Satoshi Kojo

IL-21–induced Bε cell apoptosis mediated by natural killer T cells suppresses IgE responses

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Vα14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bɛ cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2–modifying factor, resulting in Bɛ cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell–dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

A common polymorphism in the interleukin 8 gene promoter is associated with Clostridium difficile diarrhea.

In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25+CD4+ regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25+CD4+ T cells increased proliferative response of CD4+ T cells and cytotoxic T lymphocyte induction of CD8+ T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25+CD4+ population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25+CD4+ regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction. Liver Transpl 12:1112–1118, 2006.

Vα14 NK T cell–triggered IFN-γ production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets

Pancreatic islet transplantation is a highly promising approach for the treatment of insulin-dependent diabetes mellitus. However, the procedure remains experimental for several reasons, including its low efficiency caused by the early graft loss of transplanted islets. We demonstrate that Gr-1+CD11b+ cells generated by transplantation and their IFN-γ production triggered by Vα14 NKT cells are an essential component and a major cause of early graft loss of pancreatic islet transplants. Gr-1+CD11b+ cells from Vα14 NKT cell–deficient (Jα281−/−) mice failed to produce IFN-γ, resulting in efficient islet graft acceptance. Early graft loss was successfully prevented through the repeated administration of α-galactosylceramide, a specific ligand for Vα14 NKT cells, resulting in dramatically reduced IFN-γ production by Gr-1+CD11b+ cells, as well as Vα14 NKT cells. Our study elucidates, for the first time, the crucial role of Gr-1+CD11b+ cells and the IFN-γ they produce in islet graft rejection and suggests a novel approach to improving transplantation efficiency through the modulation of Vα14 NKT cell function.

Induction of Regulatory Properties in Dendritic Cells by Vα14 NKT Cells

Vα14 NKT cells exhibit various immune regulatory properties in vivo, but their precise mechanisms remain to be solved. In this study, we demonstrate the mechanisms of generation of regulatory dendritic cells (DCs) by stimulation of Vα14 NKT cells in vivo. After repeated injection of α-galactosylceramide (α-GalCer) into mice, splenic DCs acquired properties of regulatory DCs in IL-10-dependent fashion, such as nonmatured phenotypes and increased IL-10 but reduced IL-12 production. The unique cytokine profile in these DCs appears to be regulated by ERK1/2 and IκBNS. These DCs also showed an ability to suppress the development of experimental allergic encephalomyelitis by generating IL-10-producing regulatory CD4 T cells in vivo. These findings contribute to explaining how Vα14 NKT cells regulate the immune responses in vivo.

Cutting Edge: Critical Role of CXCL16/CXCR6 in NKT Cell Trafficking in Allograft Tolerance

It is well-documented that certain chemokines or their receptors play important roles in the graft rejection. However, the roles of chemokines and their receptors in the maintenance of transplantation tolerance remain unclear. In this study, we demonstrate that blocking of the interaction between the chemokine receptor, CXCR6, highly expressed on Vα14+ NKT cells and its ligand, CXCL16, resulted in the failure to maintain graft tolerance and thus in the induction of acceleration of graft rejection. In a mouse transplant tolerance model, the expression of CXCL16 was up-regulated in the tolerated allografts, and anti-CXCL16 mAb inhibited intragraft accumulation of NKT cells. In vitro experiments further showed that blocking of CXCL16/CXCR6 interaction significantly affected not only chemotaxis but also cell adhesion of NKT cells. These results demonstrate the unique role of CXCL16 and CXCR6 molecules in the maintenance of cardiac allograft tolerance mediated by NKT cells.

Establishment of an Improved Mouse Model for Infantile Neuroaxonal Dystrophy That Shows Early Disease Onset and Bears a Point Mutation in Pla2g6

Calcium-independent group VIA phospholipase A(2) (iPLA(2)beta), encoded by PLA2G6, has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Mutations in the PLA2G6 gene have been recently identified in patients with infantile neuroaxonal dystrophy (INAD). Subsequently, it was reported that similar neurological impairment occurs in gene-targeted mice with a null mutation of iPLA(2)beta, whose disease onset became apparent approximately 1 to 2 years after birth. Here, we report the establishment of an improved mouse model for INAD that bears a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethyl-N-nitrosourea mutagenesis. These mutant mice developed severe motor dysfunction, including abnormal gait and poor performance in the hanging grip test, as early as 7 to 8 weeks of age, in a manner following Mendelian law. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. Because of the significantly early onset of the disease, this mouse mutant (Pla2g6-inad) could be highly useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration.

Mechanisms of NKT cell anergy induction involve Cbl-b-promoted monoubiquitination of CARMA1.

Repeated injection of α-galactosylceramide, an agonistic ligand for natural killer T (NKT) cells, results in long-term unresponsiveness or anergy, which severely limits its clinical application. However, the molecular mechanisms leading to NKT anergy induction remain unclear. We show here that the decreased IFN-γ production and failed tumor rejection observed in anergized NKT cells are rescued by Cbl-b deficiency. Cbl-b E3 ligase activity is critical for the anergy induction, as revealed by the similarity between Cbl-b−/− and its RING finger mutant NKT cells. Cbl-b binds and promotes monoubiquitination to CARMA1, a critical signaling molecule in NFκB activation. Ubiquitin conjugation to CARMA1 disrupts its complex formation with Bcl10 without affecting its protein stability. In addition, CARMA1−/− NKT cells are defective in IFN-γ production. The study identifies an important signaling pathway linking Cbl-b-induced monoubiquitination to NFκB activation in NKT cell anergy induction, which may help design approaches for human cancer therapy.

Mechanism of NKT cell-mediated transplant tolerance.

The mechanism by which CD1d‐restricted Vα14 natural killer T (NKT) cells participate in transplant tolerance has yet to be completely clarified. Recently, we showed that repeated activation of NKT cells by their specific glycolipid ligand, α‐galactosylceramide, leads to a change in function to an immune regulatory role with IL‐10 production. Moreover, these cells were shown to be able to induce regulatory dendritic cells (DCs). In this study, we showed that NKT cells from transplant tolerant recipients of cardiac allograft produced higher levels of IL‐10, which is required for the maintenance of tolerance; this was proved by adoptive transfer experiments. In addition, DCs from wild‐type (WT) tolerant recipients but not NKT cell‐deficient recipients showed a higher IL‐10‐producing profile, a more immature phenotype, and tolerogenic capability. CD4 T cells from WT tolerant recipients but not NKT cell‐deficient recipients also produced higher levels of IL‐10 upon alloantigen stimulation and showed lower proliferative activity that was reversed by blocking the IL‐10 receptor. These data indicate the existence of IL‐10‐dependent immune regulatory interplay among NKT cells, DCs, and CD4 T cells, even in the absence of artificial stimulation of NKT cells with synthetic glicolipids, which is required for the maintenance of transplant tolerance.

Successful differentiation to T cells, but unsuccessful B-cell generation, from B-cell-derived induced pluripotent stem cells

Forced expression of certain transcription factors in somatic cells results in generation of induced pluripotent stem (iPS) cells, which differentiate into various cell types. We investigated T-cell and B-cell lineage differentiation from iPS cells in vitro. To evaluate the impact of iPS cell source, murine splenic B-cell-derived iPS (B-iPS) cells were generated after retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc). B-iPS cells were identical to embryonic stem (ES) cells and mouse embryonic fibroblast (MEF)-derived iPS cells in morphology, ES cell marker expression as well as teratoma and chimera mouse formation. Both B-iPS and MEF-derived iPS cells differentiated into lymphocytes in OP9 co-culture systems. Both efficiently differentiated into T-cell lineage that produced IFN-γ on T-cell receptor stimulation. However, iPS cells including B-iPS cells were relatively resistant to B-cell lineage differentiation. One of the reasons of the failure of B-cell lineage differentiation seemed due to a defect of Pax5 expression in the differentiated cells. Therefore, current in vitro differentiation systems using iPS cells are sufficient for inducing T-cell but not B-cell lineage.

Side population is increased in paclitaxel-resistant ovarian cancer cell lines regardless of resistance to cisplatin

OBJECTIVES In recent years, cancer stem cells (CSCs) have been reported to be correlated with chemoresistance and may also be enriched in side populations (SPs). In this study, the relationship between resistance to paclitaxel (PTX) and cisplatin (CDDP) and side populations was examined in three parental PTX- and CDDP-sensitive ovarian cancer cell lines (2008, KF28, and TU-OM-1) and several other cell lines derived from these as well as the additional effects of interferon-alpha (INF-α). METHODS SP of three different parental cell lines and PTX- and/or CDDP-resistant cell lines derived from these was analyzed with flow cytometry. The expression of ABCB1 and ABCG2 in KF28 and its derived cell lines was examined. Additional cell-death effect of INF-α with PTX was also examined. RESULTS In the three parental cell lines and the PTX-sensitive cell lines derived from these lines, SP was very low. Conversely, in PTX-resistant cell lines, regardless of CDDP resistance, SP increased. ABCB1 was strongly expressed in the PTX-resistant cells, but not in their parental lines, which are sensitive to PTX. While INF-α showed only slight enhancement of the cell-death effect of PTX in PTX-sensitive cells, INF-α itself strongly induced apoptosis in PTX-resistant cells regardless of PTX concentration. CONCLUSIONS The SP could be correlated with resistance to PTX. SP could be a target of INF-α, and resistance to PTX might be overcome by INF-α.