Evaluation of Positive B- and T-Cell Gene Rearrangement Studies in Patients With Negative Morphology, Flow Cytometry, and Immunohistochemistry.

CONTEXT.­: The significance of positive immunoglobulin (IG) or T-cell receptor (TCR) gene rearrangement studies in the context of otherwise normal ancillary findings is unknown. OBJECTIVE.­: To examine long-term hematologic outcomes of individuals with positive gene rearrangement studies with otherwise unremarkable blood or bone marrow studies in parallel. DESIGN.­: Data from patients who underwent IG or TCR gene rearrangement testing at the authors' affiliated Veterans Affairs Hospital January 1, 2013 to July 6, 2018 were extracted from medical records. Date of testing, specimen source, and morphologic, flow cytometric, immunohistochemical, and cytogenetic characterization of the tissue source were recorded. Gene rearrangement results were categorized as test positive/phenotype positive (T+/P+), test positive/phenotype negative (T+/P-), test negative/phenotype negative (T-/P-), or test negative/phenotype positive (T-/P+) based on comparison to other studies and/or final diagnosis. Patient records were reviewed for subsequent diagnosis of hematologic malignancy for patients with positive gene rearrangements but no other evidence for a disease process. RESULTS.­: A total of 136 patients with 203 gene rearrangement studies were analyzed. For TCR studies, there were 2 T+/P- and 1 T-/P+ results in 47 peripheral blood assays, as well as 7 T+/P- and 1 T-/P+ results in 54 bone marrow assays. Regarding IG studies, 3 T+/P- and 12 T-/P+ results in 99 bone marrow studies were identified. None of the 12 patients with T+/P- TCR or IG gene rearrangement studies later developed a lymphoproliferative disorder. CONCLUSIONS.­: Positive IG/TCR gene rearrangement studies in the context of otherwise negative bone marrow or peripheral blood findings are not predictive of lymphoproliferative disorders.

Conformational diversity facilitates antibody mutation trajectories and discrimination between foreign and self-antigens.

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.

A Bayesian phylogenetic hidden Markov model for B cell receptor sequence analysis.

The human body generates a diverse set of high affinity antibodies, the soluble form of B cell receptors (BCRs), that bind to and neutralize invading pathogens. The natural development of BCRs must be understood in order to design vaccines for highly mutable pathogens such as influenza and HIV. BCR diversity is induced by naturally occurring combinatorial "V(D)J" rearrangement, mutation, and selection processes. Most current methods for BCR sequence analysis focus on separately modeling the above processes. Statistical phylogenetic methods are often used to model the mutational dynamics of BCR sequence data, but these techniques do not consider all the complexities associated with B cell diversification such as the V(D)J rearrangement process. In particular, standard phylogenetic approaches assume the DNA bases of the progenitor (or "naive") sequence arise independently and according to the same distribution, ignoring the complexities of V(D)J rearrangement. In this paper, we introduce a novel approach to Bayesian phylogenetic inference for BCR sequences that is based on a phylogenetic hidden Markov model (phylo-HMM). This technique not only integrates a naive rearrangement model with a phylogenetic model for BCR sequence evolution but also naturally accounts for uncertainty in all unobserved variables, including the phylogenetic tree, via posterior distribution sampling.

Tumour necrosis as assessed with 18F-FDG PET is a potential prognostic marker in diffuse large B cell lymphoma independent of MYC rearrangements.

OBJECTIVES: MYC gene rearrangements in diffuse large B cell lymphomas (DLBCLs) result in high proliferation rates and are associated with a poor prognosis. Strong proliferation is associated with high metabolic demand and tumour necrosis. The aim of this study was to investigate differences in the presence of necrosis and semiquantitative 18F-FDG PET metrics between DLBCL cases with or without a MYC rearrangement. The prognostic impact of necrosis and semiquantitative 18F-FDG PET parameters was investigated in an explorative survival analysis. METHODS: Fluorescence in situ hybridisation analysis for MYC rearrangements, visual assesment, semiquantitative analysis of 18F-FDG PET scans and patient survival analysis were performed in 61 DLBCL patients, treated at a single referral hospital between 2008 and 2015. RESULTS: Of 61 tumours, 21 (34%) had a MYC rearrangement (MYC+). MYC status was neither associated with the presence of necrosis on 18F-FDG PET scans (necrosisPET; p = 1.0) nor associated with the investigated semiquantitative parameters maximum standard uptake value (SUVmax; p = 0.43), single highest SUVmax (p = 0.49), metabolic active tumour volume (MATV; p = 0.68) or total lesion glycolysis (TLG; p = 0.62). A multivariate patient survival analysis of the entire cohort showed necrosisPET as an independent prognostic marker for disease-specific survival (DSS) (HR = 13.9; 95% CI 3.0-65; p = 0.001). CONCLUSIONS: MYC rearrangements in DLBCL have no influence on the visual parameter necrosisPET or the semi-quantiative parameters SUVmax, MATV and TLG. Irrespective of MYC rearrangements, necrosisPET is an independent, adverse prognostic factor for DSS. KEY POINTS: • Retrospective analysis indicates that MYC rearrangement is not associated with necrosis on 18 F-FDG PET (necrosis PET ) scans or semiquantitative 18 F-FDG PET parameters. • Necrosis PET is a potential independent adverse prognostic factor for disease-specific survival in patients with DLBCL and is not influenced by the presence of MYC rearrangements.

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Human monoclonal antibodies isolated from a primary pneumococcal conjugate Vaccinee demonstrates the expansion of an antigen-driven Hypermutated memory B cell response.

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.

Control of B-1a cell development by instructive BCR signaling.

B-1a cells remain one of the most enigmatic lymphocyte subsets. In this review, we discuss recent advances in our understanding of the development of these cells and their regulation by the transcription factors Bhlhe41 and Arid3a as well as by the RNA-binding protein Lin28b. A large body of literature supports an instructive role of BCR signaling in B-1a cell development and lineage commitment, which is initiated only after signaling from an autoreactive BCR. While both fetal and adult hematopoiesis can generate B-1a cells, the contribution of adult hematopoiesis to the B-1a cell compartment is low under physiological conditions. We discuss several models that can reconcile the instructive role of BCR signaling with this fetal bias in B-1a cell development.

The 11q-Gain/Loss Aberration Occurs Recurrently in MYC-Negative Burkitt-like Lymphoma With 11q Aberration, as Well as MYC-Positive Burkitt Lymphoma and MYC-Positive High-Grade B-Cell Lymphoma, NOS.

OBJECTIVES: The latest revision of lymphoma's World Health Organization classification describes the new provisional entity "Burkitt-like lymphoma with 11q aberration" (BLL, 11q) as lacking MYC rearrangement, but harboring the specific11q-gain/loss aberration. We report genetic characteristics of 11 lymphoma cases with this aberration. METHODS: Classical cytogenetics, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism/array comparative genomic hybridization. RESULTS: The 11q aberrations were described as duplication, inversion, and deletion. Array comparative genomic hybridization showed two types of duplication: bigger than 50 megabase pairs (Mbp) and smaller than 20 Mbp, which were associated with bulky tumor larger than 20 cm and amplification of the 11q23.3 region, including KMT2A. Six cases revealed a normal FISH status of MYC and were diagnosed as BLL,11q. Five cases showed MYC rearrangement and were diagnosed as Burkitt lymphoma (BL) or high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS). CONCLUSIONS: The 11q-gain/loss is not specific for BLL, 11q, but occurs recurrently in MYC-positive BL and MYC-positive HGBL.

The BTG2-PRMT1 module limits pre-B cell expansion by regulating the CDK4-Cyclin-D3 complex.

Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.

Development of a Bioinformatics Framework for the Detection of Gene Conversion and the Analysis of Combinatorial Diversity in Immunoglobulin Heavy Chains in Four Cattle Breeds.

We have developed a new bioinformatics framework for the analysis of rearranged bovine heavy chain immunoglobulin (Ig) variable regions by combining and refining widely used alignment algorithms. This bioinformatics framework allowed us to investigate alignments of heavy chain framework regions (FRHs) and the separate alignments of FRHs and heavy chain complementarity determining regions (CDRHs) to determine their germline origin in the four cattle breeds Aubrac, German Black Pied, German Simmental, and Holstein Friesian. Now it is also possible to specifically analyze Ig heavy chains possessing exceptionally long CDR3Hs. In order to gain more insight into breed specific differences in Ig combinatorial diversity, somatic hypermutations and putative gene conversions of IgG, we compared the dominantly transcribed variable (IGHV), diversity (IGHD), and joining (IGHJ) segments and their recombination in the four cattle breeds. The analysis revealed the use of 15 different IGHV segments, 21 IGHD segments, and two IGHJ segments with significant different transcription levels within the breeds. Furthermore, there are preferred rearrangements within the three groups of CDR3H lengths. In the sequences of group 2 (CDR3H lengths (L) of 11-47 amino acid residues (aa)) a higher number of recombination was observed than in sequences of group 1 (L≤10 aa) and 3 (L≥48 aa). The combinatorial diversity of germline IGHV, IGHD, and IGHJ-segments revealed 162 rearrangements that were significantly different. The few preferably rearranged gene segments within group 3 CDR3H regions may indicate specialized antibodies because this length is unique in cattle. The most important finding of this study, which was enabled by using the bioinformatics framework, is the discovery of strong evidence for gene conversion as a rare event using pseudogenes fulfilling all definitions for this particular diversification mechanism.

Likelihood-Based Inference of B Cell Clonal Families.

The human immune system depends on a highly diverse collection of antibody-making B cells. B cell receptor sequence diversity is generated by a random recombination process called "rearrangement" forming progenitor B cells, then a Darwinian process of lineage diversification and selection called "affinity maturation." The resulting receptors can be sequenced in high throughput for research and diagnostics. Such a collection of sequences contains a mixture of various lineages, each of which may be quite numerous, or may consist of only a single member. As a step to understanding the process and result of this diversification, one may wish to reconstruct lineage membership, i.e. to cluster sampled sequences according to which came from the same rearrangement events. We call this clustering problem "clonal family inference." In this paper we describe and validate a likelihood-based framework for clonal family inference based on a multi-hidden Markov Model (multi-HMM) framework for B cell receptor sequences. We describe an agglomerative algorithm to find a maximum likelihood clustering, two approximate algorithms with various trade-offs of speed versus accuracy, and a third, fast algorithm for finding specific lineages. We show that under simulation these algorithms greatly improve upon existing clonal family inference methods, and that they also give significantly different clusters than previous methods when applied to two real data sets.

Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells.

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.

EBV-negative aggressive B-cell lymphomas of donor origin after allogeneic hematopoietic stem cell transplantation: a report of three cases.

Post-transplant lymphoproliferative disorders (PTLD) develop as a consequence of iatrogenic immunosuppression, and the majority is associated with EBV. PTLD after allogeneic hematopoietic stem cell transplantation (allo-HCT) are rare. Most cases are donor-derived, reflecting immune reconstitution by malignant transformed donor cells, and are EBV-positive. We report three unusual cases of aggressive EBV-negative PTLD of monomorphic type after allo-HCT. All cases were of donor origin and arose with long latency, 4-12 years after HCT. The patients had a history of severe graft-versus-host disease (GVHD) resulting in prolonged immunosuppression before the diagnosis of lymphoma. In one case, the temporal evolution of the malignant clone was analyzed by clone-specific PCR targeting the immunoglobulin heavy chain rearrangement. A tumor-specific product was already detected 3 years before lymphoma development. This indicates that chronic antigenic stimulation and reduced immune surveillance, may promote the outgrowth of premalignant donor-derived clones after acquisition of additional genetic alterations.

Consistency of VDJ Rearrangement and Substitution Parameters Enables Accurate B Cell Receptor Sequence Annotation.

VDJ rearrangement and somatic hypermutation work together to produce antibody-coding B cell receptor (BCR) sequences for a remarkable diversity of antigens. It is now possible to sequence these BCRs in high throughput; analysis of these sequences is bringing new insight into how antibodies develop, in particular for broadly-neutralizing antibodies against HIV and influenza. A fundamental step in such sequence analysis is to annotate each base as coming from a specific one of the V, D, or J genes, or from an N-addition (a.k.a. non-templated insertion). Previous work has used simple parametric distributions to model transitions from state to state in a hidden Markov model (HMM) of VDJ recombination, and assumed that mutations occur via the same process across sites. However, codon frame and other effects have been observed to violate these parametric assumptions for such coding sequences, suggesting that a non-parametric approach to modeling the recombination process could be useful. In our paper, we find that indeed large modern data sets suggest a model using parameter-rich per-allele categorical distributions for HMM transition probabilities and per-allele-per-position mutation probabilities, and that using such a model for inference leads to significantly improved results. We present an accurate and efficient BCR sequence annotation software package using a novel HMM "factorization" strategy. This package, called partis (https://github.com/psathyrella/partis/), is built on a new general-purpose HMM compiler that can perform efficient inference given a simple text description of an HMM.

B Lymphoblastic Leukemia With a Novel t(11;15) (q23;q15) and Unique Burkittoid Morphologic and Immunophenotypic Findings in a 9-Year-Old Boy.

B lymphoblastic leukemia is a B progenitor cell neoplasm with a range of immature immunophenotypes and several associated cytogenetic lesions. In contrast, Burkitt leukemia/lymphoma is a mature B lymphocyte neoplasm with a characteristic germinal center immunophenotype and MYC rearrangement. With modern immunophenotyping and cytogenetic methods, the distinction between these 2 entities is seldom ambiguous. Herein, we report a case of a 9-year-old white boy with circulating leukemic cells that demonstrate morphologic overlap between Burkitt leukemia and B lymphoblastic leukemia. Flow cytometry and immunohistochemical stains demonstrated expression of sets of markers with overlap between immature and mature immunophenotypes. While the leukemic cells tested positive for terminal deoxynucleotidyl transferase (TdT), they expressed CD20, BCL6 (in a subset), and lambda-restricted surface light chain. Molecular studies confirmed a true clonal light chain rearrangement, whereas fluorescent in situ hybridization (FISH) results were negative for MYC rearrangement. Metaphase cytogenetics identified a novel gene rearrangement, t(11;15)(q23;q15), that does not involve the MLL gene. This unique cytogenetic abnormality involves the loss of INO80, an adenosine triphosphatase (ATPase) with DNA binding ability. This cytogenetic abnormality may represent a unique feature of this overlap entity of B lymphoblastic lymphoma that expresses markers of maturity and demonstrates Burkitt-like morphology.

Recurrent genomic rearrangements in primary testicular lymphoma.

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune-privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin-fixed, paraffin-embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base-pair resolution in a rare, aggressive lymphoma. Our data suggest immune-checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements.

Metachronous/concomitant B-cell neoplasms with discordant light-chain or heavy-chain isotype restrictions: evidence of distinct B-cell neoplasms rather than clonal evolutions.

Metachronous/concomitant B-cell neoplasms with distinct morphology are usually considered clonally related. We retrospectively analyzed 4 cases of metachronous/concomitant B-cell neoplasms with discordant light-chain/heavy-chain restrictions. The primary diagnoses included chronic lymphocytic leukemia (CLL; n = 2), lymphoplasmacytic lymphoma (n = 1), and pediatric follicular lymphoma (FL; n = 1). The respective secondary diagnoses included diffuse large B-cell lymphoma (DLBCL; n = 2), plasmablastic myeloma, and pediatric FL. The secondary B-cell neoplasm occurred after the primary diagnosis in 3 cases, with the median interval of 120 months (range, 21-216), whereas the remaining 1 case had the 2 neoplasms (CLL/DLBCL) diagnosed concurrently. Histology suggested aggressive transformation in 3 cases and recurrence in 1 case (FL). Nonetheless, 3 cases showed discordant light-chain restrictions between the 2 B-cell neoplasms, whereas in the remaining case (lymphoplasmacytic lymphoma/plasmablastic myeloma), the 2 neoplasms shared κ light-chain restriction but expressed different heavy-chain isotypes (IgM versus IgA). The 2 CLL/DLBCL cases had polymerase chain reaction-based IGH/K gene rearrangement study and amplicon sequence analysis performed, which demonstrated distinct clonal amplicons between the 2 B-cell neoplasms in each case. Concomitant/metachronous B-cell neoplasms may be clonally unrelated, which can be confirmed by immunoglobulin isotype analysis and/or genotypic studies. We advocate analysis of clonal identities in large cell transformation or recurrent disease compared with primary indolent B-cell neoplasm because of a potential difference in prognosis between clonally related and unrelated secondary B-cell neoplasms.

The survival and differentiation of pro-B and pre-B cells in the bone marrow is dependent on IL-7Rα Tyr449.

IL-7 is critical for murine T and B cell development and survival and plays a significant role in lymphoblastic leukemia in both humans and mice. We evaluated the role of the IL-7Rα Tyr(449) cytoplasmic SH2-binding motif in IL-7-mediated B cell development using a knock-in mouse with a Tyr to Phe mutation (IL-7Rα(449F/449F) mouse). IL-7Rα(449F/449F) and IL-7Rα(-/-) mice showed no defect in the number of pre-pro-B cells, although IL-7Rα(449F/449F) mice had decreased Ebf1 in pre-pro-B cells and impairment in B cell-committed CLPs. We identified that IL-7Rα Tyr(449) was critical for both pro-B and pre-B stages of development in the bone marrow. IL-7Rα(449F/449F) and IL-7Rα(-/-) mice had comparable precursor B cell defects, indicating that signaling from the IL-7Rα required this motif. Although the defect in IL-7Rα(449F/449F) pro-B cells was associated with loss of STAT5 activation and diminished expression of Mcl1, this was not rescued by overexpression of Bcl-2. IL-7Rα(449F/449F) and IL-7Rα(-/-) pre-B cells also showed defective cyto-Igµ and CD25 expression, associated with reduced levels of Rag1, Rag2, and Irf4. Pre-B cells from IL-7Rα(449F/449F) mice also failed to proliferate, perhaps as a result of the failure to rearrange Igµ. Our data suggest that IL-7Rα Tyr(449) was essential for IL-7Rα signaling in bone marrow B cell development and survival.

Staying innate: transcription factor maintenance of innate lymphoid cell identity.

Innate and adaptive lymphocytes are characterized by phenotypic and functional characteristics that result from genomic rearrangements (in the case of antigen-specific B and T cells) coupled with selective gene expression patterns that are generated in a context-dependent fashion. Cell-intrinsic expression of transcription factors (TFs) play a critical role in the regulation of gene expression that establish the distinct lymphoid subsets but also have been proposed to play an ongoing role in the maintenance of lineage-associated transcriptional signatures that comprise lymphocyte identity. This is the case for CD19(+) B cells that require Pax5 expression throughout their lifespan, as well as for diverse T-helper subsets that have specialized immune functions. Innate lymphoid cells (ILCs) comprise diverse effectors cells that differentiate under TF control and have critical roles in the early stages of immune responses. In this review, ILC development is reviewed and the requirement for persistent TF expression in the maintenance of transcriptional signatures that define ILC identity is explored.

B-cell receptor signaling in lymphoid malignancies and autoimmunity.

The B-cell receptor (BCR) for antigen is a key sensor required for B-cell development, survival, and activation. Rigorous selection checkpoints ensure that the mature B-cell compartment in the periphery is largely purged of self-reactive B cells. However, autoreactive B cells escape selection and persist in the periphery as anergic or clonally ignorant B cells. Under the influence of genetic or environmental factors, which are not completely understood, autoreactive B cells may be activated. Similar activation can also occur at different stages of B-cell maturation in the bone marrow or in peripheral lymphoid organs and give rise to malignant B cells. The pathology that typifies neoplastic lymphocytes and autoreactive B cells differs: malignant B cells proliferate and occupy niches otherwise taken up by healthy leukocytes or erythrocytes, while autoreactive B cells produce pathogenic antibodies or present self-antigen to T cells. However, both malignant and autoreactive B cells share the commonality of deregulated BCR pathways as principal contributors to pathogenicity. We first summarize current views of BCR activation. We then explore how anomalous BCR pathways correlate with malignancies and autoimmunity. We also elaborate on the activation of TLR pathways in abnormal B cells and how they contribute to maintenance of pathology. Finally, we outline the benefits and emergence of mouse models generated by somatic cell nuclear transfer to study B-cell function in manners for which current transgenic models may be less well suited.