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.

and 1D6partement d'Immunologie de l'Institut Pasteur, 28. rue du Docteur Roux, 75724 Paris Cedex 15, France Communicated by M.FougereauHeavy 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 germline 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.

From BALB/c mice immunized with anti-GAT monoclonal antibody (mAb) G5, we have obtained anti-idiotypic mAb against individual (or private) idiotopes, expressed by G5 as well as anti-GAT mAb, that are heteroclitic because they recognize poly-(Glu50, Tyr50) (GT) better than poly(Glu60, Ala30, Tyr10) (GAT). From BALB/c mice immunized with BALB/c polyclonal anti-GAT antibodies, anti-idiotypic mAb directed against public idiotopes expressed following GAT immunization in all individuals of all mouse strains tested have been obtained. Nine anti-idiotypic mAb were studied in detail. One of these mAb recognizes only polyclonal anti-GAT antibodies; the other eight recognize polyclonal anti-GAT antibodies and anti-GAT mAb. The distribution of the structures recognized by the different anti-idiotypic mAb on a battery of 20 anti-GAT mAb allows definition of two families of public idiotopes.

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.