Analysis of somatic hypermutations of immunoglobulin gene rearrangements in childhood acute lymphoblastic leukemia.

The current study investigated the presence, frequency, and status of somatic hypermutations as well as their role in children with B lineage ALL. The obtained sequences were analyzed using IMGT/V-QUEST. Totally, 150 IGH sequences were evaluated; 139 from the 111 patients at the time of diagnosis and 11 from 8/111 patients at the time of relapse. The findings of the current report revealed the presence of somatically mutated V genes in childhood B lineage ALL. A higher frequency of somatic hypermutations was noted in unproductive rearrangements and was generally attributed to nucleotide mutation type, region, and IGHV gene subgroup biases.

T cell diversity and TcR repertoires in teleost fish.

In vertebrates, the diverse and extended range of antigenic motifs is matched to large populations of lymphocytes. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors--IG and TR--required for the recognition specificity. Immune repertoires have become useful tools to describe lymphocyte and receptor populations during the immune system development and in pathological situations. In teleosts, the presence of conventional T cells was first proposed to explain graft rejection and optimized specific antibody production. The discovery of TR genes definitely established the reality of conventional T cells in fish. The development of genomic and EST databases recently led to the description of several key T cell markers including CD4, CD8, CD3, CD28, CTLA4, as well as important cytokines, suggesting the existence of different T helper (Th) subtypes, similar to the mammalian Th1, Th2 and Th17. Over the last decade, repertoire studies have demonstrated that both public and private responses occur in fish as they do in mammals, and in vitro specific cytotoxicity assays have been established. While such typical features of T cells are similar in both fish and mammals, the structure of particular repertoires such as the one of gut intra-epithelial lymphocytes seems to be very different. Future studies will further reveal the particular characteristics of teleost T cell repertoires and adaptive responses.

B lymphocytes of xeroderma pigmentosum or Cockayne syndrome patients with inherited defects in nucleotide excision repair are fully capable of somatic hypermutation of immunoglobulin genes.

Recent experiments have strongly suggested that the process of somatic mutation is linked to transcription initiation. It was postulated that a mutator factor loads onto the RNA polymerase and, during elongation, causes transcriptional arrest that activates DNA repair, thus occasionally causing errors in the DNA sequence. We report the analysis of the role of one of the known DNA repair systems, nucleotide excision repair (NER), in somatic mutation. Epstein-Barrvirus-transformed B cells from patients with defects in NER (XP-B, XP-D, XP-V, and CS-A) were studied. Their heavy and light chain genes show a high frequency of point mutations in the variable (V), but not in the constant (C) regions. This suggests that these B cells can undergo somatic hypermutation despite significant defects in NER. Thus, it is doubtful that NER is an essential part of the mechanism of somatic hypermutation of Ig genes. As an aside, NER seems also not involved in Ig gene switch recombination.

A unique V-J-C-rearranged gene encodes a gamma protein expressed on the majority of CD3+ T cell receptor-alpha/beta- circulating lymphocytes.

We have recently described an mAb, anti-Ti gamma A, that recognizes an antigenic determinant carried by a TCR gamma chain. This antibody binds to approximately 3% of human PBLs and delineates a CD2+, CD3+, TCR-alpha/beta-, CD4-, CD8+/-, CD5+, NKH1-, and HLA class II- subset. The present study was designed to identify the gene encoding the Ti gamma A epitope. A first analysis was carried out on a previously characterized TCR gamma + fetal-cloned cell line termed F6C7. It was found that F6C7 cells have one gamma rearrangement on each chromosome: one joins V gamma 3 to J gamma 1, and the second joins V gamma 9 to J gamma P. Because only the latter allele appeared to be transcribed in the F6C7 lymphocytes, these data strongly suggested that anti-Ti gamma A mAb is specific for either a V gamma 9 or a V gamma 9-J gamma P-encoded peptide. To confirm this point, we studied an additional series of 13 randomly selected Ti gamma A+ cloned cells derived from peripheral blood of three distinct adult individuals. Each one of these lymphocytes was shown to both possess and transcribe a V gamma 9-J gamma P-C gamma 1-rearranged gene. It is therefore concluded that a predominant subpopulation of CD3+ TCR-alpha/beta- human circulating T lymphocytes (namely, the subset defined by anti-Ti gamma A mAb) surface expresses a gamma protein with a limited potential of variability from one cell to another.

Rearrangements of immunoglobulin and T cell receptor beta and gamma genes are associated with terminal deoxynucleotidyl transferase expression in acute myeloid leukemia.

The cell origin of the rare terminal deoxynucleotidyl transferase (TdT)-positive acute myeloid leukemias (AML) was investigated at the molecular level, by examining the configuration of the Ig H (Igh) and L (Ig kappa, Ig lambda) chain gene regions, and of the T cell receptor (TCR) beta and T cell rearranging (TRG) gamma loci. In 8 of the 10 TdT+ AML analyzed (classified as myeloid according to morphological and cytochemical criteria, and to the reactivity with one or more antimyeloid mAbs), a rearrangement of the Igh chain gene was found. In TdT- AML, evidence of an Igh gene reorganization was instead observed only in 2 of the 42 patients studied. Furthermore, evidence of TCR-beta and/or TRG-gamma gene rearrangement was observed in four AML, all of which belonged to the Igh-rearranged TdT+ group. In three cases (one TdT+ and two TdT-), the Ig kappa L chain gene was also in a rearranged position. These findings demonstrate a highly significant correlation between TdT expression and DNA rearrangements at the Igh and TCR chain gene regions and support the view that this enzyme plays an important role in the V-(D)-J recombination machinery. Overall, the genomic configuration, i.e., JH gene rearrangement sometimes coupled to a kappa L chain and TCR gene reorganization, similar to that found in non-T-ALL, suggests that in most cases of TdT+ AML, the neoplastic clone, despite the expression of myeloid-related features, is characterized by cells molecularly committed along the B cell lineage.

The human T-cell receptor gamma (TRG) genes.

The human T-cell receptor gamma (TRG) chain genes, like those encoding the T-cell receptor alpha- and beta-polypeptides, undergo rearrangements specifically in T cells. The human TRG locus, which has been completely mapped, is composed of two constant region genes (TRGC), five joining segments (TRGJ) and at least 14 variable gamma-genes (TRGV). Eight variable genes are functional and belong to four different subgroups. The product of the rearranged TRG gene is the gamma-chain which is expressed, along with the delta-chain, at the surface of a subset of T lymphocytes. Although some gamma delta + cells display a cytolytic activity, their precise function remains to be elucidated.

Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation.

Influence of intron length on alternative splicing of CD44.

Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

IMGT, the international ImMunoGeneTics database.

IMGT, the international ImMunoGeneTics database, freely available at http://imgt.cines.fr:8104, was created in 1989 at the Université Montpellier II, CNRS, Montpellier, France, and is a high quality integrated information system specialising in immunoglobulins, T cell receptors and major histocompatibility complex molecules of human and other vertebrates. IMGT provides researchers and clinicians with a common access to all nucleotide, protein, genetic and structural immunogenetics data. This information is of high value for medical and veterinary research, biotechnology related to antibody and T cell receptor engineering, genome diversity and evolution studies of the immune response.

Sequence and evolution of the human germline V lambda repertoire.

We recently completed a map of the human immunoglobulin lambda (IGL) locus on chromosome 22q11.2 and showed that the V lambda genes are arranged in three distinct clusters, each containing members of different V lambda families. We have now sequenced each of these V lambda genes and determined which are functional by comparison with the expressed repertoire. Our analysis indicates that there are approximately 30 functional V lambda genes, depending on the haplotype, that belong to ten V lambda families (five V lambda 1, five V lambda 2, eight V lambda 3, three V lambda 4, three V lambda 5, one V lambda 6, two V lambda 7, one V lambda 8, one V lambda 9 and one V lambda 10). V lambda genes related to the major human V lambda families (V lambda 1, V lambda 2 and V lambda 3) predominate in species that express mainly lambda light chains.

IMGT databases, web resources and tools for immunoglobulin and T cell receptor sequence analysis, http://imgt.cines.fr.

IMGT, the international ImMunoGeneTics database((R)) (http://imgt.cines.fr), is a high-quality integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates, created in 1989, by LIGM, at the Université Montpellier II, CNRS, Montpellier, France. IMGT provides a common access to standardized data which include nucleotide and protein sequences, oligonucleotide primers, gene maps, genetic polymorphisms, specificities, 2D and 3D structures. IMGT includes several databases (IMGT/LIGM-DB, IMGT/3Dstructure-DB, IMGT/HLA-DB), Web resources ('IMGT Marie-Paule page') and interactive tools (IMGT/V-QUEST, IMGT/JunctionAnalysis). IMGT expertly annotated data and tools described in this paper are particularly useful for the analysis of the IG and TR rearrangements in leukemia, lymphoma and myeloma, and in translocations involving the antigen receptor loci. IMGT is freely available at http://imgt.cines.fr.

Analysis of the B-cell compartment in plasma cell leukemia and multiple myeloma: immunoglobulin gene rearrangement of EBV-infected B-cell lines.

Multiple myeloma (MM) is defined as a tumoral expansion of plasma cells occurring in the bone marrow and sometimes in the peripheral blood (plasma-cell leukemia, PCL). Many reports have demonstrated a clonal expansion of B cells bearing the same idiotypic determinants as the myeloma protein (idiotypic B cells) in MM, suggesting that they could belong to the malignant clone. In order to investigate whether the B-cell population is a malignant component or not, either in the peripheral blood of patients with PCL or in the bone marrow of patients with MM, we derived B-cell lines by infecting, with the Epstein-Barr virus (EBV), cultures in limiting dilution of mononuclear cells from six patients. A limiting dilution culture was used to prevent the elimination of slowly proliferating clones by the more rapidly dividing ones, and thus to get the most exact representation of the B-cell repertoire of these patients. The cloning efficiency of the EBV-infected cells was similar in patients and healthy individuals (range: 1 in 100 to 1 in 1650 B cells). All of the clones obtained from a single patient exhibited different clonal immunoglobulin gene rearrangements (IGR), proving the validity of our cloning technique. No tumoral clones (61 clones analysed) showed the IGR pattern specific of autologous myeloma cells. These results indicate that malignant plasma cells cannot be immortalized with EBV. These results show that, if malignant B cells (pre-switch or post-switch) exist, they could be present only in a minor population, and the corollary of this is that there is a major population of non-malignant B cells in the sites of tumoral proliferation of patients with MM. This is remarkable in view of numerous reports showing a profound defect of the polyclonal B lymphopoiesis in these patients, and even an absence of B lymphocytes. Thus, these results challenge the existence of a major compartment of malignant idiotypic B cells and favor the hypothesis of non-malignant B cells sharing cross-reactive idiotypes with the autologous myeloma protein.

Residual disease in B-cell chronic lymphocytic leukemia patients and prognostic value.

Twenty-two B-cell chronic lymphocytic leukemia (CLL) patients were investigated to evaluate residual disease in clinico-hematological remission. Residual disease was determined by monotypy of surface light-chain expression and by dual-color staining with CD5 and CD19 markers. Samples were analyzed on flow cytometer. Total CD19+ cells above 25%, the CD5+CD19+/total CD19+ cells ratio above 0.25, clonal excess above 0.4 were considered positive for residual disease. According to these immunological criteria, only four cases achieved phenotypic remission. Our data confirm that dual marker analysis is more sensitive than clonal excess and may predict an early relapse. Ig gene rearrangements were studied by Southern blot analysis using IGHJ and IGKC probes in fifteen cases. All 12 cases that retained a detectable rearrangement displayed a phenotypic residual disease. Conversely, in two cases, DNA analysis failed to detect the residual disease characterized by flow cytometry. In conclusion, this study suggests that in B-CLL, dual marker analysis is sensitive in predicting an early relapse in sequential evaluations of residual disease, whereas rearranged bands are undetectable when the proportion of malignant cells is low.

Rearrangements of immunoglobulin light and heavy chain genes and correlation with phenotypic markers in B-cell chronic lymphocytic leukemia.

Twenty-five patients with B-cell chronic lymphocytic leukemia (B-CLL) were investigated to correlate the immunological phenotype with the description of the Ig gene rearrangements of the B-cell clone. All patients were positive for the CD19 antigen and one pan B-antigen, markers of late cells (CD20, CD37 or Y2955). Twenty-four of the 25 patients tested expressed monoclonal cell surface immunoglobulin (SIg). The CD5 antigen was present in 21 of the 25 tested patients. Immunoglobulin gene rearrangements were detected by hybridization of the BamHI, EcoRI, BgIII, and HindIII digested genomic DNAs to the IGHJ, IGKC, IGLC, and IGLJ2 probes. Twenty-four of 25 patients had two rearranged IGH loci. The IGKC rearrangements were observed in 20 patients. In four patients, the IGK loci were deleted on both chromosomes. One patient without SIg displayed a germline pattern. All six patients with lambda producing B-CLL showed a lambda gene rearranged band, although the use of IGL polymorphism to investigate IGL rearrangements must be noted. These clonal rearrangements of IGL genes, together with the detection of either the kappa or lambda light chain of SIg, confirm that patients with B-CLL meet the developmental scheme of ordered light chain gene rearrangements.

Genomic organisation of 34 kb of the human immunoglobulin lambda locus (IGLV): restriction map and sequences of new V lambda III genes.

In order to improve our knowledge of the human immunoglobulin variable lambda locus (IGLV), we mapped one cosmid clone (designated as C40.2) isolated by screening a Colo320HSR genomic library. The 34 kb insert of the C40.2 clone was shown to contain six genes. One gene, IGLV2S1, belongs to the V lambda II subgroup. Four genes belong to the V lambda III subgroup. Two of them, IGLV3S1 and IGLV3S2, are potentially functional whereas the two others are pseudogenes. The size of the IGLV3S2 leader intron is four times longer than the classical intron size of 110 bp. The cosmid also contains a vestigial sequence lambda vg2. All these genes share the same orientation of transcription. Pulsed field gel electrophoresis analysis of the IGLV locus shows that most of the V lambda I subgroup genes are located at the 5' end of the locus.

The human gamma/delta + and alpha/beta + T cells: a branched pathway of differentiation.

In human peripheral blood, most of the CD3+ cells express the alpha/beta T cell receptor. A smaller fraction of CD3+ T cells express the gamma/delta T cell receptor (from 1 to 10% depending the individuals, with an average of 3-5%). Interestingly, although the alpha/beta + T cells never express the gamma chain at the cell surface, most of them (about 98%) rearrange the gamma locus on both alleles, the remaining 2% alpha/beta + T cells have one rearranged TRG locus. We previously proposed that V-J joinings in the human TRG locus occurred sequentially and we recently demonstrated that two successive rearrangements may occur on the same chromosome [Alexandre et al. (Int. Immunol, 3, 973-982, 1991)]. In this paper, we discuss the implications of these sequential rearrangements on the relatedness of the human gamma/delta + and alpha/beta + T cell lineages.

Inhibition of T cell activation with a humanized anti-beta 1 integrin chain mAb.

The murine anti-CD29 mAb K20 (Mu-K20) is known to bind to the beta 1 chain of the human integrins and to inhibit activation and proliferation of T cells, implying an important potential for in vivo immunosuppression. However, use of K20 as an immunosuppressant drug would be impaired by the immunogenicity of mouse mAbs in man. We have therefore engineered K20 into (1) a mouse/human chimeric mAb (Ch-K20) that comprises the human kappa/gamma 1C regions and the K20 V regions; and (2) a humanized mAb (Hu-K20) combining the complementarity-determining regions (CDRs) of the K20 mAb with human framework (FR) and kappa/gamma 1 C regions. Both chimeric and humanized Abs were able to reproduce a range of functional properties of the original mouse mAb K20 (Mu-K20), namely, specific binding of CD29, inhibition of T cell proliferation and elevation of second messenger phosphatidic acid (PA) induced via CD3 in a soluble form, and activation of T cell proliferation in a cross-linked form. When compared to Ch-K20, the avidity of Hu-K20 was only slightly reduced. This demonstrates the feasibility of a successful humanization performed on the sole basis of the primary amino acid sequence analysis of the original mouse antibody V regions.

DNA sequence variability of IGHG3 alleles associated to the main G3m haplotypes in human populations.

The present study investigates the molecular basis of the G3m polymorphism expressed by the heavy constant domains of human immunoglobulins gamma 3 chains. By using a new protocol allowing the specific cloning of IGHG3 genes, a total of 51 full-length IGHG3 genomic sequences (about 2 kb) isolated from African, Siberian, West Asian and European population samples were sequenced. IGHG3 sequences were assigned precise G3m haplotypes on the basis of specific associations between G3m allotypes and IGHG3 RFLPs. Specific DNA substitutions involved in the expression of G3m(5), G3m(6), G3m(15), G3m(16), G3m(21), G3m(24) and G3m(28) allotypes were then deduced, elucidating almost completely the determination of the G3m polymorphism at the DNA level. The molecular evolution of G3m haplotypes was investigated by a maximum likelihood phylogeny of IGHG3 sequences. Sequence clusters are shown to be G3m haplotype-specific, corroborating the Gm molecular model deduced from serology, and showing that populations differentiation is much more recent than G3m haplotypes differentiation. The widely distributed G3m(5,10,11,13,14) haplotype is likely to be ancestral to the other G3m haplotypes presently found at high frequencies in different continental areas.

Assembly and extension of yeast artificial chromosomes to build up a large locus.

For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their regions of homology. By site-specific recombination a YAC of 410 kb was created accommodating the major part of the human lambda light chain locus in authentic configuration with 28 variable (V) genes, all joining (J) segments, all constant (C) genes and the downstream enhancer. A contiguous region was first constructed from three overlapping cosmids. Each of these was linearized at unique sites in the vectors and YAC arms were ligated to the 5' and 3' ends. After cells of Saccharomyces cerevisiae were transformed with the three cosmids, YACs of 120 kb were obtained which contained the reassembled 3' J-C region in authentic configuration. The assembled YAC was further extended by mitotic recombination with a YAC containing a 280-kb region of the C-proximal part of the V gene cluster with a 15-kb 3' overlap. For this, a simple three-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and right YAC arms. Rare recombination events between two overlapping YACs could be identified in yeast clones able to grow in lysine- and adenine-deficient medium in the presence of 5-fluoro-orotic acid which is toxic for yeast cells containing a YAC with a functional URA3 gene. This approach made it possible to assemble and extend large YACs from an unlimited number of smaller overlapping YACs by positive-negative selection.

Human immunoglobulin heavy-chain multigene deletions in healthy individuals.

Extensive multigene deletions have been described in the human immunoglobulin heavy-chain constant region genes, some of them encompassing perhaps more than 100 kilobases. These deletions have all been observed in healthy individuals although these individuals lacked several immunoglobulin class or subclasses, being either homozygous for one deletion or heterozygous for two different deletions. The high frequency of consanguinity in the Tunisian population accounts for the high frequency of individuals displaying one or the other of these deletions in a homozygous state.