Gérard Sommé

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.

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.

Genetic control of the immune response to the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)--IV. Heterogeneity of idiotype GAT-715.

Abstract A major idiotype GAT-715 was defined in mice by a rabbit anti-idiotypic antiserum raised against BALB/c anti-poly (Glu 60 -A!a 30 -Tyr 10 ) (GAT) antibodies. Corresponding idiotypic specificities were found to be present on anti-GAT antibodies from responder and non responder mice regardless of their allotypic markers. Some of the idiotypic specificities that we have termed highly conserved idiotypic specificities are expressed by rat and guinea pig after immunization with GAT. In this paper we report experiments that show that idiotype GAT-715 can be further analysed and subdivided into public and private idiotypic specificities. All mice tested express the public specificities that represent 80% of idiotype GAT-715. Some strains also express private specificities; no genetic linkage is observed between the inheritance of heavy chain allotypic markers and private specificities. In addition, public and highly conserved specificities have been compared. A structural model for GAT-715 idiotype is discussed.

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: Preferential effect on the IgG1 response

Abstract We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu 60 Ala 30 Tyr 10 ) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF 1 /52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF 1 /E 3 was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG 1 , IgG 2a and IgG 2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG 1 response than the IgG 2a and IgG 2b responses.

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 others 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.

Idiotype expression and fine specificity of Glu60Ala30Tyr10-specific T proliferating cells

The fine specificity of anti-Glu60Ala30Tyr10 (GAT) and anti-Glu60Ala40 (GA) proliferating cells was studied. T cells primed with GAT proliferate both to GAT and GA and GA-primed T cells proliferate also to GA and GAT. This cross-reactivity was unexpected given the results previously reported on the fine specificity of anti-GAT antibodies. The effect on the proliferation of BALB/c lymph node cells (LNC) of a syngeneic anti-idiotypic serum, prepared in BALB/c against anti-GAT antibodies, was studied. Two major points are made in this paper: (i) the in vitro addition of the anti-idiotypic serum in cultures containing GAT-primed LNC and GAT enhances the proliferation of GAT-specific T cells; (ii) the anti-idiotypic serum is effective in priming in vivo LNC which then acquire the capacity to proliferate specifically with GAT in vitro. These results further confirm the existence of idiotype-like determinants on T cells.

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.

A study of possible reciprocal influences within the respective allotypy of the polypeptidic chains constituting the immunoglobulin molecule—II: The rabbit b series allotypic specificities

Abstract We did not reveal any difference in the expression of the b series allotypic patterns (carried by the constant region of the κ light chains) either with anti-allotypic sera prepared against IgG or with anti-allotypic sera prepared against light chains when the light chains carrying a given allotypic pattern were respectively associated with allotypically different heavy chains to form an IgG molecule. Very probably, the b series allotypic determinants are relatively far away from the association region of the light and the heavy chains in the IgG molecule. These two kinds of antisera reveal the same set of determinants on IgG and on their isolated light chains, but this set seems to have a different configuration on the isolated light chains and on the same light chain included in the IgG.

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.