Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

4-Thiofuranoid Glycal: Versatile Glycosyl Donor for the Selective Synthesis of -anomer of 4’-thionucleoside and its biological activities

: The first highly diastereoselective synthesis of -anomers of 4’-thionucleosides has been carried out by means of electrophilic glycosidation utilizing 3,5-O-(di-t-butylsilylene) (DTBS)-4-thiofuranoid glycal as a glycosyl donor. The resulting glycosides were transformed into ribo-, 2’-deoxy- and arabinofuranosyl nucleosides through a chemical transformation of the 2’-substituent. The additive Pummerer reaction of the glycal S-oxide gave 1,2-di-O-acetyl-3,5-O-DTBS-4-thioribofuranose. The utility of the DTBS-protected 4-thioribofuranose has been demonstrated by the preparation of 4’-thio analogues of pyrimidine- and purine-4’-thioribonucleosides on the basis of the Vorbrüggen glycosidation. Synthesis of 4’-thio-counterpart of C-nucleoside antibiotic tiazofurin has also been carried out. -Face selective hydroboration of 1-C-aryl- or 1-C-heteroaryl-glycals obtained by cross-coupling of 1-tributylstannylglycal has furnished the respective -anomer of 4’-thio-C-ribonucleosides including 4’-thio analogue of nucleoside antibiotic pseudouridine and 9-deazaadenosine. On the basis of lithiation chemistry, 1-C- and 2-C-carbon-carbon-substituted 3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3- diyl) (TIPDS)- 4-thiofuranoid glycal were synthesized. These glycals enabled us to prepare 1’-C- and 2’--C-carbon-substituted 2’-deoxy-4’-thionucleosides which include thio-counterpart of antitumor nucleoside antibiotic angustmycin C. Furthermore, 1’-C-methyl-4’-thiothymidine emerged as potent inhibitor of angiogenesis. In addition, 1’-C-methyl-4’-thiothymidine exhibited inhibitory activity against thymidine kinase deficient mutant of herpes virus more potent than that of ganciclovir. Among the 4’-substituted 4’-thiothymidines, the 4’-C-cyano- and 4’-C-ethynyl derivatives inhibited replication of HIV variant resistant to 3TC (HIVM184V) as potent as to those of the HIV-1IIIB. In terms of the value of selectivity index (SI), 4’-C-cyano-4’-thiothymidine showed 3-fold selective index (SI) than that of the corresponding thymidine derivative. Furthermore, 4’-C-ethynyl-2’-deoxy-4’-thioguanosine has a 20-fold better value (>18,200) than that of 2’-deoxyguanosine counterpart (933). Furthermore,4’-azido-4’-thiothymidine was emerged as selective and potent anti-EBV agent. In terms of antineoplastic activity, 4’-azido- and 4’-C-fluoromethyl-2’-deoxy-4’-thiocytidine inhibited proliferation of human B-cell (CCRF-SB) and T-cell leukemia (Molt-4) cell lines although the parent compound 2’-deoxy-4’-thiocytidine did not show any cytotoxicity up to 100 M. These facts concerning the biological activities suggested that replacement of the furanose oxygen with sulfur atom is a promising approach for development of less toxic antiviral and antineoplastic nucleoside antimetabolites. 4’-Thionucleoside has also superior biological properties as monomer for oligonucleotides (ONs) therapeutics. Therefore, this review provides a wide range of potential monomer for antisense ON and siRNA.

Chromatin Content Capture Reveals Acute Leukaemia Oncogenic Vulnerability Point in Human B Cell Development

Human B cell development in adult human bone marrow (BM) is tightly regulated through well-defined stages to produce adaptive immune cells with assembled and functional B cell antigen receptor (BCR)(Martin et al., 2016). To produce mature B cells with functional immunoglobulin receptors, B cell progenitors must undergo multiple stages of highly regulated chromatin remodelling and transcriptional reprogramming which correspond to unique patterns of surface protein expression (Nutt and Kee, 2007). This complex process is frequently dysregulated in B cell neoplasia such as B cell Acute Lymphoblastic Leukemia (B-ALL). B-ALL is highly heterogenous in its phenotypic and clinical presentation, as well as in its underlying molecular features such as DNA methylation patterns and genetic aberrations (Cobaleda and Sánchez-García, 2009). The lack of general mechanism of leukemogenesis has made it difficult to identify when and where adult and pediatric B-ALL blasts diverge from normal B cell development. Here we show that across 5 B-ALL patients and 3 cell lines with diverse phenotypic and clinical presentations, blasts are epigenetically arrested at a conserved point within healthy human B cell development. First, we sought to establish a trajectory of normal B cell development to delineate the phenotypic and concomitant epigenetic changes occurring in BM progenitors as they differentiate into naïve B cells. To capture phenotype, function, and epigenetic state via single cell chromatin content (chromotype) of developing B cells in BM, we developed a multiplexed, high throughput, single cell proteomic method (chromotyping) to simultaneously measure cell surface markers, intracellular regulators such as transcription factors and chromatin structure regulators such as histone post-translational modifications (i.e. H3K4me3, H3K27me3, H2AK119ubi) and chromatin re-modelers (i.e. CTCF, DNMT1, MLL1). Using these surrogates for single cell, global chromatin content, we notably identified 3 coordinated epigenetic inflection or switch (S) points in healthy B cell development corresponding to previously characterized phenotypic landmarks of STAT5 signalling and active re-arrangement of IgH loci (S1), CD24 expression-linked high translation and proliferation (S2), and IgM and CD20 expression-linked BCR assembly completion (S3) (Bendall et al., 2014). To determine how these coordinated chromotypes translated to chromatin accessibility and primed gene regulation networks, we isolated BM B cell population from these chromatin content transition points and analysed them with our modified ATAC-seq protocol, InTAC-seq (Baskar et al., 2021). Strikingly, the chromatin accessibility landscape revealed putative oncogenic priming with high activity of leukemic TFs such as PAX5, TCF3, ZEB1 and ID4 predominantly at S2 and some at S3 switch points. By integrating our InTAC-seq data with publicly available single cell ATAC and RNA seq data on BM, we located this oncogenic primed state as existing from S2 to before S3 (IgH rearranged, late pro- / Pre-B cell stage) in healthy B cell development. This integration further associated this state with high activity of ASCL1 (role in chromatin remodelling) and high expression of STMN1 (Leukaemia-associated phosphoprotein 18). Finally we showed that across B-ALL patients (n=5) and cell lines (REH, NALM6, SUBP15), chromatin accessibility of neoplastic B cells indeed continue to occupy this point of oncogenic vulnerability in the B cell developmental space from S2 to right before S3 in our integrated scATAC map, despite variable immunophenotypes. This corresponds to a coordinated minima in our chromotyping map (lowest, coordinated abundance of chromatin structure regulators across trajectory). Further analysis of B-ALL patients reinforced the divergence between immunophenotypic and epigenetic heterogeneity within and between samples. Taken together, our findings identify key epigenetic switch points in B cell development and their underlying chromatin accessibility and gene expression patterns. Consequently, we reveal a point of epigenetic vulnerability in healthy B cell development that could be predisposed to leukemic transformation. This work opens up the possibility for new diagnostic strategies for B-ALL utilizing chromatin content and could pave the way for epigenetic modulation-based treatments beyond DNA methylation inhibition. Disclosures Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Shared and Distinct Genetic Features in Human and Canine B-Cell Lymphomas

Introduction Animal models of human cancers are an important tool for the development and preclinical evaluation of new treatments. Canine B-cell lymphoma (cBCL) is an appealing alternative to murine preclinical models due to its frequent, spontaneous incidence and its clinical and histological similarity to human B-cell non-Hodgkin lymphoma (NHL). The potential utility of cBCL as a veterinary model of human B-cell lymphomas would be bolstered by a more complete understanding of the genetic features found in cBCL. Methods To study the genetics of cBCL, we obtained fresh frozen and matched plasma/serum from 86 patients from the Canine Comparative Oncology Genomic Consortium(CCOGC) with 65 confirmed as B-cell lymphomas by immunophenotyping. Tumor DNA was prepared into libraries using the QIAseq FX DNA Library Kit (Qiagen). Plasma and serum DNA was prepared into libraries using the NebNext Ultra II DNA Library Prep Kit. Targeted hybridization enrichment was performed on the libraries using our custom baits and sequencing reads were aligned to canFam3.1 using Geneious and each mutation was visually confirmed. Variants were annotated with Variant Effect Predictor and human-dog pairwise alignments were extracted from Ensembl to identify the orthologous human amino acid for all canine variants. Results Our analysis confirmed the previously reported high frequency of mutations in TRAF3 and FBXW7. We also observed mutations in POT1, TP53, and SETD2 at similar frequencies to those reported in previous studies. DDX3X was mutated in 20% of cases, which is substantially higher than previously reported. MYC mutations were also more frequent (13%) than has been previously described in cBCL. In human lymphomas, MYC is commonly deregulated by translocation to a potent enhancer and these events are often associated with point mutations in MYC that are induced by aberrant somatic hypermutation (aSHM). Interestingly, we identified a more focal pattern of MYC mutations in cBCL that implies they do not result from aSHM and are likely functional. This finding implicates the conserved MYC phosphodegron sequence, a motif commonly mutated among additional aSHM-associated mutations, as the target of bona fide driver mutations in both human and cBCLs. Mutations in FBXW7 primarily affected the substrate recognition domain responsible for MYC degradation. The observation that MYC and FBXW7 mutations did not co-occur in any canine patient is consistent with the notion that FBXW7 mutations operate as an alternative path to MYC stabilization which is not frequently observed in human NHL. DDX3X was one of the most frequently mutated genes in our cohort (20%). DDX3X mutations are common in human Burkitt lymphoma and, though less abundant in hDLBCL, tend to be observed in samples with MYC translocations. In Burkitt lymphoma, these mutations display a sex-specific pattern, wherein females show mainly missense mutations, while males are affected by loss-of-function mutations. Interestingly, all DDX3X mutations in cBCL are missense variants and are presumed to be dominant acting. This lack of sex difference in DDX3X mutations is an important distinction between human and canine B-cell lymphomas that warrants further exploration. Conclusions Our study has revealed key differences in the mutational profiles of canine and human B-cell lymphomas and provides an impetus for enhanced genomic characterization of canine lymphomas as a model for human NHL, particularly in clinical trial settings. Disclosures Grande: Sage Bionetworks: Current Employment. Alcaide: GA Diagnostics AB: Current Employment. Morin: Celgene: Consultancy; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Epizyme: Patents & Royalties. Coyle: Allakos, Inc.: Consultancy.

Bridging the B Cell Gap: Novel Technologies to Study Antigen-Specific Human B Cell Responses

The generation of high affinity antibodies is a crucial aspect of immunity induced by vaccination or infection. Investigation into the B cells that produce these antibodies grants key insights into the effectiveness of novel immunogens to induce a lasting protective response against endemic or pandemic pathogens, such as influenza viruses, human immunodeficiency virus, or severe acute respiratory syndrome coronavirus-2. However, humoral immunity has largely been studied at the serological level, limiting our knowledge on the specificity and function of B cells recruited to respond to pathogens. In this review, we cover a number of recent innovations in the field that have increased our ability to connect B cell function to the B cell repertoire and antigen specificity. Moreover, we will highlight recent advances in the development of both ex vivo and in vivo models to study human B cell responses. Together, the technologies highlighted in this review can be used to help design and validate new vaccine designs and platforms.

The dynamic epigenetic regulation of the inactive X chromosome in healthy human B cells is dysregulated in lupus patients

Systemic lupus erythematous (SLE) is a female-predominant disease characterized by autoimmune B cells and pathogenic autoantibody production. Individuals with two or more X chromosomes are at increased risk for SLE, suggesting that X-linked genes contribute to the observed sex bias of this disease. To normalize X-linked gene expression between sexes, one X in female cells is randomly selected for transcriptional silencing through X-chromosome inactivation (XCI), resulting in allele-specific enrichment of epigenetic modifications, including histone methylation and the long noncoding RNA XIST/Xist on the inactive X (Xi). As we have previously shown that epigenetic regulation of the Xi in female lymphocytes from mice is unexpectedly dynamic, we used RNA fluorescence in situ hybridization and immunofluorescence to profile epigenetic features of the Xi at the single-cell level in human B cell subsets from pediatric and adult SLE patients and healthy controls. Our data reveal that abnormal XCI maintenance in B cells is a feature of SLE. Using single-cell and bulk-cell RNA sequencing datasets, we found that X-linked immunity genes escape XCI in specific healthy human B cell subsets and that human SLE B cells exhibit aberrant expression of X-linked genes and XIST RNA interactome genes. Our data reveal that mislocalized XIST RNA, coupled with a dramatic reduction in heterochromatic modifications at the Xi in SLE, predispose for aberrant X-linked gene expression from the Xi, thus defining a genetic and epigenetic pathway that affects X-linked gene expression in human SLE B cells and likely contributes to the female bias in SLE.