Glioma Stem-Like Cells Can Be Targeted in Boron Neutron Capture Therapy with Boronophenylalanine.

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.

Endoplasmic Reticulum Associated Degradation of Spinocerebellar Ataxia-Related CD10 Cysteine Mutant.

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.

Roles of the mitochondrial Na(+)-Ca(2+) exchanger, NCLX, in B lymphocyte chemotaxis.

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.

Modulation of Igß is essential for the B cell selection in germinal center.

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.

IgG1 cytoplasmic tail is essential for cell surface expression in Igß down-regulated cells.

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.

Down-regulation of PLCγ2-ß-catenin pathway promotes activation and expansion of myeloid-derived suppressor cells in cancer.

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.

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory.

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.

CIN85 drives B cell responses by linking BCR signals to the canonical NF-kappaB pathway.

CIN85, an adaptor protein which binds the C-terminal domain of tyrosine phosphorylated Cbl and Cbl-b, has been thought to be involved in the internalization and subsequent degradation of receptors. However, its physiological function remains unclear. To determine its role in B cells, we used Mb1-cre to generate mice with a B cell-specific deletion of CIN85. These mice had impaired T cell-independent type II antibody responses in vivo and diminished IKK-ß activation and cellular responses to B cell receptor (BCR) cross-linking in vitro. Introduction of a constitutively active IKK-ß construct corrected the defective antibody responses as well as cellular responses in the mutant mice. Together, our results suggest that CIN85 links the BCR to IKK-ß activation, thereby contributing to T cell-independent immune responses.

Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.

The transcription factor Bcl6 is essential for the development of germinal center (GC) B cells and follicular helper T (Tfh) cells. However, little is known about in vivo dynamics of Bcl6 protein expression during and after development of these cells. By using a Bcl6 reporter mouse strain, we found that antigen-engaged B cells upregulated Bcl6 before clustering in GCs. Two-photon microscopic analysis indicated that Bcl6 upregulation in pre-GC B cells contributed to sustaining their interactions with helper T cells and was required for their entry to GC clusters. Our data also suggested that Tfh cells gradually downmodulated Bcl6 protein over weeks after development. The Bcl6-low Tfh cells rapidly terminated proliferation and upregulated IL-7 receptor. These results clarify the role of Bcl6 in pre-GC B cell dynamics and highlight the modulation of Bcl6 expression in Tfh cells that persist in the late phase of the antibody response.

A lysosomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3zeta-chain degradation.

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

Essential roles of mgcRacGAP in multilineage differentiation and survival of murine hematopoietic cells.

MgcRacGAP, a negative regulator for Rho family GTPases, has been shown to play important roles in cytokinesis using several cell lines. However, the physiological role of mgcRacGAP in multilineage hematopoietic development remains unclear. Here, we conditionally ablated mgcRacGAP in vivo to clarify this issue. As the result, we found that normal hematopoietic development including proliferation and survival requires mgcRacGAP. We also found that depletion of mgcRacGAP in hematopoietic cells results in a marked decrease in c-Kit(+)Sca-1(+)Lin(-) cells, suggesting that mgcRacGAP is required for the maintenance of the hematopoietic stem cells. In addition, B cells in which mgcRacGAP had been selectively ablated showed proliferation failure and fell into apoptosis. Taken together, mgcRacGAP is now shown to play a indispensable role in the development of hematopoietic cells in vivo.

Phospholipase C-gamma2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen.

B cell receptor (BCR) recognition of membrane-bound antigen initiates a spreading and contraction response, the extent of which is controlled through the formation of signaling-active BCR-antigen microclusters and ultimately affects the outcome of B cell activation. We followed a genetic approach to define the molecular requirements of BCR-induced spreading and microcluster formation. We identify a key role for phospholipase C-gamma2 (PLCgamma2), Vav, B cell linker, and Bruton's tyrosine kinase in the formation of highly coordinated "microsignalosomes," the efficient assembly of which is absolutely dependent on Lyn and Syk. Using total internal reflection fluorescence microscopy, we examine at high resolution the recruitment of PLCgamma2 and Vav to microsignalosomes, establishing a novel synergistic relationship between the two. Thus, we demonstrate the importance of cooperation between components of the microsignalosome in the amplification of signaling and propagation of B cell spreading, which is critical for appropriate B cell activation.

Peptidoglycan and lipopolysaccharide activate PLCgamma2, leading to enhanced cytokine production in macrophages and dendritic cells.

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

Distinct regulatory functions of SLP-76 and MIST in NK cell cytotoxicity and IFN-gamma production.

Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN-gamma production. Although NKG2D-mediated IFN-gamma production depends on phospholipase C (PLC) gamma 2, YAC-1 cell-triggered IFN-gamma production is PLC gamma 2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN-gamma production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN-gamma production.

Extracellular signal-regulated protein kinase 2 is required for efficient generation of B cells bearing antigen-specific immunoglobulin G.

Activation of extracellular signal-regulated protein kinase (ERK) has been implicated in proliferation as well as differentiation in a wide variety of cell types. Using B-cell-specific gene-targeted mice, we report here that in T-cell-dependent immune responses, ERK2 is required to generate efficient immunoglobulin G (IgG) production. In its absence, the proportion of antigen-specific surface IgG1-bearing cells and the subsequent number of IgG1 antibody-secreting cells were decreased, despite apparently unimpaired class switch recombination. Notably, this defect was countered by overexpression of the antiapoptotic factor Bcl-2. Together, our results suggest that ERK2 plays a key role in efficient generation of antigen-specific IgG-bearing B cells by promoting their survival.

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.

Regulation of cytokinesis by mgcRacGAP in B lymphocytes is independent of GAP activity.

In cytokinesis, several molecules including small G proteins and their regulators are known to have important roles. One of these regulators, mgcRacGAP has GTPase activating protein (GAP) activity for Rac, Cdc42 and Rho. MgcRacGAP has also been shown to be involved in cytokinesis using various cell types. However, the requirement of mgcRacGAP for cytokinesis and survival in B lymphocytes has not been fully examined. Here, we demonstrate that normal cytokinesis in B lymphocytes requires the GAP and NH2 terminal domains but not GAP activity of mgcRacGAP. In addition, we report that apoptosis induced by conditional ablation of mgcRacGAP in the B cell line is fully rescued by the introduction of a GAP-inactive mutant, suggesting that the survival defect in mgcRacGAP-deficient B cells is also independent of GAP activity.

Coupling between B cell receptor and phospholipase C-gamma2 is essential for mature B cell development.

Two signaling pathways known to be essential for progression from immature to mature B cells are BAFF receptor (BAFF-R) and the B cell receptor (BCR). Here, we first show that phospholipase C (PLC)-gamma2 is required for a BAFF-R-mediated survival signal. Then, we have examined the question of whether the reduced number of mature B cells in PLC-gamma2-/- mice is caused by a defect in either BCR or BAFF-R signaling. We find that a PLC-gamma2 SH2 mutant, which inhibits coupling between BCR and PLC-gamma2, fails to restore B cell maturation, despite supporting BAFF-dependent survival. Therefore, our data suggest that the BAFF-R-mediated survival signal, provided by PLC-gamma2, is not sufficient to promote B cell maturation, and that, in addition, activation of PLC-gamma2 by BCR is required for B cell development.

Regulation of Vav localization in membrane rafts by adaptor molecules Grb2 and BLNK.

Despite the importance of the Vav family proteins for B cell receptor (BCR) signaling, their activation mechanisms remain poorly understood. We demonstrate here that adaptor molecules Grb2 and BLNK, in addition to Vav, are required for efficient Rac1 activation in response to BCR stimulation. Loss of either Grb2 or BLNK results in decreased translocation of Vav3 to membrane rafts. By expression of Vav3 as a raft-targeted construct, the defective Rac1 activation in Grb2- or BLNK-deficient B cells is restored. Hence, our findings suggest that Grb2 and BLNK cooperate to localize Vav into membrane rafts, thereby contributing to optimal activation of Vav in B cells.

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.