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Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

Alzheimer's disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved. As promising approach passive immunization strategies have thereby come forth. In this study, we focused on naturally occurring autoantibodies against the AD-associated peptide amyloid-β. These antibodies have already reported to show beneficial functions in vitro and in mouse models of AD. However, their availability is limited due to their low abundance in peripheral blood. In a recent study, we were able to generate four recombinant antibodies against amyloid-β. In the present study, we tested these antibodies in ELISA and SPR assays for their binding behavior and by aggregation- and phagocytosis assays as functional evidences to characterize their amyloid-β-related neutralizing and clearance abilities. Further ex vivo assay on organotypic hippocampal slice cultures gave first evidence of microglial activation and inflammatory features. The tested recombinant antibodies in IgG format showed, in comparison to naturally occurring autoantibodies against amyloid-β, insufficient binding capacities and -affinities. However, after conversion of one antibody into a single chain format multimerization of the scFv-Fc construct, the investigated binding capacity and -affinity showed improvements. Further functional assays predict a protective effect of this antibody. Although, all four recombinant antibodies showed binding to amyloid-β, promising features were only detectable after conversion into a multimeric format. The multimeric scFv-Fc antibody exhibited thereby strong impact on amyloid-β clearance and inhibition of oligomerization.

Single-cell RNAseq/VDJseq of tumor cells and normal residual B (NRB) cells from peripheral blood of chronic lymphocytic leukemia (CLL) patients identified three distinct tumor subsets according to phenotype, transcriptome, and immunoglobulin-V-gene (IgV)-mutations. Two major subsets share a typical CLL phenotype but differ in signaling, metabolism and cell cycle control, indicating that the circulating CLL pool is shaped by two states of activity. The third CLL subset shows the phenotype, proliferation capacity and extensive IgV-mutation diversity of normal CD5+ memory B cells. This previously unrecognized CLL tumor subset, which intermingles with NRB cells, was confirmed in 33 IgV-mutated (M-CLL) and IgV-unmutated (U-CLL) cases. Longitudinal IgV-mutation phylogenetics suggest that these NRB-associated CLL cells are generated pathogenetically early, mostly in germinal center reactions, and archive the individual IgV-diversification program, which is conserved throughout CLL course. Our study suggests that diversity is established early in CLL, that each tumor is composed of multiple subclonal expansions, and subclonal evolution can be depicted by IgV-mutation phylogenetics.

We performed neutron imaging of ferromagnetic transitions in Ni(3)Al and HgCr(2)Se(4) crystals. These neutron depolarization measurements revealed bulk magnetic inhomogeneities in the ferromagnetic transition temperature with spatial resolution of about 100 μm. To obtain such spatial resolution, we employed a novel neutron microscope equipped with Wolter mirrors as a neutron image-forming lens and a focusing neutron guide as a neutron condenser lens. The images of Ni(3)Al show that the sample does not homogeneously go through the ferromagnetic transition; the improved resolution allowed us to identify a distribution of small grains with slightly off-stoichiometric composition. Additionally, neutron depolarization imaging experiments on the chrome spinel, HgCr(2)Se(4), under pressures up to 15 kbar highlight the advantages of the new technique especially for small samples or sample environments with restricted sample space. The improved spatial resolution enables one to observe domain formation in the sample while decreasing the acquisition time despite having a bulky pressure cell in the beam.

Kibler et al. show that circulating memory B cells are systematically and comprehensively archived in the human spleen throughout life. The random, dynamic priming of archive members determines the composition of human splenic marginal zone B cells., Human memory B cells (MBCs) are generated and diversified in secondary lymphoid tissues throughout the organism. A paired immunoglobulin (Ig)-gene repertoire analysis of peripheral blood (PB) and splenic MBCs from infant, adult, and elderly humans revealed that throughout life, circulating MBCs are comprehensively archived in the spleen. Archive MBC clones are systematically preserved and uncoupled from class-switching. Clonality in the spleen increases steadily, but boosts at midlife, thereby outcompeting small clones. The splenic marginal zone (sMZ) represents a primed MBC compartment, generated from a stochastic exchange within the archive memory pool. This is supported by functional assays, showing that PB and splenic CD21+ MBCs acquire transient CD21high expression upon NOTCH2-stimulation. Our study provides insight that the human MBC system in PB and spleen is composed of three interwoven compartments: the dynamic relationship of circulating, archive, and its subset of primed (sMZ) memory changes with age, thereby contributing to immune aging., Graphical Abstract

Bruton tyrosine kinase (BTK) inhibitors are highly active drugs for the treatment of chronic lymphocytic leukemia (CLL). To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines (pS), threonines (pT), and tyrosines (pY) (pS:pT:pY). Expression of 83 proteins differed between unmutated immunoglobulin heavy-chain variable region (IGHV) CLL (UM-CLL) and mutated IGHV CLL (M-CLL). Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition, and survival. One protein, myristoylated alanine-rich C-kinase substrate (MARCKS), showed striking differences in expression and phosphorylation level in UM-CLL vs M-CLL. MARCKS sequesters phosphatidylinositol-4,5-bisphosphate, thereby affecting central signaling pathways and clustering of the B-cell receptor (BCR). Genetically induced loss of MARCKS significantly increased AKT signaling and migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in patients with CLL treated with acalabrutinib.

The human infant B cell system is considered premature or impaired. Here we show that most cord blood B cells are mature and functional as seen in adults, albeit with distinct transcriptional programs providing accelerated responsiveness to T cell-independent and T cell-dependent stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into antibody-secreting cells, thereby presumably limiting memory B cell formation. The neonatal Ig-repertoire is highly variable, but conserved, showing recurrent B cell receptor (BCR) clonotypes frequently shared between neonates. Our study demonstrates that cord blood B cells are not impaired but differ from their adult counterpart in a conserved BCR repertoire and rapid but transient response dynamics. These properties may account for the sensitivity of neonates to infections and limited effectivity of vaccination strategies. Humanized mice suggest that the distinctness of cord blood versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice. Still, our findings reveal overall limited comparability of human cord blood B cells and murine B-1 cells.Significance StatementNeonates and infants suffer from enhanced susceptibility to infections. Our study contrasts with the current concept of a premature or impaired B cell system in neonates, by showing that most cord blood B cells are mature and functional. However, their responses are rapid but provide only short-term protection, which may help to improve infant vaccination strategies. We propose an altered perspective on the early human B cell system, which looks similar to but functions differently from the adult counterpart. Finally, our analysis indicates that cord blood- and adult B cell development occur layered as in mice, but certain mouse models still may offer a limited view on human neonatal B cell immunity.

Background In cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset, and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations. Methods We developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation. Results Methyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level. Conclusions Methyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.

We have designed the chopper configuration for the future multi-purpose neutron-imaging instrument ODIN at the European Spallation Source (ESS). The configuration is based on the concept of wavelength frame multiplication (WFM) using a pair of optically blind choppers, which results in a constant wavelength resolution over the utilized spectrum. When using the WFM mode, the wavelength resolution can be adjusted in the range 0 . 19 % ≤ Δ λ ∕ λ ≤ 0 . 84 % and the instrument will feature a spectrum in the range 1.0 A ≤ λ ≤ 8 . 7 A where either the full bandwidth or half of it can be accessed. Additionally, a mode utilizing the natural resolution in the range 2 % ≤ Δ λ ∕ λ ≤ 10 % given by the instrument length can be realized by parking the WFM choppers in an open position. After an initial analytical specification of the chopper system, simulations have been performed using the Monte Carlo software package McStas in order to optimize the parameters. In this paper we discuss the proposed WFM chopper concept and present an analytical description of the chopper discs involved. Furthermore, we present simulation results demonstrating the performance of the chopper system, implemented in the neutron guide system that is being realized for the imaging beam line ODIN at the ESS.

Immunoglobulin (IG) gene remodeling by V(D)J recombination plays a central role in the generation of normal B cells, and somatic hypermutation and class switching of IG genes are key processes during antigen-driven B cell differentiation. However, errors of these processes are involved in the development of B cell lymphomas. IG locus-associated translocations of proto-oncogenes are a hallmark of many B cell malignancies. Additional transforming events include inactivating mutations in various tumor suppressor genes and also latent infection of B cells with viruses, such as Epstein-Barr virus. Many B cell lymphomas require B cell antigen receptor expression, and in several instances, chronic antigenic stimulation plays a role in lymphoma development and/or sustaining tumor growth. Often, survival and proliferation signals provided by other cells in the microenvironment are a further critical factor in lymphoma development and pathophysiology. Many B cell malignancies derive from germinal center B cells, most likely because of the high proliferation rate of these cells and the high activity of mutagenic processes.

We performed neutron imaging of ferromagnetic transitions in Ni$_3$Al and HgCr$_2$Se$_4$ crystals. These neutron depolarization measurements revealed bulk magnetic inhomogeneities in the ferromagnetic transition temperature with spatial resolution of about 100~$\mu$m. To obtain such spatial resolution, we employed a novel neutron microscope equipped with Wolter mirrors as a neutron image-forming lens and a focusing neutron guide as a neutron condenser lens. The images of Ni$_3$Al show that the sample does not homogeneously go through the ferromagnetic transition; the improved resolution allowed us to identify a distribution of small grains with slightly off-stoichiometric composition. Additionally, neutron depolarization imaging experiments on the chrome spinel, HgCr$_2$Se$_4$, under pressures up to 15~kbar highlight the advantages of the new technique especially for small samples or sample environments with restricted sample space. The improved spatial resolution enables one to observe domain formation in the sample while decreasing the acquisition time despite having a bulky pressure cell in the beam.

The pathogenesis of T-cell large granular lymphocytic leukemia (T-LGL) is poorly understood, as STAT3 mutations are the only known frequent genetic lesions. Here, we identified non-synonymous alterations in the TNFAIP3 tumor suppressor gene in 3 of 39 T-LGL. In two cases these were somatic mutations, in one case the somatic origin was likely. A further case harbored a SNP that is a known risk allele for autoimmune diseases and B cell lymphomas. Thus, TNFAIP3 mutations represent recurrent genetic lesions in T-LGL that affect about 8% of cases, likely contributing to deregulated NF-κB activity in this leukemia.

Very few B cells in germinal centers (GCs) and extrafollicular (EF) regions of lymph nodes express CD30. Their specific features and relationship to CD30-expressing Hodgkin and Reed/Sternberg (HRS) cells of Hodgkin lymphoma are unclear but highly relevant, because numerous patients with lymphoma are currently treated with an anti-CD30 immunotoxin. We performed a comprehensive analysis of human CD30+ B cells. Phenotypic and IgV gene analyses indicated that CD30+ GC B lymphocytes represent typical GC B cells, and that CD30+ EF B cells are mostly post-GC B cells. The transcriptomes of CD30+ GC and EF B cells largely overlapped, sharing a strong MYC signature, but were strikingly different from conventional GC B cells and memory B and plasma cells, respectively. CD30+ GC B cells represent MYC+ centrocytes redifferentiating into centroblasts; CD30+ EF B cells represent active, proliferating memory B cells. HRS cells shared typical transcriptome patterns with CD30+ B cells, suggesting that they originate from these lymphocytes or acquire their characteristic features during lymphomagenesis. By comparing HRS to normal CD30+ B cells we redefined aberrant and disease-specific features of HRS cells. A remarkable downregulation of genes regulating genomic stability and cytokinesis in HRS cells may explain their genomic instability and multinuclearity.

Most human B cell lymphomas originate from germinal center (GC) B cells. This is partly caused by the high proliferative activity of GC B cells and the remodeling processes acting at the immunoglobulin (Ig) loci of these cells, i.e., somatic hypermutation and class-switching. Mistargeting of these processes can cause chromosomal translocations, and the hypermutation machinery may also target non-Ig genes. As somatic hypermutation is exclusively active in GC B cells, the presence of somatic mutations in rearranged IgV genes is a standard criterium for a GC or post-GC B cell origin of lymphomas. Beyond this, ongoing somatic hypermutation during lymphoma clone expansion indicates that the lymphoma has an active GC B cell differentiation program. The proto-oncogene BCL6 is specifically expressed in GC B cells and also acquires somatic mutations as a physiological by-product of the somatic hypermutation process, albeit at a lower level than IgV genes. Thus, detection of BCL6 mutations is a further genetic trait of a GC experience of a B cell lymphoma. Typically, B cell lymphomas retain key features of their specific cells of origin, including a differentiation stage-specific gene expression pattern. This is at least partly due to genetic lesions, which "freeze" the lymphoma cells at the differentiation stage at which the transformation occurred. Therefore, identification of the normal B cell subset with the most similar gene expression pattern to a particular type of B cell lymphoma has been instrumental to deduce the precise cell of origin of lymphomas.We present here protocols to analyze human B cell lymphomas for a potential origin from GC B cells by determining the presence of mutations in rearranged IgV genes and the BCL6 gene, and by comparing the gene expression pattern of lymphoma cells with those of normal B cell subsets by genechip or RNA-sequencing analysis.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive post-thymic T-cell malignancy characterized by the recurrent inv(14)(q11q32)/t(14;14)(q11;q32) or t(X;14)(q28;q11) leading to activation of either the TCL1 or MTCP1 gene, respectively. However, these primary genetic events are insufficient to drive leukemogenesis. Recently, activating mutations in JAK3 have been identified in other T-cell malignancies. Since JAK3 is essential for T-cell maturation, we analyzed a cohort of 32 T-PLL patients for mutational hot spots in the JAK3 gene using a step-wise screening approach. We identified 14 mutations in 11 of 32 patients (34%). The most frequently detected mutation in our cohort was M511I (seen in 57% of cases) previously described as an activating change in other T-cell malignancies. Three patients carried two mutations in JAK3. In two patients M511I and R657Q were simultaneously detected and in another patient V674F and V678L. In the latter case we could demonstrate that the mutations were on the same allele in cis. Protein modeling and homology analyses of mutations present in other members of the JAK family suggested that these mutations likely activate JAK3, possibly by disrupting the activation loop and the interface between N and C lobes, increasing the accessibility of the catalytic loop. In addition, four of the 21 patients lacking a JAK3 point mutation presented an aberrant karyotype involving the chromosomal band 19p13 harboring the JAK3 locus. The finding of recurrent activating JAK3 mutations in patients with T-PLL could enable the use of JAK3 inhibitors to treat patients with this unfavorable malignancy who otherwise have a very poor prognosis.

Most human B cell lymphomas originate from germinal center (GC) B cells. This is partly caused by the high proliferative activity of GC B cells and the remodeling processes acting at the immunoglobulin (Ig) loci of these cells, i.e., somatic hypermutation and class-switching. Mistargeting of these processes can cause chromosomal translocations, and the hypermutation machinery may also target non-Ig genes. As somatic hypermutation is exclusively active in GC B cells, the presence of somatic mutations in rearranged IgV genes is a standard criterium for a GC or post-GC B cell origin of lymphomas. Beyond this, ongoing somatic hypermutation during lymphoma clone expansion indicates that the lymphoma has an active GC B cell differentiation program. The proto-oncogene BCL6 is specifically expressed in GC B cells and also acquires somatic mutations as a physiological by-product of the somatic hypermutation process, albeit at a lower level than IgV genes. Thus, detection of BCL6 mutations is a further genetic trait of a GC experience of a B cell lymphoma. Typically, B cell lymphomas retain key features of their specific cells of origin, including a differentiation stage-specific gene expression pattern. This is at least partly due to genetic lesions, which "freeze" the lymphoma cells at the differentiation stage at which the transformation occurred. Therefore, identification of the normal B cell subset with the most similar gene expression pattern to a particular type of B cell lymphoma has been instrumental to deduce the precise cell of origin of lymphomas.We present here protocols to analyze human B cell lymphomas for a potential origin from GC B cells by determining the presence of mutations in rearranged IgV genes and the BCL6 gene, and by comparing the gene expression pattern of lymphoma cells with those of normal B cell subsets by genechip or RNA-sequencing analysis.

Array-based DNA methylation analysis in classical Hodgkin lymphoma reveals new insights into the mechanisms underlying silencing of B cell-specific genes

Normal B cells undergo extensive epigenetic programming during normal differentiation and distinct B cell differentiation stages represent unique DNA methylation patterns. Chronic Lymphocytic Leukemia (CLL) originates from rapidly differentiating B cells and their DNA methylation signature is stably propagated in CLL. Consequently, CLL methylome data can be used to infer the putative cell-of-origin (COO) for each individual CLL case. We define the COO of CLL as the cell that has acquired a first oncogenic hit and which will initiate tumorigenic growth if one or more additional hits have been acquired. This means that two factors contribute to the epigenetic profile of CLL cells: first, the epigenetic profile of the founder B cell at the time of malignant transformation and second, CLL-specific epigenetic alterations that are acquired during leukemogenesis and progression of the disease. Previous studies using peripheral blood CD19+ B cells as a reference for aberrant methylation calls completely neglected the massive epigenetic programming that occurs during normal B cell differentiation. Thus, novel strategies aiming at identifying truly CLL-specific methylation changes considering the highly dynamic methylome during normal B cell differentiation were urgently needed. Here we outline a new analytical framework to delineate CLL-specific DNA methylation. We demonstrate how this approach can be applied to detect epigenetically deregulated transcripts in CLL. Firstly, we modeled the epigenome dynamics occurring during normal B cell differentiation using linear regression. The DNA methylomes of CLL cells were then precisely positioned onto the normal B cell differentiation trajectory to define the closest normal B cell methylome for every CLL patient, the COO. The epigenome of the COO then served as a reference for aberrant DNA methylation calls. We dissected two categories of CLL-specific methylation events: those occurring at sites undergoing epigenetic programming during B cell differentiation and those that normally do not change during B cell differentiation. The first group was further subdivided into class A and B, displaying exaggerated methylation loss or gain, respectively, and class C showing both hyper- and hypomethylation relative to the normal differentiation. The second group was classified into class D displaying hypo- and class E showing hypermethylation. Overall, only 1.6% of the CpG-sites (7,248 CpGs) represented on the Illumina 450k array were affected by disease-specific methylation programming, mostly hypomethylation (6,680 CpGs). Next, the molecular programs underlying the CLL-specific methylation patterns were investigated. We tested enrichment of chromatin states and of transcription factor binding sites (TFBS) as identified in an immortalized B cell line (GM12878). This indicated that disease-specific methylation events target transcriptionally relevant cis-regulatory elements in CLL (enhancers, weak and poised promoters and insulator regions). In line with this, CLL-specific differentially methylated regions affected TFBS associated with signaling pathways known to be important in normal B-cell differentiation (i.e. BATF, EBF1). We also observed altered methylation at CTCF binding sites suggesting their involvement in CLL pathogenesis. In the present work, we dissected CLL methylomes to distinguish between normal B cell differentiation-associated methylation patterns and CLL-specific methylation events. We showed that this approach is indispensable to identify key pathogenic events driving CLL pathogenesis. The relevance of our approach was demonstrated by contrasting the number of epigenetically deregulated miRNAs and protein-coding genes to those determined with a classic analysis using CD19+ B cells as controls. This highlights the extent of overcalling of CLL-specific methylation patterns in previous studies (~30-fold for protein-coding genes and ~10-fold for miRNAs) and stresses the importance to consider normal differentiation trajectories for the identification of aberrant DNA methylation events. Here we propose 11 protein-coding genes (e.g. DOK2, CLLU1) and 4 miRNAs (e.g. miR-486, miR-195) as being epigenetically deregulated in CLL. Our analytical approach provides a general framework for the identification of disease-specific epigenomic changes that should be applicable to other cancers in the future. Disclosures Küppers: the Takeda Advisory Board: Membership on an entity's Board of Directors or advisory committees. Stilgenbauer:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype–specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell–like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

The origin of IgM(+)CD27(+) B lymphocytes with mutated IgV genes, which account for approximately 20% of human peripheral blood (PB) B cells, is controversially discussed. A generation in a primary diversification pathway, in T cell-independent immune responses, or in T cell-dependent germinal center (GC) reactions has been proposed. We show here that IgM(+)IgD(+)CD27(+) and IgM(+)IgD(-/low)CD27(+) B cell subsets carry, like class-switched memory B cells, mutations in the Bcl6 gene as a genetic trait of a GC experience. Moreover, the identification of PB IgM(+)IgD(+)CD27(+) B cells clonally related to GC-derived IgG(+) memory B cells with shared and distinct IgV gene mutations demonstrates the GC origin also of the former subset. These findings provide genetic evidence for a GC derivation of somatically mutated IgM(+) B cells and indicate that adult humans harbor a large population of IgM(+)IgD(+) post-GC memory B cells. Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction. This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.

A key feature of the adaptive immune system is the generation of memory B and T cells and long-lived plasma cells, providing protective immunity against recurring infectious agents. Memory B cells are generated in germinal center (GC) reactions in the course of T cell-dependent immune responses and are distinguished from naive B cells by an increased lifespan, faster and stronger response to stimulation and expression of somatically mutated and affinity matured immunoglobulin (Ig) genes. Approximately 40% of human B cells in adults are memory B cells, and several subsets were identified. Besides IgG+ and IgA+ memory B cells, ∼50% of peripheral blood memory B cells express IgM with or without IgD. Further smaller subpopulations have additionally been described. These various subsets share typical memory B cell features, but likely also fulfill distinct functions. IgM memory B cells appear to have the propensity for refined adaptation upon restimulation in additional GC reactions, whereas reactivated IgG B cells rather differentiate directly into plasma cells. The human memory B-cell pool is characterized by (sometimes amazingly large) clonal expansions, often showing extensive intraclonal IgV gene diversity. Moreover, memory B-cell clones are frequently composed of members of various subsets, showing that from a single GC B-cell clone a variety of memory B cells with distinct functions is generated. Thus, the human memory B-cell compartment is highly diverse and flexible. Several B-cell malignancies display features suggesting a derivation from memory B cells. This includes a subset of chronic lymphocytic leukemia, hairy cell leukemia and marginal zone lymphomas. The exposure of memory B cells to oncogenic events during their generation in the GC, the longevity of these B cells and the ease to activate them may be key determinants for their malignant transformation.

Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM(+) and IgG(+) memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM(+)(IgD(+)) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells--including IgM(+)IgD(+)CD27(+) B cells--are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM(+)(IgD(+)) and class-switched memory B cells.

B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1−/− mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1−/− mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses., Antibody responses are regulated by selective survival of B cells with proper antigen specificity. Here the authors show that CEACAM1 is critical for B-cell survival during homeostasis and antiviral responses.

The generation and functions of human peripheral blood (PB) IgM(+)IgD(+)CD27(+) B lymphocytes with somatically mutated IgV genes are controversially discussed. We determined their differential gene expression to naive B cells and to IgM-only and IgG(+) memory B cells. This analysis revealed a high similarity of IgM(+)(IgD(+))CD27(+) and IgG(+) memory B cells but also pointed at distinct functional capacities of both subsets. In vitro analyses revealed a tendency of activated IgM(+)IgD(+)CD27(+) B cells to migrate to B-cell follicles and undergo germinal center (GC) B-cell differentiation, whereas activated IgG(+) memory B cells preferentially showed a plasma cell (PC) fate. This observation was supported by reverse regulation of B-cell lymphoma 6 and PR domain containing 1 and differential BTB and CNC homology 1, basic leucine zipper transcription factor 2 expression. Moreover, IgM(+)IgD(+)CD27(+) B lymphocytes preferentially responded to neutrophil-derived cytokines. Costimulation with catecholamines, carcinoembryonic antigen cell adhesion molecule 8 (CEACAM8), and IFN-γ caused differentiation of IgM(+)IgD(+)CD27(+) B cells into PCs, induced class switching to IgG2, and was reproducible in cocultures with neutrophils. In conclusion, this study substantiates memory B-cell characteristics of human IgM(+)IgD(+)CD27(+) B cells in that they share typical memory B-cell transcription patterns with IgG(+) post-GC B cells and show a faster and more vigorous restimulation potential, a hallmark of immune memory. Moreover, this work reveals a functional plasticity of human IgM memory B cells by showing their propensity to undergo secondary GC reactions upon reactivation, but also by their special role in early inflammation via interaction with immunomodulatory neutrophils.

In 1989 and 1990, spruce cones were harvested at 29 sites distributed over the five main geographic regions of Switzerland, i.e. Jura, Central Plateau, North, Central and South Alps. The presence of insects and the species-spe- cific consumption of seeds was determined for each cone by examination of the median longitudinal section. Seven seed-feeding species (Assara terebrella, Cydia strobilella, Dioryctria abietella, Eupithecia abietaria, Megastigmus strobi- lobius, Plemeliella abietina, Strobilomyia anthracina) and the spruce cone gall midge Kaltenbachiola strobi were found in the samples. The proportions of infested cones ranged from 36 to 100%. The regions did not show significant dif- ferences in terms of infestation rates, whereas differences between sites within the regions were highly significant. Seed loss ranged from 2 to 100%, its geographical distribution exhibiting a similar pattern to the infestation rates. Considering the number of infested cones, C. strobilella was the most abundant species, followed by K. strobi and P. abietina. Cones infested by conospermatophagous species (C. strobilella, D. abietella, E. abietaria) showed higher seed losses than those infested by spermatophages (M. strobilobius, P. abietina). Due to the ubiquitous occurrence of C. strobilella, its high infestation rates and the resulting seed losses caused by its offspring, this species has to be con- sidered as the most important insect seed predator in Switzerland. A multiple linear regression analysis was carried out for each region to simultaneously explore the impact of site and tree variables as well as the effect of insect attack on the yield of viable seeds. The results indicate that site and tree conditions contributed more to the variability of seed yield than insect presence in a cone. The model predicted significant effects of insect attack mainly for cones with total seed numbers that were higher than the average.

The spruce bark beetle Ips typographus (L.) is one of the most important forest pests in Central Europe, but despite this the effects of temperature on life history and population growth are largely unknown. This study examines the effects of temperature on reproduction and intrinsic demographic statistics. 2. Laboratory experiments on oviposition were carried out at six temperatures in the range 12-33 ∞C, using the so-called sandwich rearing technique for bark beetles. 3. A linear relationship between oviposition rates and temperatures in the range 15-25 ∞C was used to estimate the lower temperature threshold for oviposition as 11.4 ∞C. With a nonlinear model fitted to the data across the whole range of experimental temperatures, the lower and upper limiting temperatures and optimum temperature were found to be 7.9, 33.7, and 28.9 ∞C, respectively. A model for daily oviposition rate was fitted, which describes the pattern of oviposition over the entire oviposition period. 4. The analysis of life tables, combining developmental rates, reproduction, mortality, and sex ratio, suggests maximum population growth (rm) at near 30 ∞C. 5. After generating a first brood, spruce bark beetles often re-emerge from the tree and produce other sister broods. The effects of temperature and number of sister broods on demography were evaluated using age-specific life-table analyses. It is hypothesized that sister broods play an important role in regions where I. typographus is monovoltine, but have only moderate significance where this species has more than one generation per season.

Following individual development by means of the ‘sandwich method’ the duration of egg, larval, and pupal stages, as well as of adult maturation feeding of the spruce bark beetle (Ips typographus) was measured at constant temperatures in the range between 12° and 33° C. At 20° C complete development from egg to adult emerging from pupa averaged 29 days. The proportion of the duration of larval development relative to total preimaginal development increased with temperature. Developmental rates, i.e. the speed of development increased linearly with temperature in a range between 15° and 25°C. Based on linear regressions, lower developmental thresholds were calculated to be 10.6°C (eggs), 8.2°C (larvae), 9.9°C (pupae), and 8.3°C (preimaginal development egg to pupa), respectively. Differing heat sums reported in the literature matched ours when recalculated with our developmental thresholds. A nonlinear model (Logan/Lactin) was fitted to the data which allowed to describe development in the entire temperature range. It further permits to identify lower and upper (≅ 40°C) developmental thresholds as well as optimum temperatures (30–33°C) of the instars.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive post-thymic T-cell malignancy characterized by the recurrent inv(14)(q11q32)/t(14;14)(q11;q32) or t(X;14)(q28;q11) leading to activation of either the TCL1 or MTCP1 gene, respectively. However, these primary genetic events are insufficient to drive leukemogenesis. Recently, activating mutations in JAK3 have been identified in other T-cell malignancies. Since JAK3 is essential for T-cell maturation, we analyzed a cohort of 32 T-PLL patients for mutational hot spots in the JAK3 gene using a step-wise screening approach. We identified 14 mutations in 11 of 32 patients (34%). The most frequently detected mutation in our cohort was M511I (seen in 57% of cases) previously described as an activating change in other T-cell malignancies. Three patients carried two mutations in JAK3. In two patients M511I and R657Q were simultaneously detected and in another patient V674F and V678L. In the latter case we could demonstrate that the mutations were on the same allele in cis. Protein modeling and homology analyses of mutations present in other members of the JAK family suggested that these mutations likely activate JAK3, possibly by disrupting the activation loop and the interface between N and C lobes, increasing the accessibility of the catalytic loop. In addition, four of the 21 patients lacking a JAK3 point mutation presented an aberrant karyotype involving the chromosomal band 19p13 harboring the JAK3 locus. The finding of recurrent activating JAK3 mutations in patients with T-PLL could enable the use of JAK3 inhibitors to treat patients with this unfavorable malignancy who otherwise have a very poor prognosis.

Unmutated CLL derives from unmutated mature CD5+ B cells and mutated CLL derives from CD5+CD27+ post–germinal center B cells., The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5+ B cells and mutated CLL derives from a distinct, previously unrecognized CD5+CD27+ post–germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5+ B cells, providing independent evidence for a CD5+ B cell derivation of CLL. Notably, these CD5+ B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.

In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length (ΔTL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p = 0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r = 0.509, p < 0.001). With respect to prognosis, a threshold ΔTL of −4.2 kb was the best predictor for progression-free and overall survival. ΔTL was not significantly altered over time or with therapy. The correlation between the mutational load in IGHV genes and the ΔTL in CLL might reflect the initial telomere length of the putative cell of origin (pre- versus post-germinal center B cells). In conclusion, the ΔTL is a reliable prognostic marker for patients with CLL. Short telomeres and high telomerase activity as occurs in some patients with CLL with a worse prognosis might be an ideal target for treatment with telomerase inhibitors.

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell–specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non–B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Background: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of geneassociated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.

Human peripheral blood (PB) B cells expressing only IgD and tonsillar IgD-secreting plasma cells carry highly mutated V(H) genes and show preferential Iglambda usage. To further characterize these peculiar cells and gain insight into their generation, we analysed rearranged V(H) and V(L) genes of single IgD-only lambda(+) PB B cells and IgD(+) plasma cells from four individuals each. We demonstrate that the high somatic hypermutation activity in these cells is not restricted to V(H) genes but also present in V(L) genes. Moreover, not only PB IgD-only B cells, as reported earlier, but also IgD-expressing plasma cells often belong to very large clones. Surprisingly, the V(H)3-30 gene segment was used in each PB donor by >30% of IgD-only cells and in 2 tonsils by >50% of IgD plasma cells, whereas it was used less frequent in other B cells. All these features fit to a model in which IgD-only cells develop in superantigen-driven germinal center reactions, in which B cells are activated by binding of antigens to constant parts of Cdelta and often lambda light chains and the V(H)3-30 segment, and are selected for deletion of Cmu. IgD-only B cells may hence represent a unique B lineage subset generated in response to particular antigens.

The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner. It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes. Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL. Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.

Knowledge of the cell-of-origin is essential for the full understanding of the causes of a malignant disease and for the rational design of targeted therapies. The B cell compartment is composed of a highly complex mixture of subtypes, each with distinct phenotypes and roles within the immune system. In chronic lymphocytic leukemia (CLL), heterogeneity in the biology and clinical course of the disease is thought to be linked to divergent cellular origins. We and others have previously shown that the epigenome of CLL, as measured by the global pattering of DNA methylation, is highly clonal and remarkably stable over time and thus represents a powerful approach to trace founder subtype populations. Here we combine epigenomic and transcriptomic analysis using next-generation sequencing approaches to compare CLL cells to highly purified and specific B cell subpopulations at various stages of maturation. We find that B cell maturation involves substantial unidirectional epigenetic programming that occurs as a continuum throughout the transition between naïve to fully-mature memory B cell subpopulations. Combining 258 CLL cases using Illumina 450K analysis reveals that all CLLs arise from a discrete window within the spectrum of B cell maturation that is more similar to mature B cells, with the majority of cases clustering at two distinct points correlating with unmutated IGHV versus highly mutated ( Citation Format: Christopher C. Oakes, Marc Seifert, Assenov Yassen, Lei Gu, Martina Przekopowitz, Amy Ruppert, Andrius Serva, Sandra Koser, David Brocks, Daniel Lipka, Olga Bogatyrova, Daniel Mertens, Marc Zapatka, Peter Lichter, Hartmut Doehner, Ralf Kueppers, Thorsten Zenz, Stephan Stilgenbauer, John Byrd, Christoph Plass. Progressive epigenetic programming during B cell maturation yields a continuum of clonal disease phenotypes with distinct etiologies in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B25.