Immunoglobulin heavy chain expression shapes the B cell receptor repertoire in human B cell development.

Developing B cells must pass a series of checkpoints that are regulated by membrane-bound Ig(mu) through the Igalpha-Igbeta signal transducers. To determine how Ig(mu) expression affects B cell development and Ab selection in humans we analyzed Ig gene rearrangements in pro-B cells from two patients who are unable to produce Ig(mu) proteins. We find that Ig(mu) expression does not affect V(H), D, or J(H) segment usage and is not required for human Igkappa and Iglambda recombination or expression. However, the heavy and light chains found in pro-B cells differed from those in peripheral B cells in that they showed unusually long CDR3s. In addition, the Igkappa repertoire in Ig(mu)-deficient pro-B cells was skewed to downstream Jkappas and upstream Vkappas, consistent with persistent secondary V(D)J rearrangements. Thus, Ig(mu) expression is not required for secondary V(D)J recombination in pro-B cells. However, B cell receptor expression shapes the Ab repertoire in humans and is essential for selection against Ab's with long CDR3s.

Composite exon structure of an unusual Ig lambda-like gene located at human 22q11 position.

The surrogate light chain, composed of the VpreB and the lambda-like proteins, plays a critical role in controlling the early stages of B lymphocyte development. The lambda-like locus, located on the q11. 2-q11.3 region of human Chromosome (Chr) 22, contains three genes (14.1 Flambda-1, and 16.1) among which only the 14.1 is functional. This gene contains three exons, whereas the others lack exon 1. We have isolated in fetal liver a transcript of the Flambda-1 gene that contains the exon 3 sequence and a long non-Ig related sequence upstream. We show that this sequence resulted from the splicing of three new exons located telomeric to the Flambda-1 gene, highly homologous to beta-glucuronidase exon 11 (Chr 7), to the ABR exon 8 (Chr 17), and to an Expressed Sequence Tag (EST), respectively. We also show that this chimeric transcript is expressed in cells or tissues from various origins. This composite gene structure appears to be a new example of human genome flexibility, which can be explained by mechanisms such as exon shuffling and which results in the emergence of new transcription units inserted in regions involved in translocations.

The VDJ repertoire expressed in human preB cells reflects the selection of bona fide heavy chains.

In early steps of B cell differentiation, mu chains are transiently expressed in association with a surrogate light chain (psi L) composed of the lambda-like and VpreB monomorphic polypeptides, thus forming a putative preB receptor. Using a monoclonal anti-VpreB antibody, preB cells were isolated from two adult human bone marrow samples and their VDJ repertoire analyzed at the transcription level. All VH families were identified and further analysis focused on VH3 sequence analysis of 37 distinct VDJ cDNA clones. The VH3 genes expressed in the two bone marrow samples were also encountered in fetal liver and adult peripheral blood lymphocytes with a roughly similar contribution of 3.30, 3.23, 3.9 and 3.53. The characteristic features of the preB repertoire as compared to the activation B repertoire include the quasi absence of somatic mutations, limited N diversity and a shorter third complementarity-determining region (CDR3). It also significantly differs from the fetal repertoire, which makes higher usage of DQ52 and has CDR3 of even shorter lengths. The almost constant presence of glycine residues in the CDR3 and predominance of JH4 with a low level of DQ52 DH usage, suggest that preB cell clones are submitted to an initial selective pressure which should be antigen independent. The bona fide heavy chains would be merely selected for their ability to interact with the surrogate light chains, thus shaping the repertoire that will be co-expressed with immunoglobulin light chains in IgM molecules.

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.

Interleukin-7 regulates c-myc expression in murine T cells and thymocytes: a role for tyrosine kinase(s) and calcium mobilization.

Interleukin-7 (IL-7) was originally identified as a pre-B cell growth factor whose proliferating activity has been extended to numerous target cells including T lymphocytes. We investigated c-myc mRNA expression, an oncogene associated with proliferation, in the murine T cell line D10 G4.1 and freshly isolated thymocytes since both target cells proliferate in response to IL-7. We find that blockade of the tyrosine kinase pathway by genistein, a potent tyrosine kinase inhibitor, inhibits both IL-7-dependent D10 G4.1 cell proliferation and c-myc mRNA expression which appears to involve de novo mRNA synthesis and to be under the control of short-lived protein repressor(s). We have also examined possible signal transduction pathways which might regulate c-myc mRNA expression in the murine T cell line. IL-7 biological activity is not affected by stimulation of the protein kinase C pathway by phorbol esters. Thus, IL-7 regulates c-myc mRNA expression in a protein kinase C-independent manner and these data are strengthened by protein kinase C depletion which does not modify IL-7 c-myc mRNA responsiveness. In contrast and independent of protein kinase C activation, intracellular calcium mobilization by means of ionomycin reduces IL-7 induction of c-myc mRNA expression and may represent a physiological mechanism whereby IL-7 bioactivity is regulated. The activity of IL-7 on c-myc mRNA expression has been extended to freshly isolated thymocytes and we find a synergistic effect of IL-7 with concanavalin A. Taken together our results illuminate the molecular mechanism of IL-7 c-myc induction in the T lineage by ascribing a role for tyrosine kinase and increase in intracellular calcium in both IL-7 induced gene induction and cell proliferation.

Induction by anti-CD40 antibody or soluble CD40 ligand and cytokines of IgG, IgA and IgE production by B cells from patients with X-linked hyper IgM syndrome.

We studied the ability of B lymphocytes from patients with X-linked hyper IgM syndrome (HIGM1) to be activated via the CD40 membrane receptor. HIGM1 is caused by a CD40 ligand gene mutation, leading to defective expression on the membrane of activated T lymphocytes. We found that triggering of B cells by an anti-CD40 monoclonal antibody or the soluble CD40 ligand plus interleukin (IL)-4 or IL-10 led to B cell proliferation and/or differentiation towards IgG, IgA and IgE secretion. This was reflected by transcription of C gamma, alpha and epsilon membrane isotype expression and IgG, IgA and IgE production. These results confirm the integrity of B cells in patients with the HIGM1 immunodeficiency and open up new therapeutic possibilities.

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.

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.

Organization and expression of the lambda-like genes that contribute to the mu-psi light chain complex in human pre-B cells.

Lambda-like genes encode a polypeptide chain that associates with VpreB and mu chain in the so-called mu-psi light chain complex specifically expressed in pre-B cells. In humans, the lambda-like gene cluster contains three genes, termed 14.1, 16.1, and F lambda 1. The 14.1 gene contains three exons and was previously sequenced. The 16.1 and F lambda 1 have been isolated from cosmid libraries. They both contain only the exons 2 and 3 that appear highly homologous to their 14.1 counterparts. The lambda-like gene cluster has been mapped on chromosome 22, close and distal to the BCR gene, in the order (14.1; F lambda 1) and 16.1. Hybridization with the 14.1 exon 1 probe did not detect an equivalent on 16.1 and F lambda 1 genes, but instead, only revealed the X6 gene, that was previously identified 5 kb upstream of the IGLC locus. It appears, therefore, that the gene organization of the lambda and lambda-like loci is strikingly similar, with a three-exon-containing gene 5' of a series of J lambda-C lambda or J lambda-C lambda-like tandemly organized genes. Analysis of the transcripts in a 8 week old human fetal liver clearly indicated that 14.1 was the only functional gene of the lambda-like cluster. Various forms of transcripts resulting from alternate splicing have been characterized. The major component consisted of a full-length (exons 1-2-3) mRNA, whereas a minor (1-3) transcript was identified. Only the full-length transcript could encode a functional polypeptide chain corresponding to the 22 kDa light chain surrogate.

Lambda-like and V pre-B genes expression: an early B-lineage marker of human leukemias.

V pre-B and lambda-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a mu-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these "pre-B-specific" genes in correlation with the status (rearranged v germline) of Ig gene loci (H, kappa, lambda) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and lambda-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of V kappa or V lambda loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon kappa gene rearrangement, the mu-pseudolight chain complex was actually replaced by the classical mu-kappa molecule. Finally, V pre-B and lambda-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and lambda-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements.

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.

Rapid expansion of human immunoglobulin repertoire (VH, V kappa, V lambda) expressed in early fetal bone marrow.

We have isolated pre-B- and B-cell clones after transformation by Epstein-Barr virus (EBV) of human fetal bone marrow cells between weeks 8 and 13 of gestation. These clones were characterized for immunoglobulin (Ig) chain synthesis, the status (rearranged versus germ line) of the heavy (H) chain, kappa, and lambda loci, and of the Ig mRNA transcripts by using specific variable (V), VH, V kappa, and V lambda probes covering the almost complete IgV repertoire. Mature B cells could already be identified in the 8-week-old bone marrow, with both kappa and lambda isotypes being present. Nevertheless, at this stage, most of the clones had Xhe characteristics of pre-B cells, as indicated by the presence of mu transcripts (either functional or sterile) in the absence of L chains. The kinetics of gene rearrangements were compatible with the classical scheme H----kappa----lambda. A rapid expansion of the expressed repertoire occurred between the weeks 8 and 11, with 95% of the EBV clones having the characteristics of mature B cells. V gene family usage was analyzed for the three loci and compared with the pattern of expression observed at 30 weeks of gestation and in adult clones. The "adult" pattern was rapidly acquired for the H and kappa loci, with the major subgroups being VH3 and V kappa 1. When the expression of the repertoire was "normalized," the pattern of V usage correlated fairly well with the estimated number of VH genes, but differed noticeably for the kappa chains, suggesting that the VH and V kappa repertoires are not regulated by similar processes.

The immunoglobulin lambda-like gene cluster (14.1, 16.1 and F lambda 1) contains gene(s) selectively expressed in pre-B cells and is the human counterpart of the mouse lambda 5 gene.

A human immunoglobulin (Ig)-related gene, covering approximately 8 kb, was isolated from a cosmid genomic library, by hybridization with a C lambda probe and with a lambda-like probe. This gene was identified as 14.1 It belongs to the human lambda-like cluster which is composed of three genes (14.1, 16.1 and F lambda 1) that do not rearrange. Sequence data indicate that 14.1 is organized similarly to the mouse lambda 5 gene. It contains three exons with lengths of 69, 38, and 106 codons as compared with 65, 38, and 106 for exons 1, 2, and 3 of mouse lambda 5, respectively. The corresponding homology values were 61, 66 and 75.5%. Using a 14.1 specific probe containing exon 1, we showed that this gene was selectively expressed in human pre-B cell lines. It is likely to encode a 213-amino acid lambda-like light chain that would associate with mu chains and play an important role in the early steps of B cell differentiation.

Isolation of early immunoglobulin lambda-like gene transcripts in human fetal liver.

In an attempt to identify early events of human Ig gene expression, we have screened a human fetal liver cDNA library (less than 90 days of gestation) with C mu-, C gamma-, C kappa-, C lambda-specific probes and we report the characterization of two clones, F lambda 1 and F lambda 8, that hybridized with a human C lambda gene. These two clones, which are only 85% homologous to the functional C lambda genes, were shown to be additional nonallelic members of the 14.1/16.1 C lambda-like family. Using pulsed field gel electrophoresis these three C lambda-like genes were shown to be present on a 200-kb DNA fragment, defining a cluster distinct from that of the C lambda one. F lambda 1 and F lambda 8 contained an identical C lambda-like region, and differed from each other by a splicing event which joins a J lambda-like to the C lambda-like exon in the F lambda 1 clone in the absence of any rearrangement. Homologies observed between F lambda 1 and the mouse lambda 5 gene suggest that this human clone may contain the exon 2 and 3 equivalents of lambda 5. Since lambda 5 is selectively expressed in pre-B cells, our proposal is also supported by the early expression of this clone, together with the presence of full-length mu and gamma transcripts and the absence of functional Ig light chain transcripts. The presence of one nucleotide deletion in the C region of F lambda 1 conferring it a pseudogene status, the actual lambda 5 equivalent might be either one of the 14.1 or 16.1 human C lambda-like genes, the function of which is so far unknown.

Molecular polymorphism of various HLA-D subregions and rheumatoid arthritis.

In Caucasian populations, rheumatoid arthritis (RA) is generally associated with serologic HLA-DR4 specificity. In order to refine this correlation in the HLA-D region, we used six different probes pertaining to this locus: DR beta, DQ beta, DQ alpha, DO beta, DP beta and DP alpha. In this step, pooled RA and control DNA were hybridized with DR beta and DQ beta probes after digestion with 12 different endonucleases. Some bands appeared specific in the RA pool. In fact, with genomic DNA from 13 unrelated typed RA patients and 12 matched or partially matched control cells, these bands were revealed to be related to DR4 and/or DR1, with DR beta and DQ beta probes hybridizing BamHI, EcoRV, PvuII and StuI digests. With other probes, no differences could be related to RA disease. The polymorphism detected by these probes was suggestive of a gradient of decreasing complexity from DR beta to DO beta through DQ beta and DP beta, which could reflect discrete functions of each subregion.

Two V kappa germ-line genes related to the GAT idiotypic network (Ab1 and Ab3/Ab1') account for the major subfamilies of the mouse V kappa-1 variability subgroup.

V kappa Ig germ-line genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA. Three different clones that strongly hybridized with a V kappa-GAT-specific probe were completely characterized and sequenced. All three genes exhibited common characteristic features in their sequences encompassing the 5' to the 3' noncoding region, with coding sections 95% homologous. A comparison with other V kappa genes shows that the size of the first intron is variability subgroup specific. Moreover, a direct correlation exists between the size of this intron and the entire length of the coding region. Nucleotide sequences of these genes were compared with V kappa chains expressed at the Ab1 and Ab1' levels of the GAT idiotypic network: Ag----Ab1----Ab2----Ab3 (Ab1'). K1A5 and K5.1 genes account for V kappa chains in Ab1 and Ab1' hybridomas, respectively. The high conservation of Ab1' sequences in light chain was also recently reported for the heavy chains, suggesting that immunization with Ab2 (anti-idiotypic) antibodies preferentially stimulates the direct expression of germ-line genes. K5.1 and K1A5 genes belong to the V kappa-1 variability subgroup and encode, without any amino acid substitution, V kappa domain in myeloma TEPC 105 and MOPC 467, which are V kappa-1A and V kappa-1C subgroup prototypes, respectively. These genes are extensively used in different mouse strains and in a number of antibodies of discrete specificities, such as anti-GAT, anti-DNP, anti-flagellin, anti-phosphorylcholine, anti-digoxin, anti-phenyloxazolone, and anti-DNA.

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.

Functional and pseudogenes are similarly organized and may equally contribute to the extensive antibody diversity of the IgVHII family.

Eleven germ-line immunoglobulin VH genes have been isolated from a BALB/c genomic library, using a cDNA probe specific for the GAT/NPa variable region. Restriction fragments of all genes were sequenced: two over 800 bp, covering signals of the 5'- and 3'-non-coding regions, three encompassing the complete coding region and part of the 5', the remaining sequences covering most of the V coding region. All sequences pertained to the VHII family, and were compared with the other 13 homologous genes already published. Characteristic features defining the family are clearly visible all along the sequences analyzed, including the 5'-non-coding region, the leader fragment and the intron organization. About half of the compared genes have pseudogene characteristics, defined either by a stop codon in the coding region or the lack of an initiator codon in the leader segment. Analysis of the replacement mutations, as compared with silent ones, indicate that they are highly clustered in complementarity determining regions, for both the functional and the pseudogenes, suggesting that all genes have been submitted to similar selective pressure, and that the pseudogene repertoire may be actively used, by recombination and/or conversion process. Signals that regulate transcription are highly conserved through the family barriers. The VHII group is the largest Ig V genes family, with extreme sequence divergences reaching 22% nucleotide differences. As no two genes were found identical out of the 24 members compared, and as two genes were found to differ by as little as three nucleotides, it seems that the previous estimate of 60 members might be much too low.

Immunoglobulin diversity: analysis of the germ-line VH gene repertoire of the murine anti-GAT response.

A cDNA clone was constructed from a mRNA encoding an anti-GAT (Glu60 Ala30 Tyr10) BALB/c monoclonal antibody heavy chain. Its sequence, covering codons -5 to 162 and therefore encompassing the complete V-D-J region, was determined. Surprisingly, the sequence of the VH gene-encoded region was almost identical with that of the BALB/c VH anti-HNP (4-hydroxy-3-nitrophenyl) acetyl VH region, suggesting that the same VH germ-line might be used to encode two heavy chains contributing to antibodies of discrete specificities. A specific VH probe was derived and annealed to Eco RI and Bg1 II restriction fragments of liver (unrearranged) DNA extracted from the BALB/c, DBA/2 and C57BL/6 mouse strains that differ in their H chain allotypes. Under stringent conditions, only a few bands were identified by Southern blotting. The different patterns observed suggest that the VH anti-GAT repertoire differs between these strains even though their various anti-GAT antibodies express the same public idiotypic specificities.