Regnase-1 is essential for B cell homeostasis to prevent immunopathology.

Regnase-1 is an emerging regulator of immune responses with essential roles in the posttranscriptional control of immune cell activation. Regnase-1 is expressed in B cells; however, its B cell-specific functions remain unknown. Here, we demonstrate that Regnase-1 prevents severe autoimmune pathology and show its essential role in maintaining B cell homeostasis. Using Cre driver mice for ablation of Regnase-1 at various stages of B cell development, we demonstrate that loss of Regnase-1 leads to aberrant B cell activation and differentiation, resulting in systemic autoimmunity and early morbidity. The basis of these findings was informed by gene expression data revealing a regulatory role for Regnase-1 in the suppression of a transcriptional program that promotes B cell activation, survival, and differentiation. Overall, our study shows that Regnase-1 exerts critical control of B cell activation, which is required for prevention of immunopathology.

Quick and easy purification of murine untouched naive B cells or germinal center B cells by MACS.

Humoral immune responses depend on the generation of high-affinity antigen-specific antibodies. Germinal center (GC) B cells are the cornerstone of this response in peripheral lymphoid organs. High purities of GC B cells, and also naive B cells, are required for accurate analysis in downstream assays to yield essential knowledge on immunity. This protocol lays out quick and easy steps to purify GC B cells from spleens of immunized mice or B cells from naive animals by negative selection using MACS. For complete details on the use and execution of this protocol, please refer to Ramezani-Rad et al. (2020).

Cyclin D3 Governs Clonal Expansion of Dark Zone Germinal Center B Cells.

Germinal center (GC) B cells surge in their proliferative capacity, which poses a direct risk for B cell malignancies. G1- to S-phase transition is dependent on the expression and stability of D-type cyclins. We show that cyclin D3 expression specifically regulates dark zone (DZ) GC B cell proliferation. B cell receptor (BCR) stimulation of GC B cells downregulates cyclin D3 but induces c-Myc, which subsequently requires cyclin D3 to exert GC expansion. Control of DZ proliferation requires degradation of cyclin D3, which is dependent on phosphorylation of residue Thr283 and can be bypassed by cyclin D3T283A hyperstabilization as observed in B cell lymphoma. Thereby, selected GC B cells in the light zone potentially require disengagement from BCR signaling to accumulate cyclin D3 and undergo clonal expansion in the DZ.

E3 Ubiquitin Ligase Fbw7 Regulates the Survival of Mature B Cells.

Mature naive B cells expressing BCRs of the IgM and IgD isotypes respond to Ag in secondary lymphoid organs. However, the vast majority of B cells do not undergo productive Ag encounter and have finite life spans dependent on survival signals propagated by the BCR and the BAFFR. In this study, we show that the E3 ubiquitin ligase Fbw7 is required for the maintenance of mature B cell populations in mice. BCR stimulation of B cells induced substantial apoptosis along with proliferative and growth defects upon the loss of Fbw7. Analysis of B cell proteomes revealed aberrant signaling patterns, including lower Bcl2 and diminished NF-κB signaling. Further, excessive accumulation of Fbw7 substrate c-Myc, increased Bim expression, and loss of PI3K signaling mediated apoptosis downstream of BCR signaling. In accordance, strong prosurvival signals delivered through ectopic expression of BCL2 in B cells could largely rescue apoptotic cells in the absence of Fbw7. Overall, this study reveals an unexpected role for Fbw7 in the survival and fitness of mature B cells.

The AKT isoforms 1 and 2 drive B cell fate decisions during the germinal center response.

The PI3K pathway is integral for the germinal center (GC) response. However, the contribution of protein kinase B (AKT) as a PI3K effector in GC B cells remains unknown. Here, we show that mice lacking the AKT1 and AKT2 isoforms in B cells failed to form GCs, which undermined affinity maturation and antibody production in response to immunization. Upon B-cell receptor stimulation, AKT1/2-deficient B cells showed poor survival, reduced proliferation, and impaired mitochondrial and metabolic fitness, which collectively halted GC development. By comparison, Foxo1 T24A mutant, which cannot be inactivated by AKT1/2 phosphorylation and is sequestered in the nucleus, significantly enhanced antibody class switch recombination via induction of activation-induced cytidine deaminase (AID) expression. By contrast, repression of FOXO1 activity by AKT1/2 promoted IRF4-driven plasma cell differentiation. Last, we show that T-cell help via CD40, but not enforced expression of Bcl2, rescued the defective GC response in AKT1/2-deficient animals by restoring proliferative expansion and energy production. Overall, our study provides mechanistic insights into the key role of AKT and downstream pathways on B cell fate decisions during the GC response.

Loss of the FOXP1 Transcription Factor Leads to Deregulation of B Lymphocyte Development and Function at Multiple Stages.

The FOXP1 transcription factor is expressed throughout B cell development until its extinction just prior to terminal differentiation. Foxp1 nulls die of cardiac defects at midgestation, but adult rescue via fetal liver transfer led to a strong pre-B cell block. To circumvent these limitations and to investigate FOXP1 function at later stages of B cell differentiation, we generated and analyzed floxed (F) Foxp1 alleles deleted at pro-B, transitional (T) 1, and mature B cell stages. Mb-1cre-mediated deletion of Foxp1F/F confirmed its requirement for pro-B to pre-B transition. Cd21- and Cd19cre deletion led to significant reduction of germinal center formation and a second block in differentiation at the T2/marginal zone precursor stage. T-dependent and -independent immunization of FOXP1 mutants led to reduction of Ag-specific IgM, whereas responses of class-switched Abs were unimpaired. Yet, unexpectedly, plasmablast and plasma cell numbers were significantly increased by in vitro BCR stimulation of Foxp1F/F splenic follicular B cells but rapidly lost, as they were highly prone to apoptosis. RNA sequencing, gene set enrichment analysis, and chromatin immunoprecipitation sequencing analyses revealed strong enrichment for signatures related to downregulation of immune responses, apoptosis, and germinal center biology, including direct activation of Bcl6 and downregulation of Aicda/AID, the primary effector of somatic hypermutation, and class-switch recombination. These observations support a role for FOXP1 as a direct transcriptional regulator at key steps underlying B cell development in the mouse.

Self-reactive B cells in the GALT are actively curtailed to prevent gut inflammation.

Immune homeostasis in the gut associated lymphoid tissues (GALT) is critical to prevent the development of inadvertent pathologies. B cells as the producers of antibodies and cytokines plays an important role in maintaining the GALT homeostasis. However, the mechanism by which B cells specifically direct their responses towards non-self-antigens and become ignorant to self-antigens in the GALT is not known. Therefore, we developed a novel mouse model by expressing Duck Egg Lysozyme (DEL) in gut epithelial cells in presence of HEL reactive B cells. Notably, we observed a transient activation and rapid deletion of self-reactive B cells in Peyers Patches and Mesenteric lymph nodes upon self-antigen exposure. The survival of self-reactive B cells upon exposure to their self-antigen was partially rescued by blocking receptor editing but could be completely rescued by stronger survival signal like ectopic expression of BCL2. Importantly, rescuing the self-reactive B cells promoted production of auto-antibodies and gut inflammation. Mechanistically, we identify a specific activation of TGFß signaling in self-reactive B cells in the gut and a critical role of this pathway in maintaining peripheral tolerance. Collectively, our studies describe functional consequences and fate of self-reactive B cells in GALT and provide novel mechanistic insights governing self-tolerance of B cells in the gut.

A Brake for B Cell Proliferation: Appropriate responses to metabolic stress are crucial to maintain B cell viability and prevent malignant outgrowth.

B cell activation is accompanied by metabolic adaptations to meet the increased energetic demands of proliferation. The metabolic composition of the microenvironment is known to change during a germinal center response, in inflamed tissue and to vary significantly between different organs. To sustain cellular homeostasis B cells need to be able to dynamically adapt to changes in their environment. An inability to take up and process available nutrients can result in impaired B cell growth and a diminished humoral immune response. Furthermore, the metabolic microenvironment can affect B cell signaling and provide a means to avoid aberrant proliferation or modulate B cell function. Thus, a better understanding of the intricate interplay between cell signaling and metabolism could provide novel insight into how B cell function is regulated and have implications for the development of vaccines or treatment of autoimmune disorders and B cell derived malignancies.

New Methods To Analyze B Cell Immune Responses to Thymus-Dependent Antigen Sheep Red Blood Cells.

B cells contribute critically to an effective immune response by producing Ag-specific Abs. During the immune response to so-called "thymus-dependent Ags," activated B cells seek T cell help and form germinal centers. In contrast, thymus-independent Ags generally do not induce germinal center formation. In the germinal center, B cells undergo somatic hypermutation, affinity-based clonal expansion, and differentiation to produce plasma cells and memory B cells. Valuable insight into these processes has been gained by using model hapten-carrier complexes or SRBCs. SRBCs induce robust germinal center formation in mice. Therefore, this Ag is commonly used to study germinal center responses. In contrast to haptenated Ags, thus far it has been difficult to measure the titer of Ag-specific Abs or the expansion of Ag-specific B cells after immunization with SRBCs. We have developed new, simple methods to access these parameters, thus providing new tools to study germinal center and Ab responses.

Metabolic Regulation of the Immune Humoral Response.

Productive humoral responses require that naive B cells and their differentiated progeny move among distinct micro-environments. In this review, we discuss how studies are beginning to address the nature of these niches as well as the interplay between cellular signaling, metabolic programming, and adaptation to the locale. Recent work adds evidence to the expectation that B cells at distinct stages of development or functional subsets are influenced by the altered profiles of nutrients and metabolic by-products that distinguish these sites. Moreover, emerging findings reveal a cross-talk among the external milieu, signal transduction pathways, and transcription factors that direct B cell fate in the periphery.

Murine models of germinal center derived-lymphomas.

The germinal center (GC) reaction is an adaptive immune response to select B cells bearing high-affinity B cell receptors (BCRs) to undergo further differentiation into antibody-producing cells or memory B cells. To drive affinity maturation, (GC) B cells undergo rounds of hypermutation and rapid proliferation, which can enhance susceptibility to malignant transformation. Lymphomas frequently originate from GC B cells, but the etiology for most lymphoma subtypes is unknown. Work in the past decade has more fully documented the mutational landscape in lymphomas, but the impact of these genomic lesions is often difficult to ascertain. In addition, while mutations affecting BCR signaling are well studied, the impact of extrinsic microenvironmental factors has not been widely addressed. Murine models are useful tools to study lymphomagenesis and disease progression, as well as potential treatment in a pre-clinical setting. Herein we discuss advances in murine models of lymphoma and how they inform on key characteristics of human lymphomas.

Gsk3 is a metabolic checkpoint regulator in B cells.

B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.

Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation.

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.

The PI3K pathway in B cell metabolism.

B cell growth and proliferation is tightly regulated by signaling through the B cell receptor and by other membrane bound receptors responding to different cytokines. The PI3K signaling pathway has been shown to play a crucial role in B cell activation, differentiation and survival. Activated B cells undergo metabolic reprograming in response to changing energetic and biosynthetic demands. B cells also need to be able to coordinate metabolic activity and proliferation with nutrient availability. The PI3K signaling network has been implicated in regulating nutrient acquisition, utilization and biosynthesis, thus integrating receptor-mediated signaling with cell metabolism. In this review, we discuss the current knowledge about metabolic changes induced in activated B cells, strategies to adapt to metabolic stress and the role of PI3K signaling in these processes.

Essential Role for Survivin in the Proliferative Expansion of Progenitor and Mature B Cells.

Survivin is a member of the inhibitor of apoptosis family of proteins and a biomarker of poor prognosis in aggressive B cell non-Hodgkin's lymphoma. In addition to its role in inhibition of apoptosis, survivin also regulates mitosis. In this article, we show that deletion of survivin during early B cell development results in a complete block at the cycling pre-B stage. In the periphery, B cell homeostasis is not affected, but survivin-deficient B cells are unable to mount humoral responses. Correspondingly, we show that survivin is required for cell division in response to mitogenic stimulation. Thus, survivin is essential for proliferation of B cell progenitors and activated mature B cells, but is dispensable for B cell survival. Moreover, a small-molecule inhibitor of survivin strongly impaired the growth of representative B lymphoma lines in vitro, supporting the validity of survivin as an attractive therapeutic target for high-grade B cell non-Hodgkin's lymphoma.

Transfection of microRNA Mimics Should Be Used with Caution.

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5'- and 3'-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.

A pathway switch directs BAFF signaling to distinct NFκB transcription factors in maturing and proliferating B cells.

BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

PDK1 regulates B cell differentiation and homeostasis.

Successful B cell differentiation and prevention of cell transformation depends on balanced and fine-tuned activation of cellular signaling pathways. The phosphatidyl inositol-3 kinase (PI3K) signaling pathway has emerged as a major regulator of B lymphocyte homeostasis and function. Phosphoinositide-dependent protein kinase-1 (PDK1) is the pivotal node in the PI3K pathway, regulating the stability and activity of downstream AGC kinases (including Akt, RSK, S6K, SGK, and PKC). Although the importance of PI3K activity in B cell differentiation is well documented, the role of PDK1 and other downstream effectors is underexplored. Here we used inducible and stage-specific gene targeting approaches to elucidate the role of PDK1 in early and peripheral B cell differentiation. PDK1 ablation enhanced cell cycle entry and apoptosis of IL-7-dependent pro-B cells, blocking Ig synthesis and B cell maturation. PDK1 also was essential for the survival and activation of peripheral B cells via regulation of PKC and Akt-dependent downstream effectors, such as GSK3α/ß and Foxo1. We found that PDK1 deletion strongly impaired B cell receptor (BCR) signaling, but IL-4 costimulation was sufficient to restore BCR-induced proliferation. IL-4 also normalized PKCß activation and hexokinase II expression in BCR-stimulated cells, suggesting that this signaling pathway can act independent of PDK1 to support B cell growth. In summary, our results demonstrate that PDK1 is indispensable for B cell survival, proliferation, and growth regulation.

Context-specific BAFF-R signaling by the NF-κB and PI3K pathways.

BAFF is a soluble factor required for B cell maturation and survival. BAFF-R signals via the noncanonical NF-κB pathway regulated by the TRAF3/NIK/IKK1 axis. We show that deletion of Ikk1 during early B cell development causes a partial impairment in B cell maturation and BAFF-dependent survival, but inactivation of Ikk1 in mature B cells does not affect survival. We further show that BAFF-R employs CD19 to promote survival via phosphatidylinositol 3-kinase (PI3K), and that coinactivation of Cd19 and Ikk1 causes a profound block in B cell maturation at the transitional stage. Consistent with a role for PI3K in BAFF-R function, inactivation of PTEN mediates a partial rescue of B cell maturation and function in Baff(-/-) animals. Elevated PI3K signaling also circumvents BAFF-dependent survival in a spontaneous B cell lymphoma model. These findings indicate that the combined activities of PI3K and IKK1 drive peripheral B cell differentiation and survival in a context-dependent manner.

Requirement for Rictor in homeostasis and function of mature B lymphoid cells.

The mammalian target of rapamycin (mTOR), an essential serine/threonine kinase, functions in biochemically distinct multiprotein complexes, but little is known about roles of the complexes in B cells. The acutely rapamycin-sensitive mTOR complex 1 (mTORC1) is defined by a core subunit Raptor, whereas mTORC2 lacks Raptor and, instead, has Rictor and SIN1 as distinct essential components. We now show that homeostasis and function of B cells require Rictor. Conditional deletion of Rictor before lymphoid specification impaired generation of mature follicular, marginal zone, and B1a B lymphocytes. Induced inactivation in adult mice caused cell-autonomous defects in B lymphoid homeostasis and antibody responses in vivo, along with affecting plasma cells in bone marrow. Survival of B lymphocytes depended on Rictor, which was vital for normal induction of prosurvival genes, suppression of proapoptotic genes, nuclear factor κB induction after B-cell receptor stimulation, and B-cell activating factor-induced nuclear factor κB2/p52 generation. Collectively, the findings provide evidence that mTOR signaling affects survival and proliferation of mature B lymphocytes, and establish Rictor as an important signal relay in B-cell homeostasis, fate, and functions.