Strand breaks without DNA rearrangement in V (D)J recombination.

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.     

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.     

Propensity of adult lymphoid progenitors to progress to DN2/3 stage thymocytes with Notch receptor ligation

Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and slowly produced TCR(+) and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo. Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages were infrequent. Lin(-)c-Kit(low)GFP(+)IL-7Ralpha(+/-) prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig D(H)-J(H) rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with V(H)-D(H)J(H) rearrangement products.     

Heavy chain revision in MRL mice: a potential mechanism for the development of autoreactive B cell precursors

Abs reactive to DNA and DNA/histone complexes are distinguished by the presence of positively charged amino acids, such as arginine, in the heavy chain complementarity-determining region 3. The presence of these amino acids partly results from atypical V(H)-D-J(H) rearrangements such as D-D fusions and D inversions. Previous results in our laboratory demonstrated that newborn autoimmune MRL/MpJ-+/+ mice undergo these unusual recombinations more frequently when compared with normal C3H/HeJ controls. In addition, the heavy chain junctions in newborn MRL mice demonstrated a preferred usage of V(H)-proximal D genes and distal J(H) genes suggestive of secondary gene rearrangements. In this study we explore the possibility that adult MRL B220(+)IgM(-) pre B cells, which have not yet undergone Ag selection, exhibit similar rearrangement patterns. Indeed, MRL pre-B cells possessed more atypical rearrangements (D-D fusions) than those of C3H/HeJ mice. However, the biased use of upstream D genes and downstream J(H) genes observed in the newborn MRL mice was not present in the pre-B cell library. These results suggest that the heavy chain rearrangement process persists later during B cell life in lupus-prone mice and lead us to propose a model of heavy chain receptor revision in the periphery of autoimmune mice.     

Comparative analysis of nuclear proteins of B cells in different developmental stages

The developmental stage-specific regulation of V(D)J recombination, a gene rearrangement process of antigen receptor gene segments, is tightly controlled in cells. Here we screened proteins uniquely or differentially expressed among three developmentally distinguishable B cells (pro-B, pre-B and mature B cells) using two-dimensional gel electrophoresis and mass spectrometry. Chromatin assembly factor 1 was uniquely expressed in pro-B cells. Purine nucleotide phosphorylase, LCK, E2A and many other unidentified proteins were dominantly present in the nucleus at the early stage of B cell development where the V(D)J recombination process occurs. Also, few proteins including guanidine nucleotide binding proteins were exclusively expressed in pre-B cell. Such findings can provide some information to help understand the developmental regulation of gene rearrangement occurring during B cell development.     

Long-term retention of mature NK1.1+ NKT cells in the thymus

The NKT cell pool in the thymus contains immature (NK1.1(-)) and mature (NK1.1(+)) subsets that represent distinct linear stages of a linear developmental pathway. An unexplained paradox is why immature NK1.1(-) NKT cells are mainly exported to the periphery instead of the more mature and more abundant NK1.1(+) NKT cells. In this study we have determined that mature NK1.1(+) NKT cells are retained by the thymus to form an extremely long-lived resident population capable of rapid and prolonged production of IFN-gamma and IL-4. The retention of mature NKT cells provides an explanation for why the periphery is mainly seeded by immature NK1.1(-) cells despite mature NK1.1(+) NKT cells being more abundant in the thymus. This is the first study to identify a mature T cell subset retained within the thymus and is additional evidence of the distinct developmental pathways of mainstream T cells and NKT cells.     

Developmental changes in the human heavy chain CDR3

The CDR3 of the Ig H chain (CDR3(H)) is significantly different in fetal and adult repertoires. To understand the mechanisms involved in the developmental changes in the CDR3(H) of Ig H chains, sets of nonproductive V(H)DJ(H) rearrangements obtained from fetal, full-term neonates and adult single B cells were analyzed and compared with the corresponding productive repertoires. Analysis of the nonproductive repertoires was particularly informative in assessing developmental changes in the molecular mechanisms of V(H)DJ(H) recombination because these rearrangements did not encode a protein and therefore their distribution was not affected by selection. Although a number of differences were noted, the major reasons that fetal B cells expressed Ig H chains with shorter CDR3(H) were both diminished TdT activity in the DJ(H) junction and the preferential use of the short J(H) proximal D segment D7-27. The enhanced usage of D7-27 by fetal B cells appeared to relate to its position in the locus rather than its short length. The CDR3(H) progressively acquired a more adult phenotype during ontogeny. In fetal B cells, there was decreased recurrent DJ(H) rearrangements before V(H)-DJ(H) rearrangement and increased usage of junctional microhomologies both of which also converted to the adult pattern during ontogeny. Overall, these results indicate that the decreased length and complexity of the CDR3(H) of fetal B cells primarily reflect limited enzymatic modifications of the joins as well as a tendency to use proximal D and J(H) segments during DJ(H) rearrangements.     

Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.     

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.     

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.     

Cutting edge: TCR delta gene is frequently rearranged in adult B lymphocytes

TCR gene rearrangement generates diversity of T lymphocytes by V(D)J recombination. Ig genes are rearranged in B cells using the same enzyme machinery. Physiologically, TCR gene is postulated to rearrange exclusively in T lineage, but malignant B precursor lymphoblasts contain rearranged TCR genes in most patients. Several mechanisms by which malignant cells break the regulation of V(D)J recombination have been proposed. In this study we show that incomplete TCR delta rearrangements V2-D3 and D2-D3 occur each in up to 16% alleles in B lymphocytes of all healthy donors studied, but complete VDJ rearrangement was negative at the sensitivity limit of 1%. Data are based on real-time quantitative PCR validated by PAGE and sequencing of the cloned products. Therefore, TCR genes rearrange not exclusively in T lineage. This study opens up further questions regarding the exact extent of the "cross-lineage" TCR or Ig rearrangements in normal lymphocytes, specific subsets in which the cross-lineage rearrangements occur, and the physiological importance of these rearrangements.     

Emergence of committed B lymphoid progenitors in the developing chicken embryo.

The formation of B lymphoid restricted progenitors was followed during chicken embryonic development by monitoring the appearance of the various Ig gene rearrangements (DJH, VHDJH, V lambda J lambda), as a sensitivity that allows the detection of a single rearranged cell. By quantifying the DJH committed progenitor populations, we describe their evolution in different compartments at different developmental stages. The yolk sac is the first site where DJH-positive cells are observed (at days 5-6 of development); via the general circulation, they then seed the various organs while undergoing VHDJH and V lambda J lambda rearrangements, which occur simultaneously but lag behind DJH by one to several days. These progenitor populations decline with time in most lymphoid sites and only expand in the bursa. RAG-1 expression is observed in the bursa in the absence of ongoing rearrangement activity and thus appears to be an improper marker of rearrangement in the chicken. Commitment to the B cell lineage seems to result from an intrinsic cell program, but the survival and expansion of the committed B progenitors require the specific microenvironment of the bursa.     

Intermolecular V(D)J recombination is prohibited specifically at the joining step

V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements.     

The NF-kappaB canonical pathway is involved in the control of the exonucleolytic processing of coding ends during V(D)J recombination

V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-kappaB-binding sites are present in the human and mouse IgH, Igkappa, and Iglambda enhancer modules and RAG expression is controlled by NF-kappaB, it is not known whether NF-kappaB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-kappaB in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-kappaB essential modulator (NEMO/Ikkgamma). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkgamma-mediated canonical NF-kappaB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3(H) from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-kappaB pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-kappaB in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.     

CCR7 ligands, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are chemoattractants for CD56(+)CD16(-) NK cells and late stage lymphoid progenitors

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.