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.

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-

Cholera toxin B (CTB) is a subunit of cholera toxin, a bacterial enterotoxin secreted by Vibrio cholerae and also functions as an immune adjuvant. However, it remains unclear how CTB activates immune cells. We here evaluated whether or how CTB induces production of a pro-inflammatory cytokine, interleukin-1β (IL-1β). CTB induced IL-1β production not only from bone marrow-derived macrophages (BMMs) but also from resident peritoneal macrophages in synergy with O111:B4-derived lipopolysaccharide (LPS O111:B4) that can bind to CTB. Meanwhile, when prestimulated with O55:B5-derived LPS (LPS O55:B5) that fails to bind to CTB, resident peritoneal macrophages, but not BMMs, produced IL-1β in response to CTB. The CTB-induced IL-1β production in synergy with LPS in both peritoneal macrophages and BMMs was dependent on ganglioside GM1, which is required for internalization of CTB. Notably, not only the NLRP3 inflammasome but also the pyrin inflammasome were involved in CTB-induced IL-1β production from resident peritoneal macrophages, while only the NLRP3 inflammasome was involved in that from BMMs. In response to CTB, a Rho family small GTPase, RhoA, which activates pyrin inflammasome upon various kinds of biochemical modification, increased its phosphorylation at serine-188 in a GM1-dependent manner. This phosphorylation as well as CTB-induced IL-1β productions were dependent on protein kinase A (PKA), indicating critical involvement of PKA-dependent RhoA phosphorylation in CTB-induced IL-1β production. Taken together, these results suggest that CTB, incorporated through GM1, can activate resident peritoneal macrophages to produce IL-1β in synergy with LPS through novel mechanisms in which pyrin as well as NLRP3 inflammasomes are involved.