IGH@ translocations co-exist with other primary rearrangements in B-cell precursor acute lymphoblastic leukemia.

Primary established genetic abnormalities in B-cell precursor acute lymphoblastic leukemia include high hyperdiploidy (51-65 chromosomes), the translocations t(12;21)(p13;q22)/ETV6-RUNX1 fusion and t(9;22)(q34;q11)/BCR-ABL1 fusion, MLL rearrangements and intrachromosomal amplification of chromosome 21. These rearrangements are of prognostic and therapeutic relevance and are usually mutually exclusive. We identified 28 patients at diagnosis with both a primary genetic rearrangement and an immunoglobulin heavy chain locus translocation using chromosomal analysis and fluorescence in situ hybridization. Among these patients, the immunoglobulin heavy chain locus translocation partner gene was identified in six (CRLF2, CEBPA, CEBPB, TRA/D@, IGF2BP1 and IGK@). Clonal architecture was investigated in 17 patients using multiple color interphase fluorescence in situ hybridization analysis, which showed that the translocation was acquired as a secondary abnormality in ten patients, in four patients the etiology was undetermined and in three patients it was observed in a separate clone from the primary chromosomal rearrangement. These findings demonstrate the co-existence of immunoglobulin heavy chain locus translocations with other primary chromosomal rearrangements either in the same or separate clones, which may have prognostic significance in B-cell precursor acute lymphoblastic leukemia. Clinical trials: UKALLXII: Study ID n. ISRCTN77346223 and ALL2003: Study ID n. ISRCTN07355119.

A minority of concurrent monoclonal lymphocytes and plasmacytic cells sharing light chains are genetically related in putative lymphoplasmacytic lymphoma.

Flow cytometric cell sorting combined with molecular gene rearrangement analysis was used to detect and to further characterize simultaneously occurring phenotypically distinct B cell monoclonal lymphoid and monoclonal plasma cell populations from 38 individual specimens. By sorting and subsequent gene rearrangement analysis, separate or identical monoclonality genotypes could be revealed and confirmed. In only 13 of 38 specimens, the B lymphoid cells and plasma cell populations showed an identical genotypic profile, while 25 had non-identical profiles (including 4 process control specimens). The majority of the genotypically identical group had a phenotype consistent with Waldenström's/lymphoplasmacytic lymphoma (WM/LPL), while WM/LPL phenotype was present in 16/25 of the non-identical cases. Proof of an identical monoclonal genotype for plasmacytic and B-lymphoid cell populations must be used to define WM/LPL as a distinct entity in the clinical setting of monoclonal lymphoid and plasma cells expressing the same light chains. Conversely, the confirmation of genotypically distinct populations can significantly improve confidence in diagnostic and prognostic decisions in specimens with B lymphoid lymphomas and a concurrent, possibly smoldering myeloma or multiple myeloma. These techniques are requisite in future clinical studies for diagnosis and prognosis in these diseases.

B cell receptor editing in tolerance and autoimmunity.

Receptor editing is the process of ongoing antibody gene rearrangement in a lymphocyte that already has a functional antigen receptor. The expression of a functional antigen receptor will normally terminate further rearrangement (allelic exclusion). However, lymphocytes with autoreactive receptors have a chance at escaping negative regulation by "editing" the specificities of their receptors with additional antibody gene rearrangements. As such, editing complicates the Clonal Selection Hypothesis because edited cells are not simply endowed for life with a single, invariant antigen receptor. Furthermore, if the initial immunoglobulin gene is not inactivated during the editing process, allelic exclusion is violated and the B cell can exhibit two specificities. Here, we describe the discovery of editing, the pathways of receptor editing at the heavy (H) and light (L) chain loci, and current evidence regarding how and where editing happens and what effects it has on the antibody repertoire.

Sin1-mTORC2 suppresses rag and il7r gene expression through Akt2 in B cells.

Mammalian target of rapamycin (mTOR) is an important mediator of phosphoinositol-3-kinase (PI3K) signaling. PI3K signaling regulates B cell development, homeostasis, and immune responses. However, the function and molecular mechanism of mTOR-mediated PI3K signaling in B cells has not been fully elucidated. Here we show that Sin1, an essential component of mTOR complex 2 (mTORC2), regulates B cell development. Sin1 deficiency results in increased IL-7 receptor (il7r) and RAG recombinase (rag1 and rag2) gene expression, leading to enhanced pro-B cell survival and augmented V(D)J recombinase activity. We further show that Akt2 specifically mediates the Sin1-mTORC2 dependent suppression of il7r and rag gene expression in B cells by regulating FoxO1 phosphorylation. Finally, we demonstrate that the mTOR inhibitor rapamycin induces rag expression and promotes V(D)J recombination in B cells. Our study reveals that the Sin1/mTORC2-Akt2 signaling axis is a key regulator of FoxO1 transcriptional activity in B cells.

NF-kappaB family of transcription factor facilitates gene conversion in chicken B cells.

Activation-induced cytidine deaminase (AID) is critical for immunoglobulin (Ig) diversification in B cells. The majority of evidence supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines into uracils. The mutagenic activity is largely restricted to Ig genes to avoid genomic damage in general, but the underlying mechanism is not understood. We addressed this question in chicken B cell line DT40. We characterized a regulatory region within the Iglambda locus. This regulatory region is important for AID-mediated gene conversion at the Iglambda locus, and is capable of targeting AID activity to ectopic loci. This regulatory region contains binding sites for transcription factors NF-kappaB, Mef2 and octamer binding proteins. Mutation of these binding sites or ablation of NF-kappaB family member, p50 or c-Rel, impairs the AID targeting function of this regulatory region. These results suggest that NF-kappaB family of transcription factors contribute to AID-mediated gene conversion.

Recovering probabilities for nucleotide trimming processes for T cell receptor TRA and TRG V-J junctions analyzed with IMGT tools.

BACKGROUND: Nucleotides are trimmed from the ends of variable (V), diversity (D) and joining (J) genes during immunoglobulin (IG) and T cell receptor (TR) rearrangements in B cells and T cells of the immune system. This trimming is followed by addition of nucleotides at random, forming the N regions (N for nucleotides) of the V-J and V-D-J junctions. These processes are crucial for creating diversity in the immune response since the number of trimmed nucleotides and the number of added nucleotides vary in each B or T cell. IMGT sequence analysis tools, IMGT/V-QUEST and IMGT/JunctionAnalysis, are able to provide detailed and accurate analysis of the final observed junction nucleotide sequences (tool "output"). However, as trimmed nucleotides can potentially be replaced by identical N region nucleotides during the process, the observed "output" represents a biased estimate of the "true trimming process." RESULTS: A probabilistic approach based on an analysis of the standardized tool "output" is proposed to infer the probability distribution of the "true trimmming process" and to provide plausible biological hypotheses explaining this process. We collated a benchmark dataset of TR alpha (TRA) and TR gamma (TRG) V-J rearranged sequences and junctions analysed with IMGT/V-QUEST and IMGT/JunctionAnalysis, the nucleotide sequence analysis tools from IMGT, the international ImMunoGeneTics information system, http://imgt.cines.fr. The standardized description of the tool output is based on the IMGT-ONTOLOGY axioms and concepts. We propose a simple first-order model that attempts to transform the observed "output" probability distribution into an estimate closer to the "true trimming process" probability distribution. We use this estimate to test the hypothesis that Poisson processes are involved in trimming. This hypothesis was not rejected at standard confidence levels for three of the four trimming processes: TRAV, TRAJ and TRGV. CONCLUSION: By using trimming of rearranged TR genes as a benchmark, we show that a probabilistic approach, applied to IMGT standardized tool "outputs" opens the way to plausible hypotheses on the events involved in the "true trimming process" and eventually to an exact quantification of trimming itself. With increasing high-throughput of standardized immunogenetics data, similar probabilistic approaches will improve understanding of processes so far only characterized by the "output" of standardized tools.

Yin Yang 1 is a critical regulator of B-cell development.

The role of the transcription factor Yin Yang 1 (YY1) in development is largely unknown. Here we show that specific ablation of YY1 in mouse B cells caused a defect in somatic rearrangement in the immunoglobulin heavy-chain (IgH) locus and a block in the progenitor-B-to-precursor-B-cell transition, which was partially rescued by a prerearranged IgH transgene. Three-dimensional DNA fluorescence in situ hybridization analysis revealed an important function for YY1 in IgH locus contraction, a process indispensable for distal V(H) to D(H)J(H) recombination. We provide evidence that YY1 binds the intronic Ei mu enhancer within the IgH locus, consistent with a direct role for YY1 in V(H)D(H)J(H) recombination. These findings identified YY1 as a critical regulator of early B-cell development.

Antigen specificity of clonally expanded and receptor edited cerebrospinal fluid B cells from patients with relapsing remitting MS.

We re-engineered the immunoglobulin rearrangements from clonally expanded CSF B cells of three Multiple Sclerosis patients as Fab fragments, and used three methods to test for their antigen (Ag) specificity. Nine out of ten Fab fragments were reactive to Myelin Basic Protein (MBP). The one Fab that did not react to MBP was a product of receptor editing. Two of the nine MBP reactive Fabs were also reactive to GFAP and CNPase, indicating that these clones were polyreactive. Targeting the mechanisms that allow these self-reactive B cells to reside in the CSF of MS patients may prove to be a potent immunotherapeutic strategy.

Pre-BCR signals and the control of Ig gene rearrangements.

Progenitor B lymphocytes that successfully assemble a heavy chain gene encoding an immunoglobulin capable of pairing with surrogate light chain proteins trigger their own further differentiation by signaling via the pre-BCR complex. The pre-BCR signals several rounds of proliferation and, in this expanded population, directs a complex, B cell-specific set of epigenetic changes resulting in allelic exclusion of the heavy chain locus and activation of the light chain loci for V(D)J recombination.

E47 is required for V(D)J recombinase activity in common lymphoid progenitors.

Common lymphoid progenitors (CLPs) are the first bone marrow precursors in which V(D)J recombinase activity is up-regulated. Here, we show that loss of the transcription factor E47 produces a reduced CLP population that lacks V(D)J recombinase activity and D-J(H) rearrangements in vivo. Apart from a profound arrest before the pro-B cell stage, other downstream lymphoid progeny of CLPs are still intact in these mice albeit at reduced numbers. In contrast to the inhibition of recombinase activity in early B lineage precursors in E47-deficient animals, loss of either E47 or its cis-acting target Erag (enhancer of rag transcription) has little effect on recombinase activity in thymic T lineage precursors. Taken together, this work defines a role for E47 in regulating lineage progression at the CLP stage in vivo and describes the first transcription factor required for lineage-specific recombinase activity.

Birth pangs: the stressful origins of lymphocytes.

Inositol-requiring enzyme 1 (IRE1) is a transmembrane protein that signals from the ER and contributes to the generation of an active spliced form of the transcriptional regulator X-box-binding protein 1 (XBP1). XBP1 is required for the terminal differentiation of B lymphocytes into plasma cells, and IRE1 also participates in this differentiation event. A study in this issue of the JCI reveals, quite unexpectedly, that IRE1 is also required early in B lymphocyte development for the induction of the machinery that mediates Ig gene rearrangement.

The unfolded protein response sensor IRE1alpha is required at 2 distinct steps in B cell lymphopoiesis.

B lymphocyte differentiation is coordinated with the induction of high-level Ig secretion and expansion of the secretory pathway. Upon accumulation of unfolded proteins in the lumen of the ER, cells activate an intracellular signaling pathway termed the unfolded protein response (UPR). Two major proximal sensors of the UPR are inositol-requiring enzyme 1alpha (IRE1alpha), an ER transmembrane protein kinase/endoribonuclease, and ER-resident eukaryotic translation initiation factor 2alpha (eIF2alpha) kinase (PERK). To elucidate whether the UPR plays an important role in lymphopoiesis, we carried out reconstitution of recombinase-activating gene 2-deficient (rag2-/-) mice with hematopoietic cells defective in either IRE1alpha- or PERK-mediated signaling. IRE1alpha-deficient (ire1alpha-/-) HSCs can proliferate and give rise to pro-B cells that home to bone marrow. However, IRE1alpha, but not its catalytic activities, is required for Ig gene rearrangement and production of B cell receptors (BCRs). Analysis of rag2-/- mice transplanted with IRE1alpha trans-dominant-negative bone marrow cells demonstrated an additional requirement for IRE1alpha in B lymphopoiesis: both the IRE1alpha kinase and RNase catalytic activities are required to splice the mRNA encoding X-box-binding protein 1 (XBP1) for terminal differentiation of mature B cells into antibody-secreting plasma cells. Furthermore, UPR-mediated translational control through eIF2alpha phosphorylation is not required for B lymphocyte maturation and/or plasma cell differentiation. These results suggest specific requirements of the IRE1alpha-mediated UPR subpathway in the early and late stages of B lymphopoiesis.

CD19 regulates positive selection and maturation in B lymphopoiesis: lack of CD19 imposes developmental arrest of immature B cells and consequential stimulation of receptor editing.

Ligand-independent signals that are produced by the B-cell antigen receptor (BCR) confer an important positive selection checkpoint for immature B cells. Generation of inappropriate signals imposes developmental arrest of immature B cells, though the fate of these cells has not been investigated. Studies have shown that the lack of CD19 results in inappropriate signaling. In immunoglobulin transgenic mice, this inappropriate signaling impairs positive selection and stimulates receptor editing. Here, we studied the extent and significance of receptor editing in CD19-regulated positive selection of normal, nontransgenic B lymphopoiesis, using our bone marrow culture system. We found that the lack of CD19 resulted in elevated tonic signaling and impaired maturation, as revealed by surface marker expression and by functional assays. Immature CD19-/- B cells did not suppress RAG and underwent intensive receptor editing attempts in culture. Finally, in vivo analysis of light-chain isotype expression and Jkappa use in CD19-/- mice validated our in vitro observations. Our results suggest that CD19 has an important function in regulating positive selection and maturation of nontransgenic B-cell precursors and that receptor editing is an important salvage mechanism for immature B cells that fail positive selection.

The role of the non-homologous end-joining pathway in lymphocyte development.

One of the most toxic insults a cell can incur is a disruption of its linear DNA in the form of a double-strand break (DSB). Left unrepaired, or repaired improperly, these lesions can result in cell death or neoplastic transformation. Despite these dangers, lymphoid cells purposely introduce DSBs into their genome to maximize the diversity and effector functions of their antigen receptor genes. While the generation of breaks requires distinct lymphoid-specific factors, their resolution requires various ubiquitously expressed DNA-repair proteins, known collectively as the non-homologous end-joining pathway. In this review, we discuss the factors that constitute this pathway as well as the evidence of their involvement in two lymphoid-specific DNA recombination events.

Sibling rivalry: competition between Pol X family members in V(D)J recombination and general double strand break repair.

The nonhomologous end-joining pathway is a major means for repairing double-strand breaks (DSBs) in all mitotic cell types. This repair pathway is also the only efficient means for resolving DSB intermediates in V(D)J recombination, a lymphocyte-specific genome rearrangement required for assembly of antigen receptors. A role for polymerases in end-joining has been well established. They are a major factor in determining the character of repair junctions but, in contrast to 'core' end-joining factors, typically appear to have a subtle impact on the efficiency of end-joining. Recent work implicates several members of the Pol X family in end-joining and suggests surprising complexity in the control of how these different polymerases are employed in this pathway.

Artemis sheds new light on V(D)J recombination.

V(D)J recombination represents one of the three mechanisms that contribute to the diversity of the immune repertoire of B lymphocytes and T lymphocytes. It also constitutes a major checkpoint during the development of the immune system. Indeed, any V(D)J recombination deficiency leads to a block of B-cell and T-cell maturation in humans and animal models, leading to severe combined immunodeficiency (T-B-SCID). Nine factors have been identified so far to participate in V(D)J recombination. The discovery of Artemis, mutated in a subset of T-B-SCID, provided some new information regarding one of the missing V(D)J recombinase activities: hairpin opening at coding ends prior to DNA repair of the recombination activating genes 1/2-generated DNA double-strand break. New conditions of immune deficiency in humans are now under investigations and should lead to the identification of additional V(D)J recombination/DNA repair factors.

Pax5 activates immunoglobulin heavy chain V to DJ rearrangement in transgenic thymocytes.

Mice deficient for the B cell-restricted transcription factor Pax5 show a defect in the VH to DJH rearrangement step of immunoglobulin heavy chain gene assembly even though the expression of the V(D)J recombinase is not diminished in Pax5-/- pro-B cells. To investigate whether Pax5 is limiting for VH to DJH rearrangement, we generated transgenic mice which express Pax5 in developing thymocytes. We show that enforced expression of Pax5 in thymocytes results in a partial block in T cell development due to defective pre-TCR signaling in beta-selection. Moreover, our results demonstrate that expression of Pax5 in early thymocytes is sufficient to induce VH to DJH rearrangements in CD4+CD8+ T cells and lead us to suggest that Pax5 may play a direct role in the lineage-specific regulation of immunoglobulin heavy chain gene rearrangement.

Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and development.

Although V(D)J recombination is thought to be regulated by changes in the accessibility of chromatin to the recombinase machinery, the mechanisms responsible for establishing "open" chromatin are poorly understood. We performed a detailed study of the acetylation status of histones associated with 11 V(H) gene segments, their flanking regions, and various intergenic elements during B-cell development and ontogeny, when V(D)J recombination is highly regulated. Histone H4 shows higher and more-regulated acetylation than does histone H3 in the V(H) locus. In adult pro-B cells, V(H) gene segments are acetylated prior to V(D)J rearrangement, with higher acetylation associated with J(H)-distal V(H) gene segments. While large regions of the V(H) locus have similar patterns of histone acetylation, acetylation is narrowly confined to the gene segments, their flanking promoters, and recombinase signal sequence elements. Thus, histone acetylation in the V(H) locus is both locally and globally regulated. Increased histone acetylation accompanies preferential recombination of J(H)-proximal V(H) gene segments in early B-cell ontogeny, and decreased histone acetylation accompanies inhibition of V-DJ recombination in a transgenic model of immunoglobulin heavy-chain allelic exclusion. Thus, changes in histone acetylation appear to be important for both promotion and inhibition of V-DJ rearrangement during B-cell ontogeny and development.

Transcriptional control of B-cell development.

Significant progress has recently been made in our understanding of how transcription factors such as PU.1, Notch1, E2A, EBF, Pax5, Bcl6, Blimp1 and XBP1 control different developmental decisions during the onset and terminal phase of B-lymphopoiesis. One emerging theme is that negative regulatory networks play an important role in suppressing alternative gene programs and their corresponding cell fates throughout B-cell development.