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 in the germinal center reaction. 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. Most 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. Most B cell malignancies derive from germinal center B cells, most likely due to the high proliferative activity of these B cells and aberrant mutations caused by their naturally active mutagenic processes.

Background Rare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging. Methods To address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes. Results We found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities. Conclusions Altogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers.

The development of a fatal, clonal, autonomously proliferating CD4-CD8- chimeric antigen receptor (CAR)+ peripheral T-cell lymphoma (PTCL) occurred 1 month after a patient received treatment with tisagenlecleucel for relapsed primary central nervous system lymphoma. The PTCL had a clonal T-cell receptor rearrangement, which was already detectable in the apheresis product for CAR T-cell manufacturing and 7 months earlier for autologous transplantation. Somatic DNMT3A and TET2 mutations in CD34+ stem cells and their progeny were detected in the PTCL, in the apheresis specimen that was obtained for CAR T-cell production, and in the autotransplant. The PTCL harbored an additional somatic TET2 mutation, which was already detectable in the CAR T-cell apheresis product and the final CAR T-cell product at very low frequencies, providing evidence that clonal hematopoiesis had contributed to lymphomagenesis. Shortly after a patient received CAR T-cell therapy for relapsed CNS lymphoma, an aggressive CAR+ T-cell lymphoma was diagnosed. Subsequent analysis revealed the presence of an abnormal clone in an early apheresis specimen.

The redirection of T cells has emerged as an attractive therapeutic principle in B cell non-Hodgkin lymphoma (B-NHL). However, a detailed characterization of lymphoma-infiltrating T cells across B-NHL entities is missing. Here we present an in-depth T cell reference map of nodal B-NHL, based on cellular indexing of transcriptomes and epitopes, T cell receptor sequencing, flow cytometry and multiplexed immunofluorescence applied to 101 lymph nodes from patients with diffuse large B cell, mantle cell, follicular or marginal zone lymphoma, and from healthy controls. This multimodal resource revealed quantitative and spatial aberrations of the T cell microenvironment across and within B-NHL entities. Quantitative differences in PD1 + TCF7 − cytotoxic T cells, T follicular helper cells or IKZF3 + regulatory T cells were linked to their clonal expansion. The abundance of PD1 + TCF7 − cytotoxic T cells was associated with poor survival. Our study portrays lymphoma-infiltrating T cells with unprecedented comprehensiveness and provides a unique resource for the investigation of lymphoma biology and prognosis.

The epiCMIT (epigenetically determined Cumulative MIToses) mitotic clock traces B-cell mitotic history via DNA methylation changes in heterochromatin and H3K27me3-containing chromatin. While high scores correlated with poor outcomes in CLL and MCL, its prognostic significance in SMZL remains unknown. Derived from 142 SMZL cases using DNA methylation microarrays, epiCMIT values were correlated with genomic, transcriptomic, and clinical data. EpiCMIT as a continuous variable was significantly higher in females (p=0.02), patients with IGHV1-2*04 allele usage (p<0001), intermediate IGHV somatic hypermutation load (97-99.9% identity, p=0.04), elevated mutational burden (25 vs. 17 mut/Mb, p=0.001), driver gene mutations [KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01), KMT2D (p<0.001)], and del(7q) (p=0.01). Negative correlation between epiCMIT and telomere length (r=-0.29 p<0.001) supported the association between cumulated proliferation and telomere attrition. While univariate analysis highlighted epiCMIT as robust predictor of shorter treatment-free survival (TFS), multivariate analysis confirmed epiCMIT as an independent marker for shorter TFS. In summary, our matched multi-omic datasets facilitate the clinico-biological characterization of SMZL and introduces epiCMIT as a strong prognostic marker, identifying high-risk patients and predicting reduced treatment-free survival, hence providing a new tool for risk-adapted patient management.### Competing Interest StatementR.R. had received honoraria from AbbVie, AstraZeneca, Illumina, Janssen and Roche. L.S. had received consultancy from AbbVie, AstraZeneca, BeiGene, Janssen and Lilly. P.G had received honoraria , consultancy and research funding from AbbVie, AstraZeneca, BeiGene, BMS, Lilly, Janssen, MSD and Roche. D.R. had received honoraria and research funding from AbbVie, AstraZeneca, BeiGene, BMS, Gilead, Janssen, Lilly and Kyte. M.A had received research funding from Pfizer. F.F had received honoraria from AbbVie, AstraZeneca, BeiGene and Janssen. R.W had received honoraria from AbbVie, AstraZeneca, BeiGene, Janssen and Secura bio. C.T. had received honoraria and/or consultancy from BMS, Janssen, Novartis, Roche, Abbvie, Gilead, Hospira, Amgen, Cellectis, Kyte, Takeda, Incyte, Bayer. All other authors have declared no competing interests.### Funding StatementThe study was funded by research grants from the Kay Kendall Leukemia Fund (873, 1104), and Cancer Research UK (ECRIN-M3 accelerator award C42023/A29370). MDF is supported by a postdoctoral grant from the Spanish Association Against Cancer. R.R is supported by grants from the Swedish Cancer Society, the Swedish Research Council, Region Stockholm, and Radiumhemmets Forskningsfonder, Stockholm. ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:NHS South Central- Hampshire B Research ethics committee granted ethical approval for this workI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Intratumor heterogeneity is intrinsic to cancer evolution and treatment resistance, although it is typically considered distinct from the differentiation processes driving cell-type and cancer-type diversity. Nodal B cell non-Hodgkin lymphomas are tied to distinct stages of B cell maturation. Through a single-cell multi-omic and spatial atlas of diffuse large B cell, mantle cell, follicular, and marginal zone lymphomas in addition to reactive lymph nodes in 51 patients, we uncovered co-existing B cell maturation states within the same tumors. These intratumor maturation states emerged from the same clone, revealing ongoing differentiation from the cell-of-origin. Maturation state composition varied across and within disease entities, with tumors encompassing mixed cell-of-origin clinical subtypes. Intratumor maturation states inhabited unique spatial niches, maintaining their maturation-associated cellular interactions and regulatory networks while harboring distinct genetic variant expression patterns. Our findings show that cell-type differentiation remains plastic in cancer and is central to tumor variation and evolution. ![Figure][1] ### Competing Interest Statement G.P.N. is a co-founder and stockholder of Akoya Biosciences, Inc. and inventor on patent US9909167 (On-slide staining by primer extension). [1]: pending:yes

Introduction: PAP is due to accumulation of phospholipids and lipoproteins in the alveoli. WLL is still the treatment of choice. Light microscopy examination of WLL fluid reveals a moderate to high lymphocytosis but lymphocyte subpopulations have not been further characterized at yet. Aims: To investigate T lymphocyte subsets in WLL fluid from patients with PAP. Methods: From the first recovered fluid portion during WLL, samples of 50 mL each were collected and analyzed fresh. Pancoll density-gradient centrifugation was performed to separate mononuclear (MN) cells. MACS® enrichment was used to separate T lymphocytes from remaining MN cells and acellular particles after adding anti-CD3 microbeads to the MN cell layer. Flow cytometry for T cell immunophenotyping was performed by using a 9-color antibody panel and a FACSAria™ Fusion cytometer (Becton Dickinson, Germany). Zombie UV™ fluorescent dye was used to assess cell viability. T lymphocyte counts were expressed as proportion of CD3+ viable lymphocyte counts. Results: WLL fluid from 3 consecutive PAP patients (2 M, 1 F, age 44±12) who underwent therapeutic WLL was analyzed. The mean T lymphocyte count was 87±1%. CD4+ T cells were the dominant T cell subset across all samples (73±7%), followed by CD8+ cytotoxic T cells (19±7%). Th17.1 cells were the most frequent Th cell subset (33±14%), followed by Th1 cells (29±2%), Tregs (17±6%), Th17 cells (4±2%), and Th2 cells (0.7±0.2%). Conclusions: Our study identified different T lymphocyte subsets, mostly related to autoimmunity, in WLL fluid obtained from PAP patients. Flow cytometry may be used to unravel pathogenetic mechanisms of this rare disease.

Improved classification of rare lymphoid neoplasms would be aided by a deeper understanding of their underlying molecular features and is important for diagnosis, prognosis and therapy. Tumor entities classified within the WHO category of splenic B cell lymphomas and leukemias often exhibit heterogenous, transecting features, and include hairy cell leukemia (HCL), splenic diffuse red pulp lymphoma (SDRPL), splenic marginal zone lymphoma (SMZL), and the newly described WHO entity, splenic B cell lymphoma/leukemia with prominent nucleoli (SBLPN); the latter including patients formerly classified as HCL-variant (HCL-V). Genome-wide epigenetic information provides a tumor cell fingerprint combining cell-of-origin and tumor-specific events. Here we used DNA methylation to perform an unbiased molecular subclassification and to explore novel biological aspects of these patients. Samples from patients with a pathological diagnosis of HCL, HCL-V, SDRPL and SMZL (made prior to the 5 th WHO revision and ICC classifications) were obtained from 19 institutions across 9 countries, totaling 367 patients. Cells were FACS-purified where necessary and DNA was analyzed by 450/850K Illumina DNA methylation arrays. Genetic mutations were assessed by whole-exome or targeted sequencing, IGHV-D-J sequences by Sanger sequencing, and copy number alterations (CNAs) by Illumina arrays. The 1000 most variable CpG methylation sites were used for k-means clustering. Recursive feature elimination/random forest algorithms were used to develop a classifier for DNA methylation-based subgroups with 98% accuracy. Unsupervised clustering of 197 patients diagnosed with HCL, HCL-V or SDRPL revealed 5 distinct DNA methylation (M) subgroups ( Figure 1). Subgroup assignment was stable throughout longitudinal sampling (including pre/post-treatment) and consistent between splenic, bone marrow and PBMC derived cells. A subgroup with universally clonal BRAF-V600E mutations and majority diagnosed as HCL was termed the M-HCL subgroup ( Table 1). Four other groups termed M-SBLPN1-4 contained all HCL-V and SDRPL diagnosed samples and were devoid of BRAF-V600E mutations. M-SBLPN1 comprised MAP2K1 mutations (91%) and was enriched for CREBBP, ARIDIA and TERT-promoter mutations. These patients displayed an HCL-like immunophenotype (64.3% CD25+) with 1/3 diagnosed as HCL. M-SBLPN2 exhibited the highest prevalence of TP53 mutations and concomitant genomic instability. Patients in M-SBLPN1,2 were enriched in unmutated IGHV4-34 rearrangements. M-SBLPN3,4 subgroups displayed an immunophenotype more dissimilar to HCL, mutated IGHV genes, and enrichment of IGLL5, SYK and BIRC3 mutations. M-SBLPN4 contained the most SDRPL samples, suggesting it may represent the SDRPL entity retained by the WHO. We next uncovered that 29/170 SMZL patients displayed DNA methylation patterns mapping to M-SBLPN2-4. These patients were phenotypically and molecularly similar to SBLPN (70% displaying villous morphology and depleted in IGHV1-2*04, NOTCH2, KLF2 mutations), likely representing SMZL patients suggested for reassignment to SBLPN in the updated WHO classification. To elucidate molecular pathways governing the biology of M-SBLPN subgroups, transcription factor motif enrichment analysis in hypomethylated genomic regions revealed selective activation of AP-1 in M-HCL along with ETS in M-SBLPN1,2. Both transcription factors are downstream of MAPK signaling, consistent with activating BRAF and MAP2K1 mutations in these subgroups. However, we observed strong ETS enrichment in the absence of MAP2K1 in M-SBLPN along with mutual exclusivity of MAP2K1 and TP53 mutations, suggesting TP53 mutations are driving ETS activation. Although lymphoid neoplasms rarely exhibit TERT promoter mutations, 83% of M-SBLPN1 patients showed the c.-124C>T mutation commonly observed in other cancers producing an ETS binding site and ectopic TERT activation. ETS activation and gain of an ETS site by mutation implies oncogenesis involves aberrant TERT activation in this subgroup. In summary, we have developed a DNA methylation-based classifier that resolves 4 SBLPN subgroups with distinct molecular features, and reclassifies a subset of SMZL and HCL patients, adding further information to the updated WHO/ICC entities. We reveal distinct biological pathways operating in M-SBLPN subgroups that may aid targeted therapy approaches.

Background Rare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging. Methods To address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3,760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes. Results We found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities. Conclusions Altogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers. ### Competing Interest Statement T.H. declares part ownership of Munich Leukemia Laboratory (MLL). Sa.H. and St.H. are employed by the MLL. ### Funding Statement This study was supported by the German Bundesministerium fuer Bildung und Forschung (BMBF) supported through the VALE (Entdeckung und Vorhersage der Wirkung von genetischen Varianten durch Artifizielle Intelligenz fuer LEukaemie Diagnose und Subtypidentifizierung) project [031L0203B to X.Q., J.G.; 031L0203C to Sa.H., St.H.; and 031L0203A to S.V., M.H.]. This work was funded via the EVUK programme ("Next-generation Al for Integrated Diagnostics") of the Free State of Bavaria [to A.J.A. and J.G.]. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All patients or their legal guardians gave written informed consent for genetic analyses and to the use of laboratory results and clinical data for research purposes according to the Declaration of Helsinki. The study was approved by the MLL's institutional review board. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the corresponding authors. * CGC : Cancer Gene Census LRP1B : Low-Density Lipoprotein Receptor-related protein 1B MLL : Munich Leukemia Laboratory NB-act : Negative Binomial activation RNA-Seq : total RNA sequencing VEP : Ensembl variant effect predictor WGS : Whole Genome Sequencing

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eμ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.

INTRODUCTION:Naturally occurring autoantibodies (nAbs) against the pathologic isoform of amyloid beta (Aβ42 ) were found in body fluids and indicate a systemic B cell response that may prevent Alzheimer's disease (AD) onset. N-glycans attached to immunoglobulin G-Fab/Fc fragments are features that influence their mechanism of action. The aim was to study the role of N-glycans in nAbs-Aβ42 . METHODS:nAbs-Aβ42 were isolated from AD patients and age-/sex-matched controls (n = 40) and immunoglobulin preparations. Glycosylated/deglycosylated nAbs-Aβ42 were analyzed for their effect on Aβ42 's aggregation, toxicity, and phagocytosis. Glycan structure was analyzed using matrix assisted laser desorption ionization time of flight mass spectrometry. RESULTS:Deglycosylation of nAbs-Aβ42 had a major impact on Aβ42 's aggregation/toxicity/phagocytosis. The glycan structure showed considerable differences between AD and controls. We were able to predict disease status with a sensitivity/specificity of 95% (confidence interval [CI]: 76.4-99.7%)/100% (CI: 83.9-100%). DISCUSSION:N-glycosylation has been identified as a critical attribute maintaining the beneficial effects of autoreactive Aβ antibodies. These data have consequences for the development of monocloncal Aβ antibodies and may open new avenues for diagnostics.

In this review, we will summarize the growing body of knowledge on the age-related changes of human splenic B cell composition and molecular evidence of immune maturation and discuss the contribution of these changes on splenic protective function. From birth on, the splenic marginal zone (sMZ) contains a specialized B cell subpopulation, which recruits and archives memory B cells from immune responses throughout the organism. The quality of sMZ B cell responses is augmented by germinal center (GC)-dependent maturation of memory B cells during childhood, however, in old age, these mechanisms likely contribute to waning of splenic protective function.

Intratumor heterogeneity (ITH) refers to the coexistence of distinct cancer cell subpopulations within a single tumor, each with unique molecular and functional properties. Understanding the dynamics and evolution of ITH is crucial for predicting tumor progression and the development of therapy resistance. Here, we conducted a comprehensive investigation of ITH in Chronic Lymphocytic Leukemia (CLL) and identified a subpopulation that carried the CLL B-cell receptor rearrangement but contrasted from the CLL main population by low CD5 and high CD20 expression and highly mutated Ig-genes. This CLL-related memory B-cell population shared somatic driver mutations with the main CLL population but also exhibited exclusive somatic mutations. Phylogenetic analysis suggested a pathogenically early generation of these CLL-related B cells before monoclonal B lymphocytosis or CLL manifestation. Our data indicated that CLL-related B cells have self-replenishing potential, as they diminish upon treatment but recover indistinguishably on relapse. This contrasts with the main CLL population, which mostly represents a selective and novel expansion of the CLL-related B cells. This differentiation capacity into conventional CLL cells, and the expression of leukemic stem cell signatures further supported their tumorigenic capacity. We propose that these CLL-related B cells represent a pool of highly diversified, early-stage CLL precursor cells, which persist in the shape of “malignant memory B cells”. Longitudinal analyses of these CLL precursor cells suggested that they form a reservoir of malignant, leukemia-originating cells which contribute during disease progression to CLL outgrowth and clonal evolution. ### Competing Interest Statement The authors have declared no competing interest.

Topic: 20. Lymphoma Biology & Translational Research Background: Intratumor heterogeneity (ITH) is increasingly recognized as an essential factor in the variability of B cell non-Hodgkin lymphoma (B-NHL) patient outcomes. It is well established that distinct nodal B cell non-Hodgkin lymphoma (NBNHL) entities originate from different stages of the humoral affinity maturation process, but the drivers of ITH are not well understood. Aims: In this study, we aimed to characterize ITH in B-NHL and investigate its relationship with the nodal B cell affinity maturation process. Methods: 51 diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and non-malignant reactive lymph node (rLN) samples from distinct patients were sequenced with CITE-Seq (coupling single-cell RNA-seq with surface epitope profiling). Transcription factor activities and copy number variants (validated against targeted DNA sequencing) were inferred computationally from gene expression, while B cell receptor clones were determined with VDJ profiling. Spatial relationships were determined with highly multiplexed immunochemistry (CODEX). Informed by gene expression signatures from RNA sequencing of FACS-identified nodal B cell maturation states, a CITE-Seq reference map was constructed in reactive lymph nodes. This reference map was used to classify maturation states in the malignant samples with logistic regression, which were verified and refined by their maturation marker profiles. Using the shared features between CITE-Seq and CODEX panels, maturation states were similarly classified in the spatial data and their microenvironments were characterized with cellular neighborhood analysis. (Fig A) Results: As in the non-malignant context, variability in maturation states was also observed among malignant cells in most NBNHL tumors. BCR-sequencing and light chain restriction confirmed that different intra-tumor maturation states observed in samples were monoclonal, and therefore arose from the same cell of origin. Although commonalities in composition existed between samples within entities, such as an abundance of germinal center (GC) states in DLBCL and memory states in MZL, we observed high variability in maturation state proportions between samples. For example, ABC DLBCL and FL tumors frequently contained both GC and post-GC subpopulations. Transcription factor mediators of the B cell maturation process remained active in malignancy. Copy number variation was observed between maturation states of the same tumor, indicating genetic variants can arise at sequential stages of the B cell maturation process in existent malignancy. Intratumor maturation states typically occupied distinct spatial niches. Although nodal architecture disruption and microenvironmental variation were observed across tumors, tumor cells often maintained proximity with follicular dendritic and follicular helper T cells, key mediators of the affinity maturation process. Summary/Conclusion: Our findings reveal that B cell maturation is not halted during malignancy, but remains a continuous and divergent process central to inter- and intratumor variation. Intratumor maturation states can be subject to genetic variation and occupy distinct spatial niches while retaining the cellular interactions and signals which enable the maturation process. (Fig B)Keywords: B cell development, B cell lymphoma, B cell subsets, OMICS

Young children and older adults suffer from enhanced susceptibility to infections with blood-borne pathogens. An essential step towards immunity is the establishment of a splenic marginal zone (sMZ), which is immature at below 2 years of age. At approximately 5 years of age, an adult level of protection is reached but wanes again in older adults. Although the infant sMZ is thought to contain mostly naïve B cells, memory B cells are recruited to and recirculate from the sMZ throughout life, and class-switched sMZ B cells dominate in older adults. For a better resolution of naïve versus memory B-cell subset accumulation in the sMZ, we performed a single cell-based gene expression analysis of (CD21highIgMhigh) sMZ B cells among five healthy donors (age 3 to 48 years) and validated the sMZ B-cell subset composition by flow cytometry of 147 spleen biopsies (age 0 to 82 years). We identified a major sMZ B-cell subpopulation, which is abundant at birth but decreases with age. These cells lack CD27 expression but carry a weak-to-intermediate memory B-cell signature. These CD27neg sMZ B cells are either IGHV-unmutated or carry only a few IGHV mutations early in life but show average memory B-cell IGHV mutation frequencies (>3%) in adults. The activation and proliferation potential of CD27neg sMZ B cells is significantly above that of non-sMZ B cells already in children. Our study suggests that the human sMZ B-cell pool changes with age, encompassing a major population of lowly Ig-mutated CD27neg but antigen-experienced B cells early in life.

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.

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.

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.

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.

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.