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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., (© 2022. The Author(s).)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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., (© 2016 de Valle et al.)

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.

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.

To segregate the many contributions that B cell receptor (BCR)-mediated signals make to immune responses, we have analyzed here B cells deficient in the 'pan-leukocyte' marker CD45. BCR ligation of Cd45-/- B cells failed to activate phosphatidylinositol-3-OH kinase, NF-kappaB, Erk1 or Erk2 kinases or to upregulate cell survival proteins and instead induced apoptosis. Immunization of Cd45-/- B cell chimeras induced germinal centers and antigen-specific immunoglobulin G1 antibody-forming cells early, but both cellular compartments decreased by day 14. Proliferation of Cd45-/- B cells induced by CD40 ligand in vitro was impaired as a result of abrogation by BCR ligation of the upregulation of prosurvival proteins. In contrast, enforced expression of the antiapoptotic factor Bcl-xL prevented the collapse of Cd45-/- B cell germinal centers. These results show mechanistic differences in B cell survival during germinal center initiation and propagation; CD40 signaling is sufficient for the former, whereas the latter requires signaling from the BCR.