The histone acetyltransferase KAT6B is required for hematopoietic stem cell development and function.

The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.

An arrayed CRISPR screen of primary B cells reveals the essential elements of the antibody secretion pathway.

Background: Humoral immunity depends on the differentiation of B cells into antibody secreting cells (ASCs). Excess or inappropriate ASC differentiation can lead to antibody-mediated autoimmune diseases, while impaired differentiation results in immunodeficiency. Methods: We have used CRISPR/Cas9 technology in primary B cells to screen for regulators of terminal differentiation and antibody production. Results: We identified several new positive (Sec61a1, Hspa5) and negative (Arhgef18, Pold1, Pax5, Ets1) regulators that impacted on the differentiation process. Other genes limited the proliferative capacity of activated B cells (Sumo2, Vcp, Selk). The largest number of genes identified in this screen (35) were required for antibody secretion. These included genes involved in endoplasmic reticulum-associated degradation and the unfolded protein response, as well as post-translational protein modifications. Discussion: The genes identified in this study represent weak links in the antibody-secretion pathway that are potential drug targets for antibody-mediated diseases, as well as candidates for genes whose mutation results in primary immune deficiency.

Epigenetic modulators of B cell fate identified through coupled phenotype-transcriptome analysis.

High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.

OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

IRF4 Activity Is Required in Established Plasma Cells to Regulate Gene Transcription and Mitochondrial Homeostasis.

The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.

New players in the gene regulatory network controlling late B cell differentiation.

The differentiation of B cells into antibody-secreting plasma cells is associated with profound changes in morphology, lifespan, and cellular metabolism that are needed to support high rates of antibody production. These processes are driven by dramatic alterations to the transcriptional program and to the organization of the nucleus itself that in turn are regulated by the activity of a select group of transcription factors and epigenetic regulators. Although the core differentiation program is conserved in all mature B cells, subset-specific regulators, such as those found in B1 or memory B cells, provide additional complexity. Here, we review the key components of the gene regulatory network controlling B-cell terminal differentiation, with an emphasis on the new players and processes that have emerged in recent years.

A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets.

NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCRB6C14R strain. Ly5.1C14R NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46C14R in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1C14R mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a+ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function. Significance Innate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells in vivo. This Ncr1 C14R mutation impairs NKp46 surface expression resulting in destabilization of Ncr1 and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46+ ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively.

IMiDs prime myeloma cells for daratumumab-mediated cytotoxicity through loss of Ikaros and Aiolos.

Recent studies have demonstrated that the immunomodulatory drugs (IMiDs) lead to the degradation of the transcription factors Ikaros and Aiolos. However, why their loss subsequently leads to multiple myeloma (MM) cell death remains unclear. Using CRISPR-Cas9 genome editing, we have deleted IKZF1/Ikaros and IKZF3/Aiolos in human MM cell lines to gain further insight into their downstream gene regulatory networks. Inactivation of either factor alone recapitulates the cell intrinsic action of the IMiDs, resulting in cell cycle arrest and induction of apoptosis. Furthermore, evaluation of the transcriptional changes resulting from their loss demonstrates striking overlap with lenalidomide treatment. This was not dependent on reduction of the IRF4-MYC "axis," as neither protein was consistently downregulated, despite cell death occurring, and overexpression of either factor failed to rescue for Ikaros loss. Importantly, Ikaros and Aiolos repress the expression of interferon-stimulated genes (ISGs), including CD38, and their loss led to the activation of an interferon-like response, contributing to MM cell death. Ikaros/Aiolos repressed CD38 expression through interaction with the nucleosome remodeling and deacetylase complex in MM. IMiD-induced loss of Ikaros or treatment with interferon resulted in an upregulation of CD38 surface expression on MM cells, priming for daratumumab-induced NK cell-mediated antibody-dependent cellular cytotoxicity. These results give further insight into the mechanism of action of the IMiDs and provide mechanistic rationale for combination with anti-CD38 monoclonal antibodies.

Mining the Plasma Cell Transcriptome for Novel Cell Surface Proteins.

Antibody Secreting Cells (ASCs) are a fundamental component of humoral immunity, however, deregulated or excessive antibody production contributes to the pathology of autoimmune diseases, while transformation of ASCs results in the malignancy Multiple Myeloma (MM). Despite substantial recent improvements in treating these conditions, there is as yet no widely used ASC-specific therapeutic approach, highlighting a critical need to identify novel methods of targeting normal and malignant ASCs. Surface molecules specifically expressed by the target cell population represent ideal candidates for a monoclonal antibody-based therapy. By interrogating the ASC gene signature that we previously defined we identified three surface proteins, Plpp5, Clptm1l and Itm2c, which represent potential targets for novel MM treatments. Plpp5, Clptm1l and Itm2c are highly and selectively expressed by mouse and human ASCs as well as MM cells. To investigate the function of these proteins within the humoral immune system we have generated three novel mouse strains, each carrying a loss-of-function mutation in either Plpp5, Clptm1l or Itm2c. Through analysis of these novel strains, we have shown that Plpp5, Clptm1l and Itm2c are dispensable for the development, maturation and differentiation of B-lymphocytes, and for the production of antibodies by ASCs. As adult mice lacking either protein showed no apparent disease phenotypes, it is likely that targeting these molecules on ASCs will have minimal on-target adverse effects.

Environmental sensing by mature B cells is controlled by the transcription factors PU.1 and SpiB.

Humoral immunity requires B cells to respond to multiple stimuli, including antigen, membrane and soluble ligands, and microbial products. Ets family transcription factors regulate many aspects of haematopoiesis, although their functions in humoral immunity are difficult to decipher as a result of redundancy between the family members. Here we show that mice lacking both PU.1 and SpiB in mature B cells do not generate germinal centers and high-affinity antibody after protein immunization. PU.1 and SpiB double-deficient B cells have a survival defect after engagement of CD40 or Toll-like receptors (TLR), despite paradoxically enhanced plasma cell differentiation. PU.1 and SpiB regulate the expression of many components of the B cell receptor signaling pathway and the receptors for CD40L, BAFF and TLR ligands. Thus, PU.1 and SpiB enable B cells to appropriately respond to environmental cues.

NFκB1 is essential to prevent the development of multiorgan autoimmunity by limiting IL-6 production in follicular B cells.

We examined the role of NFκB1 in the homeostasis and function of peripheral follicular (Fo) B cells. Aging mice lacking NFκB1 (Nfκb1(-/-)) develop lymphoproliferative and multiorgan autoimmune disease attributed in large part to the deregulated activity of Nfκb1(-/-)Fo B cells that produce excessive levels of the proinflammatory cytokine interleukin 6 (IL-6). Despite enhanced germinal center (GC) B cell differentiation, the formation of GC structures was severely disrupted in the Nfκb1(-/-)mice. Bone marrow chimeric mice revealed that the Fo B cell-intrinsic loss of NFκB1 led to the spontaneous generation of GC B cells. This was primarily the result of an increase in IL-6 levels, which promotes the differentiation of Fo helper CD4(+)T cells and acts in an autocrine manner to reduce antigen receptor and toll-like receptor activation thresholds in a population of proliferating IgM(+)Nfκb1(-/-)Fo B cells. We demonstrate that p50-NFκB1 represses Il-6 transcription in Fo B cells, with the loss of NFκB1 also resulting in the uncontrolled RELA-driven transcription of Il-6.Collectively, our findings identify a previously unrecognized role for NFκB1 in preventing multiorgan autoimmunity through its negative regulation of Il-6 gene expression in Fo B cells.

Investigating the Antigen Specificity of Multiple Sclerosis Central Nervous System-Derived Immunoglobulins.

The central nervous system (CNS) of patients with multiple sclerosis (MS) is the site where disease pathology is evident. Damaged CNS tissue is commonly associated with immune cell infiltration. This infiltrate often includes B cells that are found in multiple locations throughout the CNS, including the cerebrospinal fluid (CSF), parenchyma, and the meninges, frequently forming tertiary lymphoid structures in the latter. Several groups, including our own, have shown that B cells from distinct locations within the MS CNS are clonally related and display the characteristics of an antigen-driven response. However, the antigen(s) driving this response have yet to be conclusively defined. To explore the antigen specificity of the MS B cell response, we produced recombinant human immunoglobulin (rIgG) from a series of expanded B cell clones that we isolated from the CNS tissue of six MS brains. The specificity of these MS-derived rIgG and control rIgG derived from non-MS tissues was then examined using multiple methodologies that included testing individual candidate antigens, screening with high-throughput antigen arrays and evaluating binding to CNS-derived cell lines. We report that while several MS-derived rIgG recognized particular antigens, including neurofilament light and a protocadherin isoform, none were unique to MS, as non-MS-derived rIgG used as controls invariably displayed similar binding specificities. We conclude that while MS CNS resident B cells display the characteristics of an antigen-driven B cell response, the antigen(s) driving this response remain at large.

Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells.

When B cells encounter an antigen, they alter their physiological state and anatomical localization and initiate a differentiation process that ultimately produces antibody-secreting cells (ASCs). We have defined the transcriptomes of many mature B cell populations and stages of plasma cell differentiation in mice. We provide a molecular signature of ASCs that highlights the stark transcriptional divide between B cells and plasma cells and enables the demarcation of ASCs on the basis of location and maturity. Changes in gene expression correlated with cell-division history and the acquisition of permissive histone modifications, and they included many regulators that had not been previously implicated in B cell differentiation. These findings both highlight and expand the core program that guides B cell terminal differentiation and the production of antibodies.

The transcription factors IRF8 and PU.1 negatively regulate plasma cell differentiation.

Activated B cells undergo immunoglobulin class-switch recombination (CSR) and differentiate into antibody-secreting plasma cells. The distinct transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors: those that maintain the B cell program, including BCL6 and PAX5, and plasma cell-promoting factors, such as IRF4 and BLIMP-1. We show that the complex of IRF8 and PU.1 controls the propensity of B cells to undergo CSR and plasma cell differentiation by concurrently promoting the expression of BCL6 and PAX5 and repressing AID and BLIMP-1. As the PU.1-IRF8 complex functions in a reciprocal manner to IRF4, we propose that concentration-dependent competition between these factors controls B cell terminal differentiation.

Transcription factor IRF4 regulates germinal center cell formation through a B cell-intrinsic mechanism.

In response to antigenic stimulation, mature B cells interact with follicular helper T cells in specialized structures called germinal centers (GCs), which leads to the development of memory B cells and Ab-secreting plasma cells. The transcription factor IFN regulatory factor 4 (IRF4) is essential for the formation of follicular helper T cells and thus GCs, although whether IRF4 plays a distinct role in GC B cells remains contentious. RNAseq analysis on ex vivo-derived mouse B cell populations showed that Irf4 was lowly expressed in naive B cells, highly expressed in plasma cells, but absent from GC B cells. In this study, we used conditional deletion of Irf4 in mature B cells as well as wild-type and Irf4-deficient mixed bone marrow chimeric mice to investigate how and where IRF4 plays its essential role in GC formation. Strikingly, GC formation was severely impaired in mice in which Irf4 was conditionally deleted in mature B cells, after immunization with protein Ags or infection with Leishmania major. This effect was evident as early as day 5 following immunization, before the development of GCs, indicating that Irf4 was required for the development of early GC B cells. This defect was B cell intrinsic because Irf4-deficient B cells in chimeric mice failed to participate in the GC in response to L. major or influenza virus infection. Taken together, these data demonstrate a B cell-intrinsic requirement for IRF4 for not only the development of Ab secreting plasma cells but also for GC formation.

Protein array-based profiling of CSF identifies RBPJ as an autoantigen in multiple sclerosis.

OBJECTIVE: To profile the reactivity of CSF-derived immunoglobulin from patients with multiple sclerosis (MS) against a large panel of antigens, to identify disease-specific reactivities. METHODS: CSF from subjects with MS with elevated immunoglobulin G and CSF from control subjects presenting with other inflammatory neurologic disease were screened against a protein array consisting of 9,393 proteins. Reactivity to a candidate protein identified using these arrays was confirmed with ELISA and immunocytochemistry. RESULTS: Autoantibodies against one protein on the array, recombination signal binding protein for immunoglobulin kappa J region (RBPJ), discriminated between patients with MS and controls (p = 0.0052). Using a large validation cohort, we found a higher prevalence of autoantibodies against RBPJ in the CSF of patients with MS (12.5%) compared with the CSF of patients with other neurologic diseases (1.6%; p = 0.02) by ELISA. This difference in reactivity was restricted to the CSF as serum reactivity against RBPJ did not differ between patients and controls. The presence of CSF autoantibodies against RBPJ was further confirmed by immunocytochemistry. CONCLUSIONS: These data indicate that RBPJ, a ubiquitous protein of the Notch signaling pathway that plays an important role in Epstein-Barr virus infection, is a novel MS autoantigen candidate that is recognized by CSF-derived immunoglobulin G in a subset of patients with MS.

Autoantibodies produced at the site of tissue damage provide evidence of humoral autoimmunity in inclusion body myositis.

Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens.

Related B cell clones that populate the CSF and CNS of patients with multiple sclerosis produce CSF immunoglobulin.

We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.

Related B cell clones populate the meninges and parenchyma of patients with multiple sclerosis.

In the central nervous system of patients with multiple sclerosis, B cell aggregates populate the meninges, raising the central question as to whether these structures relate to the B cell infiltrates found in parenchymal lesions or instead, represent a separate central nervous system immune compartment. We characterized the repertoires derived from meningeal B cell aggregates and the corresponding parenchymal infiltrates from brain tissue derived primarily from patients with progressive multiple sclerosis. The majority of expanded antigen-experienced B cell clones derived from meningeal aggregates were also present in the parenchyma. We extended this investigation to include 20 grey matter specimens containing meninges, 26 inflammatory plaques, 19 areas of normal appearing white matter and cerebral spinal fluid. Analysis of 1833 B cell receptor heavy chain variable region sequences demonstrated that antigen-experienced clones were consistently shared among these distinct compartments. This study establishes a relationship between extraparenchymal lymphoid tissue and parenchymal infiltrates and defines the arrangement of B cell clones that populate the central nervous system of patients with multiple sclerosis.