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.immune exhaustion | CD8T cells | antitumor immunity | tumor microenvironment | multifunctionality

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1-and PKR2-gene-disrupted mice (Pkr1 ؊/؊ and Pkr2 ؊/؊ , respectively). Phenotypic analysis indicated that not Pkr1 ؊/؊ but Pkr2 ؊/؊ mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2 ؊/؊ mice. In addition, the Pkr2 ؊/؊ mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2 ؊/؊ mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2 ؊/؊ mice. The phenotype of the Pkr2 ؊/؊ mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

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.

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 (CD103 hi ) DCs, which immigrate from the skin, and resident (CD8α hi ) 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.dendritic cell | CD8 + T cell | cross-presentation | intravital two-photon imaging | photoconversion

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.

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-Bdeficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this un-

Pseudomonas sp. strain SCT is capable of using iodate (IO 3 − ) as a terminal electron acceptor for anaerobic respiration. A possible key enzyme, periplasmic iodate reductase (Idr), was visualized by active staining on non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that at least four proteins, designated as IdrA, IdrB, IdrP 1 , and IdrP 2 , were involved in Idr. IdrA and IdrB were homologues of catalytic and electron transfer subunits of respiratory arsenite oxidase (Aio); however, IdrA defined a novel clade within the dimethylsulfoxide (DMSO) reductase family. IdrP 1 and IdrP 2 were closely related to each other and distantly related to cytochrome c peroxidase. The idr genes (idrABP 1 P 2 ) formed an operon-like structure, and their transcription was upregulated under iodate-respiring conditions. Comparative proteomic analysis also revealed that Idr proteins and high affinity terminal oxidases (Cbb 3 and Cyd), various H 2 O 2 scavengers, and chlorite (ClO 2 − ) dismutase-like proteins were expressed specifically or abundantly under iodate-respiring conditions. These results suggest that Idr is a respiratory iodate reductase, and that both O 2 and H 2 O 2 are formed as by-products of iodate respiration. We propose an electron transport chain model of strain SCT, in which iodate, H 2 O 2 , and O 2 are used as terminal electron acceptors.

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.