201095121
Antigen Complexed with a TLR9 Agonist Bolsters c-Myc and mTORC1 Activity in Germinal Center B Lymphocytes
The germinal center (GC) is the anatomical site where humoral immunity evolves. B cells undergo cycles of proliferation and selection to produce high-affinity Abs against Ag. Direct linkage of a TLR9 agonist (CpG) to a T-dependent Ag increases the number of GC B cells. We used a T-dependent Ag complexed with CpG and a genetic model for ablating the TLR9 signaling adaptor molecule MyD88 specifically in B cells (B-MyD88− mice) together with transcriptomics to determine how this innate pathway positively regulates the GC. GC B cells from complex Ag-immunized B-MyD88− mice were defective in inducing gene expression signatures downstream of c-Myc and mTORC1. In agreement with the latter gene signature, ribosomal protein S6 phosphorylation was increased in GC B cells from wild-type mice compared with B-MyD88− mice. However, GC B cell expression of a c-Myc protein reporter was enhanced by CpG attached to Ag in both wild-type and B-MyD88− mice, indicating a B cell–extrinsic effect on c-Myc protein expression combined with a B cell–intrinsic enhancement of gene expression downstream of c-Myc. Both mTORC1 activity and c-Myc are directly induced by T cell help, indicating that TLR9 signaling in GC B cells either enhances their access to T cell help or directly influences these pathways to further enhance the effect of T cell help. Taken together, these findings indicate that TLR9 signaling in the GC could provide a surrogate prosurvival stimulus, “TLR help,” thus lowering the threshold for selection and increasing the magnitude of the GC response.
39224694
Intracellular toll-like receptors.
Foreign nucleic acids, the signature of invading viruses and certain bacteria, are sensed intracellularly. The nucleic acid-specific Toll-like receptors (TLRs) detect and signal within endolysosomal compartments, triggering the induction of cytokines essential for the innate immune response. These cytokines include proinflammatory molecules produced mainly by macrophages and conventional dendritic cells, as well as type I interferons, which are produced in great quantities by plasmacytoid dendritic cells. The cellular and molecular pathways by which nucleic acids and TLRs meet within the endosome assure host protection yet also place the host at risk for the development of autoimmunity. Here, we review the latest findings on the intracellular TLRs, with special emphasis on ligand uptake, receptor trafficking, signaling, and regulation.
9111199
Adjuvant-Enhanced Antibody Responses in the Absence of Toll-Like Receptor Signaling
Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell–dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.
4399604
Control of B-cell responses by Toll-like receptors
Toll-like receptors (TLRs) detect microbial infection and have an essential role in the induction of immune responses. TLRs can directly induce innate host defence responses, but the mechanisms of TLR-mediated control of adaptive immunity are not fully understood. Although TLR-induced dendritic cell maturation is required for activation of T-helper (TH) cells, the role of TLRs in B-cell activation and antibody production in vivo is not yet known. Here we show that activation and differentiation of TH cells is not sufficient for the induction of T-dependent B-cell responses. We find that, in addition to CD4+ T-cell help, generation of T-dependent antigen-specific antibody responses requires activation of TLRs in B cells.
10521323
BCR-mediated uptake of antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation.
The activation of Toll-like receptor 9 (TLR9) expressed within B cells is associated with enhanced humoral immunity. However the role of TLR9 in the stimulation of B-cell responses, and more specifically in shaping the outcome of B-cell differentiation, remains unclear. Here, we observed that immunization with the TLR9 agonist CpG linked to protein antigen gave rise to enhanced production of antigen-specific class-switched antibodies in vivo. Unlike dendritic cells, B cells are unable to acquire these conjugates by macropinocytosis and instead depend on uptake through a signaling-competent B-cell receptor (BCR), provided the overall BCR-antigen avidity exceeds a defined threshold. The resultant stimulation of intrinsic TLR9 leads to enhanced antigen-specific B-cell proliferation and differentiation to form extrafollicular plasma cells. Thus, the direct conjugation of antigen and CpG reveals a mechanism that may operate during the initiation of primary immune responses, and may prove useful as a strategy for the design of adjuvants suitable for vaccinations.
35795982
Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the antiviral germinal center response.
The contribution of Toll-like receptor (TLR) signaling to T cell-dependent (TD) antibody responses was assessed by using mice lacking the TLR signaling adaptor MyD88 in individual cell types. When a soluble TLR9 ligand was used as adjuvant for a protein antigen, MyD88 was required in dendritic cells but not in B cells to enhance the TD antibody response, regardless of the inherent immunogenicity of the antigen. In contrast, a TLR9 ligand contained within a virus-like particle substantially augmented the TD germinal center IgG antibody response, and this augmentation required B cell MyD88. The ability of B cells to discriminate between antigens based on the physical form of a TLR ligand probably reflects an adaptation to facilitate strong antiviral antibody responses.
12096226
TLR9 Signaling in B Cells Determines Class Switch Recombination to IgG2a
Although IgG2a is the most potent Ab isotype in the host response to viral and bacterial infections, the regulation of class switch recombination to IgG2a in vivo is not yet well understood. Recognition of pathogen-associated molecular patterns by dendritic cells expressing TLRs, like TLR7, recognizing ssRNA, or TLR9, recognizing DNA rich in nonmethylated CG motifs (CpG), favors induction of Th1 responses. It is generally assumed that these Th1 responses are responsible for the TLR-mediated induction of IgG2a. Using virus-like particles loaded with CpGs, we show here that TLR9 ligands can directly stimulate B cells to undergo isotype switching to IgG2a. Unexpectedly, TLR9 expression in non-B cells did not affect isotype switching in the Ab response against virus-like particles. Thus, TLR9 can regulate isotype switching to IgG2a directly by interacting with B cells rather than indirectly by inducing Th1 responses.
29719273
B Cell–Intrinsic TLR7 Signaling Is Required for Optimal B Cell Responses during Chronic Viral Infection
The importance for activation of innate immunity by pattern recognition receptors in forming an effective adaptive immune response is well known. TLRs were demonstrated to be critical for Ab responses to a variety of immunizations. In particular, recent evidence suggests that B cell–intrinsic TLR signaling is required for optimal responses to virus-like Ags, but the mechanisms by which TLR signaling impacts Ab responses during infection in vivo is unclear. In the current study, we demonstrate that deficiency of TLR7 in B cells alone is sufficient to significantly impact Ab responses in mice during chronic viral infection. This effect was independent of T follicular helper cells and resulted in a loss of plasma cells generated later, but not early, in the response. The defect in plasma cell formation appeared to be secondary to a qualitative effect of TLR signaling on the germinal center (GC) B cell response. GC B cells in TLR7-deficient mice proliferated to a lesser extent and had a greater proportion of cells with phenotypic characteristics of light zone, relative to dark zone, GC B cells. These results suggest that B cell–intrinsic TLR signaling in vivo likely affects plasma cell output by altered selection of Ag-specific B cells in the GC.
5499479
The B cell receptor governs the subcellular location of Toll-like receptor 9 leading to hyperresponses to DNA-containing antigens.
Synergistic engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9) in response to DNA-containing antigens underlies the production of many autoantibodies in systemic autoimmune diseases. However, the molecular basis of this synergistic engagement is not known. Given that these receptors are spatially segregated, with the BCR on the cell surface and TLR9 in endocytic vesicles, achieving synergy must involve unique mechanisms. We show that upon antigen binding, the BCR initiates signaling at the plasma membrane and continues to signal to activate MAP kinases as it traffics to autophagosome-like compartments. The internalized BCR signals through a phospholipase-D-dependent pathway to recruit TLR9-containing endosomes to the autophagosome via the microtubular network. The recruitment of TLR9 to the autophagosomes was necessary for hyperactivation of MAP kinases. This unique mechanism for BCR-induced TLR9 recruitment resulting in B cells hyperresponses may provide new targets for therapeutics for autoimmune diseases.
24323715
Endocytic sequestration of the B cell antigen receptor and toll-like receptor 9 in anergic cells
In autoimmune prone murine strains, sequential engagement of the B cell antigen receptor (BCR) on the cell surface and toll-like receptors (TLRs) in late endosomes is necessary and sufficient for secretion of autoantibodies. However, ubiquitous nucleoprotein self-antigens fail to elicit productive TLR activation, and break self-tolerance in anergic DNA-reactive B cells. The mechanisms limiting TLR activation in these cells are largely unknown. Here, we demonstrate that in anergic 3H9/Vκ8 and Ars/A1 B cells the normal endocytic transit of both the ligated BCR and TLR9 into late endosomes is abrogated. The BCR and TLR9 arrest together just outside late endosomes, indicating that they enter this compartment along a single, regulated endocytic route. Access to late endosomes could be restored by reversing anergy through several methods, including conferring genetic susceptibility to autoimmunity, complementing proximal BCR signaling or by preventing BCR binding to self-antigen. Downstream of the BCR, JNK, which is activated in naive but not anergic B cells, regulated entry into late endosomes. Restoration of BCR and TLR9 endocytic trafficking rescued TLR9 activation by BCR-captured ligands. These results indicate that B cell anergy is reinforced by the exclusion of both TLRs and their BCR captured ligands from subcellular environments necessary for TLR activation.
29843114
Altered B cell signalling in autoimmunity
Recent work has provided new insights into how altered B cell-intrinsic signals — through the B cell receptor (BCR) and key co-receptors — function together to promote the pathogenesis of autoimmunity. These combined signals affect B cells at two distinct stages: first, in the selection of the naive repertoire; and second, during extrafollicular or germinal centre activation responses. Thus, dysregulated signalling can lead to both an altered naive BCR repertoire and the generation of autoantibody-producing B cells. Strikingly, high-affinity autoantibodies predate and predict disease in several autoimmune disorders, including type 1 diabetes and systemic lupus erythematosus. This Review summarizes how, rather than being a downstream consequence of autoreactive T cell activation, dysregulated B cell signalling can function as a primary driver of many human autoimmune diseases.
36307611
Toll-like receptor 9 signaling acts on multiple elements of the germinal center to enhance antibody responses
Significance The antibody response of B lymphocytes proceeds in two phases, a rapid low-affinity response and a slower germinal center (GC) response that is responsible for high-affinity antibody, long-lived antibody-secreting cells, and high-affinity memory B cells. We have examined how immune cell recognition of foreign nucleic acid by Toll-like receptors enhances the GC response and found that it does so by multiple complementary mechanisms. Recognition of foreign nucleic acid by dendritic cells led to increased numbers of helper T cells and GC B cells. In contrast, recognition by the antigen-specific B cells promoted the selection of high-affinity B cells and their differentiation into memory B cells, both of which are central goals for vaccination. Recent studies have demonstrated important roles of nucleic acid-sensing Toll-like receptors (TLRs) in promoting protective antibody responses against several viruses. To dissect how recognition of nucleic acids by TLRs enhances germinal center (GC) responses, mice selectively deleted for myeloid differentiation primary-response protein 88 (MyD88) in B cells or dendritic cells (DCs) were immunized with a haptenated protein antigen bound to a TLR9 ligand. TLR9 signaling in DCs led to greater numbers of follicular helper T (TFH) cells and GC B cells, and accelerated production of broad-affinity antihapten IgG. In addition to modulating GC selection by increasing inducible costimulator (ICOS) expression on TFH cells and reducing the number of follicular regulatory T cells, MyD88-dependent signaling in B cells enhanced GC output by augmenting a class switch to IgG2a, affinity maturation, and the memory antibody response. Thus, attachment of a TLR9 ligand to an oligovalent antigen acted on DCs and B cells to coordinate changes in the T-cell compartment and also promoted B cell-intrinsic effects that ultimately programmed a more potent GC response.
207435669
B Cell–Intrinsic MyD88 Signaling Promotes Initial Cell Proliferation and Differentiation To Enhance the Germinal Center Response to a Virus-like Particle
Although TLR signaling in B cells has been implicated in the germinal center (GC) responses during viral infections and autoimmune diseases, the underlying mechanism is unclear. Bacterial phage Qβ-derived virus-like particle (Qβ-VLP) contains TLR ligands, which can enhance Qβ-VLP-induced Ab response, including GC response, through TLR/MyD88 signaling in B cells. In this study, by examining Ag-specific B cell response to Qβ-VLP, we found that lack of B cell MyD88 from the beginning of the immune response led to a more severe defect in the GC scale than abolishing MyD88 at later time points of the immune response. Consistently, B cell–intrinsic MyD88 signaling significantly enhanced the initial proliferation of Ag-specific B cells, which was accompanied with a dramatic increase of plasma cell generation and induction of Bcl-6+ GC B cell precursors. In addition, B cell–intrinsic MyD88 signaling promoted strong T-bet expression independent of IFN-γ and led to the preferential isotype switching to IgG2a/c. Thus, by promoting the initial Ag-specific B cell proliferation and differentiation, B cell–intrinsic MyD88 signaling enhanced both T-independent and T-dependent Ab responses elicited by Qβ-VLP. This finding will provide additional insight into the role of TLR signaling in antiviral immunity, autoimmune diseases, and vaccine design.
4579450
T cell‐dependent antigen adjuvanted with DOTAP‐CpG‐B but not DOTAP‐CpG‐A induces robust germinal center responses and high affinity antibodies in mice
The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. Here we compared the outcome of immunization with the T‐cell dependent antigen, NP‐conjugated to chicken gamma globulin (NP‐CGG) adjuvanted with the toll‐like receptor 9 (TLR9) ligands, CpG‐A or CpG‐B, alone or conjugated with the cationic lipid carrier, DOTAP. We provide evidence that only NP‐CGG adjuvanted with DOTAP‐CpG‐B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP‐specific antibodies. The effectiveness of DOTAP‐CpG‐B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP‐CpG‐B but not DOTAP‐CpG‐A is an effective adjuvant for T cell‐dependent protein antigen‐based vaccines.
3704537
Autoreactive helper T cells alleviate the need for intrinsic TLR signaling in autoreactive B cell activation.
T cells play a significant role in the pathogenesis of systemic autoimmune diseases, including systemic lupus erythematosus; however, there is relatively little information on the nature and specificity of autoreactive T cells. Identifying such cells has been technically difficult because they are likely to be rare and low affinity. Here, we report a method for identifying autoreactive T cell clones that recognize proteins contained in autoantibody immune complexes, providing direct evidence that functional autoreactive helper T cells exist in the periphery of normal mice. These T cells significantly enhanced autoreactive B cell proliferation and altered B cell differentiation in vivo. Most importantly, these autoreactive T cells were able to rescue many aspects of the TLR-deficient AM14 (anti-IgG2a rheumatoid factor) B cell response, suggesting that TLR requirements can be bypassed. This result has implications for the efficacy of TLR-targeted therapy in the treatment of ongoing disease.
13932426
MYC is essential for the formation and maintenance of germinal centers
Germinal centers (GCs) are sites of intense B cell proliferation and are central for T cell–dependent antibody responses. However, the role of c-Myc, a key cell-cycle regulator, in this process has been questioned. Here we identified c-Myc+ B cell subpopulations in immature and mature GCs and found, by genetic ablation of Myc, that they had indispensable roles in the formation and maintenance of GCs. The identification of these functionally critical cellular subsets has implications for human B cell lymphomagenesis, which originates mostly from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the c-Myc+ GC subpopulations may be at a particularly high risk for malignant transformation.
195683609
The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry
After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.
205426908
Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase
Summary During antibody affinity maturation, germinal center (GC) B cells cycle between affinity‐driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen‐dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell‐cycle progression itself and must be regulated dynamically during cyclic re‐entry to ensure efficient affinity‐based selection. Graphical Abstract Figure. No Caption available. HighlightsPositive selection of GC B cells triggers mTORC1 activation prior to clonal expansionmTORC1 is required for biomass accumulation but dispensable for cell‐cycle progressionDeregulated mTORC1 promotes DZ phenotype and leads to loss of GC competitivenessRapamycin‐resistant mouse strain is used for cell‐intrinsic mTORC1 inhibition &NA; During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst.
12365992
MicroRNAs 24 and 27 Suppress Allergic Inflammation and Target a Network of Regulators of T Helper 2 Cell-Associated Cytokine Production.
MicroRNAs (miRNAs) are important regulators of cell fate decisions in immune responses. They act by coordinate repression of multiple target genes, a property that we exploited to uncover regulatory networks that govern T helper-2 (Th2) cells. A functional screen of individual miRNAs in primary T cells uncovered multiple miRNAs that inhibited Th2 cell differentiation. Among these were miR-24 and miR-27, miRNAs coexpressed from two genomic clusters, which each functioned independently to limit interleukin-4 (IL-4) production. Mice lacking both clusters in T cells displayed increased Th2 cell responses and tissue pathology in a mouse model of asthma. Gene expression and pathway analyses placed miR-27 upstream of genes known to regulate Th2 cells. They also identified targets not previously associated with Th2 cell biology which regulated IL-4 production in unbiased functional testing. Thus, elucidating the biological function and target repertoire of miR-24 and miR-27 reveals regulators of Th2 cell biology.
5515073
Magnetic-based purification of untouched mouse germinal center B cells for ex vivo manipulation and biochemical analysis
Detailed biochemical analysis of unmanipulated germinal center (GC) B cells has not been achieved. Previously, we designed and used a simple, economical and new magnetic bead separation scheme for the purification of 'untouched' mature GC and non-GC B cells from the spleens of immunized mice and reported the first biochemical assessment of the signaling cascades that contribute to cyclin D stability and GC B cell proliferation. Here we provide a detailed protocol for the method we used, which involves preparing single-cell suspension from the spleens of immunized mice, followed by labeling of nontarget cells with biotinylated antibodies specific for CD43, CD11c and IgD (for GC enrichment) or GL7 (for non-GC enrichment); these steps are followed by cell depletion using standard magnetic bead technology. This protocol can yield GC and non-GC B cells with purities exceeding 90%. The sorting process can be carried out in ∼1 h and provides a population of GC B cells of sufficient purity and quantity to allow ex vivo manipulation, including biochemical and genetic analysis as well as cell culture.
2374637
Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles
Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.
49666458
Second signals rescue B cells from activation-induced mitochondrial dysfunction and death
B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response–activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.B cells need at least two signals to terminally differentiate into antibody-secreting cells. Pierce and colleagues show that persistent exposure to antigen in the absence of T cell help or ‘pathogen pattern motifs’ leads to B cell death via a calcium-dependent ‘metabolic timer’.
3999015
B Cell–Intrinsic mTORC1 Promotes Germinal Center–Defining Transcription Factor Gene Expression, Somatic Hypermutation, and Memory B Cell Generation in Humoral Immunity
B lymphocytes migrate among varied microenvironmental niches during diversification, selection, and conversion to memory or Ab-secreting plasma cells. Aspects of the nutrient milieu differ within these lymphoid microenvironments and can influence signaling molecules such as the mechanistic target of rapamycin (mTOR). However, much remains to be elucidated as to the B cell–intrinsic functions of nutrient-sensing signal transducers that modulate B cell differentiation or Ab affinity. We now show that the amino acid–sensing mTOR complex 1 (mTORC1) is vital for induction of Bcl6—a key transcriptional regulator of the germinal center (GC) fate—in activated B lymphocytes. Accordingly, disruption of mTORC1 after B cell development and activation led to reduced populations of Ag-specific memory B cells as well as plasma cells and GC B cells. In addition, induction of the germ line transcript that guides activation-induced deaminase in selection of the IgG1 H chain region during class switching required mTORC1. Expression of the somatic mutator activation-induced deaminase was reduced by a lack of mTORC1 in B cells, whereas point mutation frequencies in Ag-specific GC-phenotype B cells were only halved. These effects culminated in a B cell–intrinsic defect that impacted an antiviral Ab response and drastically impaired generation of high-affinity IgG1. Collectively, these data establish that mTORC1 governs critical B cell–intrinsic mechanisms essential for establishment of GC differentiation and effective Ab production.
54479018
The mTORC1/4E-BP/eIF4E Axis Promotes Antibody Class Switching in B Lymphocytes
During an adaptive immune response, activated mature B cells give rise to Ab-secreting plasma cells to fight infection. B cells undergo Ab class switching to produce different classes of Abs with varying effector functions. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is activated during this process, and disrupting mTOR complex 1 (mTORC1) in B cells impairs class switching by a poorly understood mechanism. In particular, it is unclear which mTORC1 downstream substrates control this process. In this study, we used an in vitro murine model in which the mTORC1 inhibitor rapamycin, when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Investigation of mTORC1 substrates revealed a role for eukaryotic translation initiation factor 4E (eIF4E) and eIF4E-binding proteins in class switching. Mechanistically, we show that genetic or pharmacological disruption of eIF4E binding to eIF4G reduced cap-dependent translation, which specifically affected the expression of activation-induced cytidine deaminase protein but not Aicda mRNA. This translational impairment decreased Ab class switching independently of proliferation. These results uncover a previously undescribed role for mTORC1 and the eIF4E-binding proteins/eIF4E axis in activation-induced cytidine deaminase protein expression and Ab class switching in mouse B cells, suggesting that cap-dependent translation regulates key steps in B cell differentiation.
17926527
A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens
Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27– human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-&ggr; promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.
9145997
Dynamic regulation of c‐Myc proto‐oncogene expression during lymphocyte development revealed by a GFP‐c‐Myc knock‐in mouse
c‐Myc induces widely varying cellular effects, including cell proliferation and cell death. These different cellular effects are determined, in part, by c‐Myc protein expression levels, which are regulated through several transcriptional and post‐transcriptional pathways. c‐Myc transcripts can be detected in cells at all stages of B and T lymphocyte development. However, little is known about c‐Myc protein expression, and how it varies, in developing lymphocytes. Here mice have been generated in which the endogenous c‐Myc locus has been modified (c‐MycG) so that it encodes a GFP‐c‐Myc fusion protein. c‐MycG/G mice are viable, appear normal and exhibit grossly normal lymphocyte development. Flow cytometric analyses revealed significant heterogeneity in c‐Myc protein expression levels in developing c‐MycG/G B and T lymphocytes. GFP‐c‐Myc expression levels were highest in proliferating lymphocytes, suggesting that c‐Myc up‐regulation is important for promoting lymphocyte cell division, and demonstrating that GFP‐c‐Myc expression is a marker of proliferating lymphocytes in vivo.
19340553
mTOR has distinct functions in generating versus sustaining humoral immunity.
Little is known about the role of mTOR signaling in plasma cell differentiation and function. Furthermore, for reasons not understood, mTOR inhibition reverses antibody-associated disease in a murine model of systemic lupus erythematosus. Here, we have demonstrated that induced B lineage-specific deletion of the gene encoding RAPTOR, an essential signaling adaptor for rapamycin-sensitive mTOR complex 1 (mTORC1), abrogated the generation of antibody-secreting plasma cells in mice. Acute treatment with rapamycin recapitulated the effects of RAPTOR deficiency, and both strategies led to the ablation of newly formed plasma cells in the spleen and bone marrow while also obliterating preexisting germinal centers. Surprisingly, although perturbing mTOR activity caused a profound decline in serum antibodies that were specific for exogenous antigen or DNA, frequencies of long-lived bone marrow plasma cells were unaffected. Instead, mTORC1 inhibition led to decreased expression of immunoglobulin-binding protein (BiP) and other factors needed for robust protein synthesis. Consequently, blockade of antibody synthesis was rapidly reversed after termination of rapamycin treatment. We conclude that mTOR signaling plays critical but diverse roles in early and late phases of antibody responses and plasma cell differentiation.
25653551
B Cell–Specific Deficiencies in mTOR Limit Humoral Immune Responses
Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP–specific or anti-Pn14–specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A–specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell–specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.
3411138
B Cell Receptor and CD40 Signaling Are Rewired for Synergistic Induction of the c‐Myc Transcription Factor in Germinal Center B Cells
Summary Positive selection of germinal center (GC) B cells is driven by B cell receptor (BCR) affinity and requires help from follicular T helper cells. The transcription factors c‐Myc and Foxo1 are critical for GC B cell selection and survival. However, how different affinity‐related signaling events control these transcription factors in a manner that links to selection is unknown. Here we showed that GC B cells reprogram CD40 and BCR signaling to transduce via NF‐&kgr;B and Foxo1, respectively, whereas naive B cells propagate both signals downstream of either receptor. Although either BCR or CD40 ligation induced c‐Myc in naive B cells, both signals were required to highly induce c‐Myc, a critical mediator of GC B cell survival and cell cycle reentry. Thus, GC B cells rewire their signaling to enhance selection stringency via a requirement for both antigen receptor‐ and T cell‐mediated signals to induce mediators of positive selection. Graphical Abstract Figure. No Caption available. HighlightsIn GC B cells, CD40 signals via NF‐&kgr;B but not PI3K, while BCR does not activate NF‐&kgr;BWeak BCR signals in GC B cells propagate to Akt‐dependent phosphorylation of Foxo1In GC B cells, both CD40 and BCR signals are required to synergistically induce c‐MycCombined CD40 and BCR signals induce p‐S6 in GC B cells, permitting cell cycle entry &NA; Luo et al. show that CD40 and BCR signaling in GC B cells is rewired to control very different pathways, and both signals are required for optimal induction of c‐Myc, suggesting a mechanism of signaling‐directed positive selection of GC B cells.
206662761
The microanatomic segregation of selection by apoptosis in the germinal center
Light- and dark-zone death dynamics Germinal centers (GCs) are areas within lymphoid organs where mature B cells expand and differentiate during normal immune responses. GCs are separated into two anatomic compartments: the dark zone, where B cells divide and undergo somatic hypermutation, and the light zone, where they are selected for affinity-enhancing mutations after interacting with T follicular helper cells. Mayer et al. studied apoptosis reporter mice and found that both GC zones experience very high rates of apoptosis (see the Perspective by Bryant and Hodgkin). However, the underlying mechanisms were distinct and microanatomically segregated. Light-zo ne B cells underwent apoptosis by default unless they were rescued by positive selection. In contrast, apoptotic dark-zone B cells were highly enriched among cells with genes damaged by random antibody-gene mutations. Science, this issue p. eaao2602; see also p. 171 The selection of germinal center B cells by apoptosis is regulated by microanatomically distinct mechanisms. INTRODUCTION Germinal centers (GCs) are transient microanatomic structures that form in lymphoid organs during an immune response. They are the sites of B cell clonal expansion and affinity maturation, a process that leads to the production of high-affinity antibodies. GCs are highly dynamic and contain activated B cells, specialized T follicular helper (TFH) cells, and antigen-trapping follicular dendritic cells. GCs are organized into two functionally distinct compartments: a dark zone (DZ) and a light zone (LZ). The DZ is the site of rapid cell division and random antibody-gene mutation, which is initiated by activation-induced cytidine deaminase (AID). The mutation process leads to the accumulation of a large number of closely related B cells that carry receptors with distinct antigen-binding properties. Once they stop dividing, DZ B cells migrate to the LZ, where their newly generated B cell receptors (BCRs) are tested: GC B cells with relatively higher-affinity receptors capture and process more antigen, leading to positive selection by interaction with TFH cells. The positively selected LZ B cells return to the DZ, where they undergo further cycles of division and mutation. Concomitantly, small numbers of memory B cells and antibody-secreting plasma cells exit the GC. Together, these processes provide the mechanistic basis for affinity maturation, which is essential for effective vaccination and protection from infections. RATIONALE In addition to producing antibody variants, AID expression is also a threat to the genome. AID can produce double-strand breaks that are substrates for chromosome translocations. It can also produce immunoglobulin (Ig) gene missense mutations and deletions or create self-reactive antibodies. These deleterious mutations need to be selected against. Indeed, histologists have long appreciated large numbers of apoptotic nuclei in the specialized tingible body macrophages found in GCs. However, beyond histology, little is known about the exact rate of GC B cell apoptosis and whether it differs in the DZ and LZ of the GC. Moreover, the mechanisms that cause apoptosis, their relative importance in each GC compartment, and their role in GC B cell selection have not been defined. To examine these questions, we created fluorescent apoptosis-indicator mice and used them to enumerate, isolate, and characterize dying cells in the GC. RESULTS We found that apoptosis is prevalent in both the DZ and LZ compartments of GCs throughout the immune response: up to 50% of GC B cells undergo programmed cell death every 6 hours. Single dying GC B cells were isolated, and their antibody genes were cloned, expressed by transient transfection, and tested for antigen binding and other properties. Apoptotic DZ cells were highly enriched for Ig genes damaged by AID, including missense mutations and deletions. By contrast, dying LZ cells primarily expressed intact antibodies with a range of affinities indistinguishable from GC B cells in the live LZ compartment. By experimentally blocking positive selection and by using reporter mice for Myc, a proto-oncogene, as an indicator of positive selection, we found that apoptosis is the default fate for LZ GC B cells that are not actively positively selected. Thus, LZ GC B cells carrying low-affinity BCRs do not preferentially undergo apoptosis. Instead, apoptosis occurs irrespective of BCR-affinity, and LZ B cells carrying high-affinity BCRs are simply more likely to be positively selected. CONCLUSION Apoptosis is a major feature of GC B cell biology and is required to counterbalance the high rate of proliferation and purge B cells that carry deleterious mutations. Although apoptosis occurs in both the DZ and LZ, the underlying mechanisms of apoptosis in each zone are distinct and microanatomically segregated. These insights into GC B biology are relevant for vaccine design, particularly for pathogens that normally evade effective antibody responses. Germinal center B cells expressing an apoptosis indicator. Intravital two-photon microscopy of GC B cells in popliteal lymph nodes of immunized mice (GC B cells, yellow and green; follicular dendritic cell networks, red). The fluorescence resonance energy transfer–based INDIA reporter was used to visualize and purify dying GC B cells. B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.
7443822
ERK activation drives intestinal tumorigenesis in Apcmin/+ mice
Toll-like receptor (TLR) signaling is essential for intestinal tumorigenesis in Apcmin/+ mice, but the mechanisms by which Apc enhances tumor growth are unknown. Here we show that microflora-MyD88-ERK signaling in intestinal epithelial cells (IECs) promotes tumorigenesis by increasing the stability of the c-Myc oncoprotein. Activation of ERK (extracellular signal–related kinase) phosphorylates c-Myc, preventing its ubiquitination and subsequent proteasomal degradation. Accordingly, Apcmin/+/Myd88−/− mice have lower phospho-ERK (p-ERK) levels and fewer and smaller IEC tumors than Apcmin/+ mice. MyD88 (myeloid differentiation primary response gene 88)-independent activation of ERK by epidermal growth factor (EGF) increased p-ERK and c-Myc and restored the multiple intestinal neoplasia (Min) phenotype in Apcmin/+/Myd88−/− mice. Administration of an ERK inhibitor suppressed intestinal tumorigenesis in EGF-treated Apcmin/+/Myd88−/− and Apcmin/+ mice and increased their survival. Our data reveal a new facet of oncogene-environment interaction, in which microflora-induced TLR activation regulates oncogene expression and related IEC tumor growth in a susceptible host.
28017374
An overview of MYC and its interactome.
This review is intended to provide a broad outline of the biological and molecular functions of MYC as well as of the larger protein network within which MYC operates. We present a view of MYC as a sensor that integrates multiple cellular signals to mediate a broad transcriptional response controlling many aspects of cell behavior. We also describe the larger transcriptional network linked to MYC with emphasis on the MXD family of MYC antagonists. Last, we discuss evidence that the network has evolved for millions of years, dating back to the emergence of animals.
12672287
Phosphorylation regulates c-Myc's oncogenic activity in the mammary gland.
Expression of the c-Myc oncoprotein is affected by conserved threonine 58 (T58) and serine 62 (S62) phosphorylation sites that help to regulate c-Myc protein stability, and altered ratios of T58 and S62 phosphorylation have been observed in human cancer. Here, we report the development of 3 unique c-myc knock-in mice that conditionally express either c-Myc(WT) or the c-Myc(T58A) or c-Myc(S62A) phosphorylation mutant from the constitutively active ROSA26 locus in response to Cre recombinase to study the role of these phosphorylation sites in vivo. Using a mammary-specific Cre model, we found that expression of c-Myc(WT) resulted in increased mammary gland density, but normal morphology and no tumors at the level expressed from the ROSA promoter. In contrast, c-Myc(T58A) expression yielded enhanced mammary gland density, hyperplastic foci, cellular dysplasia, and mammary carcinoma, associated with increased genomic instability and suppressed apoptosis relative to c-Myc(WT). Alternatively, c-Myc(S62A) expression reduced mammary gland density relative to control glands, and this was associated with increased genomic instability and normal apoptotic function. Our results indicate that specific activities of c-Myc are differentially affected by T58 and S62 phosphorylation. This model provides a robust platform to interrogate the role that these phosphorylation sites play in c-Myc function during development and tumorigenesis.
19296961
Mammalian target of rapamycin (mTOR) orchestrates the defense program of innate immune cells
The mammalian target of rapamycin (mTOR) can be viewed as cellular master complex scoring cellular vitality and stress. Whether mTOR controls also innate immune‐defenses is currently unknown. Here we demonstrate that TLR activate mTOR via phosphoinositide 3‐kinase/Akt. mTOR physically associates with the MyD88 scaffold protein to allow activation of interferon regulatory factor‐5 and interferon regulatory factor‐7, known as master transcription factors for pro‐inflammatory cytokine‐ and type I IFN‐genes. Unexpectedly, inactivation of mTOR did not prevent but increased lethality of endotoxin‐mediated shock, which correlated with increased levels of IL‐1β. Mechanistically, mTOR suppresses caspase‐1 activation, thus inhibits release of bioactive IL‐1β. We have identified mTOR as indispensable component of PRR signal pathways, which orchestrates the defense program of innate immune cells.
35790051
Contribution of Toll‐like receptor signaling to germinal center antibody responses
Summary:  Toll‐like receptors (TLRs) have emerged as one of the most important families of innate immune receptors for initiating inflammation and also for promoting adaptive immune responses. Recent studies have examined the ability of TLRs to promote antibody responses, including T‐cell‐dependent antibody responses. Initial study suggested that TLR stimulation promotes primarily an extrafollicular antibody response, which rapidly produces moderate affinity antibodies made by short‐lived plasma cells. Recent studies, however, have shown that TLRs can also enhance the germinal center response, which produces high affinity class‐switched antibody made by long‐lived plasma cells. TLR stimulation can increase the magnitude of the latter response and also enhance selection for high affinity IgG. This review summarizes recent advances in understanding the roles of TLRs in B cells and also in other cell types for enhancement of antibody responses, with an emphasis on T‐cell‐dependent and germinal center antibody responses.
3708877
Toll-like receptor 9 antagonizes antibody affinity maturation
Key events in T cell–dependent antibody responses, including affinity maturation, are dependent on the B cell’s presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells’ capture and presentation of antigen.The presentation of antigen by germinal-center B cells to follicular T cells engenders the process of antibody affinity maturation and humoral memory. Pierce and colleagues show that TLR9 signaling in B cells antagonizes B cell–mediated antigen presentation, which leads to the enhanced generation of short-lived plasma cells and the production of lower-affinity antibodies.
25054350
Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells
Vaccines can greatly reduce the spread of and deaths from many infectious diseases. However, many infections have no successful vaccines. Better understanding of the generation of protective CD8 memory T cells by vaccination is essential for the rational design of new vaccines that aim to prime cellular immune responses. Here we demonstrate that the combination of two adjuvants that are currently licensed for use in humans can be used to prime long-lived memory CD8 T cells that protect mice from viral challenge. The universally used adjuvant, aluminum salts, primed long-lived memory CD8 T cells; however, effective cytotoxic T-cell differentiation occurred only in the presence of an additional adjuvant, monophosphoryl lipid A (MPL). MPL-induced IL-6 was required for cytotoxic differentiation. The IL-6 acted by inducing granzyme B production and reducing expression of inhibitory molecule PD1 on the surface of the primed CD8 T cells. CD8 memory T cells generated by antigen delivered with both aluminum salts and MPL provided significant protection from influenza A challenge. These adjuvants could be used in human vaccines to prime protective memory CD8 T cells.
19312912
Old and new adjuvants.
Adjuvants have been deliberately added to vaccines since the 1920's when alum was discovered to boost antibody responses, leading to better protection. The first adjuvants were discovered by accident and were used in the safer but less immunogenic subunit vaccines, supposedly by providing an antigen depot to extend antigen presentation. Since that time, much has been discovered about how these adjuvants impact cells at the tissue site to activate innate immune responses, mobilize dendritic cells and drive adaptive immunity. New approaches to vaccine construction for infectious diseases that have so far not been well addressed by conventional vaccines often attempt to induce antibodies, polyfunctional CD4+ T cells and CD8+ CTLs. The discovery of pattern recognition receptors and ligands that drive desired T cell responses has led to development of novel adjuvant strategies using immunomodulatory agents to direct appropriate immune responses.
205428920
B Cells Are the Dominant Antigen‐Presenting Cells that Activate Naive CD4+ T Cells upon Immunization with a Virus‐Derived Nanoparticle Antigen
Graphical Abstract Figure. No caption available. SUMMARY B cells can present antigens to CD4+ T cells, but it is thought that dendritic cells (DCs) are the primary initiators of naive CD4+ T cell responses. Nanoparticles, including virus‐like particles (VLPs), are attractive candidates as carriers for vaccines and drug delivery. Using RNA phage Q&bgr;‐derived VLP (Q&bgr;‐VLP) as a model antigen, we found that antigen‐specific B cells were the dominant antigen‐presenting cells that initiated naive CD4+ T cell activation. B cells were sufficient to induce T follicular helper cell development in the absence of DCs. Q&bgr;‐specific B cells promoted CD4+ T cell proliferation and differentiation via cognate interactions and through Toll‐like receptor signaling‐mediated cytokine production. Antigen‐specific B cells were also involved in initiating CD4+ T cell responses during immunization with inactivated influenza virus. These findings have implications for the rational design of nanoparticles as vaccine candidates, particularly for therapeutic vaccines that aim to break immune tolerance. HIGHLIGHTSB cells are the dominant APCs that initiate CD4+ T cell response to VLPsThe few antigen‐specific B cells in WT mice are sufficient to activate CD4+ T cellsInnate signaling in B cells is required for the VLP‐induced CD4+ T cell polarizationB cell‐mediated antigen‐presentation can break CD4+ T cell tolerance &NA; Initiation of CD4+ T cell response depends on antigen‐presenting cells. Contrary to the paradigm that dendritic cells are required for this process, Hong et al. show that antigen‐specific B cells are essential and sufficient to activate naive CD4+ T cells in response to virus‐like particles or influenza vaccination.
5750435
Metabolic Reprogramming Is Required for Antibody Production That Is Suppressed in Anergic but Exaggerated in Chronically BAFF-Exposed B Cells
B cell activation leads to proliferation and Ab production that can protect from pathogens or promote autoimmunity. Regulation of cell metabolism is essential to support the demands of lymphocyte growth and effector function and may regulate tolerance. In this study, we tested the regulation and role of glucose uptake and metabolism in the proliferation and Ab production of control, anergic, and autoimmune-prone B cells. Control B cells had a balanced increase in lactate production and oxygen consumption following activation, with proportionally increased glucose transporter Glut1 expression and mitochondrial mass upon either LPS or BCR stimulation. This contrasted with metabolic reprogramming of T cells, which had lower glycolytic flux when resting but disproportionately increased this pathway upon activation. Importantly, tolerance greatly affected B cell metabolic reprogramming. Anergic B cells remained metabolically quiescent, with only a modest increase in glycolysis and oxygen consumption with LPS stimulation. B cells chronically stimulated with elevated BAFF, however, rapidly increased glycolysis and Ab production upon stimulation. Induction of glycolysis was critical for Ab production, as glycolytic inhibition with the pyruvate dehydrogenase kinase inhibitor dichloroacetate sharply suppressed B cell proliferation and Ab secretion in vitro and in vivo. Furthermore, B cell–specific deletion of Glut1 led to reduced B cell numbers and impaired Ab production in vivo. Together, these data show that activated B cells require Glut1-dependent metabolic reprogramming to support proliferation and Ab production that is distinct from T cells and that this glycolytic reprogramming is regulated in tolerance.