The RNA-binding protein HuR is required for maintenance of the germinal centre response

The germinal centre (GC) is required for the generation of high affinity antibodies and immunological memory. Here we show that the RNA binding protein HuR has an essential function in GC B cells to sustain the GC response. In its absence, the GC reaction and production of high-affinity antibody is severely impaired. Mechanistically, HuR affects the transcriptome qualitatively and quantitatively. The expression and splicing patterns of hundreds of genes are altered in the absence of HuR. Among these genes, HuR is required for the expression of Myc and a Myc-dependent transcriptional program that controls GC B cell proliferation and Ig somatic hypermutation. Additionally, HuR regulates the splicing and abundance of mRNAs required for entry into and transition through the S phase of the cell cycle, and it modulates a gene signature associated with DNA deamination protecting GC B cells from DNA damage and cell death.

The Paradoxical Efficacy of KDM6 Inhibition in Germinal Centre B-Cell Lymphomas

Introduction Germinal centre (GC) lymphomas Follicular Lymphoma (FL) and Diffuse Large B Cell Lymphoma (DLBCL) are strikingly dependent on mutations affecting chromatin regulators. The majority of FL tumours harbour multiple mutated chromatin-regulatory genes, including loss-of-function mutations in KMT2D (80%), CREBBP (60%) and gain-of-function mutations in EZH2 (25%), whilst these are also frequently mutated within the GC B-cell (GCB) subtype of DLBCL. The FDA approval of EZH2-inhibitor Tazemetostat for relapsed/refractory FL exemplifies the potential and the need to broaden our search for novel avenues targeting epigenetic reprogramming in GC lymphomas. Hence, in this study, we sought to identify novel epigenetic vulnerabilities and biological interactions in GC B cell lymphomas. Methods and Results: 25 compounds targeting 18 histone binding/modifying enzymes were tested across 12 GCB-DLBCL cell lines harbouring differing degrees of epigenetic dysregulation. The two most potent cytotoxic compounds (GSK-J4 and KDOBA67) both target the H3K27me3 demethylase, KDM6. The KDM6 family consists of KDM6A (UTX) and KDM6B (JMJD3), both of which demethylate H3K27me3/H3K27me2 to lower methylation states, and KDM6C (UTY) which has negligible enzymatic activity. Indeed, KDM6 inhibition (KDM6i) with GSK-J4 increased levels of the inhibitory chromatin mark H3K27me3 and simultaneously reduced H3K27me1/me2, while we observed no global changes in H3K4me3. The induction of apoptosis in the cell lines following either inhibition of the 'eraser' KDM6 (leading to increased H3K27me3) or the opposing 'writer' EZH2 (lowering H3K27me3) appears paradoxical at first glance, given the EZH2 gain-of-function mutations seen in FL/DLBCL and known efficacy of EZH2 inhibitors. However, RNA-sequencing analysis of GSK-J4-treated cells revealed no significant overlap with published EZH2 mutation or EZH2 inhibition signatures from GC lymphoma models, implying that KDM6i-regulated H3K27me3 peaks do not overlay gene promoters targeted by EZH2 in the GC reaction. The most significant gene regulation by KDM6i was a significant up-regulation of 6 members of the metallothionein (MT) family within 4-hours of treatment, which was sustained for 72-hours (MT1E, MT1F, MT1G, MT1H, MT1X and MT2A at 4 hours log2FoldChange 5.1-12.1, average p adj 3.4E-06). These metal-binding proteins cluster on chromosome 16 and their up-regulation correlated with up-regulation of the zinc transporter SLC30A1, suggesting a potential biological relationship between KDM6 enzymes and zinc homeostasis, with both isoforms containing a zinc-binding domain. Targeted validation across 6 DLBCL cell lines also showed MT up-regulation correlated with sensitivity to GSK-J4, whilst siRNA-mediated knockdown of KDM6A or KDM6B individually recapitulated the up-regulation of MT genes, albeit to a lower extent. ChIP-qPCR profiling at the MT loci for H3K27me3 and H3K4me3 revealed no significant changes at 4 or 24 hours; considering that expression of MT genes increase within 60 minutes of drug exposure, suggesting these changes may be induced by an indirect mechanism of KDM6A and/or KDM6B activity. Using ENCODE data of 4 cell lines included in our series (DOHH-2, OCI-LY-1, SU-DHL-6, KARPAS-422), we observe a link between GSK-J4 sensitivity, up-regulation of MT genes and significantly lower basal enrichment for H3K27ac at the MT loci. This distinction may reflect altered 3D chromatin conformation that render only sensitive cell lines permissive to regulation by KDM6i, with H3K27ac having recently been shown to modulate enhancer positioning and interactions in DLBCL cell lines. Conclusion: Screening epigenetic-targeting compounds in GC lymphomas has identified KDM6 as a particularly promising candidate target. Overall, we describe up-regulation of MT genes as one potential mechanism of action, which correlates with response in DLBCL cell lines and suggests that KDM6 members may regulate H3K27me3 in a manner different from EZH2. In our current model we propose, that in GC-lymphomas KDM6A may have tumour suppressive roles whilst KDM6B is required for cell survival. Given KDM6A is frequently lost in cancer and Kabuki syndrome, a congenital developmental disorder caused by germline mutations in KMT2D or KDM6A, our results highlight the importance of establishing what roles the KDM6 family play in lymphoma pathogenesis. Disclosures Fitzgibbon: Epizyme: Research Funding.

Targeting Asparagine and Serine Metabolism in Germinal Centre-Derived B Cells Non-Hodgkin Lymphomas (B-NHL)

BL and DLBCL are subtypes of B-cell lymphomas that arise from germinal centre B lymphocytes. Differentiation between BL and DLBCL is critical and can be challenging, as these two types of cancer share the same morphological, immunophenotypic, and genetic characteristics. In this study, we have examined metabolism in BL and DLBCL lymphomas and found distinctive differences in serine metabolism. We show that BL cells consume significantly more extracellular asparagine than DLBCL cells. Using a tracer-based approach, we find that asparagine regulates the serine uptake and serine synthesis in BL and DLBCL cells. Calculation of Differentially Expressed Genes (DEGs) from RNAseq datasets of BL and DLBCL patients show that BL cancers express the genes involved in serine synthesis at a higher level than DLBCL. Remarkably, combined use of an inhibitor of serine biosynthesis pathway and an anticancer drug asparaginase increases the sensitivity of BL cells to extracellular asparagine deprivation without inducing a change in the sensitivity of DLBCL cells to asparaginase. In summary, our study unravels metabolic differences between BL and DLBCL with diagnostic potential which may also open new avenues for treatment.

Advances in understanding the formation and fate of B-cell memory in response to immunisation or infection

Immunological memory has the potential to provide lifelong protection against recurrent infections. As such, it has been crucial to the success of vaccines. Yet, the recent pandemic has illuminated key gaps in our knowledge related to the factors influencing effective memory formation and the inability to predict the longevity of immune protection. In recent decades, researchers have acquired a number of novel and powerful tools with which to study the factors underpinning humoral memory. These tools have been used to study the B-cell fate decisions that occur within the germinal centre, a site where responding B-cells undergo affinity maturation and is one of the major routes for memory B-cell and high-affinity long-lived plasma cell formation. The advent of single-cell sequencing technology has provided an enhanced resolution for studying fate decisions within the germinal centre and cutting-edge techniques have enabled researchers to model this reaction with more accuracy both in vitro and in silico. Moreover, modern approaches to studying memory B-cells have allowed us to gain a better appreciation for the heterogeneity and adaptability of this vital class of B-cells. Together, these studies have facilitated important breakthroughs in our understanding of how these systems operate to ensure a successful immune response. In this review, we describe recent advances in the field of germinal centre and memory B-cell biology in order to provide insight into how humoral memory is formed, as well as the potential for generating lasting immunity to novel pathogens such as SARS-CoV-2.

Comparitive Study of Human Lymph Nodes

All the lymph nodes observed were typically bean shaped irrespective of their age or sex. The cervical lymph nodes were the largest of all the four groups in size. The mean maximum short axis axial diameter was 7.19mm (Range 5.67mm - 8.12mm). The mesentericl lymph nodes were the smallest of all the four groups in size. The mean maximum short axis axial diameter was 3.89mm (Range 2.7mm -6.lmm). The lymph nodes showed well defined deep cortical regions often containing large lymphoblasts and prominent endothelial cells in small blood vessels. The thickness of the deep cortex was however reduced when compared to its foetal counterpart. The mean thickness of the deep cortical region was 423 microns. To reconfirm the age related involution of the paracortical region, the mesenteric lymph nodes were observed. In sharp· distinction, axillary lymph nodes showed fewer germinal centres which were relatively smaller in size.Mean actual diameter of the germinal centre was77 microns also had had fewer and smaller germinal centres. Mean actual diameter of the germinal centre was 68 microns.

AB0022 AUTOREACTIVE B CELLS IN RHEUMATOID ARTHRITIS DISPLAY AN ACTIVATED PHENOTYPE OF RECENT ANTIGEN EXPOSURE

Background:Rheumatoid arthritis, in particular ACPA+ RA, is characterized by frequent disease flares and poor chances to achieve DMARD-free sustained remission. Recently, we have shown that ACPA-expressing memory B cells (MBC) remain in a persistently activated state throughout disease, even in patients in DMARD-induced clinical remission.(1) The reasons why the ACPA B cell response is continuously activated are unknown, as well as why the response does not revert to a resting, ‘quiescent’ state. We hypothesized that continuous antigen exposure in germinal centres drives ACPA B cell activation, leading to a ‘recent germinal centre emigrant’ phenotype of these cells in the circulation.Objectives:To understand whether the activated phenotype of ACPA-expressing B cells could be induced by recent antigen exposure, to thereby discern the processes of immune activation that remain active in patients even in clinical remission and to argue whether these processes could be targets for therapeutic intervention.Methods:ACPA-expressing B cells were identified in peripheral blood of RA patients by flow cytometry during different stages of disease and characterized by a panel of activation- and germinal centre related markers (CD80, CD86, CD32, CD95, Ki-67). In addition, three healthy donors received a TT booster vaccination. TT-specific MBC were identified in blood at different timepoints (before vaccination and up to 22 weeks after vaccination) and analysed phenotypically over time.Results:The majority of ACPA-expressing B cells strongly expressed CD95 and the co-stimulatory marker CD80. A part was also positive for the proliferation marker Ki-67 (on average 30%), and most cells downregulated the inhibitory marker CD32. TT-specific MBC adopted a comparable phenotype after booster vaccination, but most markers returned to the pre-vaccination expression level gradually over time. These effects were antigen-dependent because the phenotype of TT-negative B cells remained unchanged. The phenotypic composition of the proliferating ACPA-positive B cell pool most closely corresponded to a stimulation history of 1-2 weeks after antigen exposure. Notably, none of the Ki-67 negative ACPA-specific MBC showed phenotypic quiescence, indicating either a short life-time (in circulation) after antigen encounter or persistent additional factors of activation.Figure 1.Ki-67 expression on ACPA-specific MBC in RA (A) and on TT-specific MBC in 3 healthy donors before and after booster vaccination (B).Conclusion:ACPA-expressing MBC phenotypically resemble TT-specific MBC after recent (1-2 weeks) booster vaccination, reflecting the phenotype of recent germinal centre emigrants, and remain activated, whereas TT-specific MBC lose this marker profile over time. These observations suggest that ACPA-expressing MBC either home to tissue or survive shortly in the circulation, or that additional factors drive or program these cells to persistent activation. Transcriptomic profiling and analysis of the homing marker profile may help to answer these questions. Furthermore, it will be important to understand the association of persistent activation of ACPA-expressing B cells in clinical remission and the risk for disease flares upon treatment discontinuation.References:[1]Kristyanto H, Blomberg NJ, Slot LM, van der Voort EIH, Kerkman PF, Bakker A, et al. Persistently activated, proliferative memory autoreactive B cells promote inflammation in rheumatoid arthritis. Sci Transl Med. 2020;12(570).Disclosure of Interests:Nienke Blomberg: None declared, Hendy Kristyanto: None declared, Thomas Huizinga Grant/research support from: Gilead, Rene Toes: None declared, Hans Ulrich Scherer Grant/research support from: Pfizer, Lilly, Sanofi, BMS