Primary B-cells Research Articles

Abstract 2260: Transcriptomic immuno-oncology investigation of B-cell lymphomas using a novel comparative workflow

Abstract Patients with B-cell lymphoma currently have limited treatment options, often with serious side effects; for example, B-cell lymphoma patients undergoing chemo- and radiation therapies experience poor health from off-target toxicity. Similarly, though CD19 CAR T-cell therapy is very effective, patients suffer lifelong B-cell aplasia as a side effect. Comparing a cancer to an autoimmune disease in the same tissue type may grant us additional insights on the immune dysregulation of the cancer, leading to development of more immune-aware cancer treatments. We introduce a new data-driven comparative workflow for the analysis of immuno-oncology attributes by comparing a cancer and an autoimmune disease in the same tissue type. As an example use case, we compare primary B-cell lymphomas to primary B-cells from systemic lupus erythematosus with the goal of delineating new mechanisms that can be leveraged to treat B-cell lymphomas. Our analysis will utilize bulk RNA-sequencing (RNA-seq) of B-cells for initial discovery, followed by single-cell RNA-sequencing (scRNA-seq) of Peripheral Blood Mononuclear Cells (PBMCs) for validation. Our analytical framework incorporates differential gene analysis, GO Terms, known protein-protein interactions, principal component analysis, and machine learning for biomarker detection. Here, we describe our novel tool in detail and share the results of our lymphoma use case to gain a better understanding of the targetable immune-related attributes of B-cell lymphoma. We expect that this novel tool, along with the results of our B-cell lymphoma use case, will contribute to the continued development of immune-focused treatment options for B-cell lymphoma and other cancers. Citation Format: Naomi Rapier-Sharman, Michael T. Told, Sehi Kim, Kim L. O'Neill, Brett E. Pickett. Transcriptomic immuno-oncology investigation of B-cell lymphomas using a novel comparative workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2260.

Abstract 7357: Comparative transcriptomic analysis reveals novel insights into targetable B-cell lymphoma mechanisms

Abstract B-cell lymphoma is a severe type of lymphatic cancer that is often misdiagnosed in adolescents. While some cases of B-cell lymphoma respond well to chemo- and radiation therapy, there are severe side effects, and many cases are refractory and fail to achieve remission. On the other hand, though CD19 CAR T-cell therapy is highly effective at treating B-cell lymphoma, its association with long-term immunocompromisation justifies the exploration of alternative treatments. Comparing B-cell lymphomas to an autoimmune disease that affects the same tissue type may grant us additional insights into immuno-oncology related treatments for B-cell lymphoma. We have developed a novel tool to compare cancer and autoimmune disease by analyzing bulk RNA-sequencing (RNA-seq) data. This algorithmic approach uses the transcriptional profile to improve our understanding of B-cell specific mechanisms that can be targeted in B-cell lymphoma. Using this tool, we compared the transcriptional signatures of primary B-cell lymphomas with primary B-cells from patients with systemic lupus erythematosus, an autoimmune disease. We identified specific genes and enriched functions associated with B-cell lymphoma’s immune signature. We expect that the results of our B-cell lymphoma analysis could be used to develop beneficial treatments alternative to current therapies, potentially mitigating side effects for B-cell lymphoma patients and increasing their quality of life. Citation Format: Sehi Kim, Naomi Rapier-Sharman, Michael T. Told, Kim L. O’Neill, Brett E. Pickett. Comparative transcriptomic analysis reveals novel insights into targetable B-cell lymphoma mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7357.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

Genome-Wide Mapping of R-Loops in Single Cells Reveals Cell-Type Specific Roles for R-Loops in Hematopoietic Cell Identity

R-loops are three-stranded structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops play critical roles in gene regulation and pathologic roles in DNA damage response. Current methods to map R-loops require at least 500,000 cells. Consequently, R-loop characterization in primary cells is limited. Furthermore, there are no genomic tools to identify R-loops in single cells. We therefore developed here methods to map genome-wide R-loops at single cell resolution. R-loop mapping in primary stem and mature hematopoietic cell populations, and individual single cells revealed striking cell-type specific R-loops. Current methods to map R-loops utilize immunoprecipitation with a monoclonal antibody (clone “S9.6”) or catalytically inactive, but R-loop binding competent, RNAseH1. Motivated by success mapping chromatin modifications in single cells through fusion of Tn5 transposase to a nanobody as well as insertion of T7 promoters to amplify DNA at sites of transposition, we combined both techniques to identify R-loops in limited cell numbers, down to the single cell level. We used a nanobody-Tn5 fusion to identify S9.6 and developed an orthogonal recombinant fusion protein of enzymatic dead RNAseH1 fused to Tn5 (Figure A). We first applied our nanobody-Tn5 method to identify R-loops bound by S9.6 in mouse lineage-negative c-Kit + bone marrow cells in biological duplicates of increasingly reduced cell numbers (64,000, 32,000, 16,000, and 8,000 cells). This revealed remarkable reproducibility of R-loop detection and characteristics. R-loops identified were sensitive to RNAseH1 and enriched at sites with G/C skew and mitochondrial DNA (a site with abundant physiologic R-loops), suggesting authentic, bona fide R-loops mapped in as few 8,000 cells. We next evaluated R-loops across FACS-isolated hematopoietic stem and mature cells from mouse and human bone marrow. Mapping of R-loops in 10,000 cells in duplicate revealed reproducible R-loops which were characteristic of each cell population. Hierarchical clustering of R-loop profiles delineated mature from stem and progenitor cells and discovered increased abundance of genic R-loops in hematopoietic stem and progenitors relative to mature cells (Figure B). Application of our enzymatic dead RNaseH1/Tn5 fusion protein to CD34 + cells revealed a similar genome-wide distribution of R-loops as with the S9.6/nanobody-Tn5 method. There was high correlation of long- and short-term HSC-specific R-loops with sites characteristic of active transcription (based on H3K4me1/2/3 as well as H3K27Ac). Transcription factor motifs activated in unique hematopoietic cell types were enriched at R-loops within the same cell types. For example, GATA motif usage was enriched at R-loops in CMP, MEP, and erythroid precursors. We also detected massive increases in R-loop abundance at the immunoglobulin heavy chain locus in primary B-cells stimulated to undergo class switch recombination. This provides further proof of our low input method to identify R-loops, as physiologic R-loops are known to occur at this locus to promote class switch recombination. Despite recent advances in single cell genomics there are no single cell maps of R-loops. We therefore profiled R-loops using our nanobody-Tn5, low-input S9.6 CUT&Tag approach in individual human CD34 + and mouse c-Kit + cells. We obtained 1,579 single-cell profiles from human and 1,716 from mouse. Uniform manifold approximation and projection (UMAP) identified 12 clusters in human and 9 mouse hematopoietic clusters. Aggregated single-cell R-loop profiles closely resembled bulk profiles indicating that single-cell data captured features of multi-cell maps. Pseudotime analyses of R-loops inferred LT-HSCs as the shared root of hematopoiesis in both mouse and human and identified seven terminal states without prior knowledge of the number of branches in the hematopoietic development. These data provide the first maps of R-loops in primary normal hematopoietic cells and identify scheduled, physiologic cell-type specific R-loops which have not previously been uncovered. The atlas of R-loops across hematopoiesis generated here will motivate future studies to understand the roles of R-loops in hematopoietic differentiation. Moreover, the methods presented map R-loop abundance at nucleotide resolution in primary and limited cell types which can be used for wide-reaching applications.

In silico Approach of Some Selected Manuka Honey Constituents as Epstein–Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) Inhibitors

Introduction: Epstein–Barr virus (EBV) is a carcinogenic cofactor for some epithelial and lymphoid cell malignancies. EBV is associated with most endemic forms of Burkitt lymphoma and nasopharyngeal carcinoma (NPC). In cell proliferation, the maintenance of the latent genome virus depends on the function of the Epstein–Barr Nuclear Antigen 1 (EBNA1) protein. EBNA1 is essential for the survival of primary B-lymphocytes after EBV infection. Manuka honey, black honey from the manuka tree, is in the spotlight for its biological composition and antiviral activity, namely flavonoids and polyphenolic components that provide antimicrobial, antiviral, and antioxidant effects. This research is an alternative therapy for NPC that causes by EBV infection. Methods: A molecular docking approach was used to evaluate the activity of 17 active manuka honeybee product compounds for the ability to inhibit the EBNA1-EBV using the Chimera 1.16 program, SPHGEN program, SPHERE_SELECTOR program, SHOWBOX program, GRID program, and ANTECHAMBER program. Results: All 17 of the ligands demonstrated good binding affinity with the receptor in different ways. Three compounds had a strong binding affinity with a good grid score and may inhibit the EBNA1-EBV and replication of the virus. Conclusion: Leptosin has potential activity as an EBNA1 inhibitor candidate compound that may have potential for treatment of EBV latent infection. This is supported by a gridscore of −61.49 kcal/mol, which is close to the drug candidate (VK-0497), which has a gridscore of −63.32 kcal/mol.

Medium supplementation with human, but not fetal calf serum facilitates endocytosis of PLGA nanoparticles by human primary B-lymphocytes via complement opsonization

The “biological identity” of nanoparticles (NPs) is governed by a shell consisting of various biomolecules that is formed upon exposure to biological media, the so-called biomolecule corona. Consequently, supplementation of cell culture media with e.g. different sera is likely to affect interactions between cells and NPs ex-vivo, especially endocytosis. We aimed to investigate the differential impact of human and fetal-bovine serum on the endocytosis of poly (lactic-co-glycolic acid) NPs by human peripheral blood mononuclear cells via flow cytometry. Furthermore, we employed different methods to inhibit endocytosis, providing mechanistic insights. The resulting biomolecule corona was characterized via denaturing gel electrophoresis. We found profound differences between human and fetal bovine serum regarding the endocytosis of fluorescently labeled PLGA nanoparticles by different classes of human leukocytes. Uptake by B-lymphocytes was particularly sensitive. We further present evidence, that these effects are mediated by a biomolecule corona. We demonstrate to our knowledge for the first time that the complement is an important contributor to the endocytosis of non-surface-engineered PLGA-nanoparticles prepared via emulsion solvent evaporation by human immune cells. Our data demonstrates that results obtained with xenogeneic culture supplements such as fetal bovine serum may have to be interpreted with caution.

Expression of integrin β-7 is epigenetically enhanced in multiple myeloma subgroups with high-risk cytogenetics

BackgroundOncogenic overexpression of integrin-β7 (ITGB7) in cases of high-risk multiple myeloma (MM) was reported to promote enhanced interactions between neoplastic plasma-B cells and stromal cells to develop cell-adhesion mediated drug resistance.MethodsExpression profiles of adhesion related genes were analyzed in a cohort of MM patients containing major IgH translocations or hyperdiploidies (HY), diagnosed at the premalignant monoclonal gammopathy of undetermined significance (MGUS; n = 103), smoldering multiple myeloma; (SMM; n = 190) or MM (MM; n = 53) stage. Differential expression was integrated with loci-specific alterations in DNA-methylation and chromatin marks in MM patients. A CRISPR-based targeted induction of DNA-methylation at the ITGB7 super-enhancer (SE) in MM.1S cells was employed to intersect the impact of cis-regulatory elements on ITGB7 expression.ResultsITGB7 was significantly (p < 0.05) upregulated in patients with t(14;16) and t(14;20) subgroups in all MGUS, SMM and MM stages, but sporadically upregulated in t(4;14) subgroup at the MM stage. We demonstrate a predetermined enhancer state on ITGB7 in primary-B cells that is maintained under bivalent chromatin, which undergoes a process of chromatin-state alterations and develops into an active enhancer in cases of the t(4;14) subgroup or SE in cases of the t(14;16) subgroup. We also demonstrate that while targeted induction of DNA-methylation at the ITGB7-SE further upregulated the gene, inhibition of ITGB7-SE-associated transcription factor bromodomain-4 downregulated expression of the gene.ConclusionsOur findings suggest an epigenetic regulation of oncogenic overexpression of ITGB7 in MM cells, which could be critical in MM progression and an attractive therapeutic target.

CD72-semaphorin3A axis: A new regulatory pathway in systemic lupus erythematosus

CD72 is a regulatory co-receptor on B cells, with a role in the pathogenesis of systemic lupus erythematosus (SLE) in both human and animal models. Semaphorin3A (sema3A) is a secreted member of the semaphorin family that can reconstruct B cells’ regulatory functions by upregulating IL-10 expression and inhibiting the pro-inflammatory activity of B and T cells in autoimmune diseases.The aim of our present study was to identify a new ligand for CD72, namely sema3A, and exploring the signal transduction pathways following its ligation in B cells. We established that CD72 functions as sema3A binding and signal-transducing receptor. These functions of CD72 are independent of neuropilin-1 (NRP-1) (the known sema3A receptor). We discovered that sema3A induces the phosphorylation of CD72 on tyrosine residues and the association of CD72 with SHP-1 and SHP-2. In addition, the binding of sema3A to CD72 on B cells inhibits the phosphorylation of STAT-4 and HDAC-1 and induces the phosphorylation of p38-MAPK and PKC-theta in B-cells derived B-lymphoblastoid (BLCL) cells, and in primary B-cells isolated from either healthy donors or SLE patients. We concluded that sema3A is a functional regulatory ligand for CD72 on B cells. The sema3A-CD72 axis is a crucial regulatory pathway in the pathogenesis of autoimmune and inflammatory diseases namely SLE, and modulation of this pathway may have a potential therapeutic value for autoimmune diseases.

A Phosphatase Membrane-Shuttle Enables Feedback Control of B-Cell Signaling

Mechanisms of local kinase recruitment to amplify signaling in lipid rafts have been extensively studied. However, mechanisms of phosphatase membrane-recruitment to terminate local signaling activity remained elusive. Here we identified CD25 (IL2Rα) as a central phosphatase membrane-shuttle and feedback regulator of B-cell receptor (BCR)-signaling in activated and transformed B-cells. This was unexpected because CD25 is known as one of three chains of the IL2-receptor on T- and NK-cells. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells recruits an inhibitory phosphatase complex for feedback control of BCR-signaling and its oncogenic mimics. Reminiscent of genetic phosphatase-ablation, conditional CD25-deletion profoundly depleted early B-cell development followed by dramatic expansion and hyperactivation of mature B-cell subsets and autoimmunity owing to imbalances of BCR-signaling. However, defects in CD25 deficient mice were not replicated in mice that express CD25 but lack expression of the IL2 cytokine, demonstrating IL2-independent functions of CD25 in B-cells. In six clinical cohorts, high expression levels of CD25 predicted poor outcomes for patients with pre-germinal center (GC), in contrast to favorable outcomes for patients with post-GC B-cell malignancies. Consistent with opposite clinical outcome-annotation, genetic CD25-deletion in B-cell precursor leukemia induced rapid cell death but accelerated proliferation in post-GC B-cell lymphomas. Mechanistically, BCR-engagement or oncogenic BCR-signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail at S268. Genetic deletion, in vitro kinase, and interactome studies revealed that PKCδ-dependent CD25-S268 phosphorylation triggered the PKCδ-scaffold RACK1 to recruit inhibitory phosphatases (SHP1, SHIP1) for membrane-translocation and feedback control of BCR-signaling. Deletion of CD25 in human B-cells induced autonomous BCR-signaling and Ca2+-oscillations: In the absence of CD25, inhibitory phosphatases were no longer recruited to the cell membrane to control duration and strength of BCR-signaling. Genetic rescue experiments identified membrane-tethered RACK1 and CD25-S268 tail-phosphorylation as central structural elements of this previously unrecognized phosphatase membrane-shuttle. As a result, CRISPR-mediated knock-in of CD25-S268A mutant that abrogates the formation of inhibitory phosphatase complexes caused accelerated proliferation with hyperactivation of BCR-signaling in primary human tonsillar germinal center B-cells. In conclusion, PKCδ-mediated phosphorylation of CD25 assembles RACK1-dependent inhibitory phosphatase complexes to enable feedback control of BCR-signaling to prevent autoimmunity and malignant transformation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The human myocardium harbors a population of naive B-cells with a distinctive gene expression signature conserved across species.

IntroductionCardiac immunology studies in murine models have identified a sizeable population of myocardial B-cells and have shown that its modulation represents a promising strategy to develop novel therapies for heart failure. However, scarce data on B-cells in the human heart leaves unclear whether findings in rodents are relevant to human biology.MethodsWe performed immunohistochemical stains to characterize the amount and distribution of B-cells in human hearts, analyzing both fresh and post-mortem tissue. To gain insight into the biology of human myocardial B-cells we analyzed publicly-available spatial transcriptomics and single-cell sequencing datasets of myocardial and peripheral blood mononuclear cells (PBMCs). We validated these findings on primary B-cells sorted from the heart and peripheral blood of left ventricular assistive device recipients. To identify biological pathways upregulated in myocardial B-cells across species, we compared differential gene expression in myocardial vs peripheral blood B-cells across the studied human datasets and published rodent datasets.ResultsIn healthy human heart samples, we found B-cells at a ratio of 1:8 compared to T-cells (2.41 ± 0.45 vs 19.36 ± 4.43, p-value <0.001). Myocardial B-cells were more abundant in the interstitium compared with the intravascular space (p-value=0.011), and also more abundant in the myocardium vs. epicardium (p-value=0.048). Single-cell gene expression analysis showed that the human myocardium harbored mostly naive B-cells with a gene expression profile distinct from that of PBMC B-cells. Cross-comparison of differentially-expressed genes in myocardial vs. PBMC B-cells across human and rodent datasets identified 703 genes with consistent differential gene expression across species (binomial p-value=2.9e-48). KEGG pathway analysis highlighted “B-cell receptor signaling pathway,” “Antigen processing and presentation,” and “Cytokine-cytokine receptor interaction” among the top pathways upregulated in cardiac B-cells (FDR <0.001) conserved between species.ConclusionsLike the murine heart, the human heart harbors naive B-cells that are both intravascular and extravascular. Human myocardial B-cells are fewer and more evenly distributed between these two compartments than rodent myocardial B-cells. However, analysis of single-gene expression data indicates that the biological function of myocardial B-cells is conserved across species.

Increased serum level of alpha-2 macroglobulin and its production by B-lymphocytes in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), the most common adult’s leukemia in the western world, is caused in 95% of the cases by uncontrolled proliferation of monoclonal B-lymphocytes. The complement system in CLL is chronically activated at a low level via the classical pathway (CP). This chronic activation is induced by IgG-hexamers, which are formed after binding to alpha-2-macroglobulin (A2M). The study investigated for the first time the serum levels of A2M in CLL patients, their association with the disease severity, and A2M production by the malignant B-lymphocytes. Blood samples were collected from 65 CLL patients and 30 normal controls (NC) subjects, and used for quantifications of the A2M levels, the complement activation marker (sC5b-9), the complement components C2, C3 and C4, and clinical biochemistry and hematology parameters. The production of A2M was studied in B-lymphocytes isolated from blood samples as well as in CLL and non-CLL cell lines.The serum A2M levels were significantly higher in CLL patients vs NCs, showing values of 3.62 ± 0.22 and 1.97 ± 0.10 mg/ml, respectively. Within the CLL group, A2M levels correlated significantly with the disease stage, with sC5b-9, and with clinical indicators of the disease severity. Increased A2M production was showed in three out of four CLL B-lymphocytic lines that were studied, as compared to non-CLL lines, to a non-lymphocytic line, and to blood-derived primary B-lymphocytes. A2M production was further increased both in primary cells and in the CLL cell-line after incubation with CLL sera, compared to NC sera. This study shows for the first time that serum A2M levels in CLL are significantly increased, likely due to A2M production by the malignant B-lymphocytes, and are correlated with the disease severity and with chronic complement activation. The moderate change in A2M production after incubation with NC sera in-vitro supports the hypothesis that inhibition of excess A2M production can be achieved, and that this may potentially down-regulate the IgG-hexamerization and the resulting chronic CP activation. This may also help restore complement system activity, and eventually improve complement activity and immunotherapy outcomes in CLL.

Proteasome inhibition induces p53 driven apoptosis in early activated B-cells

Abstract Proteasome inhibition is a useful therapeutic strategy against plasma cell disorders in autoimmunity, neoplasias, and transplant rejection. However, how bortezomib induces cell death and the transcription factors that are involved in its execution are not well characterized. In this study, we aim to address the mechanism of action of proteasome inhibitors in plasma cells and in early precursor activated B-cells. We immunized mice with the hapten-protein conjugate nitrophenyl chicken-gamma-globulin (NP-CGG) and validated depletion of plasma cells and germinal center B-cells in vivo. At steady-state, we utilized B220 expression as a surface marker for newly-formed plasma cells and determined that bortezomib primarily depletes newly-formed plasma cells. To gain more mechanistic insight on how bortezomib affects newly-formed plasma cells, we employed an in vitro culture system of murine-derived primary follicular B-cells that were stimulated with CPG, IL-4, and IL5, to promote differentiation into the plasma cell fate. We find that deletion of the ER-stress induced transcription factor CHOP rescued in vitro generated plasma cells from low-dose bortezomib treatment. In addition, we identify the transcription factor p53 as a key inducer of apoptosis in in vitro generated early activated B-cells. Deletion of p53 rescued cells from apoptosis and reduced the number of cells expressing cleaved caspase-3 in response to bortezomib treatment. In conclusion, we have identified two transcription factors that are involved in bortezomib-induced cell death in B-lineage cells. Supported by NIAID Diversity Supplement: R01AI139123

Enhancer RNA AL928768.3 from the IGH Locus Regulates MYC Expression and Controls the Proliferation and Chemoresistance of Burkitt Lymphoma Cells with IGH/MYC Translocation.

Chromosomal rearrangements leading to the relocation of proto-oncogenes into transcription-active regions are found in various types of tumors. In particular, the transfer of proto-oncogenes to the locus of heavy chains of immunoglobulins (IGH) is frequently observed in B-lymphomas. The increased expression of the MYC proto-oncogene due to IGH/MYC translocation is detected in approximately 85% of Burkitt lymphoma cases. The regulatory mechanisms affecting the oncogenes upon translocation include non-coding enhancer RNAs (eRNAs). We conducted a search for the eRNAs that may affect MYC transcription in the case of IGH/MYC translocation in Burkitt lymphoma, looking for potentially oncogenic eRNAs located at the IGH locus and predominantly expressed in B cells. Overexpression and knockdown of our primary candidate eRNA AL928768.3 led to the corresponding changes in the expression of MYC proto-oncogene in Burkitt lymphoma cells. Furthermore, we demonstrated that AL928768.3 knockdown decreased lymphoma cell proliferation and resistance to chemotherapy. Significant effects were observed only in cell lines bearing IGH/MYC abnormality but not in B-cell lines without this translocation nor primary B-cells. Our results indicate that AL928768.3 plays an important role in the development of Burkitt’s lymphoma and suggest it and similar, yet undiscovered eRNAs as potential tissue-specific targets for cancer treatment.

CellCelector™ as a platform in isolating primary B cells for antibody discovery.

ABSTRACTThe most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery. In the current study, we present a method to isolate individual antigen-specific primary B-cells using the CellCellector™ single-cell isolation platform from XenoMouse® (XM) immunized with a recombinant therapeutic protein, EGFR. We screened a subset of CD138+ B-cells and identified 238 potential EGFR-specific B-cells from 1189 antibody-secreting cells (ASCs) and isolated 94 by CellCellector. We identified a diverse set of heavy chain complementarity-determining region sequences and cloned and expressed 20 into a standard human immunoglobulin G1 antibody format. We further characterized and identified 13 recombinant antibodies that engage soluble and native forms of EGFR. By extrapolating the method to all 400 000 CD138+ B-cells extracted from one EGFR immunized XM, a potential 1196 unique EGFR-specific antibodies could be discovered. CellCelector allows for interrogating the B-cell pool directly and isolating B-cells specific to the therapeutic target of interest. Furthermore, antibody sequences recovered from isolated B-cells engage the native and recombinant target, demonstrating the CellCellector can serve as a platform in antibody discovery.

Differential Transcriptional Reprogramming by Wild Type and Lymphoma-Associated Mutant MYC Proteins as B-Cells Convert to a Lymphoma Phenotype.

Simple SummaryOncogenic transformation in response to the activation of MYC requires secondary events that reduce the role of MYC as an inducer of apoptosis and cellular senescence. We made a primary B-cell model in which such secondary processes were reduced and MYC levels could be regulated at the transcriptional level. We studied the changes in transcriptional programs as MYC levels were progressively increased, causing a simultaneous transition to a lymphoma cell phenotype. Two lymphoma-associated MYC mutants (resulting in the substitution of threonine 58 with isoleucine, T58I, or alanine, T58A) were also analyzed and compared with each other and wild type MYC. All three MYC proteins induced cell cycle entry, cell growth and cell proliferation with associated changes in gene expression programs. The MYC-regulated genes overlap with MYC target genes in B-cells during normal activation. A minority of the MYC-regulated genes (enriched in specific aspects of the cell cycle) were regulated significantly differently by the mutant MYC proteins compared to wild type MYC and in most cases between the two mutant MYC proteins. These genes did not overlap significantly with the set of genes regulated during B-cell activation, and may be specific to oncogenesis. Many target genes were more sensitive to regulation by T58A and less sensitive to T58I, compared to wild type MYC, as was also seen for the measured phenotypic processes. This, taken together with the different sets of genes that are differentially regulated by each mutant in relation to wild type MYC, indicates that the mutations augment the oncogenic activity of MYC by different mechanisms. The different effects of the mutations may result from the different effects they are predicted to have on the conformational properties of the intrinsically disordered region of the MYC protein that surrounds the threonine 58 residue.The MYC transcription factor regulates a vast number of genes and is implicated in many human malignancies. In some hematological malignancies, MYC is frequently subject to missense mutations that enhance its transformation activity. Here, we use a novel murine cell system to (i) characterize the transcriptional effects of progressively increasing MYC levels as normal primary B-cells transform to lymphoma cells and (ii) determine how this gene regulation program is modified by lymphoma-associated MYC mutations (T58A and T58I) that enhance its transformation activity. Unlike many previous studies, the cell system exploits primary B-cells that are transduced to allow regulated MYC expression under circumstances where apoptosis and senescence pathways are abrogated by the over-expression of the Bcl-xL and BMI1 proteins. In such cells, transition from a normal to a lymphoma phenotype is directly dependent on the MYC expression level, without a requirement for secondary events that are normally required during MYC-driven oncogenic transformation. A generalized linear model approach allowed an integrated analysis of RNA sequencing data to identify regulated genes in relation to both progressively increasing MYC level and wild type or mutant status. Using this design, a total of 7569 regulated genes were identified, of which the majority (n = 7263) were regulated in response to progressively increased levels of wild type MYC, while a smaller number of genes (n = 917) were differentially regulated, compared to wild type MYC, in T58A MYC- and/or T58I MYC-expressing cells. Unlike most genes that are similarly regulated by both wild type and mutant MYC genes, the set of 917 genes did not significantly overlap with known lipopolysaccharide regulated genes, which represent genes regulated by MYC in normal B cells. The genes that were differently regulated in cells expressing mutant MYC proteins were significantly enriched in DNA replication and G2 phase to mitosis transition genes. Thus, mutants affecting MYC proteins may augment quantitative oncogenic effects on the expression of normal MYC-target genes with qualitative oncogenic effects, by which sets of cell cycle genes are abnormally targeted by MYC as B cells transition into lymphoma cells. The T58A and T58I mutations augment MYC-driven transformation by distinct mechanisms.

Serotype-dependent recombinant adeno-associated vector (AAV) infection of Epstein\u2013Barr virus-positive B-cells, towards recombinant AAV-based therapy of focal EBV\u2009+\u2009lymphoproliferative disorders

BackgroundB-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD), are increased among persons afflicted by T-cell compromise. Most are Epstein–Barr virus (EBV) + and can first present with a focal lesion. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity. Despite extensive study as a vehicle for gene therapy, adeno-associated viruses (AAV) have rarely been applied to human cancer research due to technical and theoretical obstacles. Moreover, human B-cells have historically been described as resistant to AAV infection. Nonetheless, advances using different recombinant (r)AAV serotypes with unique tropisms to deliver cytotoxic therapy suggested a localized anti-tumor approach was feasible.MethodsAs a prelude to the development of a therapeutic vehicle, the ability of fifteen distinct EGFP-bearing rAAV serotypes to transduce human B-cells, including primary, immortalized, and B-cell tumor lines ± EBV was assessed by confocal microscopy, flow cytometry and subsequently cell viability assay.ResultsRank order analysis revealed augmented transduction by rAAV6.2 and closely related virions. EBV infection of EBV-negative B-cell tumor lines and EBV immortalization of primary B-cells increased susceptibility to rAAV6.2 transduction. As a proof of concept, transduction by rAAV6.2 encoding herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) eliminated TK-negative rhabdomyosarcoma cells and diminished viability of transduced B-cell lines upon incubation with ganciclovir.ConclusionsrAAV serotypes differentially transduce human B-cell lines reversing the dogma that human B-cells are refractory to AAV infection. EBV + B-cells display increased susceptibility to rAAV6.2 infection, uncovering a new method for improved nucleic acid transfer into transfection-resistant B-cell lines. The introduction of a functional suicide gene into the rAAV6.2 genome identifies a candidate vector for the development of rAAV-based oncolytic therapy targeting focal EBV-bearing B-lymphoproliferative disorders.

IL-4 Mediated Upregulation of AMPK/SIRT1/PGC-1α Signalling Pathway Is Bypassed By Mitochondrial Complex I Downregulation By NAMPT Inhibition in Venetoclax and Ibrutinib Treated CLL Cells

Introduction: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Despite significant improvement in the treatment of CLL, drug resistance is emerging when using the single agents ibrutinib or venetoclax. To achieve greater depth of response, combination treatments are being used to eradicate disease. Altered mitochondrial metabolism is a key factor in CLL survival. In order to gain insights into the underlying biology of a promising drug combination treatment, we investigated the combination of venetoclax and ibrutinib on mitochondrial function as well as the B-cell receptor (BCR), apoptotic and adenosine monophosphate activated protein kinase /silent information regulator 1 / peroxisome proliferator-activated receptor-coactivator-1α (AMPK/SIRT1/PGC-1α) signaling pathways in CLL cells. We also evaluated a proposed mechanism of resistance using interleukin-4 (IL-4) to demonstrate the role of a nicotinamide phosphoribosyltransferase (NAMPT) specific inhibitor, FK866, in order to overcome resistance in vitro.Methods: Freshly isolated primary B-cells from CLL patients were treated with venetoclax, ibrutinib or their combination in a dose- and -time responsive fashion. CLL cells were also treated with IL-4 and FK866 in the presence or the absence of the combination treatment. Flow cytometry (Novocyte) was used to assess cell viability, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). Mitochondrial respiration rates and specific substrate-dependent respiration of individual complexes of the respiratory chain were measured by respirometry (Orobooros O2k oxygraph) and ATP levels by luminometry (Lmax Luminometer, Molecular Devices). Cellular, mitochondrial, and lysosomal morphology was evaluated by Philips CM10 electron microscope and Olympus BX51 fluorescent microscope. Changes in protein levels of signaling pathways were detected by immunoblotting.Results: Each single agent venetoclax or ibrutinib reduced mitochondrial respiration profiles in CLL cells in vitro. The combined effect of these drugs on the respiration profiles, ATP, MMP, ROS and cell viability was more profound than with each agent alone. Proteins involved in 1. BCR [Bruton's tyrosine kinase (BTK); serine/threonine-specific protein kinase (AKT); phospholipase Cɣ2 (PLCɣ2) and extracellular signal-regulated kinase (ERK)], 2. Apoptotic B-cell lymphoma 2 (BCL-2); myeloid cell leukemia-1 (MCL-1) and 3. AMPK/SIRT1/PGC-1α signaling in the venetoclax and ibrutinib combination treated samples were significantly reduced when compared to DMSO and each single agent. AMPK/SIRT1/PGC-1α regulated transcription factors responsible for mitochondrial biogenesis [nuclear respiratory factor (NRF1 and NRF2)] and mitochondrial dynamics related proteins [mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1)] were preferentially downregulated by the combination treatment. These effects are seen in the morphological changes, as visualized by transmission electron microscopy demonstrating swelling of mitochondria (venetoclax) and vacuole formation (ibrutinib) in addition to the formation of multi-vesicular bodies in the combination. We also validated the impact of the mitochondria and lysosomes using immunofluorescence. In the presence of IL-4 (a secreted cytokine used to activate the BCR), the effects of the combination were negated by the addition of the NAMPT inhibitor, FK866. FK866 also preferentially decreased mitochondrial respiration rates in the presence of Complex I specific substrates and sustained this inhibition in all FK866 containing conditions regardless of IL-4.Conclusions: The combined effect of venetoclax and ibrutinib to target mitochondrial metabolism via the AMPK/SIRT1/PGC-1α signaling pathway provides a rationale for this drug combination treatment. The use of IL-4 identifies a potential path of resistance that can be overcome by NAMPT inhibition by directly targeting Complex I of the electron transport chain of the mitochondria. DisclosuresNo relevant conflicts of interest to declare.

Surface-associated antigen induces permeabilization of primary mouse B-cells and lysosome exocytosis facilitating antigen uptake and presentation to T-cells.

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

Marek's disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype.

Marek's disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.

Expression of Concern: Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells

Expression of Concern: Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells