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.     

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.     

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.     

A novel type of aberrant T cell receptor alpha-chain gene rearrangement. Implications for allelic exclusion and the V-J recombination process

In the process of analyzing the contribution of nonproductive alpha- and beta-chain gene rearrangements to the allelic exclusion of TCR gene expression, we have found a novel type of aberrant alpha-gene rearrangement. In one alpha-allele of the mouse KB5-C20 T cell clone, a J alpha gene segment has been abutted precisely to a sequence that does not display any homology to known V and D gene segment. The appended sequence originates from within the V alpha locus and is located, in the germ-line, 1 kb upstream of a member of the V alpha 2-gene segment subfamily. No recombination signal sequences have been found contiguous to the recombination point. These observations indicate that in normal T lymphocytes, TCR alpha-genes may be affected by aberrant rearrangements similar to those that predominate in human T cell tumors containing chromosome 14 inversion or translocation. Furthermore, compilation of published data and cloning and sequencing of three additional alpha-alleles has allowed us to examine the status of alpha-loci in nine mouse T cell clones expressing functional alpha beta-heterodimers. Interestingly, in contrast to the situation observed at the beta-locus, only 1 of 18 analyzed alpha-alleles has retained a germ-line unrearranged configuration. In addition, in each T cell clone, alpha-rearrangements on homologous chromosomes were unevenly distributed over the J alpha region and shown to generally involve neighboring J alpha gene segments.     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

Sites that direct nuclear compartmentalization are near the 5' end of the mouse immunoglobulin heavy-chain locus

VDJ rearrangement in the mouse immunoglobulin heavy chain (Igh) locus involves a combination of events, including a large change in its nuclear compartmentalization. Prior to rearrangement, Igh moves from its default peripheral location near the nuclear envelope to an interior compartment, and after rearrangement it returns to the periphery. To identify any sites in Igh responsible for its association with the periphery, we systematically analyzed the nuclear positions of the Igh locus in mouse non-B- and B-cell lines and, importantly, in primary splenic lipopolysaccharide-stimulated B cells and plasmablasts. We found that a broad approximately 1-Mb region in the 5' half of the variable-gene region heavy-chain (Vh) locus regularly colocalizes with the nuclear lamina. The 3' half of the Vh gene region is less frequently colocalized with the periphery, while sequences flanking the Vh gene region are infrequently so. Importantly, in plasmacytomas, VDJ rearrangements that delete most of the Vh locus, including part of the 5' half of the Vh gene region, result in loss of peripheral compartmentalization, while deletion of only the proximal half of the Vh gene region does not. In addition, when Igh-Myc translocations move the Vh genes to a new chromosome, the distal Vh gene region is still associated with the nuclear periphery. Thus, the Igh region that interacts with the nuclear periphery is localized but is likely comprised of multiple sites that are distributed over approximately 1 Mb in the 5' half of the Vh gene region. This 5' Vh gene region that produces peripheral compartmentalization is the same region that is distinguished by requirements for interleukin-7, Pax5, and Ezh2 for rearrangement of the Vh genes.     

Murine lambda gene rearrangements: the stochastic model prevails over the ordered model.

The ontogeny of the immunoglobulin (Ig) gene rearrangement in mammalian B cells seems to be ordered. Heavy chain gene segments rearrange first, followed by light chain gene segments, kappa before lambda. The genomic organization of murine lambda locus does not preclude the simultaneous expression of two subtypes from the same chromosome. In order to distinguish between an ordered and a stochastic model of rearrangement, a panel of 67 B cell hybridomas secreting either lambda 1, lambda 2, lambda 3 or lambda x (recently described) were analysed for V lambda J lambda rearrangements. The results show that in 97% of cases, a single rearrangement occurred, favouring the stochastic model over the ordered one. Strikingly, the possibility of having a productive rearrangement if the first try results in an aberrant one is rare. We propose therefore, that the lambda Ig is not necessarily required to ensure allelic and subtypic exclusion mechanisms. Moreover, in 97% of the cases, at least one kappa allele is rearranged. Furthermore, the RS recombination has been detected in 77% of the cases. This suggests that, although the stimulation of kappa precedes that of lambda locus, the RS recombination acts as a transacting albeit dispensable lambda activator.     

Ordered and coordinated rearrangement of the TCR alpha locus: role of secondary rearrangement in thymic selection

The Ag receptor of the T lymphocyte is composed of an alphabeta heterodimer. Both alpha- and beta-chains are products of the somatic rearrangement of V(D)J segments encoded on the respective loci. During T cell development, beta-chain rearrangement precedes alpha-chain rearrangement. The mechanism of allelic exclusion ensures the expression of a single beta-chain in each T cell, whereas a large number of T cells express two functional alpha-chains. Here we demonstrate evidence that TCR alpha rearrangement is initiated by rearranging a 3' Valpha segment and a 5' Jalpha segment on both chromosomes. Rearrangement then proceeds by using upstream Valpha and downstream Jalpha segments until it is terminated by successful positive selection. This ordered and coordinated rearrangement allows a single thymocyte to sequentially express multiple TCRs with different specificities to optimize the efficiency of positive selection. Thus, the lack of allelic exclusion and TCR alpha secondary rearrangement play a key role in the formation of a functional T cell repertoire.     

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.     

A T cell clone expresses two T cell receptor alpha genes but uses one alpha beta heterodimer for allorecognition and self MHC-restricted antigen recognition

All of the T cell receptor alpha- and beta-chain rearrangements present in a dual reactive T cell clone were characterized. This clone exhibits allelic exclusion of its beta-chain genes in that only one of the two alleles is productively rearranged. Unexpectedly, it displays two productive V alpha-gene rearrangements, which are both transcribed into 1.5 kb mRNA. The contribution of each of the two productive alpha genes to the dual recognition was analyzed by gene transfer. To this end, each of the two alpha genes was separately transfected with the single productively rearranged beta gene. Transfer of only one of the two alpha beta combinations restored both allogeneic MHC recognition and self MHC-restricted antigen recognition. Thus, T cell dual recognition results from the cross-reactive recognition of an allo-MHC product by a single antigen-specific and MHC-restricted alpha beta T cell receptor. Furthermore, the presence of two productively rearranged alpha-chain genes in a T cell clone raises questions concerning the level at which allelic exclusion operates in T cells.     

Molecular involvement of the pvt-1 locus in a gamma/delta T-cell leukemia bearing a variant t(8;14)(q24;q11) translocation.

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.     

Allelic exclusion of a T cell receptor-beta minilocus.

A TCR-beta minilocus in germline configuration (beta M) has previously been shown to undergo rearrangement and expression in transgenic mice. To study allelic exclusion of TCR miniloci, several beta M transgenic mouse lines were generated and crossed with mice transgenic for a functionally rearranged TCR V beta 2 gene (beta R). PCR analysis of beta M beta R double transgenic mice revealed almost complete suppression of endogenous TCR V beta gene rearrangements, but blockage of minilocus V beta rearrangements was achieved with only one of five minilocus transgenic lines. This result cannot be explained by copy number or arrangement of the multiple miniloci integrated. It appears that the minilocus is not autonomously regulated which suggests that sequences flanking the integration sites influence accessibility of the minilocus for rearrangement and allelic exclusion. However, although productively rearranged genes were formed in double transgenic mice, surface expression of minilocus-encoded beta chains was not detected. This indicates that allelic exclusion may operate at a level after gene rearrangement.     

DNA sequences 3' of the Ig H chain cluster rearrange in mouse B cell lines

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.     

Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus.

We have cloned extrachromosomal circular DNAs containing T cell receptor (TCR) delta gene segments in adult mouse thymocytes and splenocytes. We find that the frequency of circular DNA clones carrying germline delta sequences is lower than that of J alpha probe-positive clones, possibly related to increasing 5' distance from the most upstream J alpha segment. This suggests that the TCR alpha/delta locus is successively rearranged from within and that the delta-containing excision products are progressively diluted out by the subsequent cell division which includes further alpha gene rearrangements. In addition, examination of delta gene excision products revealed newly identified V delta subfamilies, the reciprocal joining of two D delta elements, J delta 2 usage in thymocytes and novel sequences homologous to the human delta-gene deleting elements.     

VHDJH formation and DJH replacement during pre-B differentiation: non-random usage of gene segments.

The Abelson murine leukemia virus (A-MuLV) transformed cell line 300-19 was derived from the bone marrow of an adult NIH/Swiss outbred mouse. The original 300-19 clonal isolate carried DHH rearrangements of both JH alleles, a molecular genotype characteristic of early pre-B cells. During propagation in culture, the 300-19 line frequently generates secondary rearrangements of its JH alleles including rearrangements which append VH segments to the pre-existing DJH complexes to form complete VHDJH variable region genes and secondary D to JH rearrangements which replace the pre-existing DJH rearrangement by joining an upstream D to a downstream JH. The two types of secondary rearrangement events occur at approximately equal frequency. Approximately 30% of the VH to DJH joins lead to the production of mu heavy chains providing support for a regulated model of allelic exclusion. Like pre-B cell lines from other origins, the 300-19 line preferentially utilized VH gene segments from the more JH-proximal (3') families to form VHDJH rearrangements. However, the VH segments preferentially employed by 300-19 were from a different family than those previously demonstrated to be utilized by pre-B lines of BALB/c origin; we relate these different utilization patterns to differences in the organization of the more 3' VH families between the two strains. The initial DJH rearrangements of the 300-19 line employed more 3' (JH-proximal) D segments; however, the DJH replacements preferentially employed the most 5' D segment. We discuss this phenomenon in the context of a mechanism which may target recombinase to regions of the chromosome more 5' to the D locus (VH-containing regions) once an initial DJH complex is formed.