Genetic basis for expression of the idiotypic network. One unique Ig VH germline gene accounts for the major family of Ab1 and Ab3 (Ab1') antibodies of the GAT system.

Ig germline genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA either in pBR328 plasmid or in EMBL 3 phage. Three clones that gave a very strong positive hybridization signal with a VH anti-GAT-specific probe were completely characterized and sequenced. All three were greater than 95% homologous, with the exception of the 5' noncoding region, which was only 85% homologous but contained characteristic regulatory signals. One of these genes, H10, had a sequence that was completely identical to that of a cDNA derived from a GAT-specific BALB/c hybridoma. Southern blot analysis using Eco RI-digested DNA from rearranged GAT-specific hybridomas revealed that the same gene was used for other GAT-specific VH regions, including one differing from the H10 sequence by 12 nucleotides, which must have been generated by a somatic mechanism. The same H10 germline gene was also used, in most cases without any nucleotide substitution, in hybridomas of the Ab1' set of the GAT idiotypic cascade, suggesting that immunization with Ab2 (antiidiotypic) antibodies preferentially stimulates the direct expression of VH germline genes. Finally, the previous hypothesis that NPa and GAT VH genes were derived from the same germline gene was definitively confirmed, both from sequence data and Southern blot analysis.

V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains.

A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level.

Activation of clones producing self-reactive antibodies by foreign antigen and antiidiotype antibody carrying the internal image of the antigen.

Because we found in previous work that a high fraction of antibodies exhibiting various specificities bound to glutamic acid 50-tyrosine50 homopolymer (GT) and expressed pGAT cross-reactive idiotype (IdX), we studied the activation of clones producing multireactive antibodies in 1-mo-old MRL/lpr and C3H/HeJ mice bearing VHJ haplotype. The activation of such clones was studied after mice were immunized with GT in CFA, HP20 (an anti-Id MAb carrying the internal image of GT in the D region), and a synthetic peptide corresponding to the D segment of HP20. Our results indicate that immunized mice produced both GT- and self-reactive antibodies. Study of the immunochemical properties of MAb showed that they exhibit multispecific properties and bind with similar-affinity constants to GT or self-antigens such as DNA, Smith antigen (Sm), and IgG2a. An important fraction of antibodies obtained from MRL/lpr mice immunized with HP20 expressed pGAT IdX and some of these antibodies share IdX expressed on anti-DNA, Sm, and rheumatoid factor (RFs) antibodies. The hybridomas producing multispecific autoantibodies use heavy-chain- (VH) and light-chain-variable region (VK) genes from various V gene families, suggesting that they do not derive from the pool of GAT precursors. Sequencing of VH and VK genes of two antibodies show that they can use closely related VHJ558, unmutated VK1, or different VK genes than those used by anti-GT antibodies. Our data demonstrate that clones producing antibodies binding to GT and self-antigens with similar-affinity constants can be activated by foreign or anti-Id antibodies carrying the internal image of the antigen or even by a synthetic peptide corresponding to the D segment of anti-Id antibodies.

A human non-XLA immunodeficiency disease characterized by blockage of B cell development at an early proB cell stage.

We report a detailed analysis of a B cell defect affecting a patient girl born from first cousin parents, characterized by a severe non-X-linked agammaglobulinemia with a total absence of CD19- cells in the periphery. In the bone marrow, CD19 expression was also highly impaired, resulting in the absence of both B and preB compartments. By contrast, CD34+CD10+, CD34psiL+, and some CD19+CD10+ mostly CD34+ early proB cells were present, although diminished. Semiquantitative RT-PCR analysis performed on mononuclear bone marrow cells indicated that lambda-like, VpreB, Rag-1, Rag-2, and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Igalpha, Igbeta, VH-Cmu, and Vkappa-Ckappa transcripts characteristic of later stages were severely depressed. This phenotype resembles that of Pax-5 knock-out mice, but since the coding sequence of the patient Pax-5 cDNA was shown to be normal, the defect might rather result from an altered regulation of this gene. All these data indicate that the patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, i.e., earlier than X-linked agammaglobulinemia, before the onset of Ig gene rearrangements.

Bone marrow cells in X-linked agammaglobulinemia express pre-B-specific genes (lambda-like and V pre-B) and present immunoglobulin V-D-J gene usage strongly biased to a fetal-like repertoire.

Expression of Ig and Ig-related genes has been studied in bone marrow cells from two patients with severe form of X-linked agammaglobulinemia (XLA). Phenotypic analysis revealed the presence of pre-B cells, in the absence of mature B cell markers. The pre-B-specific genes, lambda-like and V pre-B, were normally transcribed. Sequence analysis of 48 distinct V-D-J cDNA clones directly derived from XLA bone marrow cells indicated that they had characteristics of an early fetal pre-B repertoire. All VH families were identified, with a strong bias in the gene usage: a few VH genes were largely overexpressed, either germline or slightly mutated; most genes had been located 3' of the VH locus and were also used in fetal liver (8-13 wk of gestation). Short D regions, (resulting from D-D fusion, making usage of all D genes in both orientations with utilization of the three reading frames), restricted N diversity, and a fetal JH usage pattern were also observed. Taken together, our data suggest that the XLA defect does not alter V-D-J rearrangements nor the expression of mu, lambda-like, and V pre-B transcripts and most likely results in a poor efficiency of some critical steps of the B cell maturation.

Early occurrence of immunoglobulin isotype switching in human fetal liver.

A cDNA library prepared from a human fetal liver of the first trimester of gestation was screened with Ig C mu, C gamma, C kappa and C lambda probes. Ten heavy chain clones were isolated and characterized by restriction mapping and partial sequencing. The absence of Ig light chain clone and the presence of pre-B-specific lambda-like transcripts suggest that the immune compartment of this cDNA library was mostly derived from pre-B cells. Three transcripts of mu, gamma 2 and gamma 4 isotypes contained a V-D-J-C region with an open reading frame and used members of the VHIV, VHIII and VHI families, respectively. Seven clones were derived from sterile transcripts, one C mu and six C gamma. In addition to C mu exons, the sterile mu transcript contained the 5' flanking germline region. By contrast, the gamma sterile transcripts used a 5' sequence that was spliced from the I gamma 1 region onto the first C gamma 1 exon. In addition several of these transcripts were derived from alternative splicing. The simultaneous expression of both sterile and functional gamma transcripts suggests that the switch mechanism operates in normal fetal liver very early in ontogeny.

Idiotypic cross-reactivity of anti-GAT and anti-alprenolol antibodies: an approach to the structural correlates of the pGAT idiotypic specificity.

Most anti-GAT antibodies in the BALB/c strain express a public idiotypic specificity (pGAT), which is encoded by specific germline genes (VH10, VK5.1 and VK1A5). One or both of these germline genes, referred to as "GAT-specific genes", are also used by four anti-alprenolol antibodies. Anti-Alp and anti-GAT antibodies show no cross-reactivity for the antigens. The light chain of one anti-Alp antibody, 22C4, is encoded as the anti-GAT antibodies by a VK5.1-J2 combination and expresses part of the pGAT idiotopes, whereas the heavy chain is not "GAT"-related. Two anti-Alp using the VH10-VK5.1-J1 association do not express any of the pGAT idiotopes. Sequence comparison of the various CDR sequences points to the predominant role of the VH-CDR2 and VL-CDR3 for the constitution of the pGAT specificity. Regarding VL-CDR3, a drastic change in idiotypic determinants appears to be linked to V-J junctional diversity.

A single VH-gene associated with a variety of D- and J-segments encodes for a large family of ABPC48-related antibodies induced by antiidiotypic immunization.

Two series of monoclonal antibodies have been obtained from BALB/c mice immunized against two antiABPC48 antiidiotypic antibodies. They are divided into two serologically different classes. Class I antibodies bind only the immunizing antibody; class II antibodies display a broad binding capacity to various antiidiotypic antibodies, and some bind levan, as does ABPC48. Northern blot analyses and partial mRNA sequencing show that all class II antibodies express the VH-gene coding for ABPC48 and UPC10 antilevan antibodies associated with a variety of D- and J-segments. The third hypervariable region of the sequenced antibody with antilevan activity is structurally related to that of ABPC48 and UPC10 antibodies but has a different genetic origin. This study indicates that the identification of idiotype-related antibodies arising from antiidiotypic immunization may be misleading, if based on their antigen-binding properties; and it stresses the importance of structural approaches for the analysis of regulatory mechanisms ruling immune responses.

IGM kappa/lambda EBV human B cell clone: an early step of differentiation of fetal B cells or a distinct B lineage?

In agreement with the clonal theory, one B lymphocyte synthesizes 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 IgM kappa and IgM lambda. Structural analysis of produced IgM, indicated that lambda-containing pentamers could be considered hybrid molecules, expressing both the kappa and lambda. chains, with a kappa/lambda ratio between 5 and 10. It was also found that 60% of the lambda chains were secreted in free form, presumably as a result of a better affinity of mu chains for kappa 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 V kappa and J kappa gene which has never been described in adult myeloma protein or cDNA human sequence. This observation is suggestive of N diversity taking place in kappa chains. The possible role of Kde (kappa deleting element) recombination onto kappa/lambda locus activation was analyzed on a collection of 23 lambda clones. The status of rearrangement of kappa genes indicated that 35% of these clones had retained, at least, one kappa allele without the Kde recombination, four lambda clones had one kappa allele in germline configuration. Different hypotheses of maturation from pre-B cell to B cell with activation of light chain genes are discussed.

Structural correlates to the rabbit immunoglobulin heavy chain a100 allotype.

Three a100/a100 homozygous rabbits immunized with Micrococcus lysodeikticus produced large amounts of anti-polysaccharide antibodies of restricted heterogeneity. These antibodies were purified by either immunoabsorption or ion exchange chromatography. The almost complete sequence of one heavy chain spanning residues 1-123, with the exception of 10 residues (66-67 and 79-86), was determined. Partial sequence data were also obtained for the two other heavy chains. The identity of these three sequences in the first framework region unraveled a prototype sequence of the a100 allotype that differs from homologous sequence of VH regions that determine other allotypic specificities. The gradient of sequence conservation was found to be a100 greater than a3 greater than a1 greater than a- greater than a2. Homologies in sequence paralleled previously described serological cross-reactions observed between a100, a3 and a1 specificities. This remarkable conservation of framework residues suggests that the VH regions of the rabbit immunoglobulins represent a paucigene system, in which each basic allotypic specificity might be encoded a discrete subgroup of genes.

The human pre-B cell receptor: structural constraints for a tentative model of the pseudo-light (psi L) chain.

In human pre-B cells, the mu chain is associated with a surrogate light chain composed of the lambda-like and Vpre-B gene products. This pre-B cell receptor presumably triggers early steps of B cell differentiation, We have determined the NH2-terminal amino acid sequence of the lambda-like chain, showing that the mature chain results from the cleavage of a leader segment of 44 residues, leaving a polypeptide of 169 amino acids having partial features of the Ig light chain domains, with the exception of the first 50 amino acid NH2-terminal region. We have completed the nucleotide sequence of the Vpre-B gene, which appears to contain 126 residues in its mature form of which the 24 COOH-terminal portion was not Ig-related. Analysis of transfectants has provided direct evidence that lambda-like and Vpre-B chains assemble together even in the absence of heavy chain, prompting the search for a structural basis of this interaction. Comparison with the domain organization of the regular Ig lambda chain suggests that most of the psi L chain can be accommodated within a CL-VL-like structure, with an extra "subdomain" contributed by the non-Ig-like portions of both the lambda-like and Vpre-B polypeptides.

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.

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.

Construction of temperature and Zn-dependent human stromal cell lines that amplify hematopoietic precursors from cord blood CD34+ cells.

Forty-five human stromal cell lines were established from long-term bone marrow cultures transformed with a new vector, pNu MTSVts, which contains the Zn-inducible metallothionein promoter and the temperature-dependent SV40 T antigen from SV40 A58 mutant. Six of these cell lines were studied because of their growth capacity. All cell lines differed with respect to growth potential, expression of cell surface markers, and cytokine transcripts. Major histocompatibility complex (MHC) class I, CD29, CD49d, and CD51 were present on all stromal cell lines, MHC class II and CD34 were consistently absent, and CD11a (LFA-1), CD18 (ICAM-1R), CD54 (ICAM-1), CD58 (LFA-3) CD56 (N-CAM), CD106 (V-CAM), laminin, and collagen IV were diversely expressed. All cell lines contained interleukin (IL)-1alpha, IL-1beta, IL-2, IL-5, and macrophage colony-stimulating factor (M-CSF) transcripts, whereas granulocyte M-CSF, TNFalpha, IL-3, IL-4, and IL-7 were diversely expressed. The most characteristic feature of these cells was their varying capacity to expand cord blood CD34+ cells. One of these stromal cell lines ensured more than twofold expansion of the initial CD34+CD10-CD19- population in the first 2 weeks. Differentiation toward the B cell lineage was limited, producing only very small numbers of CD19+ cells after 6 weeks of culture.

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 (Fc epsilonRI) at the surface of RBL-2H3 rat tumor mast cells. Lyn and the Bruton's tyrosine kinase (Btk), two protein tyrosine kinases involved in Fc epsilonRI-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.

A non-XLA primary deficiency causes the earliest known defect of B cell differentiation in humans: a comparison with an XLA case.

We report a detailed comparison of B cell defects in two patients, one XLA and one non-XLA. Both had severe agammaglobulinemia with a total absence of CD19+ cells in the periphery. In the non-XLA case, CD19 expression was also highly impaired in the bone marrow, resulting in the absence of both B and preB compartments. Early proB cells were present since CD34+CD10+ and some CD19+CD10+ mostly CD34+ were identified, although diminished. By contrast, in the XLA patient the CD34+CD19+ proB cells were increased whereas the CD34-CD19+ preB cell population was low. Semi-quantitative RT-PCR analysis performed on mononuclear bone marrow cells from the non-XLA patient indicated that lambda-like, VpreB, Rag-1, Rag-2 and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Ig alpha, Ig beta, VH-C mu and V kappa-C kappa transcripts characteristic of later stages were severely depressed. By contrast in the XLA patient most of these transcripts were observed in normal amounts. The phenotype of the non-XLA patient resembles that of Pax-5 or Ig beta knock-out mice, but since the coding sequence of both cDNAs were shown to be normal, the blockage might rather result from an altered regulation of one of these genes or from defect of other genes. All these data indicate that the non-XLA patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, before the onset of Ig gene rearrangements. From all agammaglobulinemias reported so far, including XLA cases and those resulting from C mu gene defects, the non-XLA patient exhibits the earliest blockage in the B cell differentiation pathway.

Organization and expression of the pseudo-light chain genes in human B-cell ontogeny.

In pre-B cells, the mu chain is expressed at the cell surface in association with a "light chain surrogate" encoded by the V pre-B and the lambda-like genes. This mu-psi-L complex presumably triggers early steps of the B cell differentiation, possibly by controlling the Ig gene rearrangements. In the humans, the lambda-like complex contains 3 genes, located in the 22q11.2-q12.3 band, telomeric to the IGCL locus, with which they share a similar organization, pointing to a common genetic origin. Only one lambda-like gene, 14.1, is functional and specifically expressed with V pre-B in pre-B cells. This expression starts in cells which still have the IGH locus in germline configuration (pro-B stage) and ceases as soon as the IGL loci rearrange. These pre-B specific transcripts provide useful markers of cells of the B lineage in both physiological and pathological situations.

Fetal versus adult PreB or B cells: the human VH repertoire.

At the preB stage, when only the IGH locus has rearranged, mu chains become expressed in association with the psi L chains, lambda-like and VpreB, thus forming the preB receptor. By the use of a monoclonal anti VpreB antibody, preB cells were isolated from two adult bone marrow samples, and the VH repertoire was analyzed and compared to fetal, XLA (X-linked agammaglobulinemia), and adult B repertoires. Most VH genes identified were also expressed in fetal liver, XLA bone marrow, and adult PBLs, with similar predominant usage of certain germline genes. Multiple D/D fusions, limited N diversity, and preferential use of JH4 with a low level of DQ52 usage were also identified. Few mutations could be observed, not specifically localized in CDR regions, that could be interpreted as not positively selected. Conversely, a shorter length of CDR3 appeared to be the hallmark of the preB step. Thus, the association of psi L chains with mu does not bring about a bias in the VH gene usage, but a first selection on the CDR3 region could be the result of recognition by given autoantigens or ligands different for preB cells and B cells.

[Structural analysis of immunoglobulins and the problem of antibody diversity. New approaches]. / Analyse de la structure des immunoglobulines et problème de la diversité des anticorps. Nouvelles approches.

The function of the various immunoglobulins is revealed by their structure. During humoral response to antigens, the host can modify the structure of its IgG's according to requirements. This is done by genetic processes and also by selection of the appropriate lymphocyte clones.