Michel Fougereau

Tyrosine phosphorylation of the Wiskott-Aldrich syndrome protein by Lyn and Btk is regulated by CDC42.

The Wiskott‐Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (FcϵRI) at the surface of RBL‐2H3 rat tumor mast cells. Lyn and the Brutons tyrosine kinase (Btk), two protein tyrosine kinases involved in FcϵRI‐signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.

Individual germinal centres of myasthenia gravis human thymuses contain polyclonal activated B cells that express all the Vh and Vk families.

Using in situ hybridization, we analysed the immunoglobulin repertoire expressed by the B cells present in myasthenia gravis thymuses from four patients. B cells, mostly in activated state, were clustered in germinal centres, in which multiple isotypes were identified. A majority of cells expressed IgG as compared with IgM. with a roughly similar contribution of k and δ chains. Hybridization with the six VH and the 4 Vk human family probes was observed in serial sections, providing additional evidence that individual germinal centres were polyclonal. The thymic B cell repertoire closely reflected the VH and the Vk family usage of normal peripheral blood lymphocytes with the preferential utilization of VH3, Vk1 and Vk3.

The Ig germline gene repertoire: economy or wastage?

Abstract BALB/c V H and V K germline genes of the GAT system exist in large families. Here, Michel Fougereau and his colleagues report that they are used in many antibodies of discrete specificities. They propose that only a small number of genes (including V H and V K GAT) are functionally active and are used either in their germline configuration or after somatic conversion involving the large pool provided by the other genes (including pseudogenes) of the corresponding family. This hypothesis reconciles the paradox that although there appear to be many V genes, only a few are expressed.

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.

The human pre-B-specific λ-like cluster is located in the 22q11.2–22q12.3 region, distal to the IgCλ locus

The chromosomal location of the lambda-like gene cluster, a gene family selectively expressed in human pre-B cells, was analyzed by in situ hybridization with a probe specific for the lambda-like genes. This cluster mapped in the q11.2-q12.3 region of chromosome 22. The use of Burkitt lymphoma and myelogenous leukemia cell lines with translocations in the 22q11 region led to a refinement in the location according to the following order: cen----BCRL2, VpreB, IgV lambda 1----BCRL4, IgV lambda----IgC lambda----BCR----BCRL3, lambda-like----tel. Unlike those of the mouse system, the pre-B-specific genes VpreB and lambda-like do not belong to the same transcriptional unit.

NH2-terminal amino-acid sequences of poly (Glu60, Ala30, Tyr10) (GAT) and poly (Glu60, Ala40) (GA) monoclonal antibody heavy chains☆

Abstract The heavy-chain NH 2 -terminal sequences of five IgM, kappa monoclonal anti-GAT§ antibodies, and five IgG, kappa anti-GA antibodies (all derived from immune DBA/2 mice), have been determined. These monoclonal hybridoma antibodies were previously characterized with respect to their idiotypic properties. Extensive identity, covering possibly up to residue 33, was observed between the five IgM, kappa anti-GAT antibodies, whether they expressed the cross-reactive idiotypic specificities (CGAT) or not. One anti-GAT γ 1 -chain derived from (B6 × D2)F1 mice was blocked. Among the five CGAT-negative, monoclonal anti-GA antibodies from DBA/2 mice, strong homologies were observed between the four heavy-chain amino-acid sequences of the antibodies expressing the cross-reactive GA-1 idiotypic specificities. These NH 2 -terminal V H sequences differed from each other and from those of the anti-GAT monoclonal heavy chains from the DBA/2 strain by only one amino acid, on average. Conversely, of the three GA-1-negative anti-GA antibodies for which sequencing was attempted, two were blocked, and one differed sharply from the others.

IGMκ/λ EBV human B cell clone: An early step of differentiation of fetal B cells or A distinct B lineage?☆

Abstract In agreement with the clonal theory, one B lymphocyte synthetizes one antibody due to allelic and isotypic exclusion. We analyzed an EBV B-cell clone, E29.1, derived from an 11 week-old embryo, and secreting both IgMκ and IgMλ. Structural analysis of produced IgM, indicated that λ-containing pentamers could be considered hybrid molecules, expressing both the κ and the λ chains, with a κ/λ ratio between 5 and 10. It was also found that 60% of the λ chains were secreted in free form, presumably as a result of a better affinity of μ chains for κ chains. The sequence of the three transcripts had an entirely ORF (Open Reading Frame), and were very close to germline sequences, with, however, an additional codon between Vκ and Jκ gene which has never been described in adult myeloma protein or cDNA human sequence. This observation is suggestive of N diversity taking place in κ chains. The possible role of Kde (κ deleting element) recombination onto κ/λ locus activation was analyzed on a collection of 23 λ clones. The status of rearrangement of κ genes indicated that 35% of these clones had retained, at least, one κ allele without the Kde recombination, four λ clones had one κ allele in germline configuration. Different hypotheses of maturation from pre-B cell to B cell with activation of light chain genes are discussed.

Repertoire of the anti-MOPC 173 anti-idiotypic antibodies: genetical and structural features.

Abstract Anti-idiotypic antibodies against the MOPC 173 protein (IgG2a,κ) were raised in five strains of mice. Three types of responses were observed: CBA and A/J mice were all high-responder (HR) as appreciated from the haemagglutinating titers. Balb/B animals were very low or non-responders (NR). Individual antisera titers of Balb/c and C57BL/6 mice were scattered, ranging from no response to high titers. CBA and A/J antibodies (HR) gave an IEF pattern that was characteristic of each strain, which is suggestive of the expression of germ-line genes. By contrast, discrete IEF patterns of Balb/c and C57BL/6 were observed for each antiserum tested, a situation which might be compatible with either the expression of a somatic repertoire or an extremely large collection of heterogeneous germ-line V genes. Cross-reactive idiotypes were looked for by screening a collection of 115 mouse myeloma sera. Only 2 were found to be slightly inhibitory in a specific radioimmunoassay. A segregation of the NR and HR characters was observed within our Balb/c colony, which is suggestive that some level of genetic drift had taken place. Genetical characteristic of HR and NR strains, screened for the H-2 locus and the C H allotypes, suggest that the expression of these anti-idiotypic antibodies depends both on the Ig gene repertoire and on regulation mechanisms that might be encoded by the I region genes.

Analysis of the GAT B Repertoire

Synthetic polypeptides(1) were first used as structurally well-defined models to understand the antigenicity of proteins, for which the exact nature of any given epitope was and still remains far from clear, except in a very few cases.(2,3) They subsequently proved decisive tools to discover genes which regulate the immune response in the guinea pig(4) and the mouse.(5) Among synthetic polypeptides, the (Glu60Ala30Tyr10) n random terpolymer, known as “GAT,” has been extensively used. This polymer, with molecular weights usually ranging between 30,000 and 100,000, is recognized by the immune system mostly through conformational epitopes,(6) and contains a high amount of α-helix. Genetic control of the immune response to GAT has been largely documented in the mouse, allowing definition of responder and nonresponder strains,(7) the later being of the H-2 P , H-2 q , and H-2 s haplotypes. As “nonresponder” (NR) strains could be forced to make anti-GAT antibodies provided the synthetic polypeptide was coupled to a carrier, such as methylated BSA,(8) the absence of response in NR strains was not due to a deficient repertoire at the B-cell level. In fact, a more detailed analysis of anti-GAT antibodies produced by both responder and “nonresponder” strains indicated that the repertoires looked very similar with respect to idiotypic specificities identified on GAT-specific antibodies.(9)