Characterization of three monoclonal antibodies specifically directed against the ligand binding site area of the p55 subunit of the mouse interleukin-2 receptor.

Three new rat monoclonal antibodies (MAbs) (5A2, 125A8 and 135D5) directed against the mouse interleukin-2 receptor (IL-2R) were isolated. They were obtained after immunization of LOU rats with 14.1.6 T helper cell clones. These three MAbs recognize the p55 subunit of the IL-2R and compete with the binding of previously characterized MAbs AMT13 and 3C7 specific for this p55 subunit [Moreau et al. (1987) Eur. J. Immun. 15, 723-727]. They recognize the same (or closely related epitopes) since they reciprocally compete with each other's binding. Scatchard plot analysis of the data from inhibition experiments clearly indicate that they recognize with very high affinity the ligand binding site area of the p55 subunit of the IL-2R. The properties of the Fab fragment prepared from 5A2 and 135D5 indicate that at saturation one intact IgG molecule binds two IL-2R molecules.

Monoclonal anti-GAT antibodies with different fine specificities express the same public idiotype.

We have studied the idiotypic specificities expressed by 22 anti-GAT hybridoma products (HP). These antibodies, although derived from cells of mice with three distinct heavy-chain linkage groups (BALB/c, Igh-1a, DBA/2, Igh-1c and C57BL/6, Igh-1b) all express the same public idiotypic specificity, p. GAT, defined by the heterologous binding of anti-idiotypic serum 715 to C57BL/6 anti-GAT antibodies. None of these antibodies expressed the strain-restricted idiotypic specificity, s.r. GAT-1, defined by the binding of anti-idiotypic serum JL 122 to BALB/c anti-GAT antibodies. BALB/c anti-GAT HP could be shown to fall into three subsets with respect to their fine antigenic specificity for GAT, GT and GA. An individual idiotypic specificity, i1-GAT (defined by syngeneic anti-idiotypic sera raised against one of the BALB/c HP), was also found on a group of BALB/c HP which all shared a similar fine antigenic specificity pattern. Taken together, these observations suggest that the expressed mouse anti-GAT repertoire derives from a very few V-germ-line genes (VH-GAT and VK-GAT) which are highly conserved in the species, and which determine the structure resulting in the p. GAT idiotypic specificity. The variations in fine specificity and individual idiotype are likely therefore to reflect somatic variations affecting these germ-line genes.

Characterization of monoclonal antibodies against prostaglandin E2: fine specificity and neutralization of biological effects.

The specificity and heterogeneity of the immune response of BALB/c mice immunized with prostaglandin E2 (PGE2) coupled to thyroglobulin was studied. All the animals (n = 50) responded to PGB2, a transformation product of PGE2. However, following repeated injections most of the animals (n = 30) were also able to respond to PGE2. Cellular hybridizations were performed and five anti-PGE2 monoclonal antibodies were isolated and analysed. They are mainly directed against the ring and the omega-chain of PGE2 but their specificity toward the alpha-chain is more limited. The association constants are greater than to 1 X 10(9) M-1. The monoclonal antibody 8E.57.71 (Ka = 1.3 X 10(10) M-1) is particularly convenient for sensitive radioimmunoassays (detection limit 25pg/ml, when iodinated tracer is used). Anti-PGE2 monoclonal antibodies were found to neutralize the specific binding of [3H]PGE2 to rat brain hypothalamic receptors and to inhibit the PGE2 induction of rat fundus muscular contraction.

Analysis of a major rat idiotype associated with Anti-GAT antibodies.

An anti-idiotypic antiserum was raised in a rabbit against a pool of purified F.344 rat anti-GAT antibodies. GAT-13, the idiotype defined by this serum, is present in all F.344 anti-GAT sera from primary and secondary anti-GAT responses. Anti-GAT sera of 13 inbred rat strains, with different RT1 haplotypes and with different heavy- and light-chain allotypes, all express idiotypic determinants cross-reacting with GAT-13. Thus, like in mice anti-GAT antibodies from rats express public idiotypic determinants. The anti-idiotypic serum also recognizes a highly conserved idiotypic specificity present on mouse and guinea-pig anti-GAT antibodies. The mouse, rat and guinea-pig express a similar highly conserved idiotypic specificity after immunization with GAT. All anti-GAT antibodies from the mouse and guinea-pig bear this idiotypic specificity. These results confirm the existence in the anti-GAT response of interspecies cross-reactive idiotypic determinants.

Contribution of the H- and L-chains and of the binding site to the idiotypic specificities of mouse anti-GAT antibodies.

The contribution of the H- and L-chains to the structure of the main idiotype of anti-poly (Glu60-Ala30-Tyr10) (GAT) antibodies has been studied. This idiotype has been previously divided into four types of specificity: (1) the highly conserved idiotypic specificity (h.c. GAT) is expressed by anti-GAT antibodies from the guinea-pig, rat and mice; (2) the public specificity (p. GAT) is expressed in an identical form by all anti-GAT antibodies from all strains of mice tested and by all hybridoma products (HP) with anti-GAT activity; (3) the strain-restricted specificity (s.r. GAT-1) is only expressed by anti-GAT antibodies from strains with Ig-1a, Ig-1c and Ig-1c allotypic markers; and finally (4) the individual specificity i1-GAT defined on HP G5 is also expressed by most of the hybridoma protein with anti-poly (Glu50-Tyr50) (GT) activity. In this paper we show that h.c.GAT, p.GAT and i1-GAT require the interaction of H- and L-chains to be expressed: (1) isolated H- and L-chains from HP G5 did not express these specificities; and (2) recombinant molecules composed of H- and L-chains from HP with anti-GAT activity and an irrelevant myeloma protein (MOPC21) never expressed h.c.GAT, p.GAT and i1-GAT. We next investigated the relationship between the GAT binding site and the p.GAT, h.c.GAT and s.r.GAT-1 idiotypic specificities. GAT and GT were not able to inhibit the binding to s.r.GAT-1 while they inhibit the idiotypic binding to p.GAT and h.c.GAT. A GAT fragment of mol. wt 3000 was also shown to inhibit the binding of p.GAT and h.c.GAT to the appropriate sera. Thus p.GAT and h.c.GAT are very close to the GAT combining site while s.r.GAT-1 represents an idiotypic specificity located outside the GAT binding site.

Monoclonal antibodies to human epidermal filaggrin, some not recognizing profilaggrin.

To improve understanding of human profilaggrin processing to filaggrin, we produced seven monoclonal antibodies against epidermal filaggrin (AHF1-7). They were characterized on human epidermis by indirect immunofluorescence, immunogold labeling, and immunoblotting and found to be directed against seven different epitopes of (pro)filaggrin. AHF1-5 labeled the keratohyalin granules and the fibrous matrix of the lower corneocytes, and recognized filaggrin and profilaggrin. AHF6 also labeled the keratohyalin granules and the corneocyte matrix, but only recognized filaggrin. In addition to this reactivity within the upper epidermis, AHF4-6 stained the cytoplasm of the basal cells, and cross-reactivity of AHF5 and AHF6 with cytokeratin K14 was revealed on immunoblots. It is interesting that AHF7 recognized filaggrin, but not profilaggrin, and labeled only the corneocyte matrix and not the keratohyalin granules. This indicates that filaggrin and cytokeratins share several antigenic determinants and that filaggrin bears at least one epitope absent from its precursor. The original series of monoclonal antibodies described here appears to be a powerful tool for studying human profilaggrin processing in normal conditions and in the keratinization disorders in which processing is altered.

Specific detection of anti-HBc antibodies with an enzyme immunoassay using recombinant HBcAg and monoclonal antibodies.

An enzyme immunoassay for the detection of total anti-HBc antibodies in undiluted serum samples was developed. This assay utilizes an anti-HBc monoclonal antibody and a recombinant HBc antigen. The results of the clinical validation are now reported. A total of 1,301 sera were tested using both the Recombinant TOTAL HBc Ab EIA and a reference assay. The specificity was evaluated on a panel of 573 normal human sera and human sera from subjects with pathological findings unrelated to a hepatitis B virus infection. The sensitivity was studied on a total of 455 sera from HBV infected patients at different stages of infection. The final results indicate 99.8% sensitivity and 99.8% specificity. In addition, 273 sera with either isolated anti-HBc antibodies or with anti-HCV antibodies were tested. The agreement between the Recombinant and the reference assay for these two populations, 96.6 and 90.1%, respectively, is discussed.

Induction by monoclonal anti-idiotypic antibodies of an anti-poly(Glu60 Ala30 Tyr10) (GAT) immune response in GAT-responder and GAT-nonresponder mice.

Two different monoclonal anti-idiotypic (Id) antibodies, HP-Id20 and HP-Id22, recognizing two discrete idiotopes characteristic of the anti-poly(Glu60 Ala30 Tyr10) (GAT) response were used to immunize BALB/c (GAT-responder) and DBA/1 (GAT-nonresponder) mice. The monoclonals were injected either copolymerized with keyhole limpet haemocyanin or polymerized with glutaraldehyde. The specific response was studied by two assays: (a) inhibition of binding of monoclonal anti-GAT antibody G5Bb2-2 to HP-Id20 and HP-Id22 and (b) GAT binding assays. In BALB/c GAT-responder mice, HP-Id20 and HP-Id22 immunization led to the preferential stimulation of immunoglobulin idiotypically related to anti-GAT antibodies (Ab1') and expressing anti-GAT activity. The results obtained with BALB/c nu/nu mice indicated that this response is T-cell-dependent. By means of the same experimental protocol GAT-nonresponder animals could be induced to produce anti-GAT antibodies after HP-Id immunization. This last result indicates that anti-Id immunization can bypass Ir gene control and does not preferentially stimulate the induction of GAT-specific T suppressor cells.

Immune response against poly(Glu60,Ala30,Tyr10) (GAT): immunization with monoclonal anti-idiotypic antibodies leads to the predominant stimulation of idiotypically similar immunoglobulins with anti-GAT activity.

Two monoclonal anti-idiotypic antibodies (HP-Id20 and HP-Id22) recognizing two different public idiotopes expressed in the anti-poly(Glu60,Ala30,Tyr10) (GAT) response were used to immunize BALB/c and C57BL/6 mice. From these animals hybridomas were isolated. From BALB/c and C57BL/6 mice eight and seven monoclonal antibodies were characterized, respectively. The reagents were classified according to the expression of the public idiotypic specificity p.GAT (recognized by a rabbit antiserum). The anti-GAT activity and the expression of the various idiotopes characterized on anti-GAT polyclonal and monoclonal antibodies were also studied. Most of the reagents are Ab1'-type of antibody resembling anti-GAT antibodies. One anti-anti-idiotypic monoclonal antibody (Ab3) was also isolated from BALB/c mice. This suggests that in this experimental model the repertoire induced after HP-Id immunization and antigen stimulation is comparable. The idiotypic analysis of a large number of anti-GAT and of Ab1' monoclonal antibodies suggests that only two public idiotopes are involved in the anti-GAT response.

Immune response to the copolymer of L-glutamic acid50-L-tyrosine50 (GT): effect of adult thymectomy and genetic control of immune suppression.

The secondary antibody response to GT and GT-MBSA (GT coupled to methylated bovine serum albumin) and the ability to generate specific suppressor cells after GT preimmunization were examined in the auto-immune NZB strain and in normal and adult thymectomized C3H, BALB/c and CBA mice. As previously shown C3H, BALB/c and CBA mouse strains do not mount a secondary anti-GT antibody response after immunization with GT. On the contrary NZB mice where shown to develop a small but significant anti-GT antibody production. All the mice tested produced anti-GT antibodies after immunization with GT-MBSA. Adult thymectomy greatly decreased this anti-GT antibody response in BALB/c, C3H and CBA mice, suggesting the involvement of a short-lived T-cell subpopulation in the development of an optimal anti-GT-MBSA response. Preimmunization with GT suppressed the secondary antibody response to GT-MBSA in CBA and BALB/c mice but not in NZB and C3H mice, although all these strains bear the H-2-linked Is genes. This indicates an additional genetic control on the generation of GT-specific suppressor cells.

Genetic control of the immune response to the L-Glu60-L-Ala30-L-Tyr10 (GAT) terpolymer. V. Three types of idiotypic specificities on BALB/c anti-GAT antibodies.

Three types of idiotypic specificities compose the major idiotype of anti-poly (L-Glu60-L-Ala30-L-Tyr10) (GAT) antibodies from BALB/c mice (idiotype termed GAT-715). Assays have been designed to analyzed and study the distribution of these specificities. The highly conserved idiotypic specificity (h.c.GAT) has been assayed by the binding of serum 715-7A4 to radiolabeled rat anti-GAT antibodies. Guinea pig and mouse anti-GAT antisera all express the same h.c.GAT specificity. The public specificity (p.GAT) has been shown to be present in an identical form in all anti-GAT antisera from all strains of mice studied. The assay used for p.GAT was the binding of serum 715-7A4 to C57BL/6 anti-GAT antibodies that express only p.GAT. Finally, the strain-restricted specificity s.r.GAT has also been investigated by radioimmunoassay; this specificity is expressed only by strains BALB/c, BALB/b, BUB/J, DBA/2, DBA/1 and ATL. This expression is independent of known allotypic markers. However, the expression of the s.r.GAT specificity of BALB/c mice follows the genetic distribution of VH genes of BALB/c origin indicating that s.r.GAT can be considered as a genetic marker of some VH gene(s) involved in the specific immune response to the GAT terpolymer.

p55 IL-2 receptor mRNA precursors in murine T lymphocyte nuclei.

An unusual family of cDNA clones homologous to human p55 IL-2R sequences was isolated from the murine HT-2 Th cell line. These clones were mapped, partially sequenced, and compared with previously published human and mouse IL-2R sequences. They appear to consist of various combinations of exons and introns, suggesting that they are derived from p55 IL-2R mRNA precursors. The configuration of exons in the splicing intermediates indicates that the murine and human gene organizations are similar and that the 3' end of intron 3 is well conserved between the two species. RNA mapping experiments using nuclear, cytoplasmic, and total RNA and probes derived from various parts of the p55 IL-2R gene support and extend the sequence data. They indicate that detectable amounts of immature p55 IL-2R mRNA are found specifically in the cell nucleus of the HT-2 cell line. Similar data were obtained for the Th cell clone 52.3 and the cytotoxic T cell line CTLL. All these results indicate that the T cell nucleus contains significant amounts of immature p55 IL-2R mRNA.

The idiotypic network and the internal image: possible regulation of a germ-line network by paucigene encoded Ab2 (anti-idiotypic) antibodies in the GAT system.

Heavy and light chain variable regions from eight monoclonal Ab2 (anti-idiotypic) antibodies of the GAT antigen, a (Glu60 Ala30 Tyr10)n co-polymer, have been analyzed by direct mRNA sequencing. Three mAb2s were directed against private idiotopes and used various VH-D-JH and Vk-Jk combinations. By contrast, the five 'anti-public' mAb2 antibodies used a very restricted repertoire, including all gene segments. Interestingly, within their D regions, Glu-Glu-Tyr or Tyr-Tyr-Glu sequences were reminiscent of the original (GAT) antigen and may act as possible internal images. A striking observation was that two mAb2 antibodies shared the same V-D-J sequence although derived from separate fusions. As this D sequence, 33 nucleotides long, has not been described so far, it is suggested that it may be encoded by a new germ-line D gene, acting as a crucial regulatory element in a GAT germ-line idiotypic network. An alternative model that may lead to the construction of this D segment by 'odd' rearrangements from pre-existing already reported sequences is also presented.

Gene repertoire of the anti-poly(Glu60Ala30Tyr10) (GAT) immune response: comparison of VH, V kappa, and D regions used by anti-GAT antibodies and monoclonal antibodies produced after anti-idiotypic immunization.

Eight monoclonal antibodies were selected from BALB/c mice immunized with two different monoclonal anti-idiotypic antibodies recognizing two discrete idiotopes characteristic of the anti-poly(Glu60Ala30Tyr10) (GAT) antibody response. These monoclonal antibodies were previously classified as Ab1 (anti-GAT-like) and Ab3 (anti-anti-idiotype) on the basis of expression of the public idiotypic specificity (p.GAT) studied with a xenogeneic serum, anti-GAT activity, and expression of various public idiotopes. All the heavy chain variable region (VH) sequences from Ab1 are nearly identical to the VH sequences of Ab1 anti-GAT monoclonal antibodies. The same type of results has been found with the Ab1 kappa light chain variable region (V kappa) sequences. Confirming our classification, Ab3 VH and V kappa sequences were found to be completely different from Ab1 VH and V kappa sequences. The Ab1 diversity (D) regions are different from one another and different from the D regions found on monoclonal anti-GAT antibodies but function similarly. These D regions are not simply derived from already described D genes. Finally, our results suggest that in the anti-GAT response VH and V kappa sequence are mainly responsible for idiotype expression.

Idiotypy of human anti-Candida albicans antibodies: recurrence, presence of a cross-reactive autoanti-idiotypic-like activity, and role in the induction of specific in vitro antibody response.

Rabbit anti-idiotypic antibodies (L12) were raised against human anti-mannan of Candida albicans (CA) antibodies isolated from the serum of a normal donor. The absorbed anti-idiotypic antiserum bound to donor anti-CA mannan antibodies but not to control immunoglobulins. Binding was inhibited by CA mannan but not by other polysaccharide antigens. L12 was shown to cross-react with anti-CA mannan-isolated antibodies or with anti-CA antibody-containing sera from individuals unrelated to the donor. IgG fraction isolated from the donor serum was repeatedly absorbed on CA mannan Sepharose to remove anti-mannan antibodies. This IgG fraction (named autoanti-idiotypic fraction) blocked, in a dose-dependent fashion, the binding of rabbit anti-idiotype to donor anti-CA mannan antibodies. Moreover, this CP-depleted IgG fraction cross-reacted with public idiotypic determinants of unrelated anti-CA mannan antibodies. Finally, L12 induced sensitized lymphocytes to produce anti-CA mannan antibodies in vitro in the absence of antigen.

A transaldolase : An enzyme implicated in crab steroidogenesis.

In arthropods, development is controlled by cholesterol-derived steroid hormones: the ecdysteroids. In vertebrates and insects, steroidogenesis is positively regulated and this is mediated by cAMP. In crustaceans, ecdysteroid biosynthesis by steroidogenic organs (Y-organs) is negatively regulated by a neuropeptide, the Molt Inhibiting Hormone (MIH). This neuropeptide-induced inhibition occurs via cyclic nucleotides and depends on protein synthesis. In the present work, we provide evidence that a major 36.2-kDa cytosolic protein (P36; pl: 6.8) from crab Y-organs is positively correlated with steroidogenic activity. On the basis of its amino acid sequence, P36 could be related to transaldolase, an enzyme of the pentose phosphate pathway which generates NADPH. In Y-organs, the enzymatic activity ofCarcinus transaldolase increases with steroidogenic activity, and MIH treatment decreases both synthesis and activity of transaldolase. Various transaldolases have been characterized in very distantly related groups, namely bacteria, yeasts, and humans. These enzymes are highly conserved and present strong structural homologies, interestingly the crab transaldolase is closest to that enzyme characterized in human cells.

Neuropeptide-induced inhibition of steroidogenesis in crab molting glands: involvement of cGMP-dependent protein kinase.

In crustaceans, ecdysteroid production by the molting glands (Y-organs) is negatively regulated by a neuropeptide, molt-inhibiting hormone (MIH). The involvement of cyclic nucleotide-dependent kinases in the mechanism of action of this neuropeptide was investigated with regard to the steroidogenic activity of Carcinus maenas Y-organs. Regardless of the activity level, the major phosphotransferase activity measured in cytosolic fraction was cGMP-dependent, indicating a relatively high cytosolic concentration of cGMP-kinase in these cells. Phosphotransferase activity was nearly twofold higher in the intermolt (low steroidogenic activity) than in premolt (high steroidogenic activity) animals. In vitro incubation of premolt Y-organs with MIH for 1 hr increased by 3.7-fold the cGMP-kinase activity ratio (-cGMP/ +cGMP). Numerous endogenous protein substrates were predominantly phosphorylated in a cGMP-dependent manner in cytosolic, particulate, and membrane fractions. Similar phosphoprotein patterns were observed in both molting stages. By contrast, cAMP-kinase activity, which was low in intermolt Y-organs, increased significantly in the active steroidogenic premolt Y-organs. The increase in cAMP-kinase activity was accompanied by a cAMP-dependent phosphorylation of several specific endogenous proteins. Taken together these results strongly suggest that activation of cGMP-kinase and subsequent phosphorylation of an endogenous protein(s) may be responsible, at least in part, for the MIH-induced inhibition of steroidogenesis. By contrast, it is most unlikely that cAMP-kinase is involved in these processes.