Analyses of a Mutant Foxp3 Allele Reveal BATF as a Critical Transcription Factor in the Differentiation and Accumulation of Tissue Regulatory T Cells.

Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate.

Interleukin-17B Antagonizes Interleukin-25-Mediated Mucosal Inflammation.

The interleukin-17 (IL-17) family of cytokines has emerged as a critical player in inflammatory diseases. Among them, IL-25 has been shown to be important in allergic inflammation and protection against parasitic infection. Here we have demonstrated that IL-17B, a poorly understood cytokine, functions to inhibit IL-25-driven inflammation. IL-17B and IL-25, both binding to the interleukin-17 receptor B (IL-17RB), were upregulated in their expression after acute colonic inflammation. Individual inhibition of these cytokines revealed opposing functions in colon inflammation: IL-25 was pathogenic but IL-17B was protective. Similarly opposing phenotypes were observed in Citrobacter rodentium infection and allergic asthma. Moreover, IL-25 was found to promote IL-6 production from colon epithelial cells, which was inhibited by IL-17B. Therefore, our data demonstrate that IL-17B is an anti-inflammatory cytokine in the IL-17 family.

The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells.

The immunoregulatory cytokine interleukin 10 (IL-10) is expressed mainly by T helper type 2 (T(H)2) cells but also by T(H)1 cells during chronic infection. Here we observed plasticity in the expression of IL-10 and IL-13 after chronic T(H)1 stimulation; furthermore, the expression of Il10 and Il13 was regulated by the transcription factor E4BP4. Chronically stimulated E4BP4-deficient (Nfil3(-/-); called 'E4bp4(-/-)' here) T(H)1 cells, regulatory T cells (T(reg) cells) and natural killer T cells (NKT cells) had attenuated expression of IL-10 and IL-13. Enforced expression of E4bp4 initiated the production of IL-10 and IL-13 by conventional T(H)1 cells. E4bp4(-/-) T(H)2 cells showed impairment of IL-10 production with no effect on IL-13. Our results indicate that E4BP4 has multiple functions in controlling the plasticity of IL-13 in T(H)1 cells and IL-10 in T(H)1 cells, T(H)2 cells, T(reg) cells and NKT cells.

Early Activation of iNKT Cells Increased Survival Time of BALB/c Mice in a Murine Model of Melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. High interferon gamma (IFN-γ) levels in naive mice were reported to mediate protection against B. pseudomallei infection. Invariant natural killer T (iNKT) cells can produce and secrete several cytokines, including IFN-γ. When iNKT cell-knockout (KO) BALB/c mice were infected with B. pseudomallei, their survival time was significantly shorter than wild-type mice. Naive BALB/c mice pretreated intraperitoneally with α-galactosylceramide (α-GalCer), an iNKT cell activator, 24 h before infection demonstrated 62.5% survival at the early stage, with prolonged survival time compared to nonpretreated infected control mice (14 ± 1 days versus 6 ± 1 days, respectively). At 4 h after injection with α-GalCer, treated mice showed significantly higher levels of serum IFN-γ, interleukin-4 (IL-4), IL-10, and IL-12 than control mice. Interestingly, the IFN-γ levels in the α-GalCer-pretreated group were decreased at 4, 24, and 48 h after infection, while they were highly increased in the control group. At 24 h postinfection in the α-GalCer group, bacterial loads were significantly lower in blood (no growth and 1,780.00 ± 51.21, P < 0.0001), spleens (no growth and 34,300 ± 1,106.04, P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67, P < 0.0001) than in the control group, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63, P < 0.0001), and almost all were negative at 48 h postinfection. This study for the first time shows that early activation of iNKT cells by α-GalCer helps clearance of B. pseudomallei and prolongs mouse survival.

Regulation of IL-9 expression by IL-25 signaling.

The physiological regulation of the expression of interleukin (IL)-9, a cytokine traditionally regarded as being T(H)2 associated, remains unclear. Here, we show that IL-9-expressing T cells generated in vitro in the presence of transforming growth factor-beta and IL-4 express high levels of mRNA for IL-17 receptor B (IL-17RB), the receptor for IL-25. Treatment of these cells with IL-25 enhances IL-9 expression in vitro. Moreover, transgenic and retroviral overexpression of IL-17RB in T cells results in IL-25-induced IL-9 production that is IL-4 independent. In vivo, the IL-25-IL-17RB pathway regulates IL-9 expression in allergic airway inflammation. Thus, IL-25 is a newly identified regulator of IL-9 expression.

Decrease of spleen invariant natural killer T and natural killer cell numbers in Burkholderia thailandensis-infected C57BL/6 mice.

BACKGROUND: Burkholderia thailandensis is a non-pathogenic bacterium that is closely related to B. pseudomallei. Invariant natural killer T (iNKT) cells are innate lymphoid cells that play a role in bacterial infections; however the iNKT cells in B. thailandensis infections are still uncharacterized. OBJECTIVE: To study the cytokine production in α-galactosylceramide (α-GalCer)-stimulated lymphocytes from mouse organs. The numbers of spleen iNKT cells, natural killer (NK) cells, dendritic cells and macrophages in B. thailandensis- infected C57BL/6 (B6) mice were investigated. METHODS: Lymphocytes, obtained from mouse lungs, liver, and spleen, were cultured for 48 hours with α-GalCer, and their cytokine levels were determined. iNKT, dendritic, macrophage and NK cells in the spleen of B. thailandensis-infected B6 mice or iNKT knock out (KO) mice, stimulated with either phosphate-buffered saline (PBS) or α-GalCer, were analyzed by flow cytometry. This was also done in adoptive cell transfer experiments. RESULTS: Interferon gamma (IFN-γ) was predominantly produced in α-GalCer-stimulated mouse spleen and liver lymphocytes, while interleukin (IL)-13 was the main cytokine found in the lungs. B. thailandensis-infected mice had a significantly lower number of splenic iNKT, NK and dendritic cells, but not macrophages, compared to the control. Interestingly, the number of NK cells was significantly decreased in iNKT wild type and iNKT KO mice after B. thailandensis infection. The number of NK cells recovered by activation with α-GalCer or after adoptive transfer of iNKT cells into KO mice. The iNKT cell-mediated reduction of dendritic and NK cells might be related to infection by B. thailandensis. CONCLUSIONS: B. thailandensis decreased the number of iNKT and NK cells in the spleen of infected mice.

Structure Elucidation of Calyxoside B, a Bipolar Sphingolipid from a Marine Sponge Cladocroce sp. through the Use of Beckmann Rearrangement.

Marine sponges are an excellent source of biologically active secondary metabolites. We focus on deep-sea sponges for our discovery study. A marine sponge Cladocroce sp. exhibited cytotoxic activity in the bioactivity screening. From this sponge a previously unreported cytotoxic glycosphingolipid, calyxoside B, was isolated and the structure of this compound was elucidated by analyses of MS and NMR spectra and chemical derivatization. We converted the ketone in the middle of a long aliphatic chain into an oxime to which was applied Beckmann rearrangement to afford two positional isomers of amides. The products were subjected to acidic hydrolysis followed by LC-MS analysis, permitting us to assign unequivocally the position of the ketone. Calyxoside B shows cytotoxicity against HeLa cells with an IC50 value of 31 µM and also weakly stimulated the production of cytokines in mice.

Invariant Natural Killer T (iNKT) cells response in human melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In infected mice, IFN-γ can provide protection against B. pseudomallei infection. Invariant Natural Killer T (iNKT) cells are a subpopulation of T lymphocytes, activated by recognition of glycolipid ligands such as α-Galactosylceramide presented by CD1d, produce and secrete several cytokines, including IFN-γ and IL-4. The response of iNKT cells in human melioidosis was then investigated. OBJECTIVE: To determine the iNKT cells response in human melioidosis. METHODS: The number of human iNKT cells and its activation states were investigated in sepsis melioidosis patients compared with healthy controls using flow cytometry. The iNKT cells activation was confirmed in vitro using heatkilled B. pseudomallei with normal peripheral blood mononuclear cells. The components induced iNKT cell were also determined using different concentration of B. pseudomallei lipopolysaccharide (LPS), heat-killed B. pseudomallei treated with or without DNase, RNase, or proteinase. RESULTS: The number of human iNKT cells was significantly lower while the percentage of activated iNKT cells was higher in sepsis melioidosis when compared to control. In addition, B. pseudomallei can stimulate human iNKT cells in vitro. Heat-killed B. pseudomallei could activate iNKT cells but not relate to nucleic acid, proteins, or LPS. CONCLUSIONS: We found for the first time that the iNKT cells were activated during B. pseudomallei infection in human. However, the roles and the mechanism of iNKT cells during early state of infection needed to be further investigated.

An accumulation of two populations of dendritic cells in skin-draining lymph nodes in response to the expression of thymic stromal lymphopoietin in the skin.

Thymic stromal lymphopoietin (TSLP) acts on dendritic cells (DCs), which prime helper T (Th) cells to become type 2 cytokine producing cells. Recently, a different set of populations of TSLP-responsive DCs has been discovered. Here, we identified two populations of CD103loEpCAMhi migratory DCs (fraction I and fraction II) that accumulated in skin-draining lymph nodes in response to TSLP expressed in the mouse skin. Fraction I DCs with CD11b+PDL2hi expression primed naïve Th cells to differentiate into cells secreting IFN-γ, IL-17A and IL-22, while fraction II DCs with CD11bloPDL2+ expression primed naïve Th cells to differentiate into cells secreting IL-4, IL-5, IL-9, IL-13 and IL-10. Fraction I DCs migrated from the skin via IL-4Rα signaling pathway, whereas fraction II DCs migrated partially via TSLPR signaling pathway. All suggest that at least two populations of CD103loEpCAMhi DCs with distinct functions and pathways could migrate in response to TSLP expression in the skin.

Photo effect on the CD1d-binding ability of azobenzene-attached analogues of α-GalCer.

α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states.

Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress.

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders.

The role of plasmin in the pathogenesis of murine multiple myeloma.

Aside from a role in clot dissolution, the fibrinolytic factor, plasmin is implicated in tumorigenesis. Although abnormalities of coagulation and fibrinolysis have been reported in multiple myeloma patients, the biological roles of fibrinolytic factors in multiple myeloma (MM) using in vivo models have not been elucidated. In this study, we established a murine model of fulminant MM with bone marrow and extramedullar engraftment after intravenous injection of B53 cells. We found that the fibrinolytic factor expression pattern in murine B53 MM cells is similar to the expression pattern reported in primary human MM cells. Pharmacological targeting of plasmin using the plasmin inhibitors YO-2 did not change disease progression in MM cell bearing mice although systemic plasmin levels was suppressed. Our findings suggest that although plasmin has been suggested to be a driver for disease progression using clinical patient samples in MM using mostly in vitro studies, here we demonstrate that suppression of plasmin generation or inhibition of plasmin cannot alter MM progression in vivo.

KLRG+ invariant natural killer T cells are long-lived effectors.

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and γδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d(+) cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1(+) (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3ß by RNA deep sequencing demonstrated that some particular KLRG1(+) iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1(+) iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells.

Invariant natural killer T cells play dual roles in the development of experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) represents an experimental model for human endogenous uveitis, which is caused by Th1/Th17 cell-mediated inflammation. Natural killer T (NKT) cells recognize lipid antigens and produce large amounts of cytokines upon activation. To examine the role of NKT cells in the development of uveitis, EAU was elicited by immunization with a peptide from the human interphotoreceptor retinoid-binding protein (hIRBP1-20) in complete Freund's adjuvant and histopathology scores were evaluated in C57BL/6 (WT) and NKT cell-deficient mice. NKT cell-deficient mice developed more severe EAU pathology than WT mice. When WT mice were treated with ligands of the invariant subset of NKT cells (α-GalCer or RCAI-56), EAU was ameliorated in mice treated with RCAI-56 but not α-GalCer. IRBP-specific Th1/Th17 cytokines were reduced in RCAI-56-treated compared with vehicle-treated mice. Although the numbers of IRBP-specific T cells detected by hIRBP3-13/I-Ab tetramers in the spleen and the draining lymph node were the same for vehicle and RCAI-56 treatment groups, RORγt expression by tetramer-positive cells in RCAI-56-treated mice was lower than in control mice. Moreover, the eyes of RCAI-56-treated mice contained fewer IRBP-specific T cells compared with control mice. These results suggest that invariant NKT (iNKT) cells suppress the induction of Th17 cells and infiltration of IRBP-specific T cells into the eyes, thereby reducing ocular inflammation. However, in sharp contrast to the ameliorating effects of iNKT cell activation during the initiation phase of EAU, iNKT cell activation during the effector phase exacerbated disease pathology. Thus, we conclude that iNKT cells exhibit dual roles in the development of EAU.

Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell.

NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vß7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vß loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in peripheral lymphoid organs. NKT-iPSC-derived mice also showed normal development of other immune cells except for the absence of γδT cells and disturbed development of conventional CD4 αßT cells. These results suggest that the NKT-iPSC-derived mice are a better model for NKT cell development and function study rather than transgenic mouse models reported previously and also that the presence of a pre-rearranged Vα14Jα18 in the natural chromosomal context favors the developmental fate of NKT cells.

Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines.

There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T(H)2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB⁺iNKT cells are present in the thymic CD44⁺/⁻ NK1.1⁻ population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB⁻iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⁺ and IL-17RB⁻ subsets. The IL-17RB⁺iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⁺ IL-17RB⁺iNKT cells produce T(H)2 (IL-13), T(H)9 (IL-9 and IL-10), and T(H)17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4⁻ IL-17RB⁺iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⁺ subset producing T(H)17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB⁺iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB⁺iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.

Exacerbation of invasive Candida albicans infection by commensal bacteria or a glycolipid through IFN-γ produced in part by iNKT cells.

BACKGROUND: The commensal yeast Candida albicans is a major cause of invasive fungal infections. Despite treatment with antifungal agents, the mortality rate attributed to these types of infection is high. Although numerous cases have been reported regarding a poor outcome for patients with bacterial and C. albicans coinfection, the mechanisms by which the coinfecting bacteria exacerbate the C. albicans infection remain elusive. METHODS AND RESULTS: We evaluated how glycolipid-mediated activation of invariant natural killer T (iNKT) cells affects the clearance of C. albicans. Surprisingly, C. albicans-infected, glycolipid-treated mice exhibited significantly lower survival rates, increased fungal burden, and higher interleukin (IL)-6 production in the kidneys compared with control mice. Glycolipid-induced exacerbation of C. albicans infection was not observed in interferon-gamma knockout (IFN-γKO) mice. In the C. albicans-infected, glycolipid-treated mice, the number of neutrophils in the blood and bone marrow dramatically decreased in an IFN-γ-dependent manner. Furthermore, mice that were coinfected with C. albicans and nonfermentative gram-negative commensal bacteria exhibited increased fungal burden and inflammatory cytokine production in the kidneys that were dependent on IFN-γ and iNKT cells. CONCLUSIONS: Our results indicate that coinfecting commensal bacteria exacerbate C. albicans infection through IFN-γ produced, in part, by iNKT cells.

Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation.

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.8-fold, respectively, after short hairpin RNA-mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2-69 in human myeloid cell lines. Supernatants derived from USP2-KD cells induced IL6 (∼6-fold) and SAA3 (∼15-fold) in 3T3-L1 adipocytes to suggest the anti-inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2-69 transgenic mice fed a high-fat diet for 14 wk compared with that in their C57BL/6 littermates (P<0.01), which was consistent with a ∼40% decrease in transcription of aP2 and PAI-1. The aP2 locus exhibited elevated chromatin accessibility (>2.1-fold), methylation of histone H3 lysine 4 (>4.5-fold), and acetylation of histone H4 (>2.5-fold) in USP2-KD cells. Transfection of isopeptidase-mutated USP2-69 did not alter chromatin conformation on the aP2 locus in USP2-KD cells. Our results suggest that USP2-69 suppresses meta-inflammatory molecules involved in the development of type-2 diabetes.

Synthesis of RCAI-172 (C6 epimer of RCAI-147) and its biological activity.

RCAI-147 is one of the hydroxylated analogues of KRN7000 which is known as a ligand for the activation of CD1d mediated invariant natural killer T cells (iNKT cells) and releases both T helper 1 (Th1) cytokines such as IFN-γ and T helper 2 (Th2) cytokines such as IL-4. KRN7000 has been anticipated as an antitumor drug or an adjuvant for viral infection such as influenza, because of its strong secretion of IFN-γ. In an interesting twist, it has been obvious in our previous paper that RCAI-147 induces much more Th2 cytokines (IL-4) than Th1 cytokines (IFN-γ) from iNKT cells compared to KRN7000, and shows fairly good result in the experimental autoimmune encephalomyelitis (EAE) test. Therefore, synthesis of RCAI-172 (C6-OH epimer of RCAI-147) was attempted to examine the biological activity. As a result, RCAI-172 was synthesized and its biological activity biased to Th2 response largely compared to that of KRN7000. However, this level decreased to approximately 61% compared to that of RCAI-147. And the clinical score of RCAI-172 for EAE suppression was disappointing. There exist seven chiral centers in the aglycon part of RCAI-172, and even though the change of configuration is just one position (C6-OH), the effect on both Th1/Th2 response and EAE test is fairly large.

RCAI-61 and related 6'-modified analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce interferon-γ in vivo.

We synthesized ten new analogs of 6'-modified KRN7000 (A): RCAI-58, 61, 64, 83, 85-87, 113, 119, and 125. They could be synthesized by α-selective galactosylation of ceramide 9 with the 6-modified D-galactopyranosyl fluorides (8a-8f) or L-arabinopyranosyl fluoride (17), or by etherification of the known alcohol 19. Bioassay of the ten analogs demonstrated that RCAI-61 (1, 6'-O-methylated analog of A) was the most potent immunostimulant among them, and could induce the production of a large amount of IFN-γ even at a low concentration in mice in vivo.