First T cell receptor alpha gene rearrangements during T cell ontogeny skew to the 5' region of the J alpha locus

Fetal, neonatal, and early postnatal thymi were assessed for TCR J alpha gene rearrangements. Gene probes spanning the distance from 5' to 3' regions of the J alpha locus were used to determine the approximate location of gene rearrangements within hybridomas representing each of the early thymocyte populations. The predominant location of rearrangements was within the 5' region of the J alpha locus. Among the several cells in which rearrangements were found on only one chromosome, the one rearrangements was always in the 5' region. When two rearrangements were found, the rearrangements on homologous chromosomes were usually in the same region. The overall pattern among thymocytes was in great contrast to that previously observed among hybridomas derived from stimulated adult spleen cells within which rearrangements fell mostly to the 3' side of the alpha-locus. Results reveal the nonrandom nature of the TCR-alpha gene rearrangement event and may reflect an incidence of multiple V-J alpha joining events on each chromosome during T cell development in vivo. Due to the fact that most mature cells bear two J alpha joins, the allelic exclusion of alpha-chains cannot be explained by a mechanism whereby a functional rearrangement on one chromosome inhibits subsequent rearrangement on the second. Instead allelic exclusion may rely on a low frequency of productive vs nonproductive rearrangement events and an incompatibility between multiple alpha- and beta-protein pairs.     

Nonrandom rearrangement of T cell receptor J alpha genes in bone marrow T cell differentiation cultures

TCR J alpha genes span a distance of approximately 65 kb on mouse chromosome 14. Due to the existence of 50 to 100 discrete J genes, a potential for great diversity exists within the V-J-C alpha gene products and within the ultimate repertoire of alpha beta TCR. We have prepared hybridomas from an in vitro system that supports T cell differentiation among bone marrow cells. We have examined the J alpha genes among these cells and categorized rearrangements according to their location within the J alpha locus. It was found that alpha rearrangements were always present among the hybridomas bearing beta gene rearrangements. When two bone marrow-derived alpha-bearing chromosomes could be demonstrated in these hybridomas, both were always rearranged and rearrangements on homologous chromosomes were shown to reside in similar regions of the J alpha locus. Most surprisingly, when hybridomas were categorized by the culture from which they derived, cells from the same culture (designated as a set) demonstrated a skewing of alpha rearrangements to restricted segments of J alpha genes. In one hybridoma, rearrangements on homologous chromosomes involved J alpha genes that were either identical or situated within a 1-kb segment of DNA. The skewing within sets could not be due to clonal identity between hybridomas as the beta and gamma rearrangements in all hybridomas were different. Results suggested that skewing of J alpha gene rearrangements occurred during the course of T cell development in vitro. Should the same situation occur in vivo, the number of distinct TCR J alpha sequences available for expression in early development may be far less than that predicted by gene number alone.     

Rearrangement of immunoglobulin heavy chain genes in human T leukaemic cells shows preferential utilization of the D segment (DQ52) nearest to the J region.

The DNA rearrangements leading to the assembly of genes coding for the immunoglobulin heavy chain (IgH) in B cells and the T cell receptor for antigen in T cells are not completely lineage specific. This probably reflects the use of a common recombinase by IgH and the T cell receptor. This paper describes novel observations on the nature of these cross-lineage rearrangements. A high proportion (though not all) IgH rearrangements in human T leukaemic cells involve the D segment nearest to the J region (DQ52). This same D segment is not involved in B cell IgH rearrangements with one important exception, namely a proportion of B cell leukaemic clones with the most primitive B cell precursor phenotype. These observations have potentially important implications for early lymphoid cell differentiation and in particular support the idea that the 3' D plus J region might lie within a limited window of accessibility of the IgH gene in precursor lymphocytes.     

Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture

Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.     

V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer.

We have assessed the importance of the immunoglobulin heavy chain (IgH) intron enhancer for recombination of variable gene segments (V, D and J) during B cell development. We generated chimeric mice with embryonic stem cells lacking the intron enhancer from one of their IgH loci. The IgH intron enhancer was substituted by a short oligonucleotide through homologous recombination using the 'Hit and Run' procedure. V(D)J recombination occurred less frequently on mutant alleles, but was not blocked completely. Quantitative polymerase chain reaction analyses demonstrated that 15-30% of the mutated loci in mature B cells were unrearranged, in striking contrast to the wild-type alleles. The remainder of the mutated loci underwent D-J (65-80%) as well as V-DJ rearrangements, although the latter were less frequent (3-6%). These results are in line with previous data which showed that the V(D)J recombination machinery is modulated through cis-regulatory elements within the intron enhancer. However, our data predict the existence of additional cis-regulatory element(s) which, together with the intron enhancer, are required to activate the V(D)J recombination machinery fully. Such cis-regulatory element(s) might function as an enhancer of recombination or as a locus control region regulating the accessibility of the IgH locus.     

Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.     

Complex rearrangements within the human J delta-C delta/J alpha-C alpha locus and aberrant recombination between J alpha segments.

We have examined DNA rearrangements within a 120 kb cloned region of the human T cell receptor J delta-C delta/J alpha-C alpha locus. Three types of pattern emerge from an analysis of T cell lines and clones. Firstly, cells with two rearrangements within J delta-C delta; secondly, cells with one rearrangement within J delta-C delta and one or more J alpha rearrangements, and finally, cells with rearrangements within J alpha and consequential deletion of the delta locus. Further analysis by cloning of rearrangements within the J alpha locus show that, in addition to V alpha-J alpha joins, J alpha-J alpha aberrant recombinations occur and rearrangement data indicate that such events are frequent. A model is presented to account for such recombinations.     

Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly.

Immunoglobulin genes are assembled during lymphoid development by a series of site-specific rearrangements that are tightly regulated to ensure that functional antibodies are generated in B (but not T) cells and that a unique receptor is present on each cell. Because a common V(D)J recombinase comprising RAG1 and RAG2 proteins is used for both B- and T-cell antigen receptor assembly, lineage-specific rearrangement must be modulated through differential access to sites of recombination. We show here that the C-terminus of the RAG2 protein, although dispensable for the basic recombination reaction and for Ig heavy chain DH to JH joining, is essential for efficient VH to DJH rearrangement at the IgH locus. Thus, the RAG2 protein plays a dual role in V(D)J recombination, acting both in catalysis of the reaction and in governing access to particular loci.     

Distinguishing primary and secondary translocations in multiple myeloma

Multiple myeloma (MM) is a malignant post-germinal center tumor of somatically-mutated, isotype-switched plasma cells that accumulate in the bone marrow. It often is preceded by a stable pre-malignant tumor called monoclonal gammopathy of undetermined significance (MGUS), which can sporadically progress to MM. Five recurrent primary translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 have been identified in MGUS and MM tumors. The five partner loci include 11q13, 6p21, 4p16, 16q23, and 20q12, with corresponding dysregulation of CYCLIN D1, CYCLIN D3, FGFR3/MMSET, c-MAF, and MAFB, respectively, by strong enhancers in the IgH locus. The five recurrent translocations, which are present in 40% of MM tumors, typically are simple reciprocal translocations, mostly having breakpoints within or near IgH switch regions but sometimes within or near VDJ or JH sequences. It is thought that these translocations are caused by aberrant IgH switch recombination, and possibly by aberrant somatic hypermutation in germinal center B cells, thus providing an early and perhaps initiating event in transformation. A MYC gene is dysregulated by complex translocations and insertions as a very late event during the progression of MM tumors. Since the IgH switch recombination and somatic hypermutation mechanism are turned off in plasma cells and plasma cell tumors, the MYC rearrangements are thought to be mediated by unknown mechanisms that contribute to structural genomic instability in all kinds of tumors. These rearrangements, which often but not always juxtapose MYC near one of the strong immunoglobulin enhancers, provide a paradigm for secondary translocations. It is hypothesized that secondary translocations not involving a MYC gene can occur at any stage of tumorigenesis, including in pre-malignant MGUS tumor cells.     

V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.     

Stepwise activation of the immunoglobulin mu heavy chain gene locus.

The immunoglobulin heavy chain (IgH) gene locus spans several megabases. We show that IgH activation during B-cell differentiation, as measured by histone acetylation, occurs in discrete, independently regulated domains. Initially, a 120 kb domain of germline DNA is hyperacetylated, that extends from D(FL16.1), the 5'-most D(H) gene segment, to the intergenic region between Cmu and Cdelta. Germline V(H) genes were not hyperacetylated at this stage, which accounts for D(H) to J(H) recombination occurring first during B-cell development. Subsequent activation of the V(H) locus happens in at least three differentially regulated domains: an interleukin-7-regulated domain consisting of the 5' J558 family, an intermediate domain and the 3' V(H) genes, which are hyperacetylated in response to DJ(H) recombination. These observations lead to mechanisms for two well-documented phenomena in B-cell ontogeny: the sequential rearrangement of D(H) followed by V(H) gene segments, and the preferential recombination of D(H)-proximal V(H) genes in pro-B cells. We suggest that stepwise activation may be a general mechanism by which large segments of the genome are prepared for expression.