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1.
PLoS One ; 18(2): e0279389, 2023.
Article in English | MEDLINE | ID: mdl-36763580

ABSTRACT

Immunoglobulin (Ig) G4 is an IgG subclass that can exhibit inhibitory functions under certain conditions because of its capacity to carry out Fab-arm exchange, inability to form immune complexes, and lack of antibody-dependent and complement-dependent cytotoxicity. Although several diseases have been associated with IgG4, its role in the disease pathogeneses remains unclear. Since mice do not express an IgG subclass that is identical to the human IgG4 (hIgG4), we generated hIGHG4 knock-in (KI) mice and analyzed their phenotypes. To preserve the rearrangement of the variable, diversity, and joining regions in the IGH gene, we transfected a constant region of the hIGHG4 gene into C57BL/6NCrSlc mice by using a gene targeting method. Although the mRNA expression of hIGHG4 was detected in the murine spleen, the serum level of the hIgG4 protein was low in C57BL/6-IgG4KI mice. To enhance the production of IgG4, we established an MRL/lpr-IgG4KI mice model by backcrossing. These mice showed a high IgG4 concentration in the sera and increased populations of IgG4-positive plasma cells and CD3+B220+CD138+ T cells in the spleen. Moreover, these mice showed aggravated inflammation in organs, such as the salivary glands and stomach. The MRL/lpr-IgG4KI mouse model established in the present study might be useful for studying IgG4-related disease, IgG4-type antibody-related diseases, and allergic diseases.


Subject(s)
Immunoglobulin G4-Related Disease , T-Lymphocytes , Humans , Mice , Animals , Immunoglobulin G , Mice, Inbred MRL lpr , Mice, Inbred C57BL , Spleen
2.
ACS Omega ; 6(42): 27968-27975, 2021 Oct 26.
Article in English | MEDLINE | ID: mdl-34722996

ABSTRACT

Platelet adhesion and denaturation on artificial medical implants induce thrombus formation. In this study, bioabsorbable copolymers composed of poly(l-lactide-co-glycolide) (PLGA) and poly(1,5-dioxepan-2-one) (PDXO) were synthesized and evaluated for their antiplatelet adhesive properties. The PLGA-PXO multiblock copolymer (PLGA-PDXO MBC) and its random copolymer (PLGA-PDXO RC) showed effective antiplatelet adhesive properties, and the number of adhered platelets was similar to those adhered on poly(2-methoxyethylacrylate), a known antiplatelet adhesive polymer, although a large number of denatured platelets were observed on a PLGA-poly(ε-caprolactone) multiblock copolymer (PLGA-PCL MBC). Using monoclonal antifibrinogen IgG antibodies, we also found that both αC and γ-chains, the binding sites of fibrinogen for platelets, were less exposed on the PLGA-PDXO MBC surface compared to PLGA-PCL MBC. Furthermore, free-standing films of PLGA-PDXO MBC were prepared by casting the polymer solution on glass plates and showed good tensile properties and slow hydrolytic degradation in phosphate-buffered saline (pH = 7.4). We expect that the unique properties of PLGA-PDXO MBC, i.e., antiplatelet adhesive behavior, good tensile strength, and hydrolytic degradation, will pave the way for the development of new bioabsorbable implanting materials suitable for application at blood-contacting sites.

3.
Int Immunol ; 33(2): 79-90, 2021 Jan 28.
Article in English | MEDLINE | ID: mdl-32889526

ABSTRACT

In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent germinal center (GC) formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B-cell repertoire from early to late stages of immune responses to T cell-dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild-type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1+ antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes; thus, Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low-affinity antibody by high-affinity antibody as the immune response progresses.

4.
Cancers (Basel) ; 12(10)2020 Oct 19.
Article in English | MEDLINE | ID: mdl-33086625

ABSTRACT

As glioma stem cells are chemo- and radio-resistant, they could be the origins of recurrent malignant glioma. Boron neutron capture therapy (BNCT) is a tumor-selective particle radiation therapy. 10B(n,α)7Li capture reaction produces alpha particles whose short paths (5-9 µm) lead to selective killing of tumor cells. P-boronophenylalanine (BPA) is a chemical compound used in clinical trials for BNCT. Here, we used mass cytometry (Cytof) to investigate whether glioma stem-like cells (GSLCs) take up BPA or not. We used GSLCs, and cells differentiated from GSLCs (DCs) by fetal bovine serum. After exposure to BPA for 24 h at 25 ppm in 5% CO2 incubator, we immune-stained them with twenty stem cell markers, anti-Ki-67, anti-BPA and anti-CD98 (heterodimer that forms the large BPA transporter) antibodies and analyzed them with Cytof. The percentage of BPA+ or CD98+ cells with stem cell markers (Oct3/4, Nestin, SOX2, Musashi-1, PDGFRα, Notch2, Nanog, STAT3 and C-myc, among others) was 2-4 times larger among GSLCs than among DCs. Analyses of in vivo orthotopic tumor also indicated that 100% of SOX2+ or Nestin+ GSLCs were BPA+, whereas only 36.9% of glial fibrillary acidic protein (GFAP)+ DCs were BPA+. Therefore, GSLCs may take up BPA and could be targeted by BNCT.

5.
Int J Mol Sci ; 21(12)2020 Jun 14.
Article in English | MEDLINE | ID: mdl-32545905

ABSTRACT

Spinocerebellar ataxia (SCA) is one of the most severe neurodegenerative diseases and is often associated with misfolded protein aggregates derived from the genetic mutation of related genes. Recently, mutations in CD10 such as C143Y have been identified as SCA type 43. CD10, also known as neprilysin or neuroendopeptidase, digests functional neuropeptides, such as amyloid beta, in the extracellular region. In this study, we explored the cellular behavior of CD10 C143Y to gain an insight into the functional relationship of the mutation and SCA pathology. We found that wild-type CD10 is expressed on the plasma membrane and exhibits endopeptidase activity in a cultured cell line. CD10 C143Y, however, forms a disulfide bond-mediated oligomer that does not appear by the wild-type CD10. Furthermore, the CD10 C143Y mutant was retained in the endoplasmic reticulum (ER) by the molecular chaperone BiP and was degraded through the ER-associated degradation (ERAD) process, in which representative ERAD factors including EDEM1, SEL1L, and Hrd1 participate in the degradation. Suppression of CD10 C143Y ERAD recovers intracellular transport but not enzymatic activity. Our results indicate that the C143Y mutation in CD10 negatively affects protein maturation and results in ER retention and following ERAD. These findings provide beneficial insight into SCA type 43 pathology.


Subject(s)
Mutation , Neprilysin/chemistry , Neprilysin/metabolism , Spinocerebellar Ataxias/genetics , Cell Membrane/metabolism , Cysteine/genetics , Disulfides/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation , HeLa Cells , Humans , Neprilysin/genetics , Oligopeptides/metabolism , Proteolysis
6.
Int Immunol ; 32(1): 17-26, 2020 01 09.
Article in English | MEDLINE | ID: mdl-31412363

ABSTRACT

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , B-Lymphocytes/immunology , Adaptor Proteins, Signal Transducing/deficiency , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout
7.
J Immunol ; 202(11): 3161-3172, 2019 06 01.
Article in English | MEDLINE | ID: mdl-31019063

ABSTRACT

A defect in TCR-proximal signaling is a major characteristic of CD4 T cells in systemic lupus erythematosus; however, it is not fully known how defects in TCR signaling lead to lupus-like systemic autoimmunity characterized by germinal center development and autoantibody production against nuclear Ags. In this study, we show that SKG mice, which develop autoimmune arthritis in a BALB/c background due to defective TCR signaling by a Zap70 mutation, develop lupus-like systemic autoimmune disease in the C57BL/6 (B6) background (B6SKG mice). B6SKG mice showed multiorgan inflammation with immune complex deposition and anti-dsDNA Ab production. Follicular helper T cells (Tfh), which help germinal center formation, were spontaneously expanded in B6SKG mice. Th cells secreting IFN-γ or IL-17 and regulatory T cells were also increased in B6SKG mice compared with wild-type B6 mice, with the regulatory T cell subpopulation losing the expression of CD25. Among the factors related to Tfh differentiation, the number of dendritic cells and the expression levels of the costimulatory molecules CD80, CD86, and ICOSL in dendritic cells but not in B cells were specifically increased in wild-type B6 mice compared with BALB/c mice. The inhibition of these costimulatory molecules suppressed Tfh development and lupus-like autoimmunity. Thus, a defect in TCR-proximal signaling leads to lupus-like systemic autoimmunity under the specific genetic background that facilitates Tfh development.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Germinal Center/immunology , Lupus Erythematosus, Systemic/genetics , Mutation/genetics , ZAP-70 Protein-Tyrosine Kinase/genetics , Animals , Antibodies, Antinuclear/metabolism , Autoimmunity , Cell Differentiation , Cells, Cultured , Cytokines/metabolism , Humans , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Antigen, T-Cell/metabolism
8.
Eur J Immunol ; 49(6): 911-917, 2019 06.
Article in English | MEDLINE | ID: mdl-30888050

ABSTRACT

The BCR plays a central role in B cell development, survival, activation, and differentiation. We have identified the B cell novel protein 1 (BCNP1) as a new regulator of BCR signaling. BCNP1 contains a pleckstrin homology domain, three proline-rich motifs, and a potential SH2 binding site, and is predominantly expressed by B cells. We found that BCNP1 overexpression in WEHI231 immature B cells potentiated α-IgM-induced apoptosis. Conversely, BCNP1-deficient WEHI231 cells, generated by CRISPR-Cas9-mediated genome editing, exhibited reduced apoptosis after BCR crosslinking. Biochemical analyses revealed that BCNP1 physically interacted with the B cell linker protein (BLNK), Grb2, and PLCγ2. Moreover, absence of BCNP1 resulted in accelerated dephosphorylation of BLNK, reduced phosphorylation of SYK and PLCγ2, and decreased Ca2+ influx after BCR crosslinking. These results demonstrate that BCNP1 promotes BCR signaling by modulating the phosphorylation of BLNK, SYK, and PLCγ2.


Subject(s)
Apoptosis Regulatory Proteins/immunology , Apoptosis/immunology , B-Lymphocytes/immunology , Receptors, Antigen, B-Cell/immunology , Signal Transduction/immunology , Animals , Apoptosis Regulatory Proteins/metabolism , B-Lymphocytes/metabolism , Cell Line , Membrane Proteins/immunology , Membrane Proteins/metabolism , Mice , Receptors, Antigen, B-Cell/metabolism
9.
PLoS One ; 12(9): e0184738, 2017.
Article in English | MEDLINE | ID: mdl-28910360

ABSTRACT

Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. TAGLN2 is also expressed on B-cells but its function in B-cells is unknown. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) of secondary lymphoid tissues and TAGLN2 mRNA was significantly upregulated after IgM+IgG stimulation in primary human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. In support of this, lymph nodes (LNs) from patients with systemic lupus erythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2+B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. In contrast, CD19+ B-cells and CD19+CD27+ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. Two-photon excitation microscopy of Raji cells demonstrated that TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. TAGLN2-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of TAGLN2-knockdown Raji cells showed decreased expression of the genes associated with immune function including CCR6 and as well as of those associated with regulation of the actin cytoskeleton including ABI2, compared to controls. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle. We also suggest that TAGLN2 could be used as a marker for activated B-cells.


Subject(s)
B-Lymphocytes/immunology , Lupus Erythematosus, Systemic/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , B-Lymphocytes/cytology , Cell Movement , Cells, Cultured , Female , Germinal Center/immunology , Humans , Kidney/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lymph Nodes/immunology , Lymphocyte Activation , Male , Up-Regulation
10.
Sci Rep ; 6: 28378, 2016 06 22.
Article in English | MEDLINE | ID: mdl-27328625

ABSTRACT

Lymphocyte chemotaxis plays important roles in immunological reactions, although the mechanism of its regulation is still unclear. We found that the cytosolic Na(+)-dependent mitochondrial Ca(2+) efflux transporter, NCLX, regulates B lymphocyte chemotaxis. Inhibiting or silencing NCLX in A20 and DT40 B lymphocytes markedly increased random migration and suppressed the chemotactic response to CXCL12. In contrast to control cells, cytosolic Ca(2+) was higher and was not increased further by CXCL12 in NCLX-knockdown A20 B lymphocytes. Chelating intracellular Ca(2+) with BAPTA-AM disturbed CXCL12-induced chemotaxis, suggesting that modulation of cytosolic Ca(2+) via NCLX, and thereby Rac1 activation and F-actin polymerization, is essential for B lymphocyte motility and chemotaxis. Mitochondrial polarization, which is necessary for directional movement, was unaltered in NCLX-knockdown cells, although CXCL12 application failed to induce enhancement of mitochondrial polarization, in contrast to control cells. Mouse spleen B lymphocytes were similar to the cell lines, in that pharmacological inhibition of NCLX by CGP-37157 diminished CXCL12-induced chemotaxis. Unexpectedly, spleen T lymphocyte chemotaxis was unaffected by CGP-37157 treatment, indicating that NCLX-mediated regulation of chemotaxis is B lymphocyte-specific, and mitochondria-endoplasmic reticulum Ca(2+) dynamics are more important in B lymphocytes than in T lymphocytes. We conclude that NCLX is pivotal for B lymphocyte motility and chemotaxis.


Subject(s)
B-Lymphocytes/cytology , Calcium/metabolism , Chemotaxis/physiology , Sodium-Calcium Exchanger/metabolism , Actins/metabolism , Animals , B-Lymphocytes/metabolism , Cell Line, Tumor , Chemokine CXCL12/metabolism , Clonazepam/analogs & derivatives , Clonazepam/pharmacology , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Mice , Mitochondria/physiology , Neuropeptides/metabolism , Sodium-Calcium Exchanger/genetics , Spleen/cytology , Spleen/immunology , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Thiazepines/pharmacology , rac1 GTP-Binding Protein/metabolism
12.
Sci Rep ; 5: 10303, 2015 May 18.
Article in English | MEDLINE | ID: mdl-25980548

ABSTRACT

The positive and negative selection of antigen-reactive B cells take place in the germinal center (GC) during an immune responses. However, the precise molecular mechanisms underlying these selection machineries, including the involvement of antigen receptor signaling molecules, remain to be elucidated. We found that expression levels of Igα and Igß, which are the essential components of B cell antigen-receptor complex, were differentially regulated in GC B cells and that the expression of Igß was more prominently down-regulated in a portion of GC B cells. The suppression of Igß down-regulation reduced the number of GL7(+)GC B cells and the affinity maturation in T-dependent responses was markedly impaired. In addition, the disease phenotypes in autoimmune-prone mice were ameliorated by blocking of Igß down-regulation. These results suggest that Igß down-regulation is involved in the normal positive selection in GC and the accumulation of autoreactive B cells in autoimmune-prone mice.


Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , CD79 Antigens/metabolism , Germinal Center/immunology , Germinal Center/metabolism , Animals , Antibody Formation , Autoimmunity , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , CD79 Antigens/genetics , Cell Membrane/metabolism , Down-Regulation , Gene Expression Regulation , Immunoglobulin Class Switching , Interleukins/metabolism , Mice , Somatic Hypermutation, Immunoglobulin
13.
Immun Inflamm Dis ; 2(1): 35-43, 2014 Jun.
Article in English | MEDLINE | ID: mdl-25400923

ABSTRACT

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology.

14.
PLoS One ; 9(4): e94324, 2014.
Article in English | MEDLINE | ID: mdl-24710513

ABSTRACT

The agonistic anti-human CD3ε antibody (Ab), OKT3, has been used to control acute transplant rejection. The in vivo administration of OKT3 was previously shown to induce the partial depletion of T cells and unresponsiveness (anergy) in the remaining CD4+ T cells. However, this therapy is also associated with the systemic release of several cytokines, which leads to a series of adverse side effects. We established a novel anti-human CD3ε Ab, 20-2b2, which recognized a close, but different determinant on the CD3ε molecule from that recognized by OKT3. 20-2b2 was non-mitogenic for human CD4+ T cells, could inhibit the activation of T cells in vitro, and induced T cell anergy in in vivo experiments using humanized mice. Cytokine release in humanized mice induced by the administration of 20-2b2 was significantly less than that induced by OKT3. Our results indicated that the CD3ε molecule is still an attractive, effective, and useful target for the modulation of T cell responses. The establishment of other Abs that recognize CD3ε, even though the determinant recognized by those Abs may be close to or different from that recognized by OKT3, may represent a novel approach for the development of safer Ab therapies using anti-CD3 Abs, in addition to the modification of OKT3 in terms of the induction of cytokine production.


Subject(s)
Antibodies, Monoclonal/immunology , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/immunology , Animals , CD4-Positive T-Lymphocytes/metabolism , Clonal Anergy , Cytokines/biosynthesis , Female , Humans , Lymphocyte Activation , Mice
15.
Eur J Immunol ; 44(6): 1770-80, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24595757

ABSTRACT

The activation of T cells is known to be accompanied by the temporary downmodulation of the TCR/CD3 complex on the cell surface. Here, we established a novel monoclonal antibody, Dow2, that temporarily induces downmodulation of the TCR/CD3 complex in mouse CD4(+) T cells without activating T cells. Dow2 recognized the determinant on CD3ε; however, differences were observed in the binding mode between Dow2 and the agonistic anti-CD3ε Ab, 145-2C11. An injection of Dow2 in vivo resulted in T-cell anergy, and prolonged the survival of cardiac allografts without a marked increase in cytokine release. The phosphorylated forms of the signaling proteins PLC-γ1 and LAT in Dow2-induced anergic T cells were markedly decreased upon stimulation. However, the levels of phosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells could be rescued in the presence of the proteasome inhibitor MG-132. These results suggest that proteasome-mediated degradation is involved in hypophosphorylated LAT and PLCγ1 in Dow2-induced anergic T cells. The novel CD3-specific Ab, Dow2, may provide us with a unique tool for inducing immunosuppression.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Antibodies, Monoclonal, Murine-Derived/pharmacology , CD3 Complex/immunology , Clonal Anergy/drug effects , Membrane Proteins/immunology , Phospholipase C gamma/immunology , Phosphoproteins/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal, Murine-Derived/immunology , Male , Mice , Mice, Inbred BALB C , Phosphorylation/drug effects , Phosphorylation/immunology , Proteasome Endopeptidase Complex/immunology , Proteolysis/drug effects
16.
Biochem Biophys Res Commun ; 445(3): 572-7, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24548411

ABSTRACT

It has been shown that cytoplasmic tail of the IgG1 B cell receptors (BCRs) are essential for the induction of T-dependent immune responses. Also it has been revealed that unique tyrosine residue in the cytoplasmic tail of IgG2a has the potential of being phosphorylated at tyrosine and that this phosphorylation modulates BCR signaling. However, it still remains unclear whether such phosphorylation of IgG cytoplasmic tail is involved in the regulation of BCR surface expression. In order to approach the issue, we established and analyzed the cell lines which express wild-type or mutated forms of IgG1 BCR. As the result, we found that IgG1 BCR expressed normally on the surface of A20 B cell line independent of the cytoplasmic tail. In contrast, IgG1 BCR whose cytoplasmic tyrosine was replaced with glutamic acid which mimics phosphorylated tyrosine, was expressed most efficiently on the surface of non-B lineage cells and Igß-down-regulated B cell lines. These results suggest that tyrosine residue in IgG cytoplasmic tail is playing a essential role for the efficient expression of IgG BCR on the cell surface when BCR associated signaling molecules, including Igß, are down-regulated.


Subject(s)
B-Lymphocytes/metabolism , CD79 Antigens/genetics , Gene Expression Regulation, Neoplastic , Immunoglobulin G/genetics , Lymphoma, B-Cell/genetics , Receptors, Antigen, B-Cell/genetics , Receptors, IgG/genetics , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Cell Line, Tumor , Down-Regulation , Humans , Immunoglobulin G/chemistry , Mice , Molecular Sequence Data , Receptors, Antigen, B-Cell/chemistry , Receptors, IgG/chemistry
17.
Dev Biol ; 386(2): 419-27, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24355749

ABSTRACT

MgcRacGAP (RACGAP1) is a GTPase Activating Protein (GAP), highly produced in the mouse embryonic brain and in the human and mouse post-natal testis. MgcRacGAP negatively controls the activity of Rac and Cdc42, which are key molecular switches acting on the microtubule and actin cytoskeleton and controlling various cell processes such as proliferation, adhesion and motility. Previous studies demonstrated that MgcRacGAP plays a critical role in the cytokinesis of somatic cells; hence homozygous inactivation of the gene in the mouse and mutation in Caenorhabditis elegans led to embryonic lethality due to the inability of MgcRacGAP-null embryos to assemble the central spindle and to complete cytokinesis. In the testis, the germ cells do not complete cytokinesis and remain connected as a syncytium throughout the entire process of spermatogenesis. Interestingly, MgcRacGAP was shown to locate to the intercellular bridges, connecting these germ cells. In order to determine the function(s) of MgcRacGAP in the male germline, we generated a conditional knock-out mouse using Stra8 promoter driven Cre recombinase to induce the specific deletion of MgcRacGAP in the pre-meiotic germ cells. We found that the absence of MgcRacGAP induced a germline depletion and male sterility. Consistent with the role of MgcRacGAP in the establishment of the cytoplasm constriction during cytokinesis of the somatic cells, we observed that MgcRacGAP deletion in the germ cells prevented the formation of the intercellular bridges and induced a proliferation arrest. While we assume that inherited homozygous loss of function mutations in MgcRacGAP would be lethal in human, de novo mutations in the testis might account for some cases of non-obstructive oligo- and/or azoo-spermia syndromes, whose genetic causes are altogether still poorly defined.


Subject(s)
GTPase-Activating Proteins/deficiency , Germ Cells/chemistry , Infertility, Male/genetics , Spermatogenesis/physiology , Testis/metabolism , Animals , Blotting, Western , Immunohistochemistry , In Situ Nick-End Labeling , Male , Mice , Mice, Transgenic , Microscopy, Electron , Spermatogenesis/genetics , Testis/ultrastructure
18.
J Exp Med ; 210(11): 2257-71, 2013 Oct 21.
Article in English | MEDLINE | ID: mdl-24127488

ABSTRACT

Myeloid-derived suppressor cells (MDSCs) favor tumor promotion, mainly by suppressing antitumor T cell responses in many cancers. Although the mechanism of T cell inhibition is established, the pathways leading to MDSC accumulation in bone marrow and secondary lymphoid organs of tumor-bearing hosts remain unclear. We demonstrate that down-regulation of PLCγ2 signaling in MDSCs is responsible for their aberrant expansion during tumor progression. PLCγ2(-/-) MDSCs show stronger immune-suppressive activity against CD8(+) T cells than WT MDSCs and potently promote tumor growth when adoptively transferred into WT mice. Mechanistically, PLCγ2(-/-) MDSCs display reduced ß-catenin levels, and restoration of ß-catenin expression decreases their expansion and tumor growth. Consistent with a negative role for ß-catenin in MDSCs, its deletion in the myeloid population leads to MDSC accumulation and supports tumor progression, whereas expression of ß-catenin constitutively active reduces MDSC numbers and protects from tumor growth. Further emphasizing the clinical relevance of these findings, MDSCs isolated from pancreatic cancer patients show reduced p-PLCγ2 and ß-catenin levels compared with healthy controls, similar to tumor-bearing mice. Thus, for the first time, we demonstrate that down-regulation of PLCγ2-ß-catenin pathway occurs in mice and humans and leads to MDSC-mediated tumor expansion, raising concerns about the efficacy of systemic ß-catenin blockade as anti-cancer therapy.


Subject(s)
Down-Regulation , Myeloid Cells/metabolism , Myeloid Cells/pathology , Neoplasms/metabolism , Neoplasms/pathology , Phospholipase C gamma/metabolism , beta Catenin/metabolism , Adoptive Transfer , Animals , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Disease Progression , Female , Humans , Immunosuppression Therapy , Mice , Mice, Inbred C57BL , Phospholipase C gamma/deficiency , Protein Stability , Signal Transduction
19.
Adv Exp Med Biol ; 961: 193-201, 2013.
Article in English | MEDLINE | ID: mdl-23224880

ABSTRACT

Mitochondria Na(+)-Ca(2+) exchange (NCX(mit)) was first discovered by Carafoli et al. in 1974. Thereafter, the mechanisms and roles of NCX(mit) have been extensively studied. We review NCX(mit) in cardiomyocytes and lymphocytes by presenting our recent studies on it. Studies of NCX(mit) in rat ventricular cells demonstrated that NCX(mit) is voltage dependent and electrogenic. A targeted knockdown and knockout of NCLX in HL-1 cardiomyocytes and B lymphocytes, respectively, significantly reduced the NCX(mit) activity, indicating that NCLX is a major component of NCX(mit) in these cells. The store-operated Ca(2+) entry was greatly attenuated in NCLX knockout lymphocytes, suggesting that substantial amount of Ca(2+) enters into mitochondria and is released to cytosol via NCX(mit). NCX(mit) or NCLX has pivotal roles in Ca(2+) handling in mitochondria and cytoplasm.


Subject(s)
B-Lymphocytes/metabolism , Calcium/metabolism , Mitochondrial Proteins/metabolism , Muscle Proteins/metabolism , Myocytes, Cardiac/metabolism , Sodium-Calcium Exchanger/metabolism , Animals , Cytoplasm/genetics , Cytoplasm/metabolism , Gene Knockdown Techniques , Humans , Ion Channel Gating/physiology , Mitochondrial Proteins/genetics , Muscle Proteins/genetics , Rats , Sodium-Calcium Exchanger/genetics
20.
J Exp Med ; 209(11): 2079-97, 2012 Oct 22.
Article in English | MEDLINE | ID: mdl-23027924

ABSTRACT

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.


Subject(s)
Antibodies/immunology , B-Lymphocytes/immunology , Germ Cells/immunology , Germinal Center/immunology , Immunologic Memory/immunology , Signal Transduction/immunology , Animals , Antibodies/genetics , Antibodies/metabolism , B-Lymphocytes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Female , Flow Cytometry , Gene Expression Profiling , Germ Cells/metabolism , Germinal Center/metabolism , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Immunoglobulin Variable Region/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-bcl-6 , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Signal Transduction/genetics
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