B lymphoblastoid cell lines with rearranged T cell receptor beta-chain genes retain conventional B cell surface antigen and Ig expression

Transformation of peripheral blood lymphocytes by co-incubation with EBV produces B lymphoblastoid cell lines, but rearrangement of TCR beta-chain genes was observed in three different cell lines derived from two individuals. Because rearrangement of TCR genes in B lymphocytes is considered a rare event, these B lymphoblastoid cell lines with rearranged TCR beta-genes were examined in detail to determine whether there were any additional characteristics to distinguish them from B lymphoblastoid cell lines with germ-line TCR beta-genes. All B lymphoblastoid cell lines contained rearranged Ig H and kappa L chain genes, secreted Ig, and expressed B and not T cell surface markers. Cell lines with rearranged TCR beta-genes had rearranged both IgH genes and had rearranged and subsequently deleted both kappa C region genes. Furthermore all three B lymphoblastoid cell lines with rearranged TCR beta-genes produced small amounts of Ig with lambda-L chains. Although the cellular mechanisms maintaining lineage-specific rearrangement events remain unknown, extensive Ig gene rearrangement and inefficient Ig production by B cells may be indicators of a cellular status where normally stringent lineage-specific control elements fail to function efficiently.     

Antigenic stimulation induces recombination activating gene 1 and terminal deoxynucleotidyl transferase expression in a murine T-cell hybridoma

Secondary rearrangements of the T cell receptor (TCR) represent a genetic correction mechanism which changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. Murine T-cell hybridoma A1.1 was employed to investigate whether antigenic stimulation induced re-expression of recombinase genes and altered TCR Vβ expression. Following repeated antigenic stimulation, A1.1 cells were induced to re-express recombination activating gene (RAG)1 and terminal deoxynucleotidyl transferase (TdT) which are generally considered prerequisite to TCR gene rearrangement. Accompanied with the significant changes in TCR mRNA levels over time, it is suggested that secondary rearrangements may be induced in A1.1 cells, which represent a mature T cell clone capable of re-expressing RAG genes and possesses the prerequisite for secondary V(D)J rearrangement.     

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.     

Productive coupling of accessible Vbeta14 segments and DJbeta complexes determines the frequency of Vbeta14 rearrangement

To elucidate mechanisms that regulate Vbeta rearrangement, we generated and analyzed mice with a V(D)J recombination reporter cassette of germline Dbeta-Jbeta segments inserted into the endogenous Vbeta14 locus (Vbeta14(Rep)). As a control, we first generated and analyzed mice with the same Dbeta-Jbeta cassette targeted into the generally expressed c-myc locus (c-myc(Rep)). Substantial c-myc(Rep) recombination occurred in both T and B cells and initiated concurrently with endogenous Dbeta to Jbeta rearrangements in thymocytes. In contrast, Vbeta14(Rep) recombination was restricted to T cells and initiated after endogenous Dbeta to Jbeta rearrangements, but concurrently with endogenous Vbeta14 rearrangements. Thus, the local chromatin environment imparts lineage and developmental stage-specific accessibility upon the inserted reporter. Although Vbeta14 rearrangements occur on only 5% of endogenous TCRbeta alleles, the Vbeta14(Rep) cassette underwent rearrangement on 80-90% of alleles, supporting the suggestion that productive coupling of accessible Vbeta14 segments and DJbeta complexes influence the frequency of Vbeta14 rearrangements. Strikingly, Vbeta14(Rep) recombination also occurs on TCRbeta alleles lacking endogenous Vbeta to DJbeta rearrangements, indicating that Vbeta14 accessibility per se is not subject to allelic exclusion.     

TCR delta gene rearrangements revealed by fine structure of the recombination junction in mice

The standard products of V(D)J recombination of immunoglobulin and T cell receptor genes are two kinds of DNA junction, a coding joint and a signal joint. TCR delta V-D and D-D signal joints in adult mouse thymocytes were sequenced following PCR amplification. We observed differential nucleotide insertions at the V delta-D delta signal joints, depending on the V delta and D delta gene usage in the developmental stage. Nucleotide insertions at the V delta-D delta 1 signal joints were less frequent for the V delta 4, 5 genes preferentially utilized in adult thymocytes than for the V delta 3, 6 genes, infrequently rearranged to D delta 1. In addition to standard signal joints, unexpectedly, novel nonstandard products, "replacement joints" of D delta 1 substituted downstream by the recombination signal sequence of V delta were also found. However, no D delta 2-associated replacement joints other than V delta 5 were found. The other replacement joints of D delta 1-D delta 2 recombination were also observed. The mutation in TCR beta gene affected the frequency of nucleotide insertions at the V delta-D delta signal joints and inhibited the formation of replacement joint. Recombination mechanism generating the replacement joint and the possible role of TCR beta in up-regulation of TCR delta gene rearrangements are discussed.     

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.     

Immunoglobulin and T cell receptor gene rearrangements in lymphoproliferative disorders

Thirty-one samples representing Hodgkin's and non-Hodgkin's lymphomas, angioimmunoblastic lymphadenopathy (AILD), and benign follicular hyperplasia in HIV infections were examined for rearrangements of the immunoglobulin (Ig) and T cell receptor (TcR) beta-chain gene loci. In 11 of 12 non-Hodgkin's lymphomas (classified as Burkitt lymphoma (2), centrocytic lymphoma (1), centrocytic-centroblastic lymphoma (5), centroblastic lymphoma (3], only rearranged Ig genes could be detected. The exceptional case was an unclassified high-grade lymphoma, which represented a rearrangement of the TcR beta-chain. We also examined DNA from lymphoid neoplasms in which the lineage of the malignant cell was still controversial. Rearrangement of the TcR could exclusively be demonstrated in all 3 cases of AILD. One Ig gene rearrangement and 4 TcR beta-chain rearrangements were found in 13 samples of Hodgkin's lymphomas (11 lymph nodes, 1 pleura effusion and 1 bone biopsy with proven infiltration). Examination of 3 cases of benign follicular hyperplasia in HIV infection represented one Ig rearrangement.     

Transrearrangements between antigen receptor genes in normal human lymphoid tissues and in ataxia telangiectasia.

The polymerase chain reaction (PCR) was used on DNA obtained from various normal lymphoid tissues to amplify chimeric TCR gene rearrangements involving J segments of the beta gene and V segments of the gamma or delta genes. As found previously for the transrearrangements between the gamma and delta genes, transrearrangements involving the beta gene were more abundant in DNA of the thymus than in DNA of the spleen, lymph node, bone marrow, or PBL. In addition, transrearrangements between Ig H chain V region segment and J segment of TCR delta chain were also found in DNA of normal thymus. Sequence analysis of the trans-rearrangement PCR products revealed structures closely resembling normal intragenic rearrangements, with N insertions and often D segments at the junctions between segments. The sequences analyzed suggest that transrearrangements arise through the action of normal lymphocyte recombinase, involve trans recognition of heptamer/nonamer recombination signals, and follow the 12 + 23 spacer rule. To test whether transrearrangements result from chromosomal rearrangements with breakpoints at the sites of Ag receptor genes, PCR was performed on the DNA of PBL from patients with ataxia telangiectasia, a disorder in which circulating lymphocytes often have numerous karyotypic abnormalities with breakpoints at the cytogenetic positions of these genes. Comparison of the results of PCR on this DNA and that of normal tissues demonstrated a substantially increased frequency for most types of transrearrangements investigated. These results support the interpretation that transrearrangement among TCR genes may occur by chromosomal rearrangement.     

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.     

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.     

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.     

T-cell specific rearrangement of T-cell receptor beta transgenes in mice.

To study rearrangement of T cell receptor (TCR) genes, transgenic mice were generated with a TCR beta minilocus in germline configuration, containing three V beta, two D beta, fourteen J beta and two C beta gene segments and the TCR beta enhancer. Using the polymerase chain reaction as an analytical tool both partial DJ as well as complete VDJ rearrangements were seen, indicating that the minilocus contained all sequence elements required for rearrangment. Rearrangements of minilocus gene segments were restricted to T cells in the thymus and the periphery and did not occur in B cells. V beta 8.3 and V beta 5 sequences encoded by the minilocus were expressed on the surface of peripheral T cells at high frequencies. Transgenic mice with TCR minilocus genes will be a useful system to identify DNA sequence elements required for regulation of rearrangement in vivo.     

Irradiation-induced rescue of thymocyte differentiation and V(D)J recombination in mice lacking the catalytic subunit of DNA-dependent protein kinase

Scid mice express a truncated form of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and are unable to properly rearrange their Ig and TCR genes, resulting in a severe combined immunodeficiency that is characterized by arrested differentiation of B and T lymphocytes. Treatment of scid mice with low doses of gamma irradiation rescues rearrangements at several TCR loci and promotes limited thymocyte differentiation. The machinery responsible for sensing DNA damage and the mechanism by which irradiation compensates for the scid defect in TCR recombination remain unknown. Because DNA-PKcs is present in scid thymocytes, it may mediate some or all of the irradiation effects. To test this hypothesis, we examined the effects of irradiation on DNA-PKcs-deficient (slip) mice. Our data provide the first evidence that DNA-PKcs is not required for limited rescue of thymocyte differentiation or TCR rearrangements.