A mathematical model on germinal center kinetics and termination.

We devise a mathematical model to study germinal center (GC) kinetics. Earlier models for GC kinetics are extended by explicitly modeling 1) the cell division history of centroblasts, 2) the Ag uptake by centrocytes, and 3) T cell dynamics. Allowing for T cell kinetics and T-B cell interactions, we study the role of GC T cells in GC kinetics, GC termination, and B cell selection. We find that GC T cells play a major role in GC formation, but that the maintenance of established GC reactions requires very few T cells only. The results therefore suggest that the termination of a GC reaction is largely caused by lack of Ag on the follicular dendritic cells and is hardly influenced by Th cells. Ag consumption by centrocytes is the major factor determining the decay rate of the antigenic stimulus during a GC reaction. Investigating the effect of the Ag dose on GC kinetics, we find that both the total size of the GC and its duration are hardly influenced by the initial amount of Ag. In the model this is due to a buffering effect by competition for limited T cell help and/or competition between proliferating centroblasts.     

LKB1 inhibition of NF‐κB in B cells prevents T follicular helper cell differentiation and germinal center formation

T-cell-dependent antigenic stimulation drives the differentiation of B cells into antibody-secreting plasma cells and memory B cells, but how B cells regulate this process is unclear. We show that LKB1 expression in B cells maintains B-cell quiescence and prevents the premature formation of germinal centers (GCs). Lkb1-deficient B cells (BKO) undergo spontaneous B-cell activation and secretion of multiple inflammatory cytokines, which leads to splenomegaly caused by an unexpected expansion of T cells. Within this cytokine response, increased IL-6 production results from heightened activation of NF-κB, which is suppressed by active LKB1. Secreted IL-6 drives T-cell activation and IL-21 production, promoting T follicular helper (T_FH) cell differentiation and expansion to support a ∽100-fold increase in steady-state GC B cells. Blockade of IL-6 secretion by BKO B cells inhibits IL-21 expression, a known inducer of T_FH-cell differentiation and expansion. Together, these data reveal cell intrinsic and surprising cell extrinsic roles for LKB1 in B cells that control T_FH-cell differentiation and GC formation, and place LKB1 as a central regulator of T-cell-dependent humoral immunity.     

LKB1 regulates development and the stress response in Dictyostelium

The serine/threonine kinase LKB1 is a master kinase that regulates a number of critical events such as cell transformation, polarization, development, stress response, and energy metabolism in metazoa. After multiple unsuccessful attempts of generating Dictyostelium lkb1-null cells, an RNAi-based knockdown approach proved effective. Depletion of lkb1 with a knockdown construct displayed severe reduction in prespore cell differentiation and precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3. Similar to gsk3⁻ cells, lkb1 depleted cells displayed lower GSK3 activity than wild type cells during development and compromised cAMP-mediated inhibition of the DIF-1 mediated ecmB induction. In response to stress insult, the kinase activity of LKB1, but not that of GSK3, increased. Therefore, LKB1 positively functions at the upstream of GSK3 during development and responds to stress insults independently from GSK3.     

Follicular dendritic cell secreted protein (FDC-SP) regulates germinal center and antibody responses

We previously identified follicular dendritic cell secreted protein (FDC-SP), a small secreted protein of unknown function expressed in human tonsillar germinal centers (GC). To assess potential in vivo activities of FDC-SP, transgenic mice were generated to constitutively express FDC-SP in lymphoid tissues. FDC-SP transgenic mice show relatively normal development of immune cell populations, with the exception of a small increase in mature follicular B cells, and normal lymphoid tissue architecture. Upon immunization with a T-dependent Ag, FDC-SP transgenic mice were capable of producing an Ag-specific Ab; however, the titers of Ag-specific IgG2a and IgE were significantly reduced. GC responses after immunization were markedly diminished, with transgenic mice showing decreased numbers and sizes of GCs but normal development of follicular dendritic cell networks and normal positioning of GCs. FDC-SP transgenic mice also showed reduced production of Ag-specific IgG3 Ab after immunization with a type II T-independent Ag, suggesting that the FDC-SP can also regulate the induction of B cell responses outside the GC. Purified FDC-SP transgenic B cells function normally in vitro, with the exception of blunted chemotaxis responses to CXCL12 and CXCL13. FDC-SP can induce the chemotaxis of CD40-stimulated nontransgenic B cells and can significantly enhance B cell migration in combination with chemokines, indicating that FDC-SP may function in part by regulating B cell chemotaxis. These results provide the first evidence for immunomodulatory activities of FDC-SP and implicate this molecule as a regulator of B cell responses.     

Nak1, an essential germinal center (GC) kinase regulates cell morphology and growth in Schizosaccharomyces pombe

We have identified and characterized Nak1, a 652- amino acid NH(2)-terminal kinase belonging to the group II germinal center kinase (GCK) family, in Schizosaccharomyces pombe. We found that nak1 is essential for cell proliferation. Furthermore, partial repression of nak1, under regulation of an integrated nmt1 promoter, resulted in an aberrant round cellular morphology, actin and microtubule mislocalization, slow growth, and cell division defects. Overexpression of either a kinase-inactive mutant (Nak1(K39R)) or the non-catalytic domain resulted in similar phenotypes, suggesting dominant-negative effects. By deletion analysis, we mapped the region responsible for this dominant-negative effect to the COOH-terminal 99 residues. Furthermore, we found that deletion of the COOH-terminal 99 residues inhibited Nak1 autophosphorylation, and expression of a partially inactive (Nak1(T171A)) or truncated (Nak1(1-562)) protein only weakly suppressed morphological and growth phenotypes, indicating that both kinase and COOH-terminal regions are important for Nak1 function. GFP-Nak1 localized uniformly throughout the cytoplasm, unlike many other proteins which influence cell polarity that preferentially localize to cell ends. Together, our results implicate Nak1 in the regulation of cell polarity, growth, and division and suggest that the COOH-terminal end plays an important role in the regulation of this kinase.     

Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase

The endophilin family of proteins function in clathrin-mediated endocytosis. Here, we have identified and cloned the rat germinal center kinase-like kinase (rGLK), a member of the GCK (germinal center kinase) family of c-Jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin I-binding partner. The interaction occurs both in vitro and in cells and is mediated by the Src homology 3 domain of endophilin I and a region of rGLK containing the endophilin consensus-binding sequence PPRPPPPR. Overlay analysis of rat brain extracts demonstrates that endophilin I is a major Src homology 3 domain-binding partner for rGLK. Overexpression of full-length endophilin I activates rGLK-mediated JNK activation, whereas N- and C-terminal fragments of endophilin I block JNK activation. Thus, endophilin I appears to have a novel function in JNK activation.     

LKB1 (XEEK1) regulates Wnt signalling in vertebrate development

Germline LKB1/STK11 mutations are associated with the cancer-prone Peutz-Jeghers syndrome (PJS) in humans, and nullizygosity provokes a poorly understood constellation of developmental perturbations in the mid-gestational mouse. To gain a better understanding of the processes regulated by LKB1, we have exploited the experimental merits of the developing Xenopus embryo. Here, specific inhibition of XEEK1, the Xenopus orthologue of LKB1, engendered developmental anomalies - shortened body axis and defective dorsoanterior patterning - associated previously with aberrant Wnt signalling. In line with this, LKB1/XEEK1 cooperates with the Wnt-beta-catenin signalling in axis induction and modulates the expression of Wnt-responsive genes in both Xenopus embryos and mammalian cells. We establish that LKB1/XEEK1 acts upstream of beta-catenin in the Wnt-beta-catenin pathway in vivo. LKB1/XEEK1 regulates glycogen synthase kinase (GSK)3beta phosphorylation and it is physically associated in vivo with GSK3beta and protein kinase C (PKC)-zeta, a known GSK3 kinase. These studies show that LKB1/XEEK1 is required for Wnt-beta-catenin signalling in frogs and mammals and provides novel insights into its role in vertebrate developmental patterning and carcinogenesis.     

Studies on the germinal center

Summary Ultrastructure of mitotic cells in human lymph node germinal centers was deliberately studied in contrast to that of plasma cells in mitosis which were rarely found in medullary cords or lymphatic sinuses of the same materials. It was demonstrated that the mitotic cells in germinal centers are evidently different from the latter in the absence of lamellary arranged endoplasmic reticulum with ribosomes and Golgi apparatus, and are quite similar to the ultrastructure of thymic lymphocytes in mitosis reported by Murray et al. It should be concluded from these findings that the cells produced locally within the germinal centers in human lymph nodes are lymphocytic as has been repeatedly suggested by the authors.Supported by Grant in Aid from the Ministry of Education of Japan (69-9254).     

FcgammaRIIB regulates autoreactive primary antibody-forming cell, but not germinal center B cell, activity

The low-affinity FcR for IgG FcgammaRIIB suppresses the development of IgG autoantibodies and autoimmune disease in normal individuals, but how this effect is mediated is incompletely understood. To investigate this issue, we created FcgammaRIIB-deficient versions of two previously described targeted BCR-transgenic lines of mice that contain follicular B cells with specificity for the hapten arsonate, but with different levels of antinuclear autoantigen reactivity. The primary development and tolerance of both types of B cells were unaltered by the absence of FcgammaRIIB. Moreover, the reduced p-azophenylarsonate-driven germinal center and memory responses characteristic of the highly autoreactive clonotype were not reversed by an intrinsic FcgammaRIIB deficiency. In contrast, the p-azophenylarsonate-driven primary Ab-forming cell responses of both clonotypes were equivalently increased by such a deficiency. In total, our data do not support the idea that FcgammaRIIB directly participates in the action of primary or germinal center tolerance checkpoints. In contrast, this receptor apparently contributes to the prevention of autoimmunity by suppressing the production of autoreactive IgGs from B cells that have breached tolerance checkpoints and entered the Ab-forming cell pathway due to spontaneous, or cross-reactive, Ag-mediated activation.     

Myocardial ischemia differentially regulates LKB1 and an alternate 5'-AMP-activated protein kinase kinase

During myocardial ischemia, activation of 5'-AMP-activated protein kinase (AMPK) leads to the stimulation of glycolysis and fatty acid oxidation. Together these metabolic changes contribute to cardiac dysfunction. Although AMPK signaling in the ischemic heart is well characterized, the relative contribution of phosphorylation by AMPK kinase (AMPKK), and positive allosterism by the ratios of AMP:ATP and creatine (Cr):phosphocreatine (PCr), in stimulating AMPK during ischemia are unknown. In hearts subjected to severe ischemia, the ratios of AMP:ATP and Cr:PCr were significantly elevated as compared with aerobic hearts. Severe ischemia stimulated AMPK signaling, as demonstrated by an increase in both AMPK activity and acetyl-CoA carboxylase phosphorylation. Although AMPK phosphorylation was increased by severe ischemia, the protein abundance and activity of the recently identified AMPKK, LKB1, were similar between aerobic and severely ischemic hearts. However, in contrast to LKB1, the activity of AMPKK was stimulated in severely ischemic hearts. To further delineate the relative roles of positive allosterism and AMPKK in the regulation of AMPK during ischemia, hearts were subjected to mild ischemia. Although mild ischemia did not alter the ratios of AMP:ATP and Cr:PCr, mild ischemia increased AMPK activity and increased AMPK phosphorylation. Mild ischemia also stimulated the activity of AMPKK. In summary, we demonstrate that myocardial ischemia stimulates AMPK via an AMPKK other than LKB1. Additionally, we show that changes in high energy phosphates are not essential for the activation of AMPK by ischemia. Our data emphasize the critical role AMPKK plays in mediating AMPK signaling during myocardial ischemia.     

Immunobiology of chicken germinal center: II. Accumulation of apoptotic cells within the germinal center

The germinal center (GC) develops after antigen stimulation and is thought to occur at the site of various immune responses. We observed apoptotic cells within the GC using in situ end labeling (TUNEL), small amount DNA ladder assay, and RT-PCR analysis of Bcl-2 mRNA expression. Apoptosis was detected within GCs at all phases of the GC reaction by both TUNEL and DNA ladder assays. The number of TUNEL⁺ nuclei within the GC did not increase over the course of the GC reaction. However, the density of DNA in the ladder assay was higher in later-phase GCs. Bcl-2 mRNA expression was detected within GCs during the early phases of the GC reaction. These results indicate that accumulation of apoptotic cells and rescue from apoptosis occur within chicken GCs. In the present paper, the reasons for the accumulation of apoptotic cells will be discussed.This work was supported by Grants-in-Aid for Scientific Research (Nos. 11670322 and 10306017) from the Ministry of Education, Science, Sport and Culture, and the Ministry of Agriculture, Forestry and Fisheries of Japan (Special Scientific Research and Pioneering Research Project in Biotechnology), as well as from the Bio-oriented Technology Research Advancement Institution (BRAIN)     

Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

Antigen‐specific B‐cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B‐cell endosomal trafficking pathways and their specific roles in B‐cell responses have not been systematically investigated. Here, we report high‐throughput identification of genes regulating B‐cell receptor (BCR)‐mediated antigen internalization using genome‐wide functional screens. We show that antigen internalization depends both on constitutive, clathrin‐mediated endocytosis and on antigen‐induced, clathrin‐independent endocytosis mediated by endophilin A2. Although endophilin A2‐mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B‐cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2‐deficient mice show defects in GC B‐cell responses and production of high‐affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin‐independent intracellular trafficking in GC B‐cell clonal expansion and antibody responses.     

The SAD-1 kinase regulates presynaptic vesicle clustering and axon termination

During synapse formation, presynaptic axon outgrowth is terminated, presynaptic clusters of vesicles are associated with active zone proteins, and active zones are aligned with postsynaptic neurotransmitter receptors. We report here the identification of a novel serine/threonine kinase, SAD-1, that regulates several aspects of presynaptic differentiation in C. elegans. In sad-1 mutant animals presynaptic vesicle clusters in sensory neurons and motor neurons are diffuse and disorganized. Sensory axons fail to terminate in sad-1 mutants, whereas overexpression of SAD-1 causes sensory axons to terminate prematurely. SAD-1 protein is expressed in the nervous system and localizes to synapse-rich regions of the axons. SAD-1 is related to PAR-1, a kinase that regulates cell polarity during asymmetric cell division. Overexpression of SAD-1 causes mislocalization of vesicle proteins to dendrites, suggesting that sad-1 affects axonal-dendritic polarity as well as synaptic development.     

Complementary effects of TNF and lymphotoxin on the formation of germinal center and follicular dendritic cells

The formation of germinal centers (GC) around follicular dendritic cells (FDC) is a critical step in the humoral immune responses that depends on the cooperative effects of B cells and T cells. Mice deficient in either TNF or lymphotoxin (LT) fail to form both GC and FDC network in B cell follicles. To test a potential complementary effect of TNF and LT, a mixture of bone marrow cells from TNF(-/-) mice and LT alpha(-/-) mice was transferred into irradiated LT alpha(-/-) mice or TNF(-/-) mice. Interestingly, the formation of both GC and FDC clusters in B cell follicles was restored in such chimeric mice, suggesting that TNF and LT from different cells could complement one another. To identify the exact contributions of each subset to the complementary effect of TNF and LT, different sources of T and B cells from LT alpha(-/-) mice or TNF(-/-) mice were used for reconstitution. Our study demonstrates that either T or B cell-derived TNF is sufficient to restore FDC/GC in the presence of LT-expressing B cells. However, TNF itself is not required for GC reactions if the FDC network is already intact. Thus, the development and maintenance of these lymphoid structures depend on a delicate interaction between TNF and LT from different subsets of lymphocytes.     

Cutting edge: cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein protects germinal center B cells from apoptosis during germinal center reactions

During germinal center (GC) reactions, follicular dendritic cells are believed to select memory B lymphocytes by switching off apoptosis in the successfully binding B cells. The cellular signals involved in this process are largely unknown. Here, we show that GC B lymphocytes have a long isoform of the cellular homologue of the viral Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (cFLIP(L)), which is capable of inhibiting death receptor-induced caspase activation. In isolated GC B cells, cFLIP(L) decays rapidly even without Fas ligation, and this results in activation of caspase activity and apoptosis. Contact with follicular dendritic cells prevents cFLIP(L) degradation and blocks all signs of apoptosis, even in the presence of anti-Fas ABS: cFLIP(L) expression is sustained by CD40 ligation as well, suggesting that at least at some stage of the GC reaction activated T cells may help selected B cells to leave the follicular dendritic cell network without becoming apoptotic.     

The BTB-kelch protein KLHL6 is involved in B-lymphocyte antigen receptor signaling and germinal center formation

BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase Cgamma2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response.     

GEFH1 binds ASAP1 and regulates podosome formation

Invadopodia are cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing a BAR domain, is a substrate of Src. ASAP1 is required for the assembly of invadopodia and podosomes, which are Src-induced structures related to invadopodia in NIH 3T3 fibroblasts. The BAR domain of ASAP1 is required for the assembly of podosomes. Using two-hybrid screening, we have identified GEFH1, a guanine nucleotide exchange factor for RhoA, as a binding partner of the BAR domain of ASAP1. We validated the interaction of endogenous GEFH1 with ASAP1 by immunoprecipitation, and found GEFH1 colocalized with ASAP1 in podosomes. The overexpression of GEFH1 inhibited podosome assembly and ASAP1 catalytic activity as a GAP. A mutant of GEFH1 lacking the domain that binds to the BAR domain of ASAP1 was less effective. Reduced expression of GEFH1, achieved with siRNA treatment, did not affect matrix degradation by podosomes but increased the rate of podosome assembly. Based on these results, we conclude that GEFH1 is a negative regulator of podosomes.