Expression Of Germinal Center Research Articles

Utility of MEF2B Immunohistochemistry in Distinguishing Follicular Lymphoma and Marginal Zone Lymphoma in Diagnostically Challenging Cases

Abstract Introduction/Objective Distinguishing follicular lymphoma (FL) from marginal zone lymphoma (MZL) can be challenging, as the morphology and immunophenotype can overlap particularly in CD10-negative cases or those with marginal zone differentiation (MZD). MEF2B (Myocyte enhancer binding factor B) is a transcription factor that controls BCL6 expression in germinal center (GC) B-cells. Previous studies report MEF2B to have a high sensitivity and specificity for FL compared to other GC markers. We investigated the expression of MEF2B in low grade B cell lymphomas (LGBCL) to evaluate its utility in classifying diagnostically challenging cases and FL with MZD. Methods/Case Report Our archives were searched from 2005-2023 to identify excisional biopsies of LGBCL with MZD, including (FL), splenic MZL (SMZL), MZL of mucosa-associated lymphoid tissue (MALT), nodal MZL (NMZL) and difficult-to-classify (DTC) cases with a differential diagnosis of MZL and FL. MEF2B immunohistochemistry was performed retrospectively (Atlas Antibodies). Two hematopathologists assigned a pre-and post-MEF2B diagnosis to DTC cases. Results (if a Case Study enter NA) 57 cases were included: 21 FL (12 with MZD), 11 SMZL, 13 MALT, 4 NMZL, and 8 DTC cases. 19/21 FL cases (90%) were positive for MEF2B, including 11/12 (92%) cases with MZD. However, MEF2B expression was restricted to the follicles and negative in MZD areas in 7/11 cases (64%), while 4 cases showed partial weak expression in the areas of MZD. 0/11 SMZL (0%), 0/13 MALT (0%), and 0/4 (0%) NMZL were positive for MEF2B. 6/8 DTC cases (75%) were positive for MEF2B. Within DTC cases, retrospective inclusion of MEF2B results helped to render a more definitive diagnosis in 5 cases (2 negative/3 positive). Conclusion MEF2B immunohistochemistry can aid in distinguishing FL from MZL in challenging cases. However, it is often negative in areas of FL with MZD so care must be taken on interpretation in small biopsies.

Abstract PR04: Characterization of an essential MYC enhancer targeted by focal genomic alterations in diffuse large B-cell lymphoma

Abstract We sought to identify mechanisms that activate MYC expression in germinal center B cell-(GCB) diffuse large B-cell lymphomas (DLBCL) in the absence of MYC rearrangement. High-throughput CRISPR-interference screens in MYC-rearranged (n=4) and non-MYC-rearranged (n=2) GCB-DLBCL cell lines revealed a distal enhancer complex 436 kb downstream of MYC, GME-1, that was uniquely essential in non-rearranged (MYC-intact) cell lines. CRISPR-interference studies confirmed growth inhibition and MYC downregulation upon GME-1 repression in 4 DLBCL cell lines. ChIP-Seq showed that GME-1 subunits were bound by OCT2, OCA-B, and MEF2B, which are known to activate germinal center-specific enhancers, and knockdown of the corresponding genes reduced MYC expression. GME-1 subunits showed GCB-specific acetylation and accessibility across normal human B cell populations in public chromatin datasets. The mouse ortholog of GME-1 showed germinal center B cell-specific accessibility. GME-1 was strongly acetylated in MYC-intact but not MYC-rearranged GCB-DLBCL cell lines and patient biopsies. Hi-C revealed significant looping of GME-1 to the MYC promoter in MYC protein+, MYC-intact DLBCL biopsies (n=2), but not MYC-rearranged DLBCL (n=2). We used sequence conservation and chromatin accessibility data to define four GME-1 subunits (195A/B and196A/B), which were cloned into luciferase enhancer reporter constructs and compared to two subunits of the BCL6 distal super-enhancer complex that had previously been identified as regulated by the OCT2/OCA-B/MEF2B complex. GME-1 subunit 195B was the only strongly transcription-activating element based on luciferase assays across three MYC-intact cells lines, where it showed activity comparable to BCL6 enhancers. In contrast, GME-1 enhancers were minimally active in reporter assays performed in two MYC-rearranged GCB-DLBCL cell lines. Deletion of a conserved OCT2 binding site or knockdown of the POU2F2 and POU2AF1 genes reduced activity of GME-1. Thus GME-1 trans-regulation is divergent from that of previously characterized GCB enhancers, despite shared dependency on the OCT2 ternary complex. Whole-genome sequencing (WGS) revealed that the GME-1 dependent cell line SUDHL5 bears a 176bp tandem duplication within GME-1 195B. This altered enhancer showed stronger luciferase reporter activity across three MYC-intact cell lines. Published WGS datasets showed isolated genomic tandem copy gains of GME-1 in 2/49 biopsies from recurrent/refractory DLBCL and 1/93 unselected DLBCL. In a cohort of 18 DLBCL with the high-risk DZSig gene expression signature, GME-1 amplification was seen in two biopsies with concurrent deletion of the PVT1 promoter (PVT1-P) and no inter-chromosomal MYC locus rearrangement. CRISPRi showed that PVT1-P repression reduced growth and MYC expression in GME-1-dependent DLBCL, but not in cell lines where rearrangements removed PVT-P from its position in cis with the MYC gene. Our findings identify GME-1 as an activator of MYC expression and target of rare but recurrent genomic copy gains in high-risk DLBCL. Citation Format: Aishwarya Gurumurthy, Ashwin R. Iyer, Shih-Chun A. Chu, Marcin P. Cieslik, Russell J.H. Ryan. Characterization of an essential MYC enhancer targeted by focal genomic alterations in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR04.

Abstract P04: Role of glucocorticoid receptor in normal and malignant germinal center B cells

Abstract We have reported that super-enhancers (SEs) are specifically hypermutated by Activation-Induced Deaminase (AID)-induced Aberrant Somatic Hypermutation in >90% of diffuse large B cell lymphoma (DLBCL) cases (Bal et al. Nature 2022). Analysis of 122 primary DLBCL biopsies and cell lines identified more than 80 SEs that are recurrently hypermutated, with an average of 12 hypermutated SE/case. Hypermutated SEs are predominantly linked to genes encoding regulators of B cell development and well-known lymphoma oncogenes, including BCL6, BCL2 and CXCR4. We showed that specific hotspot mutations prevent binding and transcriptional downregulation by transcriptional repressors leading to target gene dysregulation. Among these, hypermutation of the SEs linked to BCL2 and CXCR4 abrogates the binding of the glucocorticoid receptor (GR)/transcription factor encoded by NR3C1. As a result, BCL2 and CXCR4 escape GR-mediated transcriptional repression leading to their de-regulated expression in germinal center (GC) B cells. Together with the observation that the NR3C1 gene is genetically inactivated in a small fraction of DLBCL, and the relevance of glucocorticoid-based therapy in DLBCL, these results prompted a comprehensive analysis of the role of GR in normal and malignant B cells. We report that GR is detectable in the nucleus of the great majority of GC B cells, suggesting that it is active as a transcription factor. Similarly, DLBCL primary cases (including GCB-, ABC- and unclassified DLBCL) display GR nuclear expression. Analysis of a conditional GC-specific Nr3c1 knock-out (KO) mouse model showed that GC B cells form normally in the absence of Nr3c1. However, the transcriptional profile of Nr3c1-KO GC B cells was significantly altered compared to wild-type cells. By integrating the GR binding profile obtained in normal human GC B cells with the transcripts differentially expressed in Nr3c1-KO GC B cells, we identified a set of about 2,000 genes that are directly modulated by GR during the GC reaction. Pathway enrichment analysis showed that GR controls several signaling pathways, suggesting a modulatory role on the BCR and CXCR4 pathways. In addition, GR targets display a significant overlap with the BCL6 transcriptional network, suggesting a cooperative role in GC B cell development. Notably, GR was shown to regulate pathways involved in plasma cell differentiation, while repressing targets associated with memory B cell development. In DLBCL cases, several NR3C1 binding sites are recurrently targeted by mutations, suggesting that genetic impairment of GR activity is heterogeneous and extends beyond CXCR4 and BCL2. Together, these data support a role for the glucocorticoid pathway in GC physiology. Recurrent inactivation of the NR3C1 gene or part of its transcriptional regulatory program may contribute to DLBCL pathogenesis, with implications for presently unknown specific roles of glucocorticoids in the therapeutic regimens for DLBCL. Citation Format: Clarissa Corinaldesi, Antony B Holmes, Elodie Bal, Laura Pasqualucci, Katia Basso, Riccardo Dalla-Favera. Role of glucocorticoid receptor in normal and malignant germinal center B cells [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P04.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

IRF4 Expression in Germinal Center and Lymphoma B Cells Regulates Antigen-Dependent Immune Response

A substantial subset of follicular lymphoma (FL) patients has an early relapse with a poor outcome. We showed that this unfavorable group displays increased expression of IRF4, dysregulated immune signaling and an immunosuppressive microenvironment. At the molecular level, IRF4 controls expression of antigen presentation and immune molecules (e.g. HLA-DR, CD40, PDL1). Furthermore, silencing of IRF4 triggers anti-lymphoma immunity ( Mondello P et al, Blood 2022;140 Suppl 1:168-169). However, little is known on the molecular mechanisms whereby IRF4 controls the crosstalk between B and T cells. To address this question, first we performed RNA-seq, ATAC-seq and CUT&TAG of active (H3K27Ac, H3K4me3) and repressive (H3K27me3, H3K4me1) histone marks in TMD8 and HBL1 (lymphoma cells with high IRF4 expression) transfected with two pairs of siIRF4 or siCtr for 72 hours. Gene expression revealed a broad change in transcription, with 432 upregulated genes and 136 downregulated genes in siIRF4 compared to siCtr. Notably, the genes with increased expression following IRF4 silencing coded for antigen presentation, immune molecules and cytokines (e.g. HLA-DR, CD86, IL6, IL10). Accordingly, we observed significant enrichment of antigen presentation, interferon and immune response signatures ( Fig 1A). Integrating RNA-seq with chromatin accessibility showed a significant shift towards transcriptional upregulation at genes nearest to upregulated ATAC peaks, with significant enrichment at enhancers (n=771). Conversely, transcriptional suppression was enriched at enhancers (n=2,458) of downregulated peaks. k-means analyses of all union peaks, generated by merging ATAC with CUT&TAG peaks, revealed a significant gain of H3K4me3, with concordant loss of H3K4me1 at enhancers with upregulated gene expression. In contrast, H3K4me3 was severely decreased at suppressed peaks. TF motif analysis revealed an enrichment for PU.1 and E2A at IRF4-bound regions, suggesting their cooperation in regulating immune signaling. Since antigen presentation and other immune molecules are critical components of the immune synapse, we explored the role of IRF4 on the interaction between germinal center (GC) B and T cells by crossing irf4 fl/fl mice with conditional deletion of irf4 ( Klein et al, Nat Immunol 2006) and Cγ 1cre mice ( Cγ 1cre; irf4 fl/fl) to induce recombination in GC B cells. After 10 days of immunization (GC reaction peak), Cγ 1cre; irf4 fl/fl and Cγ 1cre; irf4 fl/+ GC B cells showed significant increase of HLA-DR and CD40 expression compared to WT. As expected, Cγ 1cre; irf4 fl/fl mice displayed a significantly skewed light-zone:dark-zone ratio in favor of increased dark-zone, which are the cells that most reflect the action of BCL6, with concordant suppression of plasma cells. Additionally, we observed a significant increase of CD4 + T cells after partial or complete deletion of irf4, with upregulation of T FH cells and decrease of T reg cells ( Fig 1B). Notably, mice with loss of irf4 showed a significant decrease in exhausted T FH cells. To test if this effect was dependent on MHC:TCR interaction, we cocultured CD4 + T cells with TMD8 cells transfected with two siIRF4 or siCtr in presence of blocking antibodies for MHC class I, MHC class II or both. Blocking one or the other MHC class I or II partially rescued the IRF4 effect on T reg and T FH cells respectively as well as reduced anti-lymphoma cytotoxicity. Further experiments blocking IRF4-dependent IL6 and IL10 identified these two cytokines as responsible for the additional effects on T cells. To confirm this as an antigen-dependent immune response we coculture sorted GC B cells from NP-OVA-immunized Cγ 1cre; irf4 fl/fl, Cγ 1cre; irf4 fl/+ and WT mice with CSFE-labeled OVA-specific OT-II CD4 + T cells at a ratio of 2:1 in an organoid system for 5 days. We observed induced antigen specific proliferation of CD4 + T cells in the presence of Cγ 1cre; irf4 fl/fl and Cγ 1cre; irf4 fl/+ GC B cellscompared to WT,as indicated by decrease in CSFE expression . We confirmed an increase of T FH cells and a decrease of T reg cells, supporting the conclusion that IRF4 directly controls the antigen-dependent immune response . In summary, IRF4 expression in GC and lymphoma B cells regulates immune signaling with PU.1 and E2A, thereby modulating antigen-dependent immune responses with potential implications for lymphomagenesis. Future studies investigating targeting of IRF4 in lymphoma are warranted.

Anti CD161 Antibody IMT-009 Is a Novel Immunotherapeutic Agent That Effectively Blocks the Inhibitory CLEC2D/CD161 Axis in CLEC2D+ B Cell Hematological Malignancies Reinvigorating T and NK Cell Function Leading to Anti-Tumor Benefit

Background: The CLEC2D/CD161 axis is a ligand-receptor pathway that has been shown to inhibit both T and NK cell function 1,2. CD161 is a C-type lectin-like receptor, which is expressed on NK cells and subsets of both CD4 + and CD8 + T cells. The tumor infiltrating CD161+ T cells have high cytotoxic potential and display an NK cell-like signature 2. Its ligand CLEC2D is expressed on the surface of both malignant cells and immune cells including germinal center B cells, activated T cells and tumor associated macrophages. Owing to its expression in germinal center B cells, CLEC2D is highly expressed in multiple types of B cell lymphomas including Diffuse Large B Cell Lymphoma (DLBCL), Follicular Lymphoma and Burkitt lymphoma 3. Furthermore, subset of infiltrated T cells in these lymphomas are CD161+ making this axis a promising novel target for immunotherapeutic intervention in hematological malignancies (Fig1). Immunitas has developed IMT-009, a monoclonal, Fc silent, fully human IgG1, high affinity antibody to CD161 that selectively blocks its interaction with CLEC2D. We show that IMT-009 effectively reverses CLEC2D-mediated inhibition and restores NK cell and T cell functions, including antigen specific T cell mediated cytotoxicity to lymphoma cell lines. Furthermore, IMT-009 shows monotherapy benefit in reducing tumor burden in a human B cell lymphoma model engrafted in humanized mice in vivo. IMT-009 is currently being evaluated in a Phase 1/2a clinical trial in both solid tumors and heme malignancies. Results: IMT-009 is a highly selective antibody to CD161 that strongly blocks its interaction with CLEC2D. In presence of CLEC2D-expressing target cells, NK cell degranulation, cytokine production and cellular cytotoxicity towards lymphoma tumor targets is highly suppressed which is overcome by treatment with IMT-009. Similarly, IMT-009 reversed CLEC2D-mediated inhibition and restored T cell activity (TCR signaling, cytokine production) in a Jurkat cell reporter system, and enhanced polyfunctionality of primary antigen-specific human T cells shown by secretion of TNF-α, IL-2, and IFN-γ, and direct T cell mediated cytotoxicity. IMT-009 also released CD161-mediated suppression of antigen recall response by PBMC derived effector memory CD161+ CD4+ T cells, resulting in an increased frequency of CD161+ IFN-γ+ cells and an increase in their proliferation. These data together show that IMT-009 is highly effective in overcoming CLEC2D mediated suppression of both T and NK cell functions. To prioritize indications that will likely benefit from IMT-009, we performed IHC for CLEC2D in a diverse set of tumor indications and found that B cell lymphomas of various subtypes have high CLEC2D protein expression by H score. Also, DLBCL was found to have the second highest median RNA expression of CLEC2D in TCGA. Furthermore, analysis of publicly available single-cell RNA-sequencing (scRNA-seq) data from B cell lymphoma including CNS lymphoma revealed CLEC2D to be highly expressed within the B lineage cells including the abundant malignant cell population (Fig2). KLRB1 (gene encoding for CD161) was also found to be expressed on a subset of tumor infiltrating CD4 and CD8 T cells in these datasets (Fig2). To confirm this, we tested CD161 expression in B cell lymphomas by immunofluorescence. Results showed CD161+ cells infiltrated in Hodgkin Lymphoma, and post-R-CHOP treated DLBCL, highlighting the potential sensitivity of these malignancies to IMT-009 treatment. Together, these results indicate that B cell lymphomas may exhibit an immunosuppressive environment mediated by the CLEC2D/CD161 axis. To test the anti-tumor benefit of IMT-009 in a human B cell lymphoma model, we treated CD34+ humanized mice implanted with Raji, a Burkitt lymphoma cell line with IMT-009. The group of mice treated with IMT-009 had a mean tumor growth inhibition of 35% and the anti-tumor response correlated with increased frequency of CD161+ T and NK cells. We further show that IMT-009 can synergize with Rituximab in NK cell mediated killing of B cell lymphoma cell lines in vitro. Conclusion: B cell malignancies tend to respond poorly to anti-PD-1 therapy 4,5. This lack of efficacy could be driven by a highly immunosuppressive environment mediated by the CLEC2D/CD161 axis. Our results support evaluation of IMT-009 as a novel cancer immunotherapy to disrupt CLEC2D/CD161 signaling, with strong therapeutic potential in lymphomas.

Smarca4 (Brg1) Controls Germinal Center B-Cell Development and Is a Haploinsufficient Tumor Suppressor in Lymphoma

SMARCA4 encodes one of two mutually exclusive ATPases present in BAF (aka SWI/SNF) chromatin remodeling complexes. SMARCA4 is dynamically expressed during normal B-cell development, with high expression in germinal center B (GCB)-cells, and is targeted by heterozygous inactivating mutations in GCB-derived lymphomas including 34% of pediatric Burkitt lymphoma (BL), 17% of adult BL, 4% of GCB-DLBCL. However, the functional role of SMARCA4 in GCB cell development and malignant transformation has not been thoroughly investigated. We generated a novel floxed Smarca4 transgenic mouse model and crossed to the Aid-Cre line for GC-specific conditional knock-out (cKO). Following immunization with sheep red blood cells (SRBCs) to induce a GC reaction, Smarca4+/- mice had a significant increase in GCB-cell abundance compared to Smarca4+/+ littermates (p<0.001) due to both an increased average GC size and number. Notably, Smarca4-/- mice displayed a significantly reduced frequency of GC B-cells (p<0.001) and sorting of residual cells revealed retention of 1 or more copies of the floxed allele, suggesting that biallelic cKO of Smarca4 is lethal to GCB cells. We therefore focused subsequent studies on comparisons between Smarca4+/- and Smarca4+/+ mice. Further analysis revealed significant polarization towards a centroblastic phenotype in Smarca4+/- mice (p<0.001), in line with the centroblastic morphology of human BL. Analysis of competitive advantage with the same GC using bone marrow chimera models showed a significant advantage of Smarca4+/- GCB cells compared to Smarca4+/+, associated with a significant increase in proliferation measured by Edu incorporation and reduction of apoptosis measured by Annexin-B. We extended upon this using single cell RNA-seq (scRNA-seq) of GCB cells from Smarca4+/- and Smarca4+/+ mice, which confirmed an increased centroblastic polarization of Smarca4+/- GCB cells and identified a significant increase in light zone to dark zone recycling cells that may underly this polarization. We validated this using the Myc-GFP allele, which confirmed increased Myc+ recycling cells in GCs from Smarca4+/- mice compared to Smarca4-/- mice. BAF complexes drive ATP-dependent mobilization and eviction of nucleosomes to convert chromatin from inactive to active states. Brg1 deficiency may therefore alter distinct BAF complexes and transcription factor programs. We therefore explored the role of Smarca4 in regulating chromatin accessibility and gene expression using ATAC-seq and RNA-seq in Smarca4+/- and Smarca4+/+ murine GCB-cells. Supervised analysis revealed predominant loss of chromatin accessibility associated with repression of nearby genes. Smarca4+/- cells showed significant loss of accessibility of genes upregulated in centrocytes, and a preferential loss of NF-ĸB target genes (GSEA FDR<0.01). MYC translocations are pathognomonic for BL, in which SMARCA4 mutations are most frequent. We therefore crossed our Smarca4 cKO mice with a cre-inducible Myc knock-in (Myc-KI) allele to mimic combined Smarca4 loss-of-function and Myc activation. The addition of Myc-KI did not lead to a significant increase in GCB cell frequency following a single SRBC immunization. However, serial re-challenge of mice with SRBCs led to lymphadenopathy, splenomegaly and infiltration of highly proliferative B-cells into the major organs (lung, liver, and kidney) in Smarca4+/- + Myc-KI mice following 3 immunizations that was not observed with Smarca4+/- or Myc-KI alleles alone. Furthermore, long term follow-up of serially-challenged mice showed a significant reduction in overall survival in Smarca4+/- + Myc-KI mice compared to Smarca4+/- or Myc-KI mice (p=0.010). Collectively, these data demonstrate a competitive advantage of GCB cells with monoallelic Smarca4 cKO, and a centroblastic GC polarization driven in part by increased recycling. Mechanistically, this is associated with loss of chromatin accessibility and gene expression of genes that are expressed in centrocytes, including NFkB target genes. Monoallelic Smarca4 cKO cooperates with Myc over-expression to drive expansion and extranodal dissemination of GCB cells following serial re-challenge, leading to reduced survival. These data provide the first functional and mechanistic insight into the role of SMARCA4 in GCB cell development and lymphomagenesis.

Mechanism of CD79A and CD79B Support for IgM+ B Cell Fitness through B Cell Receptor Surface Expression.

The BCR consists of surface-bound Ig and a heterodimeric signaling unit comprised of CD79A and CD79B. Upon cognate Ag recognition, the receptor initiates important signals for B cell development and function. The receptor also conveys Ag-independent survival signals termed tonic signaling. Although the requirement of a CD79A/CD79B heterodimer for BCR complex assembly and surface expression is well established based on mice models, few studies have investigated this in human mature B cells. In this study, we found that human tonsillar B cells with high surface expression of IgM or IgG had potentiated BCR signaling compared with BCRlow cells, and high IgM expression in germinal center B cells was associated with reduced apoptosis. We explored the mechanism for IgM surface expression by CRISPR/Cas9-induced deletion of CD79A or CD79B in four B lymphoma cell lines. Deletion of either CD79 protein caused loss of surface IgM in all cell lines and reduced fitness in three. From two cell lines, we generated stable CD79A or CD79B knockout clones and demonstrated that loss of CD79A or CD79B caused a block in N-glycan maturation and accumulation of immature proteins, compatible with retention of BCR components in the endoplasmic reticulum. Rescue experiments with CD79B wild-type restored surface expression of CD79A and IgM with mature glycosylation, whereas a naturally occurring CD79B G137S mutant disrupting CD79A/CD79B heterodimerization did not. Our study highlights that CD79A and CD79B are required for surface IgM expression in human B cells and illuminates the importance of the IgM expression level for signaling and fitness.

Abstract 2655: The GPCR OR13A1 is essential for lymphoma cells

Abstract Background. G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to endogenous (amines, cations, lipids, peptides, glycoproteins) and exogenous (light, tastants, odorants) stimuli. Altered signaling pathways associated with GPCRs are implicated in the pathophysiology of various diseases, such as CXCR3/4/5, S1PR1/2/3, GPR34 in lymphomas. Olfactory receptors (ORs) are GPCRs, initially identified and characterized for their expression in olfactory neurons but now recognized as expressed in different tissues. OR51E1 is known as prostate specific G protein coupled receptor 2 (PSGR2) due to its expression in prostate cancer, OR2B6 and OR2W3 are expressed in breast cancer, and OR51E1 in melanoma. OR13A1 has been included in the double-hit expression signature reported in diffuse large B cell lymphoma (DLBCL) (Ennishi et al 2019). Here, we studied OR13A1 in lymphoma cell lines and clinical specimens. Methods. RNA-Seq and real-time PCR for evaluating the expression of ORs in DLBCL cell lines. Gene expression data for clinical samples obtained from public dataset phs001444.v2.p1. OR13A1 silencing performed with both siRNAs (OCI-Ly1, U2932) and doxycycline-inducible shRNAs (U2932); viability assessed after 3 days using MTT and for longer with Incucyte live imaging. Computer modelling performed based on available ligands for the murine orthologue Olfr211 and similar known GPCRs structure. Results. Only one OR per cell line was usually expressed, as reported for olfactory cells. Using RNA-Seq and real-time PCR, OR13A1 gene appeared the most abundantly expressed in lymphomas cell lines compared to others ORs. Its expression was seen in 35/47 B cell lymphoma lines, with higher expression in germinal center B cell (GCB) DLBCL than activated B cell like (ABC) DLBCL (P 0.03). A similar pattern was observed in patients: higher expression in GCB than ABC and in EZB than MCD (P &amp;lt; 0.001). OR13A1 also appeared higher in normal GC cells than in naïve and memory B cells. Among GCB DLBCL patients, OR13A1 expression was associated with inferior outcome. Silencing of OR13A1 with siRNAs in GCB and ABC DLBCL cells caused &amp;gt; 50% reduction in cell viability, and pERK downregulation. Viability data were confirmed using a doxycycline-inducible shRNA system and live cell imaging instrument. Natural compounds reported as ligands for the murine orthologue Olfr211 or in silico identified to bind as agonists (ex: tyramine) increased cell line growth and compounds predicted to bind as antagonists (ex: cyclohexanone) decreased the cell viability in the presence but not in the absence of the GPCR. Conclusion. OR13A1 gene appeared more abundantly expressed in lymphomas compared to others ORs, higher in GCB than ABC DLBCL and with a possible impact on clinical outcome. Genetic and chemical experiments showed that this GPCR has an essential function in lymphoma cells and could represent a novel therapeutic target. Citation Format: Giulio Sartori, Luciano Cascione, Milos Matkovic, Hiroaki Matsunami, Andrea Cavalli, Francesco Bertoni. The GPCR OR13A1 is essential for lymphoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2655.

Single-cell analysis of human B cell maturation predicts how antibody class switching shapes selection dynamics.

Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre-germinal center state primed to undergo class switch recombination and dissect how antibody class-dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell-mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection.

A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting invivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

CD30 Expression in Germinal Center B-cell-like and non-Germinal Center B-cell-like Subtypes of Diffuse Large B-cell Lymphoma

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common and most heterogeneous type of non-Hodgkin lymphoma. With current therapeutic modalities, 30%–40% of DLBCL cases still experience recurrence. The discovery of CD30 expression in DLBCL in several studies has opened up alternative opportunities for new target therapies.&#x0D; AIM: This cross-sectional study aimed to determine CD30 expression in DLBCL and its difference in expression in germinal center B-cell such as (GCB) and non-GCB subtypes.&#x0D; METHODS: The sample consisted of 25 GCB and 25 non-GCB cases based on immunohistochemical examination performed at Cipto Mangunkusumo Hospital from 2014 to 2017. CD30 staining was carried out and assessed using tumor cells percentage with positive cutoff values of &gt;0%, &gt;10%, and &gt;20%.&#x0D; RESULTS: Positive CD30 expression was obtained in 8 (16%), 4 (8%), and 3 (6%), out of 50 DLBCL cases with positive cutoff values of &gt;0%, &gt;10%, and &gt;20%, respectively. We performed Fisher’s exact test to determine CD30 expression in the GCB and non-GCB subtypes and found no significant difference with p &gt; 0.05.&#x0D; CONCLUSION: Our study found no significant difference between CD30 expression in the GCB and non-GCB subtypes of DLBCL.

Abstract 1744: SAMHD1 expression and impact on clinical outcome in diffuse large B-cell lymphoma: a potential therapeutic target

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. While standard treatment with rituximab, cyclophosphamide, doxorubicin, vincris- tine, and prednisone (R-CHOP) can cure DLBCL, failed patients have poor outcome. Thus, a search for stand-alone or adjuvant therapy to improve patient’s outcome is important. Sam and HD domain-containing protein 1 (SAMHD1) is a well-established deoxyribonucleotide triphosphate (dNTP) triphosphohydrolase. Its dNTPase activity has been associated with cellular defence mechanism against infection by depleting cellular dNTP pool bellow level required for viral replication. SAMHD1 mutations have been associated with Aicardi Goutières (AGS) and cancers such as chronic lymphocytic leukemia (CLL), demonstrating its relevance beyond viral infection. In addition, it has been shown that SAMHD1 expression in Recently, our lab has defined a novel role for SAMHD1 in DNA end resection to facilitate DNA double strand break (DSB) repair by homologous recombination (HR). SAMHD1 expression and its diagnostic or therapeutic relevance in DLBCL is not known. Thus, we analyzed SAMHD1 expression in patients with DLBCL and determined its correlation with clinical outcome. In addition, we studied the SAMHD1 expression in germinal center B cell (GCB) and activated B cell (ABC) DLBCL cells lines. Also, we examined the possibility of depleting SAMHD1 using a virus like particles (VLPs) containing viral protein x (Vpx) in DLBCL cell lines. Finally, we determined the effect on cells sensitivity to standard DLBCL therapy, R-CHOP, and other DNA damage inducing agents. Low SAMHD1 expression correlates with improved overall survival. SAMHD1 was downregulated in ABC as compared to GCB DLBCL cell lines and SAMHD1 depletion sensitizes DLBCL cell lines to R-CHOP and other DNA damage inducing agents. These findings suggest that SAMHD1 is a potential therapeutic target to improve the DLBCL patient’s clinical outcome. Citation Format: Waaqo Daddacha. SAMHD1 expression and impact on clinical outcome in diffuse large B-cell lymphoma: a potential therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1744.

B Cell Fcγ Receptor IIb Modulates Atherosclerosis in Male and Female Mice by Controlling Adaptive Germinal Center and Innate B-1-Cell Responses.

Objective- Investigate the impact of modulating B cell FcγRIIb (Fcγ receptor IIb) expression on atherosclerosis. Approach and Results- Western diet-induced atherosclerosis was assessed in Ldlr-/- or Apoe-/- mice with B cell-specific overexpression of FcγRIIb or with an FcγRIIb promoter mutation that alters FcγRIIb expression in germinal center (GC) B cells. In males, overexpression of FcγRIIb on B cells severely reduced activated, class switched B cell responses, as indicated by reductions in GC B cells, plasma cells, and serum IgG but not IgM antibodies. Male mice overexpressing FcγRIIb developed less atherosclerosis, suggesting a pathogenic role for GC B cell IgG responses. In support of this hypothesis, male mice with a promoter polymorphism-driven reduction in FcγRIIb on GC B cells but not plasma cells have a converse phenotype of enhanced GC responses and IgG2c antibodies and enhanced atherosclerosis. IgG2c significantly enhanced TNF (tumor necrosis factor) secretion by CD11b+ CD11c+ cells expressing the high-affinity receptor FcγRIV. In females, overexpression of FcγRIIb on B cells not only reduced GC B cell responses but also substantially reduced B-1 cells and IgM antibodies, which translated into acceleration of atherosclerosis. Promoter-driven reduction in FcγRIIb did not alter GC B cell responses in females and, therefore, had no impact on atherosclerosis. Conclusions- B cell FcγRIIb differentially alters proatherogenic adaptive GC B cell and atheroprotective innate B-1 responses in male and female mice fed a western diet. Our results highlight the importance of a better understanding and ability to selectively target B cell responses in future immunotherapeutic approaches against human cardiovascular disease. Visual Overview- An online visual overview is available for this article.

Mouse Models for Immunology Research available from The Jackson Laboratory Repository

Abstract The Jackson Laboratory Repository (JAX) serves as a centralized facility for the development, distribution and cryopreservation of mouse models of human biology and disease. JAX distributes more than 10,500 strains to the scientific community, many with applications in immunology research, including mouse models for autoimmune disease, infectious disease, immunodeficient platforms for PDX studies, and multi-purpose “tool strains” (such as CRISPR cas9-expressing lines), recombinase expressing strains, as well as conditional and inducible expression lines. One of our newest strains is a conditional knock-out (KO) of STING/MYPS, a pathogen sensor involved in the activation of IL6 and IFNb. Additions to our extensive set of interleukin alleles include a SMART-17A knock-in (KI) expressing human NGFR on IL17A expressing cells and a KI/KO of Il9-expressing iCre/EYFP. The latest reporter and cre-expressing strains include an inducible cre KO/KI to Lyz2, and a KI/KO strain with dual reporters of chemokine Ccr2RFP and Cx3cr1GFPactivity, and two strains expressing the INDIA apoptosis reporter, one with widespread conditional expression and the other with expression in germinal center B cells. KOMP mice, offered via the MMRRC portion of the JAX Mouse Repository, provide a growing number of targeted immunological mutations. Donating a strain to the Repository fulfills NIH’s requirements for sharing mice. Researchers wishing to have mice considered for inclusion in the Repository are encouraged to submit strains: www.jax.org/donate-a-mouse. This work is supported by NIH, HHMI and private foundations.

A novel allele for inducible Cre expression in germinal center Bcells.

The germinal center reaction is essential for efficient humoral immunity, but it can also give rise to Bcell lymphomas. Cre/loxP-mediated conditional gene knock-out or knock-in can be used for the genetic manipulation of germinal center Bcells in vivo. Here we present a novel allele, Cγ1-CreERT2, that allows for timed activation of Cre recombinase in a small fraction of germinal center Bcells. This allele will be useful to study normal and malignant germinal center Bcell development in vivo.

Cell Cycle-Associated CXCR4 Expression in Germinal Center B Cells and Its Implications on Affinity Maturation.

Adaptation of antibody-mediated immunity occurs in germinal centers (GC). It is where affinity maturation, class switching, memory and plasma cell differentiation synergize to generate specific high-affinity antibodies that aid both to clear and protect against reinfection of invading pathogens. Within GCs, light and dark zone are two compartments instrumental in regulating this process, by segregating T cell-dependent selection and differentiation from generation of GC B cells bearing hypermutated antigen receptors. Spatial segregation of GC B cells into the two zones relies on the chemokine receptor CXCR4, with textbooks attributing high and low expression to a dark and light zone phenotype. Interestingly, this bipolarity is not reflected in the CXCR4 expression profile of GC B cells, which is highly variable and unimodal, indicating a continuum of intermediate CXCR4 levels rather than a binary dark or light zone phenotype. Here, analysis of published BrdU pulse-chase data reveals that throughout cell cycle, average CXCR4 expression in GC B cells steadily increases close to twofold, scaling with cell surface area. CXCR4 expression in recently divided GC B cells in G0/G1 or early S phase shows intermediate levels compared to cells in G2M phase, consistent with their smaller size. The lowest number of CXCR4 receptors are displayed by relatively aged GC B cells in G0/G1 or early S phase. The latter, upon progressing through S phase, however, ramp up relative CXCR4 expression twice as much as recently divided cells. Twelve hours after the BrdU pulse, labeled GC B cells, while initially in S phase, are desynchronized in terms of cell cycle and match the CXCR4 profile of unlabeled cells. A model is discussed in which CXCR4 expression in GC B cell increases with cell cycle and cell surface area, with highest levels in G2 and M phase, coinciding with GC B cell receptor signaling in G2 and immediately preceding activation-induced cytidine deaminase (AID) activity in early G1. In the model, GC B cells compete for CXCL12 expression on the basis of their CXCR4 expression, gaining a relative advantage as they progress in cell cycle, but loosing the advantage at the moment they divide.

Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia is a disease with up-regulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and CXCL12 as well as enhanced B-cell receptor signaling pathway activation demonstrated by increased PLCγ2 and SYK phosphorylation after IgM stimulation. COBLL1 expression also changes during B-cell maturation in non-malignant secondary lymphoid tissue with a higher expression in germinal center B cells than naïve and memory B cells. Our data thus suggest COBLL1 involvement not only in chronic lymphocytic leukemia but also in B-cell development. In summary, we show that expression of COBLL1, encoding novel ROR1-binding partner, defines chronic lymphocytic leukemia subgroups with a distinct response to microenvironmental stimuli, and independently predicts survival of chronic lymphocytic leukemia with unmutated IGHV.

B Cell-Intrinsic Expression of the HuR RNA-Binding Protein Is Required for the T Cell-Dependent Immune Response In Vivo.

The HuR RNA-binding protein posttranscriptionally controls expression of genes involved in cellular survival, proliferation, and differentiation. To determine roles of HuR in B cell development and function, we analyzed mice with B lineage-specific deletion of the HuR gene. These HuRΔ/Δ mice have reduced numbers of immature bone marrow and mature splenic B cells, with only the former rescued by p53 inactivation, indicating that HuR supports B lineage cells through developmental stage-specific mechanisms. Upon in vitro activation, HuRΔ/Δ B cells have a mild proliferation defect and impaired ability to produce mRNAs that encode IgH chains of secreted Abs, but no deficiencies in survival, isotype switching, or expression of germinal center (GC) markers. In contrast, HuRΔ/Δ mice have minimal serum titers of all Ab isotypes, decreased numbers of GC and plasma B cells, and few peritoneal B-1 B cells. Moreover, HuRΔ/Δ mice have severely decreased GCs, T follicular helper cells, and high-affinity Abs after immunization with a T cell-dependent Ag. This failure of HuRΔ/Δ mice to mount a T cell-dependent Ab response contrasts with the ability of HuRΔ/Δ B cells to become GC-like in vitro, indicating that HuR is essential for aspects of B cell activation unique to the in vivo environment. Consistent with this notion, we find in vitro stimulated HuRΔ/Δ B cells exhibit modestly reduced surface expression of costimulatory molecules whose expression is similarly decreased in humans with common variable immunodeficiency. HuRΔ/Δ mice provide a model to identify B cell-intrinsic factors that promote T cell-dependent immune responses in vivo.

Regulation of the HSH2 adaptor protein turnover via an indirect NF-κB-dependent mechanism is involved in determining the nature of the class switched antibody response. (IRM10P.609)

Abstract The Hematopoietic SH2 containing adaptor protein (HSH2) is differentially expressed in mature, peripheral B cell subpopulations, exhibiting low level expression in germinal center (GC) B cells versus high-level expression in B1 and marginal zone B cells. Studies have demonstrated an inverse relationship between its expression and the production of class-switched antibodies due to effects on terminal differentiation. Therefore, delineation of molecular mechanism that controls its expression is critical for understanding the class-switched antibody response. Studies demonstrated that upregulation of HSH2 following TNF family receptor or TLR-mediated signals, as well as its maintenance, requires NF-κB activity. Using both pharmacological and genetic approaches, it was shown that the canonical pathway is critical for maintenance of HSH2 expression. Inhibition of NF-kB selectively results in the rapid loss of HSH2, whereas actinomycin D and cycloheximide treatment do not. Analysis of the canonical pathway revealed that maintenance of HSH2 requires translocation of NF-κB to the nucleus. Because HSH2 mRNA levels are not altered under any conditions, it is hypothesized that NF-κB indirectly regulates the function of a short-lived protease that regulates HSH2 expression at the protein. These results reveal a potentially novel mechanism, in which NF-κB regulates HSH2 expression via one or more proteases and may represent a novel paradigm for controlling GC B cell function.