Chihiro Yamazaki

Immune-mediated antitumor effect by type 2 diabetes drug, metformin

Significance The multifunctional ability of CTLs is downregulated by interaction between immune-checkpoint molecules expressed on CTLs and their ligands expressed on cancer cells, referred to as immune exhaustion. The antibody-mediated, immune-checkpoint blockade turned out to a promising method for immunotherapy against advanced melanoma. Metformin, a drug prescribed for patients with type 2 diabetes, has been recognized to have anti-cancer effect. We found that CD8+ tumor infiltrating lymphocytes (TILs) is a target of metformin. CD8+ TILs inevitably undergo immune exhaustion, characterized by diminished production of multiple cytokines such as IL-2, TNFα, and IFNγ, followed by elimination with apoptosis. Metformin is able to counter the state. Along with conventional therapy, treatment of cancer patients with metformin may have a great advantage for cancer therapy. Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell–deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8+ tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8+ TILs capable of producing multiple cytokines were mainly PD-1−Tim-3+, an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8+ TIL multifunctionality. The adoptive transfer of antigen-specific CD8+ T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8+ T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment.

Critical Roles of a Dendritic Cell Subset Expressing a Chemokine Receptor, XCR1

Dendritic cells (DCs) consist of various subsets that play crucial roles in linking innate and adaptive immunity. In the murine spleen, CD8α+ DCs exhibit a propensity to ingest dying/dead cells, produce proinflammatory cytokines, and cross-present Ags to generate CD8+ T cell responses. To track and ablate CD8α+ DCs in vivo, we generated XCR1-venus and XCR1-DTRvenus mice, in which genes for a fluorescent protein, venus, and a fusion protein consisting of diphtheria toxin receptor and venus were knocked into the gene locus of a chemokine receptor, XCR1, which is highly expressed in CD8α+ DCs. In both mice, venus+ cells were detected in the majority of CD8α+ DCs, but they were not detected in any other cells, including splenic macrophages. Venus+CD8α+ DCs were superior to venus−CD8α+ DCs with regard to their cytokine-producing ability in response to TLR stimuli. In other tissues, venus+ cells were found primarily in lymph node (LN)-resident CD8α+, LN migratory and peripheral CD103+ DCs, which are closely related to splenic CD8α+ DCs, although some thymic CD8α−CD11b− and LN CD103−CD11b− DCs were also venus+. In response to dsRNAs, diphtheria toxin–treated XCR1-DTR mice showed impaired CD8+ T cell responses, with retained cytokine and augmented CD4+ T cell responses. Furthermore, Listeria monocytogenes infection and anti–L. monocytogenes CD8+ T cell responses were defective in diphtheria toxin–treated XCR1-DTRvenus mice. Thus, XCR1-expressing DCs were required for dsRNA- or bacteria-induced CD8+ T cell responses. XCR1-venus and XCR1-DTRvenus mice should be useful for elucidating the functions and behavior of XCR1-expressing DCs, including CD8α+ and CD103+ DCs, in lymphoid and peripheral tissues.

Spi-B is critical for plasmacytoid dendritic cell function and development

Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through the nucleic acid-sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B-deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-B-deficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this uneven distribution was likely because of defective retainment of mature nondividing pDCs in the BM. The expression pattern of cell-surface molecules in Spi-B-deficient mice indicated the involvement of Spi-B in pDC development. The developmental defects of pDCs in Spi-B-deficient mice were more prominent in the BM than in the peripheral lymphoid organs and were intrinsic to pDCs. We conclude that Spi-B plays critical roles in pDC function and development.

Transcription of Vibrio parahaemolyticus T3SS1 genes is regulated by a dual regulation system consisting of the ExsACDE regulatory cascade and H-NS.

Vibrio parahaemolyticus, one of the human pathogenic vibrios, causes gastroenteritis, wound infections and septicemia. Genomic sequencing of this organism revealed that it has two distinct type III secretion systems (T3SS1 and T3SS2). T3SS1 plays a significant role in lethal activity in a murine infection model. It was reported that expression of the T3SS1 gene is controlled by a positive regulator, ExsA, and a negative regulator, ExsD, which share a degree of sequence similarity with Pseudomonas aeruginosa ExsA and ExsD, respectively. However, it is unknown whether T3SS1 is regulated by a mechanism similar to that demonstrated for P. aeruginosa, because functional analysis of VP1701, which is homologous to ExsC, is lacking and there is no ExsE homologue in the T3SS1 region. Here, we demonstrate that vp1701 and vp1702 are functional orthologues of exsC and exsE, respectively, of P. aeruginosa. VP1701 was required for the production of T3SS1-related proteins. VP1702 was a negative regulator for T3SS1-related protein production and was secreted by T3SS1. We also found that H-NS represses T3SS1-related gene expression by suppressing exsA gene expression. These findings indicate that the transcription of V. parahaemolyticus T3SS1 genes is regulated by a dual regulatory system consisting of the ExsACDE regulatory cascade and H-NS.

Imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node

Significance This work, for the first time to our knowledge, distinctly visualizes the two different populations of dendritic cells (DCs) essential for cytotoxic T-cell generation in the skin-draining lymph nodes (SDLNs): the migratory CD103hi DCs that immigrate from other organs including the skin and the CD8αhi DCs that are resident in the SDLNs. By imaging the spatiotemporal dynamics of the migratory and resident subsets of DCs in the SDLNs, we find that these two different populations play different roles in antigen presentation, with the migratory DCs being dramatically more potent in interacting with CD8+ T cells. This work offers critical insights into several areas, including optimizing vaccines for microbes and tumors. Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8+ T cells and play critical roles in cytotoxic T-cell–mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8+ T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4+ T-cell activation by CD11b+ DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103hi) DCs, which immigrate from the skin, and resident (CD8αhi) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8+ T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8+ T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8+ T-cell activation.

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, function as inhibitors of cellular and molecular components involved in type I interferon production.

OBJECTIVE Statins, which are used as cholesterol-lowering agents, have pleiotropic immunomodulatory properties. Although beneficial effects of statins have been reported in autoimmune diseases, the mechanisms of these immunomodulatory effects are still poorly understood. Type I interferons (IFNs) and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Therefore, PDCs may be a specific target of statins in therapeutic strategies against SLE. This study was undertaken to investigate the immunomodulatory mechanisms of statins that target the IFN response in PDCs. METHODS We isolated human blood PDCs by flow cytometry and examined the effects of simvastatin and pitavastatin on PDC activation, IFNalpha production, and intracellular signaling. RESULTS Statins inhibited IFNalpha production profoundly and tumor necrosis factor alpha production modestly in human PDCs in response to Toll-like receptor ligands. The inhibitory effect on IFNalpha production was reversed by geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert their inhibitory actions through geranylgeranylated Rho inactivation. Statins inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory effect on the IFN response was through the prevention of nuclear translocation of IFN regulatory factor 7. In addition, statins had an inhibitory effect on both IFNalpha production by PDCs from SLE patients and SLE serum-induced IFNalpha production. CONCLUSION Our findings suggest a specific role of statins in controlling type I IFN production and a therapeutic potential in IFN-related autoimmune diseases such as SLE.

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8alpha(+) conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8(+) T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3(+) DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8(+) T cells through XCR1.

Crucial roles of XCR1-expressing dendritic cells and the XCR1-XCL1 chemokine axis in intestinal immune homeostasis.

Intestinal immune homeostasis requires dynamic crosstalk between innate and adaptive immune cells. Dendritic cells (DCs) exist as multiple phenotypically and functionally distinct sub-populations within tissues, where they initiate immune responses and promote homeostasis. In the gut, there exists a minor DC subset defined as CD103+CD11b− that also expresses the chemokine receptor XCR1. In other tissues, XCR1+ DCs cross-present antigen and contribute to immunity against viruses and cancer, however the roles of XCR1+ DCs and XCR1 in the intestine are unknown. We showed that mice lacking XCR1+ DCs are specifically deficient in intraepithelial and lamina propria (LP) T cell populations, with remaining T cells exhibiting an atypical phenotype and being prone to death, and are also more susceptible to chemically-induced colitis. Mice deficient in either XCR1 or its ligand, XCL1, similarly possess diminished intestinal T cell populations, and an accumulation of XCR1+ DCs in the gut. Combined with transcriptome and surface marker expression analysis, these observations lead us to hypothesise that T cell-derived XCL1 facilitates intestinal XCR1+ DC activation and migration, and that XCR1+ DCs in turn provide support for T cell survival and function. Thus XCR1+ DCs and the XCR1/XCL1 chemokine axis have previously-unappreciated roles in intestinal immune homeostasis.

Cutting Edge: Critical Role of IκB Kinase α in TLR7/9-Induced Type I IFN Production by Conventional Dendritic Cells

A plasmacytoid dendritic cell (DC) can produce large amounts of type I IFNs after sensing nucleic acids through TLR7 and TLR9. IκB kinase α (IKKα) is critically involved in this type I IFN production through its interaction with IFN regulatory factor-7. In response to TLR7/9 signaling, conventional DCs can also produce IFN-β but not IFN-α in a type I IFN-independent manner. In this study, we showed that IKKα was required for production of IFN-β, but not of proinflammatory cytokines, by TLR7/9-stimulated conventional DCs. Importantly, IKKα was dispensable for IFN-β gene upregulation by TLR4 signaling. Biochemical analyses indicated that IKKα exerted its effects through its interaction with IFN regulatory factor-1. Furthermore, IKKα was involved in TLR9-induced type I IFN-independent IFN-β production in vivo. Our results show that IKKα is a unique molecule involved in TLR7/9-MyD88–dependent type I IFN production through DC subset-specific mechanisms.

Critical role of IkappaB Kinase alpha in TLR7/9-induced type I IFN production by conventional dendritic cells.

A plasmacytoid dendritic cell (DC) can produce large amounts of type I IFNs after sensing nucleic acids through TLR7 and TLR9. IκB kinase α (IKKα) is critically involved in this type I IFN production through its interaction with IFN regulatory factor-7. In response to TLR7/9 signaling, conventional DCs can also produce IFN-β but not IFN-α in a type I IFN-independent manner. In this study, we showed that IKKα was required for production of IFN-β, but not of proinflammatory cytokines, by TLR7/9-stimulated conventional DCs. Importantly, IKKα was dispensable for IFN-β gene upregulation by TLR4 signaling. Biochemical analyses indicated that IKKα exerted its effects through its interaction with IFN regulatory factor-1. Furthermore, IKKα was involved in TLR9-induced type I IFN-independent IFN-β production in vivo. Our results show that IKKα is a unique molecule involved in TLR7/9-MyD88–dependent type I IFN production through DC subset-specific mechanisms.