A syngeneic idiotype is immunogenic when borne by IgM but tolerogenic when joined to IgG

Some syngeneic monoclonal antibodies (mAb) elicit immune responses like conventional T-dependent antigens. To find out whether the heavy chain class (isotype) plays a role for the immunogenicity of an idiotype (Id), we isolated rare subclones of an IgM mAb (termed Id3) in which the variable region of the heavy chain (V_H) is associated with a new constant region (C_H). The V_H-Id3 gene is a member of the murine 36–60 family and probably has three replacement mutations. The light chain V gene is germ-line Vλ2. IgM, IgG1, IgG2a and IgG2b variants of Id3 were purified from protein-free medium and injected without adjuvant into BALB/c mice. The parental 19S IgM mAb given subcutaneously (s.c.) elicited a vigorous humoral response against Id3; in comparison, monomeric 8S IgM was a much weaker immunogen. Unlike IgM, multiple challenges with the IgG switch variants failed to induce anti-Id3 Ab. IgG variants gained immunogenicity if they were purified from medium containing fetal calf serum, mixed with complete Freund's adjuvant or injected into mice primed with IgM-Id3. Pretreatment with 100 μg s.c. + 50 μg of the IgG2a variant extinguished the Ab response to parental IgM, but the response to adjuvant-free bovine serum albumin was intact. Therefore, the tolerance induced by the IgG2a switch variant is antigen-specific and not due to toxicity. Significant inhibition of the Ab response to parental IgM was observed after treatment with 4 μg of the IgG2a switch variant. Administration of the IgG1 and IgG2b switch variants also inhibited this response significantly. Thus, the outcome of an encounter with Id3 is strongly influenced by the C_H isotype to which the Id is joined. This suggests novel ways to minimize unwanted Ab responses against Id of human therapeutic mAb. In the context of the theory of Id networks, we suggest that dominant B cell clones can preempt anti-Id Ab responses against themselves by early switching from IgM to IgG secretion, before immunogenic IgM Ab have had time to activate anti-Id B cells.     

Induction of T cell proliferation with anti-CD3 switch-variant monoclonal antibodies: effects of heavy chain isotype in monocyte-dependent systems

Monoclonal antibodies (mAb) directed against the CD3 (T3), antigen are able to induce proliferation in resting human T lymphocytes. T cell proliferation only occurs in the presence of monocytes that carry the proper Fc receptor for the mAb used. To further analyze the role of the Fc portion of anti-CD3 mAb in proliferation induction, we isolated, starting from a gamma 1 anti-CD3-producing hybridoma, four heavy-chain isotype switch-variant antibody-secreting clones, producing gamma 2b, gamma 2a, epsilon and alpha, respectively. All variant antibodies recognize the CD3 antigen as determined by immunoprecipitation and cross-blocking experiments. With this series of isotype variant antibodies we were able, in proliferation induction experiments, to confirm the Fc receptor polymorphism for murine IgG2a, IgG2b and IgG1 on human monocytes. Moreover, we found that all 30 donors tested responded to the IgE anti-CD3 antibody, while no IgA responders could be identified. The induction of proliferation by the IgE variant antibody does not require the 72-kDa Fc receptor which is responsible for the interaction with mouse IgG2a. Nonresponsiveness to the IgG1 antibody, but not to the IgG2b or IgA variant antibodies, could be overcome by the addition of exogenous interleukin 2 to the cultures. When the switch-variant antibodies were used to induce IgM synthesis in peripheral blood mononuclear cells only low IgM synthesis was found, with the exception of the IgE variant, which induced excellent T cell help for IgM production.     

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.     

Inhibition of human IgE synthesis by anti-IgE antibodies requires divalent recognition

We used a selection of well-characterized murine monoclonal anti-IgE antibodies to investigate their effect on human in vitro IgE synthesis. We found anti-IgE antibodies that either inhibited or enhanced interleukin-4 plus anti-CD40-induced in vitro IgE synthesis in peripheral blood mononuclear cells (PBMC). This differential activity was isotype specific as neither IgM nor IgG synthesis were affected. Interestingly, only coding IgE mRNA was down-regulated, whereas germ-line ε RNA expression was not influenced by anti-IgE monoclonal antibody (mAb). On purified B cells all anti-IgE mAb inhibited interleukin-4 plus anti-CD40-induced IgE synthesis, implying a role of non-B cells for the enhancing activity observed in PBMC. Using Fab and F(ab')₂ of an inhibitory anti-IgE mAb we could show that divalent recognition was required for inhibition of IgE synthesis.     

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.     

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 production and characterisation of a chimaeric human IgE antibody, recognising the major mite allergen Der p 1, and its chimaeric human IgG1 anti-idiotype.

BACKGROUND: Two mouse monoclonal antibodies have been described, namely: mAb 2C7 (IgG2bkappa), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1kappa), which is an anti-idiotypic antibody raised against mAb 2C7. Given its broad IgE specificity, anti-idiotype mAb 2G10 could potentially have immunomodulatory applications. For example, a chimaeric human IgG version of mAb 2G10 could prove to be a useful molecule for binding to mast cell and basophil FcepsilonRI bound IgE, and in doing so co-ligating FcepsilonRI with FcgammaRIIB, which has been reported to have downregulatory effects. AIMS: To produce a chimaeric human IgE version of mAb 2C7 (mAb 2C7huE) and a chimaeric human IgG1 version of its anti-idiotype mAb 2G10 (mAb 2G10huG1). METHODS: The Vkappa and VH regions of mAb 2C7 and its anti-idiotype mAb 2G10 were engineered into human constant regions of the IgE and IgG1 isotypes, respectively. RESULTS: The production of chimaeric mAb 2C7huE and its anti-idiotype mAb 2G10huG1 confirmed that the respective mouse antibody V regions were successfully engineered into human constant regions and still retained the specificity of the original murine V regions. CONCLUSION: The newly constructed chimaeric antibodies will be useful to investigate the downregulation of IgE mediated hypersensitivity by the crosslinking of FcepsilonRI with FcgammaRIIB.     

A mouse monoclonal antibody reactive preferentially with human IgM lambda.

In analyzing mouse monoclonal antibodies (mAb) against a human IgM kappa paraprotein, we found an unusual mAb (LP4; gamma 2b kappa isotype) that reacted in an enzyme linked immunosorbent assay with all 5 IgM lambda but not with 8 IgM kappa or other myelomas. Neither isolated mu heavy nor lambda light chains were reactive with LP4 mAb. By immunofluorescence, LP4 mAb identified approximately 30% of IgM+ B cells and approximately 40% of mitogen-stimulated, IgM+ plasma cells from 4-7 normal blood samples. All LP4+ cells were IgM+. Biosynthetic analysis of the plasma cells revealed that LP4 mAb recognized most IgM lambda and a very minor proportion of IgM kappa molecules. This mAb provides a useful marker for the analysis of pre-B and B cell differentiation.     

The activation of C3 and C4 in human serum by immune complexes containing mouse monoclonal antibodies of different isotype and affinity: Effects on solubilisation

Radioimmunoassays for C3a and C4a have been used to measure the activation of complement in human serum by immune complexes containing DNP-BSA and each of 11 mouse anti-DNP monoclonal antibodies of varied isotype and affinity. When preformed complexes were added to serum, those containing IgG2 or IgM were potent activators of C4, whilst IgG1 complexes were less efficient. C3 activation in normal serum was similar for complexes containing IgG1, IgG2a, IgG2b or IgM. IgA complexes did not activate C3 or C4. Solubilisation of complexes was greatest for IgM and IgG2b and least for IgG2a and IgA. In serum containing Mg²⁺ EGTA C4 activation was abolished and the amount of C3 activation was lower for all IgG and IgM complexes. Antibodies of the same isotype did not necessarily activate complement to the same extent. Unexpectedly, three of the four IgMs activated C3 in EGTA. For IgMs, neither complement activation nor solubilisation correlated with affinity. For IgG1 antibodies, solubilisation was inversely proportional to affinity. C3 or C4 activation did not correlate with affinity.     

Natural Human Antibodies Retrieved by Phage Display Libraries from Healthy Donors: Polyreactivity and Recognition of Human Immunodeficiency Virus Type 1 gp120 Epitopes

We have characterized the human natural antibody repertoire that contains antibodies recognizing the human immunodeficiency virus type 1 (HIV-1) gp120. A panel of monovalent antigen-binding fragments (Fab) selected from IgM and IgG isotype libraries generated from peripheral blood mononuclear cells (PBMC) of a healthy, HIV-1 noninfected individual was analysed, reflecting that only IgM, but not IgG, Fab were able to recognize HIV-1 gp120. The IgM Fab antibodies were not restricted to any particular heavy chain variable region (VH) germ line gene. However, the recognition of gp120 is associated to polyreactive antibodies and all display low-affinity interaction. This correlates with the absence of any maturation process as somatic mutation or isotype switch as the nucleotide sequence analysis of the variable regions reveals they are expressed near to germline configuration. In addition, none of the antibodies showed any neutralizing activity on HIV-1-infected lymphocytes, reflecting that the natural anti-gp120 repertoire is not sufficient to neutralize HIV infection.     

CD25 Blockade protects T Cells from Activation-induced Cell Death (AICD) via Maintenance of TOSO Expression

CD25 monoclonal antibody binding to the α -chain of the Interleukin-2 (IL-2) receptor, blocks high-affinity IL-2 binding, thereby preventing complete T-cell activation and being of ample importance in transplantation medicine and potentially the treatment of autoimmune disease. However, CD25 antibodies do not only block T-cell activation but also prevent activation-induced cell death (AICD) attributing a dual function to IL-2. In this study, the modulation of the genomic expression profile of human peripheral blood mononuclear cells (PBMC) with therapeutic concentrations of humanized anti-CD25 mAb was investigated. PBMC were stimulated with CD3 antibody OKT-3 together with recombinant IL-2 in the absence or presence of anti-CD25 mAb. RNA was extracted and subjected to microarray analysis on U133A microarrays (Affymetrix). Anti-CD25 treatment inhibited several genes typically expressed during T-cell activation including granzyme B, signalling lymphocyte activation molecule, family member 1 (SLAMF1), CD40-Ligand (CD40-L), IL-9 and interferon (IFN)- γ . Interestingly, anti-CD25 mAb also blocked the expression of several genes important for susceptibility to apoptosis, such as death receptor 6 (DR6) or reversed IL-2-mediated repression of anti-apoptotic genes, such as Fas apoptotic inhibitory molecule 3 (FAIM3)/TOSO. Functional significance of DR6 and TOSO expression in IL-2-dependent T-cell activation was subsequently evaluated by RNA interference in AICD: While siRNA specifically directed against DR6 did not modulate FAS-L-mediated apoptosis induction in primary T cells, down-regulation of TOSO significantly increased susceptibility to apoptosis, emphasizing an important role for TOSO in IL-2-mediated AICD.     

Monoclonal anti-peroxidase isotype switch variants. Applications in studies of protein A binding and characterization of rat monoclonal antibodies

A series of heavy chain isotype switch variants was derived from a hybridoma cell line secreting monoclonal antibodies specific for horseradish peroxidase. By the combined use of sensitive isotype-specific ELISAs and sequential sublining IgG2b, IgG2a, IgE and Iga anti-peroxidase-producing variants were successively isolated out of IgG1-secreting parental cells. The anti-peroxidase isotype variant antibodies are particularly appropriate for use in studies of the influence of heavy chain isotype in the effector functions of immunoglobulins. The use of variant antibodies with specificity for an enzyme favors their application in immunoassays because an enzyme-conjugated second antibody is not needed. Here we describe two applications of the anti-peroxidase switch variants. First, the variants are compared with respect to their affinity for Staphylococcus protein A. While IgG1 anti-peroxidase showed weak binding, both IgG2 variants strongly bound to protein A, whereas IgE and IgA variants had no affinity for protein A. Next, the switch variants were used to determine the isotype specificity of rat monoclonal antibodies generated to murine IgE.     

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.     

Effects of insulin-like growth factors and insulin-like growth factor binding protein-2 on the in vitro proliferation of peripheral blood mononuclear cells

Increasing evidence has implicated that insulin-like growth factors (IGFs), polypeptides structurally related to proinsulin, are involved in the function and development of the immune system. To probe the relevance of IGF binding protein 2 (IGFBP-2) in T-cell activation and proliferation, we studied the role of IGFBP-2 in anti-CD3 monoclonal antibody (mAb)-activated peripheral blood mononuclear cells (PBMCs). Secretion of IGF-I, IGF-II, and IGFBP-2 by PBMCs from healthy adult donors was determined by radioimmunoassays (RIAs). The PBMC proliferative response after stimulation with anti-CD3 mAb and exposure to increasing concentrations of IGF-I, IGF-II, IGFBP-2, and anti-IGFBP-2 were determined by bromodeoxyuridine enzyme-linked immunosorbent assay. Observations were tested for significance by paired t-tests. We demonstrate an increase in IGFBP-2 secretion associated with both activation of PBMC by anti-CD3 mAb and increasing cell density. Incubation with exogenous IGFBP-2 increased the proliferation of PBMCs, whereas anti-IGFBP-2 had an antiproliferative effect on PBMCs that was reversed by simultaneous exposure to IGFBP-2. The stimulatory activity of IGFBP-2 (1-10 ng/ml) on anti-CD3 mAb-activated PBMCs was similar to that of IGF-I and IGF-II (1-100 ng/ml), with the mean increase in PBMC proliferative response ranging between 150% and 160% for IGFBP-2 (p = 0.03), 150% and 170% for IGF-I (p < 0.01), 133%-161% for IGF-II (p < 0.01), and 157% and 175% for IGF-I + IGF-II (p < 0.01). Thus, our data strongly suggest a role for IGFBP-2 as a local growth factor contributing to the proliferation and activation of mononuclear cells.     

Recombinant chimeric OKT3 scFv IgM antibodies mediate immune suppression while reducing T cell activation in vitro

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), is a potent immunosuppressive agent used in clinical transplantation to treat allograft rejection. Two major drawbacks of this therapy are the systemic release of several cytokines due to cross-linking mediated by the mAb between T cells and FcgammaR-bearing cells and the human anti-mouse antibody (HAMA) response. To overcome these side effects, three chimeric OKT3 single chain variable fragment (scFv) IgM antibodies, scOKT3-gamma DeltaIgM wt, scOKT3-gamma DeltaIgM C575S and scOKT3-gamma DeltaIgM VAEVD, were generated. They consist of the light and heavy variable binding domains of OKT3 mAb as well as the CH3 and CH4 domains of different human IgM variants linked with a human IgG3 hinge region to provide more flexibility and stability. Like the native IgM, scOKT3-gamma DeltaIgM antibodies are able to form polymeric structures, which lead to an increase in binding affinity and immunosuppressive potential compared with the parental OKT3 mAb. However, independently of their polymerization, all scOKT3-gamma DeltaIgM constructs do not induce any significant T cell proliferation or cytokine release (IL-2, TNF-alpha and IFN-gamma) in in vitro assays, while their CD3-modulating properties are retained. These results suggest that the use of scOKT3-gamma DeltaIgM antibodies may offer significant advantages over the OKT3 mAb in improving clinical immunosuppressive treatment.     

Establishment and usefulness of an anti-human CD57 IgG1 monoclonal antibody

In the present study we established a new monoclonal antibody, JNK-1, which recognizes all cells recognized by CD57/HNK-1 mAb. JNK-1 and CD57 mAbs inhibited the binding of each other, suggesting that the molecules they recognize are either identical or sufficiently close to cause steric hindrance in the binding assay. JNK-1 mAb detected the 110-kDa protein, which is identical to the protein recognized by CD57/HNK-1 mAb in Western immunoblot analysis combined with immunoprecipitation. Therefore, JNK-1 mAb appears to recognize homogeneous molecules identified by the currently available CD57 mAb. Notably, JNK-1 mAb is composed of mouse IgG1 heavy chains, and thus can be used easily in immunoprecipitation, which cannot easily be performed with the available CD57 mAb because it is an IgM isotype. Thus, JNK-1, which is an IgG isotype, may present a useful tool to elucidate the CD57 protein.     

The importance of antibody isotype in HIV-1 virus capture assay and in TZM-bl neutralization

The binding of murine IgM mAbs to five different clades of HIV-1 was examined using a modified ELISA-based virus capture assay. Two murine multispecific IgM mAbs that exhibit both lipid and gp41 epitope specificities, and one murine IgM mAb that exhibits lipid-binding specificity, were utilized. The binding of the IgG and the IgM isotypes of human mAb 2F5 to clades A through AE were also evaluated. The binding of 2F5 to HIV-1 was dependent upon the antibody isotype. Monoclonal IgM antibodies bound significantly lower amounts of HIV-1 than the corresponding IgG isotype. Although murine IgM mAbs bound HIV-1 to varying degrees in the virus capture assay, they failed to neutralize HIV-1 in a TZM-bl pseudovirus assay. In contrast, 2F5-IgM mAb bound certain HIV-1 isolates, and also neutralized them, although not as efficiently as the 2F5-IgG isotype. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus.     

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.     

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)     

Heterogeneity in the mitogenic response of peripheral blood mononuclear cells to a pan T monoclonal antibody

Peripheral blood mononuclear cells (PBMC) from 80 normal donors were studied for their capacity to proliferate in response to Pan T2, an IgG1 monoclonal antibody (MoAb), that recognizes the CD3 complex. Forty percent of this population, regardless of sex or age, were found to be non-responders. However, the binding of MoAb Pan T2 to T cells as studied by indirect immunofluorescence was positive in all the donors. The addition of IL 1 or IL 2 to Pan T2-stimulated non-responder lymphocytes did not activate T cell proliferation, while the addition of responder monocytes restored the proliferation capacity in non-responder PBMC. The data indicate the existence of a heterogeneous responsiveness among normal individuals to a mitogenic IgG1 MoAb, and are in agreement with reports obtained using other anti-T3 MoAbs of IgG1 isotype, i.e. UCHT1, Leu4 and WT31. This defect is reported to be a function of monocytes, related to a polymorphism of Fc receptors for mouse IgG1 on human monocytic cells.