The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell-dependent monocytic cells with B cell-activating ability.

Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin ß receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

Substrate recognition by Arg/Pro-rich insert domain in calcium/calmodulin-dependent protein kinase kinase for target protein kinases.

Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+ -signaling pathways. Mammalian cells expressing CaMKKα and CaMKKß lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIα and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKKα and CaMKKß inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKIα at Thr177, HA-CaMKIV at Thr196, and HA-AMPKα at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKKα and CaMKKß interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors.

Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKß) with a similar potency (Ki = 0.35 µM for CaMKKα, and Ki = 0.2 µM for CaMKKß) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 µM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

Regulation of Ca2+/calmodulin-dependent protein kinase kinase ß by cAMP signaling.

BACKGROUND: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKß is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKß into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKß at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKß in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKß. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKß is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKß-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKß at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKß at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKß in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKß into Ca2+/CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKß-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKß signaling through regulatory phosphorylation.

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase ß.

The Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKß that exhibits higher basal activity (autonomous activity), activation of the CaMKKß/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKKß appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKß activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKß at multiple residues by CaMKKß-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKKß activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKß indicated that Thr144 phosphorylation by activated AMPK converts CaMKKß into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKß mutant. CaMKKß mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKß in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKß regulates the CaMKKß/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKKß.

Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help.

To reconcile conflicting reports on the role of CD40 signaling in germinal center (GC) formation, we examined the earliest stages of murine GC B cell differentiation. Peri-follicular GC precursors first expressed intermediate levels of BCL6 while co-expressing the transcription factors RelB and IRF4, the latter known to repress Bcl6 transcription. Transition of GC precursors to the BCL6hi follicular state was associated with cell division, although the number of required cell divisions was immunogen dose dependent. Potentiating T cell help or CD40 signaling in these GC precursors actively repressed GC B cell maturation and diverted their fate towards plasmablast differentiation, whereas depletion of CD4+ T cells promoted this initial transition. Thus while CD40 signaling in B cells is necessary to generate the immediate precursors of GC B cells, transition to the BCL6hi follicular state is promoted by a regional and transient diminution of T cell help.

Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKß phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 µm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKß chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKß/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.

CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34.

With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b⁺ monocytic cells (F4/80⁺, Gr-1⁻, Ly6C⁻, I-A/E(-/lo), CD11c⁻, CD115⁺, CXCR4⁺, CCR2⁺, CX3CR1⁻) when cultured with a Lin⁻c-kit⁺ population from mouse spleen cells. The developed CD11b⁺ cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

[Creation of valuable antibodies by an in vitro antibody generation system using a hypermutating B cell line].

Monoclonal antibodies (mAb) have recently proven to be excellent biopharmaceutical agents. The generation of hybridomas from antigen-stimulated B cells has been a key technology for obtaining mAbs; however, it is a laborious and time-consuming process, and sometimes mAbs for molecules conserved between species are difficult to obtain because of immunological tolerance. Thus, it is of great importance to develop in vitro technologies for generating useful Abs as drug candidates. We have been attempting to develop a novel in vitro antibody generation system using a chicken B cell line DT40, which displays Abs and mutates Ig genes during culture, thereby generating a useful Ab library for screening mAbs. First, we generated an engineered cell line DT40-SW whose mutation machinery can be reversibly switched on and off. The Ab generation system using DT40-SW is useful in the following ways: (1) mAbs for various model antigens including antoantigens can be obtained from the DT40-SW Ab library that is free from immunological tolerance; (2) the switching device of the mutation machinery enables fixing desirable Ig mutants by stopping mutation; (3) by repeated culture and sorting of clones bearing higher affinity for target antigens, affinity maturation can be mimicked in vitro. We have also genetically improved DT40-SW cells for mutation efficiency and Ab production. The Ab generation system will be applicable for obtaining valuable Abs such as antitumor Abs.

Establishment of lymphotoxin beta receptor signaling-dependent cell lines with follicular dendritic cell phenotypes from mouse lymph nodes.

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin beta receptor mAb and TNF-alpha. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, FcgammaRIIB, lymphotoxin beta receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.

B cell selection and affinity maturation during an antibody response in the mouse with limited B cell diversity.

The quasi-monoclonal mouse has limited B cell diversity, whose major (approximately 80%) B cell Ag receptors are comprised of the knockin V(H) 17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although V(H)T/lambda1 and V(H)T/lambda2 IgM were equally produced, V(H)T/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of V(H)T (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated V(H)T-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of V(H)T/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that V(H)T/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than V(H)T/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.

Contribution of light chain rearrangement in peripheral B cells to the generation of high-affinity antibodies.

Recently, peripheral B cells have been shown to undergo secondary V(D)J rearrangement of immunoglobulin genes, but the physiological role of this event has not been fully elucidated. To investigate whether rearrangement of L chain genes in the periphery is involved in the generation of high-affinity antibodies (Ab), we used the 17.2.25 rearranged VHDJH gene (VHT)-knockin mouse whose B cell diversity is limited due to the expression of the site-directed transgene. Immunization of the mouse with p-nitrophenylacetyl (pNP)-conjugated chicken gamma-globulin preferentially led to the production of anti-pNP IgG Ab comprised of non-VHT-encoded H chains and lambda chains. lambda(+) IgG constituted a majority of high-affinity Ab to this hapten. RAG-2 mRNA and the recombination signal sequence break of the lambda1 gene increased in the draining lymph node of immunized mice, but not of nonimmunized animals. There was a close correlation between the levels of these parameters implicating lambda gene rearrangement and the production of lambda(+ )high-affinity anti-pNP IgG. These observations were reproduced in RAG-1-deficient mice that were reconstituted with the spleen cells ofthe knockin mouse. Thus, our findings suggest that L chain rearrangement that occurs in the periphery can contribute to affinity maturation of Ab.