胃癌相关抗原表位模拟短肽的筛选

目的：筛选能模拟胃癌相关抗原表位的功能短肽。方法：利用离子交换层析，对抗胃癌相关抗原单克隆抗体(mAb)进行纯化，并以此mAb为靶标，将其包被于ELISA板上，以噬菌体随机12肽库对其进行3轮亲和筛选。结果：从噬菌体随机12肽库中，筛选到12个噬菌体阳性克隆：结论：获得了具有模拟胃癌相关抗原表位的阳性噬菌体克隆，且有共同的基序。     

Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library

Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.     

Amino acid sequence requirements for the epitope recognized by a monoclonal antibody reacting with the secreted antigen GP28.5 of Toxoplasma gondii.

We have characterized in detail, the epitope of the secreted antigen GP28.5 recognized by the mouse monoclonal antibody TG 17-179 using synthetic peptides and a truncated recombinant fusion protein. The screening of a T. gondii expression library with TG17-179 mAb led to the isolation of cDNAs clones, all encoding for the C-terminal region of GP28.5 and with one encoding for only the five last C-terminal residues. Competitive ELISA with longer peptides revealed that the immunoreactivity was retained for peptides of eight residues or longer, and lost when the peptide was reduced to the six last C-terminal residues or less. Experiments with the octapeptide lacking the carboxy-terminal glutamine residue showed it to be 64-fold less active. Moreover, neither addition of residues in the carboxy-end nor substitution of COOH function changed the immunoreactivity of the epitope. Competition experiments between TG17-179 mAb and sera from infected individuals demonstrates that the epitope defined by a mouse monoclonal probe is also a major epitope for human polyclonal antibodies. These results describe the sequence requirements within a probably linear epitope and give rise to some general question concerning experimental test for epitope mapping.     

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9 / 1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9 / 1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7 / 1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.     

Analysis of the molecular mimicry between HLA-B27 and a bacterial OmpA protein using synthetic peptides.

In spite of a lack of sequence 'homology' between HLA-B27 and the bacterial OmpA outer membrane proteins, they both react with the Ye-2 monoclonal anti-HLA-B27 antibody. The Ye-2 antibody also reacted positively in ELISA with a synthetic peptide derived from the segment spanning residues 63-84 of B*2705. The critical peptide residues were determined by testing first with overlapping peptides, followed by a replacement set made according to the determined epitope. The results were compared with those with overlapping eight mers made to span a carboxyl fragment of the Escherichia coli OmpA protein. They indicate the reason why Ye-2 reacts with both sets of peptides is because it has a preference for polymers of arginine.     

Two different methods result in the selection of peptides that induce a protective antibody response to Neisseria meningitidis serogroup C

Neisseria meningitidis is a leading cause of morbidity and mortality worldwide. The presently available capsular polysaccharide vaccine is poorly immunogenic in children under the age of 2 due to its T-independent (TI) nature. Efforts to overcome the TI response elicited by the polysaccharide vaccine have led to the development of polysaccharide-protein conjugate vaccines. Although a T-dependent (TD) response can be achieved in young children, the response to the polysaccharide still retains characteristics of a TI antibody response. An alternative method of potentially inducing a TD response to a carbohydrate antigen is through peptides that mimic the capsular polysaccharide. Our laboratory, through the production of an anti-idiotypic (anti-id) monoclonal antibody, designated 6F9, has previously identified a peptide mimic of the meningococcal serogroup C polysaccharide (MCPS). Using the same selecting monoclonal antibody (mAb), 1E4, we have screened a phage display library and identified 13 unique peptides that bound specifically to mAb1E4. Two peptides, Pep1C and Pep2C, that demonstrated the highest binding to mAb1E4, were selected, complexed to proteosomes, and used to immunize Balb/c mice. Of the 13 peptide motifs, only one peptide motif, that of Pep2C, was found to resemble the immunogenic peptide sequence of the anti-id selected with the same mAb, although many contained several similar amino acid residues. Immunization with Pep2C, but not Pep1C, induced a significant and functional anti-MCPS antibody response that conferred protection from a lethal challenge with meningococci. Our results indicate that immunization with a peptide of N. meningitidis serogroup C, screened with the same mAb that selected an anti-id of MCPS, induces a functional and protective anti-MCPS immune response similar to that of the anti-id. This study demonstrates that two different selection methods, production of an anti-id and biopanning using a phage display library, can be used to select functional and protective peptides of MCPS with similar moieties.     

Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus

We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.     

Two monoclonal antibodies to precisely the same epitope of type II collagen select non-crossreactive phage clones by phage display: implications for autoimmunity and molecular mimicry

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.     

B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

应用噬菌体肽文库筛选mAb F3特异性结合肽

目的 应用噬菌体展示肽文库筛选可与汉坦病毒囊膜蛋白中和性单抗（mAb） F3特异性结合的配体肽。方法 以F3mAb为筛选配基，对噬菌体展示和随机12肽文加进行3轮生物亲和淘选；用夹心ELISA和竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果 通过3轮生物淘选，能被抗体捕获的噬菌体克隆为21／22，ELISA测定显示，筛选到的噬菌体短肽能与F3mAb特异性结合。序列分析表明，7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT；同源性分析显示，该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性。结论 本研究为基于表位水平的HFRSV特异性分子多肽疫苗的设计提供了重要的依据。     

Induction of interleukin 2 (IL2) and interferon-γ and enhancement of IL2 receptor expression by a CD26 monoclonal antibody

The ability of the 134-2C2 monoclonal antibody (mAb; CD26) to transmit an activation signal and to affect T cell proliferation has been studied. The 134-2C2 mAb, although not being mitogenic by itself, is able to increase the proliferation of purified T cells in the presence of exogenous interleukin 2 (IL2) or phorbol 12-myristate 13-acetate (PMA). No effect of our mAb was observed on the proliferation of T cells induced by other stimuli such as Sepharose-bound CD3 mAb, phytohemagglutinin or calcium ionophore. Since the co-stimulatory effect of 134-2C2 mAb on PMA-induced T cell proliferation was strongly inhibited by an anti-Tac antibody, its involvement on the IL2/IL2 receptor pathway was investigated. An increased IL2 secretion in T cells cultured with PMA plus 134-2C2 mAb was observed and Northern blot analysis showed that the mAb 134-2C2 acts synergistically with PMA favoring the induction of both IL2 and interferon-γ mRNA synthesis, as well as the enhancement of IL2 receptor and transferrin receptor mRNA expression. Studies on mechanisms implicated in signal transduction showed that 134-2C2 mAb modifies neither intracellular calcium levels nor phosphoinositide breakdown. Additionally, no effect was exerted on protein kinase C translocation. These data suggest that the CD26 antigen is involved in T cell activation in an IL2/IL2 receptor-dependent pathway.     

抗前列腺特异性膜抗原膜外区多肽单克隆抗体的研制及初步应用

目的建立前列腺特异性膜抗原（PSMA）膜外区多肽杂交瘤细胞株，并对其分泌的PSMA单克隆抗体进行初步鉴定，为PSMA的功能研究和人源化抗体的制备奠定基础，以求进一步用于前列腺癌的诊断和治疗。方法使用人工合成多肽免疫BABL／c小鼠，采用PEG融合技术建立杂交瘤细胞株，制备单克隆抗体。通过免疫荧光法、酶联免疫吸附法及斑点金标法确定单克隆抗体的交叉反应性、亲和力及免疫球蛋白的类型和亚类。结果获得两株可稳定分泌PSMA单克隆抗体的杂交瘤细胞，4F4为IsG1类，1F1为IgC3类。两株单抗均能识别LNCap细胞表达的PSMA蛋白，与不表达PSMA的PC-3、SP2／0等细胞无交叉反应。杂交瘤细胞株1F1培养上清效价为1：40。腹水效价为1：6400；而杂交瘤细胞株4F4培养上清效价为1：80。腹水效价为1：8000。结论成功地制备出两株抗PSMA单克隆抗体，均具有良好的特异性和亲和力，为进一步建立免疫分析方法。进行PSMA相关研究奠定了基础。     

Epitope mapping of apamin by means of monoclonal antibodies raised against free or carrier-coupled peptide.

A panel of 20 monoclonal antibodies raised against the bee-venom peptide apamin (18 residues, 2 disulfide bridges) was prepared. Nine monoclonal antibodies (mAb) were obtained from a mouse immunized with free apamin and 11 from a mouse immunized with a mixture of free and carrier-coupled peptide. Using a panel of 11 synthetic apamin analogs, we examined the fine antigenic specificity of each antibody. The mAb generated against free apamin preferentially bound to the central part of the peptide and less frequently recognized the N- and C-terminal regions. However, monoclonal antibodies obtained by immunization with carrier-bound apamin showed a broader range of specificities, consistent with the possibility of the entire surface of this small antigen becoming immunogenic upon coupling to the carrier.     

Probing the idiotype/anti-idiotype antibody interaction with a set of synthetic peptide homologues.

Anti-idiotypic (anti-Id) antibodies were raised against two murine monoclonal antibodies (mAb 1/1 and mAb 2/1) which recognise two distinct and well-characterised epitopes on a 24-residue synthetic peptide representing part of the haemagglutinin (HA) of influenza virus. A monoclonal anti-Id antibody, specific for mAb 2/1, could bind to mAb 2/1 when the paratope of the latter was occupied with peptide, indicating that this anti-Id antibody is directed to a framework idiotope. In contrast, an anti-Id mAb derived from mAb 1/1-immunised mice was inhibited in its binding to Id by the parent peptide and also by the heptapeptide NVPEKQT which constitutes the epitope recognised by mAb 1/1. The small size of this synthetic peptide eliminates the possibility of significant steric inhibition in the system, and establishes that this mAb is a true paratope-directed anti-Id antibody. The interaction of this anti-Id mAb with the paratope of mAb 1/1 in the presence of a set of peptide homologues of the epitope was also examined. A peptide as short as 5 residues, which contains two of the three irreplaceable residues of the epitope, could inhibit binding between the two mAbs.     

Direct kinetic assay of interactions between small peptides and immobilized antibodies using a surface plasmon resonance biosensor.

A surface plasmon resonance (SPR) protocol is described for the direct kinetic analysis of small antigenic peptides interacting with immobilized monoclonal antibodies (mAb). High peptide concentrations (up to 2.5 microM) and medium mAb surface densities (about 1.5 ng/mm(2)) are needed to ensure measurable binding levels, and fast buffer flow rates (60 microl/min) are required to minimize diffusion-controlled kinetics. Good reproducibility levels in the kinetic constants are obtained under these analysis conditions (standard deviations below 10% of the mean values). Application of this protocol to determine the antigenic ranking of viral peptides shows an excellent agreement between SPR and previous competition enzyme-linked immunosorbent assays (ELISA) on the same peptide/antibody systems.     

Monoclonal antipeptide antibodies recognize epitopes upon VP4 and VP7 of simian rotavirus SA11 in infected MA104 cells

Summary To study morphogenetic events of rotavirus SA11-infected MA104 cells with strictly defined reagents we produced monoclonal antibodies against synthetic peptides from both outer capsid proteins VP4 (aa residues 228–241: QNTRNIVPVSIVSR) and VP7 (aa residues 319–326: SAAFYYRV) of simian rotavirus SA11. Two of the selected monoclonal antibodies proved to be reactive with determinants of SA11-infected MA104 rhesus monkey kidney cells, with purified SA11 as well as with the particular peptides used for immunization. The anti-VP4 antibody had a demonstrable neutralizing titer of 200 (50% focus reduction) whereas the anti-VP7 MuMAb revealed no detectable neutralizing activity. In peptide-inhibition experiments, the corresponding peptide inhibited its MuMAb whereas the noncorresponding peptide had no effect on antibody binding to intracellular viral antigen. Localization of VP7 was preceded by VP4 as shown by immunofluorescence microscopy.     

A new peptide-affinity tag for the detection and affinity purification of recombinant proteins with a monoclonal antibody

A monoclonal anti-peptide antibody (2E11) was raised against the synthetic peptide 38 (C-L-D-K-S-G-L-P-S-D-R-F-F-A) representing a part of the variable region of the Vβ 6.2 T-cell receptor. This mAb (IgG₁, κ light chain) bound very specifically to peptide 38 as shown by ELISA but did not recognize the corresponding native Vβ 6.2 T-cell receptor on T-cells. For epitope analysis, overlapping peptides of 4–10 amino acids in length corresponding to the sequence of peptide 38 were synthesized and assayed by SPOT synthesis on cellulose sheets. The shortest peptide recognized was L-P-S-D-R. The specificity of mAb 2E11 was examined with 100 different peptides comprising other parts of the different variable Vβ domains of the human T-cell receptor that do not include the epitope region L-P-S-D-R. None of these peptides were recognized. The chemical synthesis of a peptide with the sequence L-P-S-D-R on Sepharose™ beads allowed to efficiently purify the mAb 2E11 in a single step by affinity chromatography. An equilibrium binding constant of 4.9×10⁶ l/mol was determined for mAb 2E11 by using rhodamine-green-labelled peptide 38 in fluorescence correlation spectroscopy. In order to demonstrate that peptide 38 can be used as an affinity-tag, it was fused to the carboxyl-terminus of interferon regulatory factor-1 (IRF-1). It could be shown that in vitro translated peptide 38 tagged IRF-1 was immunoprecipitated by the mAb 2E11 and that the fusion protein could be purified by immunoaffinity chromatography. Additionally peptide 38 was fused to the amino-terminus of the Taq polymerase. This recombinant protein was expressed in E. coli and specifically detected in a Dot blot and Western blot using mAb 2E11.     

Epitope characterization by modifications of antigens and by mapping on resin-bound peptides. Discriminating epitopes near the C-terminus and N-terminus of Escherichia coli ribosomal protein S13

The feasibility of using antigen modifications and synthetic resin-bound peptides to distinguish closely related epitopes has been demonstrated in this report. Epitopes recognized by five monoclonal antibodies (MAbs) specific to Escherichia coli ribosomal protein S13 have been located near the C-terminus and N-terminus of the S13 molecule; these epitopes were characterized by modifications of antigens and by utilization of peptides of increasing length synthesized on aminomethyl crosslinked polystyrene resin. Three of these MAbs react with the C-terminal peptide S13(84-117) which has five Lys residues clustered within the last 16 amino acids. Phthalylation of Lys residues almost eliminated the binding of two MAbs and reduced binding of the third by 50%. Removal of the C-terminal Lys residue(s) at the S13 C-terminus with carboxypeptidase B has no effect on the binding of these three MAbs. A 23-residue peptide corresponding to S13(95-117) was synthesized by a modified Merrifield solid phase method. Samples of resin with peptides of increasing length were obtained after each cycle of amino acid coupling. The peptides were deprotected without hydrolysis of the peptide-resin linkage and used in an enzyme immunoassay to detect the extent of MAb interaction with the lengthening peptides. The epitopes recognized by the two MAbs more sensitive to Lys modification have been localized in S13(97-117). The third MAb binds to S13(98-117) but binds more strongly when the sequence is lengthened. Two MAbs directed toward the N-terminal 22 residues of S13 were similarly characterized. Binding of one MAb, little affected by phthalylation, required the N-terminal residue of S13 to be present in the synthetic peptide. The other MAb, whose binding was inhibited by phthalylation, bound to the synthetic S13(2-22) and bound more strongly to the synthetic S13(1-22).     

Inhibition of B cell proliferation with anti-CD19 monoclonal antibodies: anti-CD19 antibodies do not interfere with early signaling events triggered by anti-IgM or interleukin 4.

The 95-kDa antigen recognized by the anti-CD19 panel of monoclonal antibodies is found on the surface of most cells of the B cell lineage. Anti-CD19 antibodies inhibit B cell proliferation in response to anti-Ig plus interleukin 4 (IL4), but enhance the response to mitogenic concentrations of either phorbol 12-myristate 13-acetate (PMA) or Epstein-Barr virus. This dichotomy in the effect of anti-CD19 antibodies suggested that the inhibitory action may be directed at the transmembrane signaling pathways utilized by anti-IgM and IL4. To investigate this hypothesis, an attempt was made to determine the mechanism of signal transduction utilized by the CD19 antigen, and elucidate its effect on transmembrane signaling invoked by anti-immunoglobulin and IL4. Binding of anti-CD19 antibody to B cells did not promote activation of either the phosphoinositide or cAMP signaling pathways. In addition, anti-CD19 antibody did not inhibit phosphatidylinositol bisphosphate (PIP2) hydrolysis induced by anti-IgM or IL4, nor did it interfere with cAMP induction by IL4. We also found that anti-CD19 antibody inhibited PMA plus calcium ionophore-induced B cell proliferation. This evidence indicates that anti-CD19 mAb interrupts the signaling cascade at a point distal to receptor-mediated breakdown of PIP2 and/or activation of adenyl cyclase. This conclusion was fully consistent with experiments in which anti-CD19 antibody was shown to inhibit DNA but not RNA synthesis, and the observation that anti-CD19 antibody must be present between 6 h and 20 h after the initiation of the culture suggesting that anti-CD19 mAb exerts its inhibitory effect in late G0 or G1, after the initial signaling events.     

Activation of human B lymphocytes through CD40 and interleukin 4

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