In cultures of monocyte-depleted resting T cells, two anti-CD3-MoAb, OKT3 and anti-Leu 4, induced a state of interleukin 2 (IL-2) receptiveness which culminated in T lymphocyte proliferation when recombinant IL-2 was provided. Evidence that Fc-receptor mediation by monocytes did not contribute to this mitogenesis was supported by studies showing that polyclonal F(ab')2 anti-mouse IgG Fc antibody did not alter the magnitude of the IL-2 driven T cell proliferative response, and by the use of T cells from donors whose monocytes were unable to assist in the induction of anti-Leu 4 (IgG1 subclass) initiated proliferation. Anti-CD3-MoAb, in the absence of IL-2, induced IL-2 receptor expression on purified T cells, and anti-IL 2 receptor antibodies inhibited T cell proliferation in the presence of this growth factor. Furthermore, following modulation of the CD3 molecular complex in the presence of monocytes, depletion of accessory cells rendered the modulated T cells mitogenically dependent on exogenous IL-2. IL-2 itself did not suffice to promote T cell proliferation in the absence of anti-CD3-MoAb. These results indicate that the binding of monoclonal antibody to CD3 is capable of initiating, in an accessory cell-independent manner, premitotic alterations in T cells which can culminate in proliferation when exogenous IL-2 is provided.     

Induction of Fc receptors for IgA on murine T cell hybridoma by human monoclonal IgA and by high molecular weight IgA in IgA nephropathy.

A reproducible immunocyto-adherence assay has been developed to study the modulation of Fc receptors for IgA (Fc alpha R), using a murine T cell hybridoma (T2D4), which expresses Fc receptors for all known isotypes of secreted immunoglobulins. By using sheep red blood cells coated with the hapten 2-4-6 trinitrophenyl (TNP), as indicator cells, and a murine monoclonal IgA (MOPC 315) antibody with anti-TNP activity, we were able to study the Fc alpha R on T2D4 cells. We found that: (a) murine Fc alpha R can bind human monoclonal IgA, and this binding is isotype specific since it was inhibited by human monoclonal IgA but not by human monoclonal IgG or IgM; (b) the expression of murine Fc alpha R is unducible by human monoclonal IgA, and this effect is isotype specific since it is not observed with human monoclonal IgM or IgG (c) sera from patients with IgA nephropathy can also induce Fc alpha R expression; by contrast, no induction was observed with normal human sera, (d) in one serum from an IgA-nephropathy patient, the inducer factor was characterized by affinity chromatography on anti-IgA-Sepharose and by gel filtration: high molecular weight IgA, probably IgA aggregates or immune complexes were recognized to be responsible for the induction of murine Fc alpha R expression.     

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.     

The Human Fc Receptor for Mouse IgG2b on Monocytes and EBV-B Cells is Functionally Inhibited by Anti-HLA Class II Antibodies

We have recently described a polymorphic Fc receptor for murine IgG2b (mIgG2b), present on human monocytes and EBV-transformed B lymphocytes. The present study shows that anti-HLA class II monoclonal antibody (MoAb) completely inhibits both the (Fc receptor-dependent) T-cell proliferation, induced by mIgG2b anti-CD3 MoAb, and rosetting with mIgG2b-sensitized erythrocytes. This inhibition is also observed with F(ab')₂ fragments of anti-HLA class II MoAb, and is therefore not Fc mediated. The Fc receptor for mIgG2b is also present on EBV-transformed B cells obtained from a patient with'bare lymphocyte syndrome', that completely lack HLA class II antigens. Therefore, the Fc receptor for mIgG2b and HLA class II antigens are not identical. Since the low affinity receptor for IgE (FcɛII; CD23) was reported to be associated at the cell surface with HLA class II antigens, we have compared both types of Fc receptor, and observed that human IgE strongly inhibits the mitogenic effect of murine IgE anti-CD3 but not of mIgG2b anti-CD3 MoAb. We conclude that the human Fc receptor for mIgG2b is strongly inhibited by anti-HLA class II MoAb, but is not identical to HLA class II or FcɛRII.     

Vasoactive intestinal peptide specifically induces human IgA1 and IgA2 production

The effects of vasoactive intestinal peptide (VIP) on human IgA1 and IgA2 production were studied. In unfractionated small resting B cells stimulated with anti-CD40 monoclonal antibody (mAb), VIP induced IgA1 and IgA2 production without affecting the production of IgG1, IgG2, IgG3, IgG4, IgM, or IgE. When small B cells were separated into sIgA1⁺, sIgA2⁺, sIgA1^- and sIgA2^- B cells, anti-CD40 mAb plus VIP induced IgA1 and IgA2 production by surface IgA1^- (sIgA1^-) and sIgA2^- B cells, respectively, while having no effect on sIgA1⁺ and sIgA2⁺ B cells. This induction by VIP was specific, since anti-CD40 mAb plus other neuropeptides, i.e., somatostatin or substance P, had no effect, and moreover, the induction was specifically blocked by a VIP antagonist. Further, anti-CD40 mAb plus various cytokines, including interleukin (IL)-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, transforming growth factor-β, low molecular weight B cell growth factor, and interferon-γ, did not induce IgA1 and IgA2 production by sIgA1^- and sIgA2^- B cells, respectively. These results indicate that in the presence of anti-CD40 mAb, VIP induces IgA1 and IgA2 production by isotype switching.     

T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction

The anti-CD3 antibody BMA030 (IgG2a isotype) induces T cell activation and proliferation if an interaction with monocytes is provided. In contrast to other anti-CD3 antibodies, it is unable to induce interleukin (IL)2 responsiveness through cross-linking by plastic-bound goat anti mouse Ig antibodies (panning). Cross-linking BMA030 with a second anti-T cell antibody is, however, able to induce IL 2 responsiveness in monocyte-depleted T cell cultures. In this report we show that a large number of different antibodies are suitable for this dual antibody stimulation, and that the extent of proliferation corresponds to the percentage of T cells expressing the respective T cell antigen. Proliferation induced by low concentrations (0.1-1 ng/ml) of other anti-CD3 antibodies requires also cross-linking with second anti-T cell antibodies. The proliferative response of monocyte-depleted T cells to two cross-linked anti-T cell antibodies plus added IL 2 is of the same magnitude as the one induced by anti-CD3 antibodies plus monocytes. On the other hand, if monocytes are present, soluble anti-CD2, -CD4, -CD8, -LFA-1 antibodies (IgG1 or F(ab')2 fragments) can inhibit OKT3 or BMA030-induced T cell activation. Anti-CD6 antibodies do not interfere with this monocyte-dependent T cell stimulation. We conclude that dual antibody stimulation mimics the physical contact of T cells with monocyte membranes, where the T cell receptor CD3 complex is cross-linked with neighboring structures (mainly so-called adhesion molecules) through the interaction with respective counter-structures on monocyte membranes. Dual antibody cross-linking bypasses this interaction and can be used to stimulate IL 2 responsiveness of antibody-defined T cells.     

Human monocyte-mediated cytotoxicity towards erythrocytes induced by hybrid mouse monoclonal antibodies: effect of antibody binding valency on IgG-Fc gamma R interaction.

In this study, we describe the ability of hybrid mouse monoclonal antibody (mAb) to induce monocyte-mediated cytotoxicity towards human IgA1-coated E (HuIgA1-E), and the effect of mAb binding valency on Fc gamma RI-mediated ADCC. All hybrid monospecific (ms) anti-HuIgA1 and bispecific (bs) anti-HuIgA1/HRP mAb were capable of inducing monocyte-mediated lysis of HuIgA1-E, in spite of differences in mAb densities essential for optimal lysis. The cytotoxicity induced by hybrid mAb which consist of one or more mIgG2a H chains was predominantly mediated via Fc gamma RI, as shown by inhibition studies on monocytes with Fc gamma RI-blocking mAb TB-3 (approximately 80% inhibition). However, partial inhibition of mIgG1-2a and mIgG2a-2b-induced cytotoxicity (20-50%) was observed by using Fc gamma RII-blocking mAb IV.3 or CIKM5. For hybrid mIgG1-1 mAb the opposite was true; the cytotoxicity was predominantly mediated via Fc gamma RII (70-80%) and less via Fc gamma RI (20-30%). Comparing the hybrid ms anti-HuIgA1 mAb-induced cytotoxicity with the cytotoxicity induced by hybrid bs anti-HuIgA1/HRP mAb of the same isotype, we observed a decrease in cytotoxicity towards HuIgA1-E sensitized with univalently bound bs anti-HuIgA1/HRP mAb. This decrease was only found for Fc gamma RI-mediated ADCC (mIgG2a-2a, mIgG1-2a and mIgG2a-2b). This diminished recognition of univalently bound IgG relative to bivalently bound IgG by Fc gamma RI was also observed with U937 effector cells. In conclusion, this work shows that hybrid mAb are able to induce monocyte-mediated cytotoxicity towards E-HuIgA1 and that there appears to be an effect of Ag-IgG binding valency on Fc gamma RI-mediated cytotoxicity.     

Receptor specific probes for the study of Fc gamma receptor specific function.

Human leukocytes express three distinct families of receptors for the Fc region of IgG (Fc gamma R). We have prepared erythrocytes (E) coated with monoclonal anti-Fc gamma R antibodies for the study of receptor specific phagocytosis using biotin and streptavidin. In this technique, both the E and the monoclonal antibody (mAb) are biotinylated and coupling of the mAb to the E occurs through the use of streptavidin. The same biotin/streptavidin principle was used to prepare E coated with human IgG. Using this technique, receptor specific probes or probes coated with natural ligand (IgG) can be prepared rapidly with the use of small amounts of mAb or IgG. Finally, we have used these receptor specific probes to demonstrate that all three families of Fc gamma R (Fc gamma RI, Fc gamma RII and Fc gamma RIII) expressed on human monocytes and human macrophages are phagocytic receptors.     

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)     

Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments

We have constructed a series of eukaryotic expression vectors that permit the rapid conversion of single chain (sc) Fv antibody fragments, derived from semi-synthetic phage display libraries, into intact fully human monoclonal antibodies (mAb) of each isotype. As a model, a scFv fragment specific for sheep red blood cells (SRBC) was isolated from a semi-synthetic phage antibody (Ab) display library, and used to produce human mAbs of IgM, IgG1-IgG4, IgA1, IgA2m(1) and IgE isotype in vitro in stably transfected cells. N-terminal protein sequence analysis of purified immunoglobulin heavy (H) and light (L) chains revealed precise proteolytic removal of the leader peptide. Biochemical analysis of purified recombinant human mAbs demonstrated that properly glycosylated molecules of the correct molecular size were produced. The IgG and IgA mAbs retained SRBC-binding activity, interacted with different Fc receptor-transfectants, and induced complement-mediated hemolysis and Ab-dependent phagocytosis of SRBC by neutrophils in a pattern consistent with the immunoglobulin (Ig) H chain isotype. We conclude that in vitro produced recombinant human mAbs constructed from phage display library-derived scFv fragments mirror their natural counterparts and may represent a source of mAbs for use in human therapy.