[SLAM family proteins as therapeutic targets in multiple myeloma].

Reports have described the excellent efficacies of new immunotherapeutic strategies, such as monoclonal antibody (mAb) therapies, in multiple myeloma (MM) patients. Signaling lymphocytic activation molecule family (SLAMF) molecules are expressed strongly on normal lymphocytes and plasma cells from MM patients. The anti-SLAMF7 mAb elotuzumab (ELO) has been approved for the treatment of relapsed/refractory MM (RRMM). In MM patients, a high serum soluble SLAMF7 (sSLAMF7) concentration is associated with aggressive clinical characteristics. This suggests a proliferative function of the SLAMF7-sSLAMF7 interaction that could be inhibited by ELO. SLAMF3 is also expressed strongly and constitutively on myeloma cells. We observed the aggressive characteristics of SLAMF3+ MM in vitro and in vivo. SLAMF3 interacts directly with the adaptor proteins SHP2 and GRB2. A gene expression analysis revealed that SLAMF3 transmits positive signals to MM cells via the MAPK/ERK signaling pathway and that sSLAMF3 levels are increased markedly in advanced MM. Thus, SLAMF3 may be a novel immunotherapeutic target in MM. SLAMF2 and SLAMF6 are also expressed strongly on MM cells, and the safety of antibody-drug conjugates that target these molecules in patients with RRMM is currently under study. Our and others' reports demonstrate the value of SLAMF molecules as promising new targets for antimyeloma immunotherapies.

Distribution of dendritic cells in the septate uterus: An immunological perspective.

PROBLEM: Septate uterus is associated with spontaneous abortion. Surgical intervention of the uterine septa (US) is frequently performed following spontaneous abortion; however, immunological mechanisms for spontaneous abortion in patients with septate uterus remain completely unknown. METHOD OF STUDY: A total of 12 women with septate uterus who underwent hysteroscopic metroplasty and 10 women with uterine leiomyoma who underwent total hysterectomy were enrolled as the experimental and control groups, respectively. Immune cells, dendritic cells (DCs), macrophages, T cells, natural killer cells, invariant natural killer cells, and chemokine receptors in US and uterine myometrium tissue (UMT) were analyzed using flow cytometry and immunohistochemical staining. Additionally, the chemokine production of macrophage inflammatory protein 1 alpha (MIP-1α), regulated upon activation normal T-cell express sequence (RANTES), and macrophage inflammatory protein 3 beta (MIP-3ß) from the viable cells obtained from the US and UMT samples was evaluated in an ex vivo study. RESULTS: The percentage of CD141+ DCs in US was significantly lower than that in UMT. Both US and UMT showed CCR1 and CCR5 expression on CD141+ DCs; however, the production of chemokines, MIP-1α, RANTES, and MIP-3ß was abundant in UMT-obtained viable cells. CONCLUSION: The accumulation of CD141+ DCs was lower in US than that in UMT. This phenomenon may be caused by low chemokine productions in US. Our findings support the benefit of surgical intervention for septate uterus-that is, the elimination of inappropriate implantation sites.

Japanese Kampo Medicine Juzentaihoto Enhances Antitumor Immunity in CD1d-/- Mice Lacking NKT Cells.

Although the Japanese traditional herbal medicine (Kampo), Juzentaihoto (JTT), has been reported to have antitumor effects in several tumor models, its role in tumor immunology remains controversial. In the present study, we tested whether oral administration of JTT enhances antitumor immunity in CD1d-/- mice, in which immunosuppression was partially relieved due to the lack of NKT cells. In a subcutaneous murine syngeneic CT26 colorectal tumor model, JTT had no impact on tumor growth in wild type (WT) BALB/c mice. However, the growth rate of tumors was significantly slower in CD1d-/- mice than in WT mice. Surprisingly, JTT significantly delayed tumor growth in such CD1d-/- mice. In vivo depletion of CD8+ T cells revealed that CD8+ T cells are required for JTT's antitumor activity. Moreover, tumor-reactive cytotoxic T-lymphocytes were detected exclusively in JTT-treated mice with well-controlled tumors. JTT did not affect the number of tumor-infiltrating CD4+ regulatory T cells. On the contrary, JTT increased the degranulation marker CD107a+ CD8+ T cells and decreased Ly6G+ Ly6Clo polymorphonuclear myeloid-derived suppressor cells in tumor-infiltrating lymphocytes, most probably contributing to the suppression of tumor growth in JTT-treated mice. Nonetheless, JTT had no impact on the proportion of monocytic myeloid-derived suppressor cells. In conclusion, our results indicate that in the absence of NKT cells, JTT augments antitumor immunity by CD8+ T cells, suggesting that this Kampo medicine is a promising anticancer adjuvant when negative immune regulation is partially relieved.

A combination of check-point blockade and α-galactosylceramide elicits long-lasting suppressive effects on murine hepatoma cell growth in vivo.

Immunotherapy for cancer cells induced by interfering with PD-1/PD-L1 engagement via check-point blockades was initiated by tumour-specific PD-1+ CD8+ cytotoxic T lymphocytes (CTLs) within a tumour mass and eliminate the tumour. Here, we used C57BL/6 (B6) mice implanted with the syngeneic hepatoma cell line Hepa1-6-1, and confirmed that the dendritic cells (DCs) within Hepa1-6-1 tumour mass were tolerogenic with downmodulated co-stimulatory molecules by tumour-derived factors. Although Hepa1-6-1 cells did not prime tumour-specific CTLs within the tumour, specific CTLs primed in the regional lymph nodes seemed to be invaded into the tumour mass. The specific CTLs gained PD-1+ expression when associated with PD-L1+ Hepa1-6-1 cells within the tumour mass. Their cytotoxic activity in vivo was revitalised after intraperitoneal (i.p.) administration of the anti-PD-1 monoclonal antibody (mAb), indicating that PD-1/PD-L1 engagement within the tumour was abrogated by check-point blockade. Nonetheless, the tolerogenic DCs within the Hepa1-6-1 tumour mass remained tolerogenic even after three shots of PD-1-blockade administration, and the suppressed Hepa1-6-1 growth was revisited. In this study, we show here an excellent therapeutic effect consisting of three injections of anti-PD1 mAb and the sequential administration of the CD1d molecule-restricted ligand α-galactosylceramide (α-GalCer), an immuno-potent lipid/glycolipid, which converts tolerogenic DCs into immunogenic DCs with upregulated expression of co-stimulatory molecules. The α-GalCer-activated DCs secreted a large amount of IL-12, which can activate tumour-specific CTLs in vivo. The check-point blockade was not sufficiently effective, but the dose needed for tumour eradication was reduced by 90% when tumour-bearing mice were also administered i.p. α-GalCer.

Induction of tumor-specific CD8+ cytotoxic T lymphocytes from naïve human T cells by using Mycobacterium-derived mycolic acid and lipoarabinomannan-stimulated dendritic cells.

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.

Suppression of R5-type of HIV-1 in CD4+ NKT cells by Vδ1+ T cells activated by flavonoid glycosides, hesperidin and linarin.

We established transfectants expressing T cell receptors (TCRs) either for Vγ1 and Vδ1 (1C116) or for Vγ2 and Vδ2 (2C21) using the TCR-deficient Jurkat T cell line J.RT3-T3.5. The amount of IL-2 secreted from these γδ T cell clones accurately indicated TCR-dependent stimulation. Clone 2C21 was specifically stimulated by previously reported ligands for Vγ2Vδ2 (Vδ2)-TCR such as isopentenyl pyrophospate (IPP), ethylamine, or risedronate. In contrast, clone 1C116 was strongly stimulated through the Vγ1Vδ1 (Vδ1)-TCR by flavonoid glycosides such as hesperidin and linarin, having both rutinose at the A ring and methoxy (-OCH3) substitution at the B ring. Additionally, hesperidin and linarin showed stimulatory activity for peripheral blood mononuclear cell (PBMC)-derived T cells expressing Vδ1-TCR; these activated Vδ1+ T cells also secreted IL-5, IL-13, MIP-1α, MIP-1ß and RANTES. Such PBMC-derived Vδ1+ T cells stimulated by hesperidin and linarin suppressed R5-HIV-1-NL(AD8) viral replication in CD4+ NKT cells in a dose-dependent manner. To the best of our knowledge, this is the first demonstration that flavonoid glycosides activate functional Vδ1+ T cells.

Neutralizing antibodies for Helicobacter pylori urease inhibit bacterial colonization in the murine stomach in vivo.

Helicobacter pylori (H. pylori) urease is a key protein for persistent infection of the bacteria in the stomach. Although H. pylori generally induce anti-H. pylori-specific antibodies (Abs), these Abs do not usually work for eradication or prevention of the H. pylori infection. In our previous study, we identified a linear epitope composed of 19-mer peptides termed UB-33, CHHLDKSIKEDVQFADSRI, within the large subunit of H. pylori urease. Anti-UB-33-specific Abs neutralized the enzymatic activity of H. pylori urease in vitro. In the present study, we evaluated the effect of immunization of BALB/c mice with H. pylori UB-33 peptide. After confirming the production of anti-UB-33-specific Abs, mice were challenged orally with H. pylori Sydney Strain-1 (SS-1). Mice producing anti-UB-33-specific Abs were not infected with SS-1, and the amount of SS-1 isolate in their stomach was significantly reduced. Also, the urease-negative mutant of H. pylori, HPP1801, did not colonize in the stomach, indicating that H. pylori urease was a critical element for infection of H. pylori in the gastric mucosa. Moreover, mice producing UB-33-specific Abs apparently suppressed H. pylori infection in the stomach where anti-UB-33 Abs were secreted in the gastric juice, indicating that H. pylori colonization was inhibited in the presence of anti-UB-33 Abs. In addition, the neutralization activity of sera from mice immunized with purified urease was less potent than that in the sera from mice immunized with UB-33. Furthermore, the recognition of epitope UB-33 was mediated through Toll-like receptor 2 (TLR2) on the B-1 cells using TLR2-knockout BALB/c mice in vivo. These results indicate that liner peptide UB-33 should be used for immunization to induce neutralizing Abs instead of purified H. pylori urease to prevent H. pylori infection and their colonization in the stomach.

Differentiation of Langerhans Cells from Monocytes and Their Specific Function in Inducing IL-22-Specific Th Cells.

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets, epidermal Langerhans cells (LCs) and dermal DCs, which can be distinguished by the expression of C-type lectin receptors, Langerin and DC-SIGN, respectively. Although peripheral blood monocytes differentiate into these distinct subsets, monocyte-derived LCs (moLCs) induced by coculture with GM-CSF, IL-4, and TGF-ß1 coexpress both Langerin and DC-SIGN, suggesting that the environmental cues remain unclear. In this study, we show that LC differentiation is TGF-ß1 dependent and that cofactors such as IL-4 and TNF-α promote TGF-ß1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-α-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs, dexamethasone-treated moLCs express CD1a, whereas monocyte-derived DCs (moDCs) express CD1b, CD1c, and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and α-galactosylceramide, respectively. Strikingly, CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis, these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin.

Innate immune cells in reproduction.

Fetomaternal immune tolerance is required to prevent fetal rejection during pregnancy; simultaneously, the maternal immune system must also protect the fetus from harmful endogenous and exogenous antigens, including those associated with viral and bacterial infections. Therefore, a delicate immune balance is critical for the maintenance of a successful pregnancy, while disruption of this balance can induce complications such as implantation failure, miscarriage, preterm birth/labor, preeclampsia and fetal growth restriction. While the adaptive immune system is critical for the development of tolerance, this review summarizes evidence that modulation of the innate immunity is also essential for achieving this delicate balance between tolerance and protection. Canonical cells of the innate immune system, including dendritic cells, macrophages, natural killer cells and invariant natural killer T cells, contribute to the modulation of the immune balance at the fetomaternal interface. The newly identified myeloid-derived suppressor cells and innate lymphoid cells also appear important for successful reproduction. Moreover, it is possible that sterile inflammation facilitates complications of pregnancy via the innate immune system. In this review, the characteristic features and functions of innate immune cells in fetomaternal immunity and their contributions to pregnancy complications related to sterile inflammation are discussed. These insights on innate immune system function in reproduction may provide new perspectives for understanding the mechanisms of fetomaternal tolerance and the etiology of pregnancy complications.

HMGB1 released from intestinal epithelia damaged by cholera toxin adjuvant contributes to activation of mucosal dendritic cells and induction of intestinal cytotoxic T lymphocytes and IgA.

Cholera toxin (CT) is a potent mucosal adjuvant and oral administration of ovalbumin (OVA) antigens plus CT induces OVA-specific CD8+ cytotoxic T lymphocytes (CTLs) and IgA production in intestinal mucosa. However, the mechanisms of induction of these immune responses remain unknown. Intestinal OVA-specific CD8+ CTLs were not induced by oral administration of the CT active (CTA) or CT binding (CTB) subunit as an adjuvant and CD11c+ DCs were involved in cross-priming of intestinal CTLs. CD8+CD103+CD11c+CD11b-DCs and DCIR2+CD103+CD11c+CD11b+ DCs were distributed in the intestinal lamina propria and mesenteric lymph nodes, both DC subsets expressed DEC-205, and the expression of co-stimulatory molecules such as CD80 and CD86 was enhanced in both DC subsets after oral administration of intact CT but not the CTA or CTB subunit. Intestinal DCs activated by the oral administration of OVA plus CT cross-presented OVA antigens and DCs that captured OVA antigen through DEC-205, but not DCIR2, could cross-present antigen. We found that oral administration of intact CT, but not the CTA or CTB subunit, enhanced cell death, cytoplasmic expression of high-mobility group box 1 protein (HMGB1) in epithelial cell adhesion molecule (EpCAM)+CD45- intestinal epithelial cells (IECs), and HMGB1 levels in fecal extracts. HMGB1 dose-dependently enhanced the expression of CD80 and CD86 on DCs in vitro, and intravenous or oral administration of glycyrrhizin, an HMGB1 inhibitor, significantly suppressed activation of mucosal DCs and induction of intestinal OVA-specific CTLs and IgA by oral CT administration. These results showed that oral administration of intact CT triggers epithelial cell death in the gut and the release of HMGB1 from damaged IECs, and that the released HMGB1 may mediate activation of mucosal DCs and induction of CTLs and IgA in the intestine.

Miscarriage induced by adoptive transfer of dendritic cells and invariant natural killer T cells into mice.

Unexpected fetal loss is one of the common complications of pregnancy; however, the pathogenesis of many miscarriages, particularly those not associated with infections, is unknown. We previously found that activated DEC-205+ dendritic cells (DCs) and NK1.1+ invariant natural killer T (iNKT) cells are recruited into the myometrium of mice when miscarriage is induced by the intraperitoneal administration of α-galactosylceramide (α-GalCer). Here we demonstrate that the adoptive transfer of DEC-205+ bone marrow-derived DCs cocultured with α-GalCer (DEC-205+ BMDCs-c/w-α-GalCer) directly induced marked fetal loss by syngeneic pregnant C57BL/6 (B6) mice and allogeneic mice (B6 (♀) × BALB/c (♂)), which was accompanied by the accumulation of activated iNKT cells in the myometrium. Further, the adoptive transfer of NK1.1+ iNKT cells obtained from B6 mice injected with α-GalCer facilitated miscarriages in syngeneic Jα18(-/-) (iNKT cell-deficient) mice. These results suggest that DEC-205+ DCs and NK1.1+ iNKT cells play crucial roles required for the initiation of fetal loss associated with stimulation by glycolipid antigens and sterile inflammation.

Anti-CD137 monoclonal antibody enhances trastuzumab-induced, natural killer cell-mediated cytotoxicity against pancreatic cancer cell lines with low human epidermal growth factor-like receptor 2 expression.

Because human epidermal growth factor-like receptor (HER) 2 is expressed on the surface of human pancreatic carcinoma cells to varying degrees, trastuzumab, an anti-HER2 monoclonal antibody (mAb), is expected to exert antibody-dependent, natural killer (NK) cell-mediated cytotoxicity (ADCC) against the cells. However, some reports found that the effect of trastuzumab against human pancreatic carcinoma cells was limited because most express only limited HER2. We examined whether anti-CD137 stimulating mAb could enhance trastuzumab-mediated ADCC against Panc-1, a human pancreatic cancer cell line with low HER2 expression, in vitro. Supplementation of anti-CD137 mAb could improve trastuzumab-mediated ADCC against Panc-1 which was insufficient without this stimulating antibody. The ADCC differed in individual cells, and this was related to the expression of CD137 on the surface of NK cells after trastuzumab stimulation in association with the Fcγ-RIIIA polymorphism. NK cells with Fcγ-RIIIA-VV/VF showed high levels of ADCC against Panc-1, but those with Fcγ-RIIIA-FF did not show optimal ADCC. In addition, trastuzumab-mediated ADCC against the human pancreatic cancer cell line Capan-1 with high HER2 expression was generally high and not affected by the Fcγ-RIIIA polymorphism. These results demonstrated that in Fcγ-RIIIA-VV/VF-carrying healthy individuals, trastuzumab plus αCD137 mAb could induce effective ADCC against HER2-low-expressing pancreatic cancer cell lines, and that such an approach may result in similar findings in patients with pancreatic cancer.

Functional expression of Tim-3 on blasts and clinical impact of its ligand galectin-9 in myelodysplastic syndromes.

T-cell immunoglobulin mucin-3 (Tim-3), an inhibitory immune checkpoint receptor, is highly expressed on acute myeloid leukemia cells and its ligand galectin-9 is reported to drive leukemic progression by binding with Tim-3. However, it remains unclear whether the Tim-3-galectin-9 pathway is associated with the pathophysiology of myelodysplastic syndromes (MDS). Thus, we investigated the expression and function of Tim-3 and the clinical impact of its ligand galectin-9 in MDS. Tim-3 expression levels on MDS blasts by CD45/side-scatter or CD34/CD45 gating were increased as MDS progressed to the advanced stage. Tim-3 expression in the MDS blasts was upregulated in the presence of the cell culture supernatant of human stromal cells or the MDS-related cytokine transforming growth factor-ß1. The proliferation of Tim-3+ MDS blasts was inhibited by the blockade of anti-Tim-3 antibody. Furthermore, plasma levels of galectin-9 were elevated as MDS progressed to the advanced stage in 70 MDS/acute leukemia transformed from MDS patients and was a prognostic factor in 40 MDS patients. Our data demonstrated that the Tim-3-galectin-9 pathway is associated with the pathogenesis and disease progression of MDS. These findings provide new insight into potential immunotherapy targeting the galectin-9-Tim-3 pathway in MDS.

Japanese Kampo medicine ninjin'yoeito synergistically enhances tumor vaccine effects mediated by CD8+ T cells.

Although Japanese traditional herbal medicine (Kampo) has been widely applied to the treatment of various diseases, including cancer, their mechanisms of action have not yet been elucidated in detail, particularly regarding their role in tumor immunology. The present study investigated the antitumor effects of the Japanese Kampo medicine, ninjin'yoeito (NYT; Ren-Shen-Yang-Rong-Tang in Chinese), which was orally administered with or without an irradiated tumor cell vaccine to a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine in a prophylactic setting significantly delayed tumor growth in mice fed a control diet, whereas a diet containing NYT alone did not exert any antitumor effects in vivo. However, the inhibition of tumor growth was significantly greater in vaccinated mice fed the NYT diet compared with in vaccinated mice given the control diet. These results suggest that NYT synergistically enhances the effects of the antitumor vaccine. The depletion of cluster of differentiation (CD)8+ T cells abrogated these effects, indicating that antitumor activity required CD8+ T cells. Furthermore, reductions in CD4+ CD25+ and forkhead box protein 3+ T regulatory cell numbers were more apparent between vaccinated mice fed the NYT diet and non-vaccinated mice fed the control diet than between vaccinated mice and non-vaccinated mice fed the control diet, suggesting that the weaker impact of T regulatory cells contributes to the augmentation of antitumor immunity by CD8+ T cells in vaccinated mice fed with NYT. Overall, these results indicate that NYT synergistically enhances the effects of the prophylactic tumor vaccine mediated by CD8+ T cells and that this Japanese Kampo medicine has potential as a useful adjuvant agent for cancer immunotherapy.

Detection Method for Aquatic Bacteria of the Fingers, as a Potential Origin of the Aqueous Solution Contamination.

　Aquatic bacteria were isolated from the hands of working staffs by an adapted culture protocol. When the sample solution obtained by the" glove juice method" was incubated for 3 days at room temperature, viable cell counts increased up to 105-fold, and the majority of the isolated colonies were shown to be Gram-negative aquatic bacteria, which carry the risk of contaminating water. Using R2A medium, coagulase-negative staphylococci were the dominant microbes immediately after recovery from the hands. Here it was revealed that bacteria of the phylum Proteobacteria isolated from the hand can be the causative bacteria of aqueous contamination. This modification in the GJ method may be useful as an effective training protocol to demonstrate the importance of hand hygiene and clean operation for aseptic manufacturing.

Distribution of invariant natural killer T cells and dendritic cells in late pre-term birth without acute chorioamnionitis.

PROBLEM: Acute chorioamnionitis (aCAM) is an important cause of pre-term birth. However, little is known about the pathogenesis of late pre-term birth without aCAM that was the most common category of pre-term birth. Here we analyze the kinetics of immune cells obtained from the decidua of women with late pre-term births with and without aCAM. METHOD OF STUDY: Deciduas were obtained from women who underwent labor with late pre-term birth without aCAM (PB-n/aCAM) or with aCAM (PB-w/aCAM). The population of DEC-205+ dendritic cells (DCs), macrophages, invariant natural killer T (iNKT) cells, NK cells, CD8+ T cells, and CD4+ T cells were analyzed by flow cytometry. RESULTS: The number of iNKT cells was higher in the decidua obtained from women with PB-n/aCAM than PB-w/aCAM. DEC-205+ DCs obtained from women with PB-n/aCAM preferentially induced iNKT cell proliferation. CONCLUSION: iNKT cell accumulation with DEC-205+ DCs in PB-n/aCAM suggests that iNKT cells contribute to the onset of PB-n/aCAM.

Suppression of murine tumour growth through CD8+ cytotoxic T lymphocytes via activated DEC-205+ dendritic cells by sequential administration of α-galactosylceramide in vivo.

Cancer immunity is mediated through the effective priming and activation of tumour-specific class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). DEC-205+ dendritic cells (DCs) can cross-present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumour-specific CD8+ CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of impaired CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8+ CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8+ CTLs with tumour-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide (α-GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC-205+ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α-GalCer every 48 hr appeared to convert tolerogenic DEC-205+ DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a stronger cross-presentation capacity, which primed CTL precursors and induced tumour-specific CD8+ CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205+ DCs within tumours into immunogenic DCs through the sequential administration of an immuno-potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co-stimulatory molecules prime and activate tumour-specific CD8+ CTLs within the tumour to control tumour growth.

Induction of langerin+ Langerhans cell-like cells expressing reduced TLR3 from CD34+ cord blood cells stimulated with GM-CSF, TGF-ß1, and TNF-α.

Langerhans cells (LCs), a subset of dendritic cells (DCs), reside in body surface presenting antigens from various pathogens and activate immune system after migrating to vicinal lymph nodes. We recently demonstrated that the E-cadherin interaction allowed peripheral blood (PB) CD14+ cells to differentiate into LC-like cells that closely resemble primary LCs. Here, with a combination of GM-CSF, TGF-ß, and TNF-α, we induced LC-like cells from umbilical cord blood (UCB)derived CD34+ cells and compared them with those induced from PB CD14+ cells. In contrast to PB CD14+ cell-derived LC-like cells with an undetectable surface level of toll-like receptor (TLR)4 and an unresponsiveness feature to bacterial lipopolysaccharide (LPS), CB CD34+ cellsderived LC like cells expressed a low, but apparent, surface level of TLR4 and a reduced level of intracellular TLR3. Consistent with this result, they responded to bacterial LPS, but poorly to poly(I:C) reflecting viral RNA. These findings suggest that LC-precursors from circulating PB CD14+ cells seem to be arranged in the outer barrier of skin, while LC-precursors from local undifferentiated UCB-derived CD34+ cells may be arranged in the inner barrier of mucosal tissues and work together to combat against external pathogens as well as internal malignancies throughout body surface.

α-Galactosylceramide-activated murine NK1.1(+) invariant-NKT cells in the myometrium induce miscarriages in mice.

Innate immunity, which is unable to discriminate self from allo-antigens, is thought to be important players in the induction of miscarriages. Here, we show that the administration of IL-12 to syngeneic-mated C57BL/6 mice on gestation day 7.5 (Gd 7.5), drives significant miscarriages in pregnant females. Furthermore, the administration on Gd 7.5 of α-galactosylceramide (α-GalCer), which is known to activate invariant natural killer T (iNKT) cells, induced miscarriages in both syngeneic-mated C57BL/6 mice and allogeneic-mated mice (C57BL/6 (♀) × BALB/c (♂)). Surprisingly, the percentages of both DEC-205(+) DCs and CD1d-restricted NK1.1(+) iNKT cells were higher in the myometrium of pregnant mice treated i.p. with α-GalCer than in the decidua. IL-12 secreted from α-GalCer-activated DEC-205(+) DCs stimulated the secretion of cytokines, including IL-2, IL-4, IFN-γ, TNF-α, perforin, and granzyme B, from the NK1.1(+) iNKT cells in the myometrium, leading to fetal loss in pregnant mice. Finally, the i.p. administration of IL-12 and/or α-GalCer in iNKT-deficient Jα18(-/-) (Jα18 KO) mice did not induce miscarriages. This study provides a new perspective on the importance of the myometrium, rather than the decidua, in regulating pregnancy and a mechanism of miscarriage mediated by activated DEC-205(+) DCs and NK1.1(+) iNKT cells in the myometrium of pregnant mice.