Detection and follow-up of fibroblast growth factor receptor 3 expression on bone marrow and circulating plasma cells by flow cytometry in patients with t(4;14) multiple myeloma.

The t(4;14)(p16;q32) translocation, found in 15% of multiple myeloma (MM) cases, indicates a poor prognosis. Plasma cells (PC) with t(4;14) ectopically express the fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase receptor, which has potential transforming activity and may represent a therapeutic target. To detect FGFR3 protein expression, bone marrow (BM) aspirate from 200 consecutive newly diagnosed (n = 116) or relapsing (n = 74) MM patients was studied by flow cytometry (FC) using anti-CD138 and anti-FGFR3 antibodies. FC data was compared to real time quantitative-polymerase chain reaction (RQ-PCR) of the IGH-MMSET and FGFR3 transcripts. An IGH-MMSET transcript was found in 24/200 patients (12%). In 20 of these, FC detected CD138(+)/FGFR3(+) cells. No expression of FGFR3 was detected in the 4 FGFR3(-) cases by RQ-PCR. FGFR3 was never expressed on PC without t(4;14). Circulating PC (CPC) were detected in patients with (11/11) and patients without (13/41) t(4;14). In 2/8 t(4;14) cases studied longitudinally, coexisting FGFR3(+) and FGFR3(-) CPC were observed. Fluorescent in situ hybridisation (FISH) analysis of the FGFR3(-) subclones showed deletion of the der(14) in one patient. In conclusion, as a supplemental method to RQ-PCR or FISH, FC analysis of FGFR3 expression is a reliable and routinely available method for the detection and management of new therapeutic approaches of t(4;14) MM.

Characterization of hematopoietic lineage-specific gene expression by ES cell in vitro differentiation induction system.

The continuous generation of mature blood cells from hematopoietic progenitor cells requires a highly complex series of molecular events. To examine lineage-specific gene expression during the differentiation process, we developed a novel method combining LacZ reporter gene analysis with in vitro hematopoietic differentiation induction from mouse embryonic stem cells. For a model system using this method, we chose the erythroid and megakaryocytic differentiation pathways. Although erythroid and megakaryocytic cells possess distinct functional and morphologic features, these 2 lineages originate from bipotential erythro-megakaryocytic progenitors and share common lineage-restricted transcription factors. A portion of the 5' flanking region of the human glycoprotein IIb (alphaIIb) integrin gene extending from base -598 to base +33 was examined in detail. As reported previously, this region is sufficient for megakaryocyte-specific gene expression. However, previous reports that used human erythro-megakaryocytic cell lines suggested that one or more negative regulatory regions were necessary for megakaryocyte-specific gene expression. Our data clearly showed that an approximately 200-base enhancer region extending from -598 to -400 was sufficient for megakaryocyte-specific gene expression. This experimental system has advantages over those using erythro-megakaryocytic cell lines because it recapitulates normal hematopoietic cell development and differentiation. Furthermore, this system is more efficient than transgenic analysis and can easily examine gene expression with null mutations of specific genes.

Accessibility control of variable region gene assembly during T-cell development.

T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR beta and TCR alpha/delta loci during T-cell development.

The CBFbeta subunit is essential for CBFalpha2 (AML1) function in vivo.

The CBFbeta subunit is the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF). CBFbeta associates with DNA-binding CBFalpha subunits and increases their affinity for DNA. Genes encoding the CBFbeta subunit (CBFB) and one of the CBFalpha subunits (CBFA2, otherwise known as AML1) are the most frequent targets of chromosomal translocations in acute leukemias in humans. We and others previously demonstrated that homozygous disruption of the mouse Cbfa2 (AML1) gene results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system and blocks fetal liver hematopoiesis. Here we demonstrate that homozygous mutation of the Cbfb gene results in the same phenotype. Our results demonstrate that the CBFbeta subunit is required for CBFalpha2 function in vivo.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the Ets-1 proto-oncogene.

The Ets-1 proto-oncogene is a member of a transcription factor family characterized by homology to the v-ets oncogene. In adult mice, Ets-1 is expressed predominantly in lymphoid cells where it has been implicated in regulating transcription of lymphocyte-specific genes. Following T-cell activation, the specific DNA binding activity of Ets-1 is inactivated by transient phosphorylation, suggesting a function in the transition from the resting to activated state. Ets-1 has also been suggested to cooperate with the AP-1 transcription factor complex to mediate cellular growth factor responses. Here we show, by using RAG-2-deficient blastocyst complementation, that Ets-1 deficiency has dramatic, but different, effects on development and function of T- and B-lineage cells. Ets-1-deficient T cells were present in reduced numbers and were highly susceptible to cell death in vitro. In contrast, Ets-1-deficient B cells were present in normal numbers but a large proportion were IgM plasma cells. Our data demonstrate that Ets-1 is essential for maintenance of the normal pool of resting T- and B-lineage cells.

Unexpected consistent involvement of V beta gene segments in inappropriate T-cell receptor beta gene rearrangements occurring in B-lineage acute lymphoblastic leukemias.

T-cell receptor beta (TCR beta) gene rearrangements occur in a third of early B-cell acute lymphoblastic leukemias (ALLs). V, D, and J segments involved in these inappropriate rearrangements remain unknown and are of interest, both because partial D beta J beta and complete V beta D beta J beta recombinations occur at distinct stages of thymic maturation and because these rearrangements are regulated differently. We have therefore studied in detail seven cases of B-lineage ALL that show inappropriate clonal TCR beta gene rearrangements. Analysis of genomic DNA by Southern hybridization with C beta, J beta 1, V beta 8, and V beta 11 probes suggested the involvement of V beta segment in tumor cell rearrangements. A complete genomic library constructed from one case was screened with a C beta probe, and the TCR beta gene rearrangement was cloned and fully sequenced to show an out of frame V beta 2.2-J beta 2.6 recombination. TCR beta gene rearrangements occurring in other cases were further analyzed by polymerase chain reaction (PCR) using J beta and V beta primers and the resulting specific PCR products were sequenced. Evidence of clonal V beta rearrangements was obtained in all cases. These unexpected findings represent the first definitive demonstration that complete V beta(D beta)J beta rearrangements can occur in B-lineage cells and contrast with the previously reported lack of V beta(D beta)J beta rearrangement in B cells from V beta-J beta-C beta-E mu transgenic mice. In the context of increasing evidence that rearrangements are linked to transcription of unrearranged gene segments, these data prompt a search in B-lineage ALL cells for the presence of germline V beta transcripts whose deregulated expression may be linked to early transforming events.

Expression of human recombination activating genes (RAG1 and RAG2) in neoplastic lymphoid cells: correlation with cell differentiation and antigen receptor expression.

Regulation of V-(D)-J recombinations that occur in antigen receptor encoding genes remains poorly understood. Recently, two genes, RAG1 and RAG2, that are able to activate rearrangement of synthetic recombination substrates were cloned in mouse and a human gene homologous to RAG1 was described. To define the differentiation stages corresponding to RAG1 and RAG2 RNA expression, we have studied a large number of B- and T-lymphoid neoplasias. First, we show that a human gene homologous to the murine RAG2 is transcribed in humans. Moreover, using a polymerase chain reaction approach, we have shown that RAG are expressed not only in T-cell receptor (TCR)-negative T-cell acute lymphoblastic leukemias (T-ALLs), but also in some cases in which a significant percentage of cells expressed surface TCR. Absence of RAG expression was shown in certain T-ALLs at variable stages of thymic differentiation. Data obtained in B-lineage ALLs show that RAG RNAs are expressed in almost all slg- B-lineage ALLs but are not transcribed in the slg+ B-cell proliferations tested, including Burkitt's ALLs, follicular center cell lymphomas, and chronic leukemias. These findings are consistent with the involvement of RAG in the control of in vivo V-(D)-J recombinations. These findings are also of interest in the delineation of potential regulatory factors acting on RAG transcription and in the understanding of the mechanisms of specific chromosomal abnormalities occurring in immature lymphoid cells.

Recombination activating genes and CD3-TCR molecules are coexpressed in a subset of human T-cell acute lymphoblastic leukemias.

The V-(D) -J recombination that occurs during lymphoid differentiation is the basic event which settles the diversity of antigen receptors. Two recently cloned genes, RAG1 and RAG2, are sufficient to induce rearrangements of synthetic substrates when transfected into fibroblasts. In order to determine at which differentiation steps where the human RAG are expressed and in order to investigate the possible impairment of their expression by the leukemic process, we have recently studied a large series of B and T lymphoid neoplasias. We show that RAG are expressed in almost all sIg- B-ALL but not in the sIg+ B cell proliferations we have tested. Furthermore, we show that RAG can be expressed not only by CD3-TCR negative but also by CD3-TCRab or gd positive T-ALL cells. We conclude that the recombinase activity is not turned off by the expression of a functionnal T-cell receptor on the cell surface. This observation is in line with recent studies on RAG expression in human thymus and are of major interest to understand the set up of the TCR repertoire.

Detection of a T cell receptor delta chain with an anti-TCR alpha chain serum.

Two types of T cell antigen-specific receptors have been described. Most peripheral blood T lymphocytes express, at their surface, an antigen receptor consisting of alpha and beta subunits, while a small subset of thymocytes and a minority of mature T lymphocytes express a heterodimeric receptor termed gamma delta. Whereas the gene segments localization corresponding to the TCR gamma and beta chains are separate, genes encoding the joining and the constant regions of TCR delta chain are located between the TCR V alpha region and the J alpha-C alpha gene cluster. To determine whether V alpha gene segments are used by delta chains, immunoprecipitations from human TCR gamma delta expressing cell clones were performed with an anti-alpha serum. The results show that a rabbit antiserum raised against the purified REX TCR alpha subunit immunoprecipitates a TCR delta chain from the cell surface of only one human T cell clone termed SO1. However, since no SO1 RNA hybridization is observed with REX TCR V alpha probe and SO1 cloned cells do react with an anti-V delta 2 monoclonal antibody, we conclude that TCR delta and alpha chains expressed a limited structural homology and that REX TCR V alpha gene do not seem to be frequently used in a functional delta chain.

Regulation of transcription of the human T cell antigen receptor delta chain gene. A T lineage-specific enhancer element is located in the J delta 3-C delta intron.

We have defined transcriptional enhancing sequences inside the TCR-delta gene locus, using transient transfections with constructs containing DNA fragments cloned upstream to a reporter gene fused to a heterologous promoter. A 14-kb DNA region extending from the J delta 3 segment to 6 kb 3' to C delta was analyzed. We show the presence of positive regulatory sequences inside the J delta 3-C delta intron and have localized these sequences to two DNA fragments of approximately 300 and 258 bp. Analysis of cell specificity of the activation of such sequences demonstrates a T cell pattern for one of the two fragments. The nucleotide sequence of the T cell-specific element shows motifs sharing homology with previously described core enhancers.

Rearrangements of the T cell receptor delta gene in T acute lymphoblastic leukemia cells are distinct from those occurring in B lineage acute lymphoblastic leukemia and preferentially involve one V delta gene segment.

Rearrangement of the TCR-delta gene was studied using J delta, C delta, and V delta probes in 61 cases of acute lymphoblastic leukemia (ALL) and several cases of chronic lymphoid neoplasms to define the specificity and the diversity of rearrangements occurring at the delta locus. TCR-delta rearrangements or deletions were found in all T (33 cases) and B lineage (28 cases) ALL but not in any case of B cell chronic proliferations (13 cases). The restriction patterns of rearrangement were clearly distinct between T and B ALL and use of one V delta probe showed that rearrangement of the V delta IDP2 gene segment which is also productively rearranged in the Peer cell line, occurred frequently in T-ALL but never in B lineage ALL. Studies of WT31 and delta TCS1 antibody reactivity showed that at least 4 of 13 CD3+ T-ALL cases expressed the delta protein. CD4 and/or CD8 Ag expression were observed in some of the gamma delta expressing T-ALL. These data show that particular TCR-delta gene rearrangements occur in neoplastic early B cells and that the combinatorial diversity of TCR-delta rearrangements in T cells is higher than initially expected. In addition this study shows that an important proportion of CD3 positive T-ALL cases express the gamma delta heterodimer.

Rearranging sequence located in the intron of the human T cell receptor gamma chain gene constant region.

Human T cell rearranging gamma genes encode membrane proteins which are expressed by a minor subset of T cells. Extensive studies have shown that TcR gamma gene rearrangements result from V gamma-J gamma recombinations. Here we analyze an unusual rearrangement involving a non-translatable sequence flanked by heptamer and nonamer signals in a Ti alpha/beta-positive T cell clone (JF1). This sequence, designated here as I gamma RS, is distinct from V, D or J elements. It is located in the first intron of the first constant region (C gamma 1) and homologous sequences are conserved in C gamma 2 introns. In JF1 cells the I gamma RS is juxtaposed to the J gamma 2 segment downstream to a V gamma 10-J gamma 1 rearrangement. The use of a cryptic splicing site induced JF1 cells to produce an aberrant large-sized transcript containing the I gamma RS 3' to the first exon of C gamma 1. Such a rearrangement occurring downstream to an inframe V gamma-J gamma junction may induce premature arrest of translation and lack of the C-terminal part of C gamma involved in gamma-protein assembly.

The V gamma locus of the human T cell receptor gamma gene. Repertoire polymorphism of the first variable gene segment subgroup.

Southern blot analysis using a genomic probe of the human TCR-gamma chain first variable gene subgroup (V gamma I) was performed on DNA samples from both parents of 36 healthy Caucasian families. Two types of polymorphisms were found in these 72 unrelated DNA samples: three repertoire polymorphisms and two restriction fragment length polymorphisms (RFLP). In all cases, Mendelian inheritance of these polymorphisms was demonstrated. The most frequent repertoire polymorphism consists in the lack of the V gamma 4 and V gamma 5 segments. In 16% of chromosomes, the Eco RI and Taq I restriction fragments corresponding to V gamma 4 and V gamma 5 were lacking, with no additional bands. In these cases, a decrease of 10 kb was observed in the Bam HI fragment containing all V gamma I segments as compared with samples containing V gamma 4-V gamma 5 segments. To better understand this polymorphism, which takes place in a previously incompletely defined region, the central part of the V gamma I region, including the polymorphic V gamma 4-V gamma 5 segments, was cloned. This allowed us to localize precisely the V gamma 5 segment and thus complete the description of the V gamma I region. A striking homology of DNA and deduced amino acid sequences is present between V gamma 2 and V gamma 4 and between V gamma 3 and V gamma 5, much higher than that observed between V gamma 2 and V gamma 3 and between V gamma 4 and V gamma 5. The differences in nucleotide sequence occur mainly in the intron and three hypervariable regions. These results strongly suggest a gene duplication relationship between the segments V gamma 2-V gamma 3 and the segments V gamma 4-V gamma 5. The most frequent RFLP documented in this study is due to the combined absence of the Eco RI and the Taq I sites located in the noncoding region between V gamma 3 and V gamma 4. The haplotypic frequence of this RFLP is 6.9% of the general population. As the gamma/delta receptor may play an important role in immunological response, the biological relevance of the high degree of polymorphism occurring in the V gamma I region, as well as its possible association with some immune disturbances, should be further explored.

The human T-cell V gamma gene locus: cloning of new segments and study of V gamma rearrangements in neoplastic T and B cells.

The authors have analyzed the involvement of V gamma and J gamma segments in TRG gamma rearrangement from a series of 40 acute lymphoblastic leukemia (ALL), including 25 T- and 15 B-lineage cases, in which TRG gamma are rearranged. Sixty-five rearranged alleles were studied. The authors first describe the cloning and sequencing of two variable segments, V gamma 11 and psi V gamma 12, which rearrange in T- and B-neoplastic cells. To date three subgroups of translatable V gamma segments have been described. The authors show that V gamma 11 is the unique member of a new fourth V gamma subgroup that also rearranges in normal polyclonal T cells and that psi V gamma 12 is located at 5-kilobase (kb) downstream to V gamma 11. As shown by DNA sequence analysis, V gamma 11 shares a 60% homology with V gamma 10 (third subgroup) and a 50% homology with V gamma 9 (second subgroup) but no appreciable homology with the V gamma segments from the first family. In contrast to psi V gamma 12, V gamma 11 is translatable. In this paper the authors have also attempted to determine which V gamma segments were rearranged in the ALL cases by hybridization with a J gamma probe and genomic probes specific of the four subgroups. In the 54 instances in which the rearrangement was consistent with J gamma 1 or J gamma 2 involvement, the authors have identified the corresponding V gamma segments and have not found any other rearrangements suggestive of the existence of further V regions. The V gamma segments, belonging to the first subgroup, were the most frequently used (41 alleles). V gamma 9, V gamma 10, V gamma 11, and psi V gamma 12 were found rearranged in cases 3, 4, 5, and 1, respectively. No cases using the pseudo psi V gamma 1, psi V gamma 5, and psi V gamma 6 segments were found. Pseudo V gamma segments were not found rearranged in T cells, while V gamma 2 and V gamma 4, segments are frequently used. In contrast to the V gamma I gene rearrangement, the involvement of the V gamma II, V gamma III, and V gamma IV subgroups was most frequently observed in T-ALL with stage II differentiation (CD7+, CD4+, and/or CD8+, CD3-), than in those with stage I (CD7+, CD4-, CD8-, CD3-), than in those with stage I (CD7+, CD4-, CD8-, CD3-) and stage III (CD7+, CD4+/-CD8+/-CD3+).(ABSTRACT TRUNCATED AT 400 WORDS)

Use of a variable alpha region to create a functional T-cell receptor delta chain.

Two categories of T-cell antigen-specific receptor have been described. Most mature T lymphocytes have, on their membrane, an antigen receptor consisting of alpha and beta subunits, while early T cells and thymocytes possess a heterodimeric receptor termed gamma-delta. The DS6 clone, isolated from the peripheral blood of a patient with immunodeficiency, is a CD3+, CD4-, CD8- human T-cell line that expresses the disulfide-linked form of the gamma-delta antigen receptor. The nucleotide sequence analysis of DS6 cDNA makes clear that its variable region is a member of an alpha variable-region gene family. We have cloned and sequenced the germ-line joining and variable regions used to create the DS6 delta mRNA. Comparison of these sequences does not show evidence of extensive somatic mutations. The major difference between the germ-line and the T-cell antigen receptor delta cDNA sequence is an insertion of three consecutive nucleotides between the variable and joining segments and is evocative of somatic diversification rather than of the use of a germ-line-encoded diversity region.