Recent advances regarding the potential roles of invariant natural killer T cells in cardiovascular diseases with immunological and inflammatory backgrounds.

Invariant natural killer T (iNKT) cells, which bear αß-type T-cell antigen-receptors (TCRs), recognize glycolipid antigens in a cluster of differentiation 1d (CD1d)-restricted manner. Regarding these cells, the unique modes of thymic selection and maturation elucidate innateness, irrespective of them also being members of the adaptive immune system as a T-cell. iNKT cells develop and differentiate into NKT1 [interferon γ (IFN-γ)-producing], NKT2 [interleukin 4 (IL-4)/IL-13-producing], or NKT17 (IL-17-producing) subsets in the thymus. After egress, NKT10 (IL-10-producing), follicular helper NKT (NKTfh; IL-21-producing), and regulatory NKT (NKTreg) subsets emerge following stimulation in the periphery. Moreover, iNKT cells have been shown to possess several physiological or pathological roles. iNKT cells exhibit dual alleviating or aggravating roles in experimentally induced immune and/or inflammatory diseases in mice. These findings indicate that the modulation of iNKT cells can be employed for therapeutic use or prevention of human diseases. In this review, we discuss the potential roles of iNKT cells in the development of immune/inflammatory diseases of the cardiovascular system, with emphasis on atherosclerosis, aortic aneurysms, and cardiac remodeling.

A new approach to analysis of intracellular proteins and subcellular localization using cellprofiler and imageJ in combination.

Analytical pipeline, which is used for various analysis application, of CellProfiler, an open-source software for cell imaging analysis, is very important. In the present study, to examine whether intracellular proteins can be discriminated using a combination of CellProfiler and ImageJ, we analyzed neuroblastoma and monocytic cell lines, and disease-specific induced pluripotent stem cell (iPSC)-derived neurons. This revealed that scattered puncta of Rab7 and transferrin in neuroblastoma lines were clearly detectable by created analytical pipelines in CellProfiler. We then constructed pipelines for measuring the distance from the center of the nucleus to allow investigation of the intracellular localization of Rab7 or transferrin. Using CellProfiler and ImageJ in combination, we confirmed that our pipelines were applicable both quantitatively and objectively to analysis of membrane trafficking of proteins such as Rab proteins and transferrin. In addition, when applied to quantitative measurement of phagocytosis, our pipelines clearly detected monocytic cell lines that had engulfed bioparticles. Finally, we developed new pipelines for analysis of disease phenotype using iPSCs from a patient with familial Parkinson's disease (PD), harboring the I2020T LRRK2 mutation (PARK8). These were able to successfully detect Rab5 puncta and Rab7 puncta in PARK8 patient iPSC-derived neurons. Interestingly, in long-term culture, we found that the numbers of Rab7 puncta in a single PARK8 patient iPSC-derived neurons were lower than that of control iPSC-derived neurons. On the other hands, at 14 days in vitro, the numbers of Rab5 puncta in PARK8 patient iPSC-derived neurons were lower than those of isogenic iPSC-derived neurons, but not Rab7 puncta. Furthermore, Rab5 puncta of PARK8 patient iPSC-derived neurons exhibited distinct localization pattern relative to isogenic iPSC-derived neurons. These present results suggest that this new analytical tool can be used as a supporting method for quantification of intracellular protein.

Adipose invariant NKT cells interact with CD1d-expressing macrophages to regulate obesity-related inflammation.

Obesity is accompanied by and accelerated with chronic inflammation in adipose tissue, especially visceral adipose tissue (VAT). This low-level inflammation predisposes the host to the development of metabolic disease, most notably type 2 diabetes. We have focused on the capacity of glycolipid-reactive, CD1d-restricted natural killer T (NKT) cells to modulate obesity and its associated metabolic sequelae. We previously reported that CD1d knockout (KO) mice are partially protected against the development of obesity-associated insulin resistance, and these findings were recapitulated in mice with an adipocyte-specific CD1d deficiency, suggesting that NKT cell-adipocyte interactions play a critical role in exacerbating disease. However, many other CD1d-expressing cells contribute to the in vivo responses of NKT cells to lipid antigens. In the present study, we examined the role of CD1d expression by macrophages (Mϕ) in the development of obesity-associated metabolic inflammation using LysMcre-cd1d1f/f mice where the CD1d1 gene is disrupted in a Mϕ-specific manner. Unexpectedly, these animals contained a higher frequency of T-bet+ CD4+ T cells in VAT with increased production of Th1 cytokines that aggravated VAT inflammation. Mϕ from mutant mice displayed increased production of IL-12p40, suggesting M1 polarization. These findings indicate that interactions of CD1d on Mϕ with NKT cells play a beneficial role in obesity-associated VAT inflammation and insulin resistance with a sharp contrast to an aggravating role of CD1d in another type of antigen-presenting cell, dendritic cells.

The protective function of invariant natural killer T cells in the relapse of experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) in mice provides a useful platform to study the pathogenesis and experimental therapeutics of human uveitis. One often used EAU model employs C57BL/6 (B6) mice sensitized with a peptide residue having 1 to 20 amino acids of human interphotoreceptor retinoid binding protein (hIRBP1-20). The model using the B6 background has permitted a liberal use of genetically engineered strains and has provided insights for understanding uveoretinitis. However, this is usually acute/monophasic and does not represent human uveoretinitis that is characterized as a chronic/recurrent disease. Several chronic/recurrent EAU models have been developed; of these, we employed administration of staphylococcal enterotoxin B (SEB) for relapse in the present study, and found that recurrence was induced at day 24 after primary immunization, which is thought to be the convalescent phase. We reported the activation of invariant natural killer T (iNKT)-cells upon primary immunization of the EAU model mice with the ligand RCAI-56, which was found to mitigate the disease in our previous study. Here, we first attempted to ameliorate EAU in the relapse model using a preventive regimen by activating iNKT cells at the same time relapse induction (day 24) or in a regimen after 3 days of relapse induction (day 27). The preventive as well as post-inductive regimens were successful in reducing histopathological scores by inhibiting the Ag-specific Th17-biased response. Collectively, activation of iNKT cells may be useful to mitigate the relapse response of EAU induced with SEB.

Natural Killer T Cells Are Involved in Atherosclerotic Plaque Instability in Apolipoprotein-E Knockout Mice.

The infiltration and activation of macrophages as well as lymphocytes within atherosclerotic lesion contribute to the pathogenesis of plaque rupture. We have demonstrated that invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that recognize glycolipid antigens, play a crucial role in atherogenesis. However, it remained unclear whether iNKT cells are also involved in plaque instability. Apolipoprotein E (apoE) knockout mice were fed a standard diet (SD) or a high-fat diet (HFD) for 8 weeks. Moreover, the SD- and the HFD-fed mice were divided into two groups according to the intraperitoneal injection of α-galactosylceramide (αGC) that specifically activates iNKT cells or phosphate-buffered saline alone (PBS). ApoE/Jα18 double knockout mice, which lack iNKT cells, were also fed an SD or HFD. Plaque instability was assessed at the brachiocephalic artery by the histological analysis. In the HFD group, αGC significantly enhanced iNKT cell infiltration and exacerbated atherosclerotic plaque instability, whereas the depletion of iNKT cells attenuated plaque instability compared to PBS-treated mice. Real-time PCR analyses in the aortic tissues showed that αGC administration significantly increased expressional levels of inflammatory genes such as IFN-γ and MMP-2, while the depletion of iNKT cells attenuated these expression levels compared to those in the PBS-treated mice. Our findings suggested that iNKT cells are involved in the exacerbation of plaque instability via the activation of inflammatory cells and upregulation of MMP-2 in the vascular tissues.

NF-κB-inducing kinase contributes to normal development of cortical thymic epithelial cells: its possible role in shaping a proper T-cell repertoire.

Nuclear factor (NF)-κB-inducing kinase (NIK) is known to be a critical regulator of multiple aspects of the immune response. Although the role of NIK in the development of medullary thymic epithelial cells (mTECs) has been well documented, the impact of NIK on the differentiation and function of cortical thymic epithelial cells (cTECs) remains ambiguous. To investigate the possible involvement of NIK in cTEC differentiation, we have compared the gene expression and function of cTECs from a NIK-mutant mouse, alymphoplasia (aly/aly) with those of cTECs from wild-type (WT) mice. Flow cytometric analyses revealed that expression levels of MHC class II, but not MHC class I or other TEC markers, were higher in aly/aly cells than in WT cells. Notably, the proportion of MHC class IIhi+ cTECs was elevated in aly/aly mice. We also demonstrated that expression of Ccl5 mRNA in the MHC class IIhi+ subset of aly/aly cTECs was decreased compared with that in WT cells, implying an abnormal pattern of gene expression in aly/aly cTECs. Analyses of bone marrow chimera using aly/aly or aly/+ mice as hosts suggested that Vß usage and CD5 expression on WT T-cells were altered when they matured in aly/aly thymi. These results collectively indicate that NIK may be involved in controlling the function of cTEC in selecting a proper T-cell repertoire.

Attenuation of experimental autoimmune uveoretinitis in mice by IKKß inhibitor IMD-0354.

Uveitis is a sight-threatening intraocular inflammatory disease that accounts for almost 10% of blindness worldwide. NF-κB signaling plays pivotal roles in inflammatory diseases. We have reported that IMD-0354, which inhibits NF-κB signaling via selective blockade of IKK-ß, suppresses inflammation in several ocular disease models. Here, we examined the therapeutic effect of IMD-0354 in an experimental autoimmune uveoretinitis (EAU) model, a well-established animal model for endogenous uveitis in humans. Systemic administration of IMD-0354 significantly suppressed the clinical and histological severity, inflammatory edema, and the translocation of NF-κB p65 into the nucleus of retinas in EAU mice. Furthermore, IMD-0354 treatment significantly inhibited the levels of several Th1/Th17-mediated pro-inflammatory cytokines in vitro. Our current data demonstrate that inhibition of IKKß with IMD-0354 ameliorates inflammatory responses in the mouse EAU model, suggesting that IMD-0354 may be a promising therapeutic agent for human endogenous uveitis.

The disruption of invariant natural killer T cells exacerbates cardiac hypertrophy and failure caused by pressure overload in mice.

NEW FINDINGS: What is the central question of this study? We questioned whether the disruption of invariant natural killer T (iNKT) cells exacerbates left ventricular (LV) remodelling and heart failure after transverse aortic constriction in mice. What are the main findings and their importance? Pressure overload induced by transverse aortic constriction increased the infiltration of iNKT cells in mouse hearts. The disruption of iNKT cells exacerbated LV remodelling and hastened the transition from hypertrophy to heart failure, in association with the activation of mitogen-activated protein kinase signalling. Activation of iNKT cells modulated the immunological balance in this process and played a protective role against LV remodelling and failure. ABSTRACT: Chronic inflammation is involved in the development of cardiac remodelling and heart failure (HF). Invariant natural killer T (iNKT) cells, a subset of T lymphocytes, have been shown to produce various cytokines and orchestrate tissue inflammation. The pathophysiological role of iNKT cells in HF caused by pressure overload has not been studied. In the present study, we investigated whether the disruption of iNKT cells affected this process in mice. Transverse aortic constriction (TAC) and a sham operation were performed in male C57BL/6J wild-type (WT) and iNKT cell-deficient Jα18 knockout (KO) mice. The infiltration of iNKT cells was increased after TAC. The disruption of iNKT cells exacerbated left ventricular (LV) remodelling and hastened the transition to HF after TAC. Histological examinations also revealed that the disruption of iNKT cells induced greater myocyte hypertrophy and a greater increase in interstitial fibrosis after TAC. The expressions of interleukin-10 and tumour necrosis factor-α mRNA and their ratio in the LV after TAC were decreased in the KO compared with WT mice, which might indicate that the disruption of iNKT cells leads to an imbalance between T-helper type 1 and type 2 cytokines. The phosphorylation of extracellular signal-regulated kinase was significantly increased in the KO mice. The disruption of iNKT cells exacerbated the development of cardiac remodelling and HF after TAC. The activation of iNKT cells might play a protective role against HF caused by pressure overload. Targeting the activation of iNKT cells might thus be a promising candidate as a new therapeutic strategy for HF.

Role of T-bet, the master regulator of Th1 cells, in the cytotoxicity of murine CD4+ T cells.

Although CD4+ T cells are generally regarded as helper T cells, some activated CD4+ T cells have cytotoxic properties. Given that CD4+ cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4+ T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4+ T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4+ T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4+ T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4+ T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas- target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 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.

Differential dependence on nuclear factor-κB-inducing kinase among natural killer T-cell subsets in their development.

Natural killer T cells (NKT cells) are comprised of several subsets. However, the possible differences in their developmental mechanisms have not been fully investigated. To evaluate the dependence of some NKT subpopulations on nuclear factor-κB-inducing kinase (NIK) for their generation, we analysed the differentiation of NKT cells, dividing them into subsets in various tissues of alymphoplasia (aly/aly), a mutant mouse strain that lacks functional NIK. The results indicated that the efficient differentiation of both invariant NKT (iNKT) and non-iNKT cells relied on NIK expression in non-haematopoietic cells; however, the dependence of non-iNKT cells was lower than that of iNKT cells. Especially, the differentiation of CD8(+) non-iNKT cells was markedly resistant to the aly mutation. The proportion of two other NKT cell subsets, NK1.1(+) γδ T cells and NK1.1(-) iNKT cells, was also significantly reduced in aly/aly mice, and this defect in their development was reversed in wild-type host mice given aly/aly bone marrow cells. In exerting effector functions, NIK in NKT-αß cells appeared dispensable, as NIK-deficient NKT-αß cells could secrete interleukin-4 or interferon-γ and exhibit cytolytic activity at a level comparable to that of aly/+ NKT-αß cells. Collectively, these results imply that the NIK in thymic stroma may be critically involved in the differentiation of most NKT cell subsets (although the level of NIK dependence may vary among the subsets), and also that NIK in NKT-αß cells may be dispensable for their effector function.

Significant involvement of nuclear factor-κB-inducing kinase in proper differentiation of αß and γδ T cells.

Nuclear factor-κB-inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune-compromised and autoimmune-prone. To investigate the role of NIK in αß T-cell repertoire formation, we analysed T-cell development in aly/aly mice bearing a transgenic T-cell receptor (TCR). Although there were no apparent abnormalities in the mature αß T cells of non-transgenic aly/aly mice, the maturation efficiency of idiotype(high+) T cells in the TCR-transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αß T-cell repertoire could be altered by the absence of functional NIK. In one strain of TCR-transgenic aly/aly mice with a negatively selecting H-2 background, the proportion of CD8(low+) idiotype(high+) cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non-transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one-half to one-fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αß T cells and that NIK in non-haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.

Activation of natural killer T cells ameliorates postinfarct cardiac remodeling and failure in mice.

RATIONALE: Chronic inflammation in the myocardium is involved in the development of left ventricular (LV) remodeling and failure after myocardial infarction (MI). Invariant natural killer T (iNKT) cells have been shown to produce inflammatory cytokines and orchestrate tissue inflammation. However, no previous studies have determined the pathophysiological role of iNKT cells in post-MI LV remodeling. OBJECTIVE: The purpose of this study was to examine whether the activation of iNKT cells might affect the development of LV remodeling and failure. METHODS AND RESULTS: After creation of MI, mice received the injection of either α-galactosylceramide (αGC; n=27), the activator of iNKT cells, or phosphate-buffered saline (n=31) 1 and 4 days after surgery, and were followed during 28 days. Survival rate was significantly higher in MI+αGC than MI+PBS (59% versus 32%, P<0.05). LV cavity dilatation and dysfunction were significantly attenuated in MI+αGC, despite comparable infarct size, accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis. The infiltration of iNKT cells were increased during early phase in noninfarcted LV from MI and αGC further enhanced them. It also enhanced LV interleukin (IL)-10 gene expression at 7 days, which persisted until 28 days. AntienIL-10 receptor antibody abrogated these protective effects of αGC on MI remodeling. The administration of αGC into iNKT cell-deficient Jα18(-/-) mice had no such effects, suggesting that αGC was a specific activator of iNKT cells. CONCLUSIONS: iNKT cells play a protective role against post-MI LV remodeling and failure through the enhanced expression of cardioprotective cytokines such as IL-10.

Contribution of NKT cells and CD1d-expressing cells in obesity-associated adipose tissue inflammation.

Natural killer T (NKT) cell are members of the innate-like T lymphocytes and recognizes lipid antigens presented by CD1d-expressing cells. Obesity-associated inflammation in adipose tissue (AT) leads to metabolic dysfunction, including insulin resistance. When cellular communication is properly regulated among AT-residing immune cells and adipocytes during inflammation, a favorable balance of Th1 and Th2 immune responses is achieved. NKT cells play crucial roles in AT inflammation, influencing the development of diet-induced obesity and insulin resistance. NKT cells interact with CD1d-expressing cells in AT, such as adipocytes, macrophages, and dendritic cells, shaping pro-inflammatory or anti-inflammatory microenvironments with distinct characteristics depending on the antigen-presenting cells. Additionally, CD1d may be involved in the inflammatory process independently of NKT cells. In this mini-review, we provide a brief overview of the current understanding of the interaction between immune cells, focusing on NKT cells and CD1d signaling, which control AT inflammation both in the presence and absence of NKT cells. We aim to enhance our understanding of the mechanisms of obesity-associated diseases.

Activation of invariant natural killer T cells by α-galactosylceramide ameliorates myocardial ischemia/reperfusion injury in mice.

Invariant natural killer T (iNKT) cells orchestrate tissue inflammation via regulating various cytokine productions. However the role of iNKT cells has not been determined in myocardial ischemia/reperfusion (I/R) injury. The purpose of this study was to examine whether the activation of iNKT cells by α-galactosylceramide (α-GC), which specifically activates iNKT cells, could affect myocardial I/R injury. I/R or sham operation was performed in male C57BL/6J mice. I/R mice received the injection of either αGC (I/R+αGC, n=48) or vehicle (I/R+vehicle, n=49) 30 min before reperfusion. After 24h, infarct size/area at risk was smaller in I/R+αGC than in I/R+vehicle (37.8 ± 2.7% vs. 47.1 ± 2.5%, P<0.05), with no significant changes in area at risk. The numbers of infiltrating myeloperoxidase- and CD3-positive cells were lower in I/R+αGC. Apoptosis evaluated by TUNEL staining and caspase-3 protein was also attenuated in I/R+αGC. Myocardial gene expression of tumor necrosis factor-α and interleukin (IL)-1ß in I/R+αGC was lower to 46% and 80% of that in I/R+vehicle, respectively, whereas IL-10, IL-4, and interferon (IFN)-γ were higher in I/R+αGC than I/R+vehicle by 2.0, 4.1, and 9.6 folds, respectively. The administration of anti-IL-10 receptor antibody into I/R+αGC abolished the protective effects of αGC on I/R injury (infarct size/area at risk: 53.1 ± 5.2% vs. 37.4 ± 3.5%, P<0.05). In contrast, anti-IL-4 and anti-IFN-γ antibodies did not exert such effects. In conclusion, activated iNKT cells by αGC play a protective role against myocardial I/R injury through the enhanced expression of IL-10. Therapies designed to activate iNKT cells might be beneficial to protect the heart from I/R injury.

Myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor-1 overexpression.

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.

CSF-1-dependent red pulp macrophages regulate CD4 T cell responses.

The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macrophages (MΦs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MΦs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MΦ (RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80(hi)Mac-1(low) MΦs residing in the red pulp showed different expression patterns of surface markers compared with F4/80(+)Mac-1(hi) monocytes/MΦs. Studies with purified cell populations demonstrated that F4/80(hi)Mac-1(low) MΦs regulated CD4(+) T cell responses by producing soluble suppressive factors, including TGF-ß and IL-10. Moreover, F4/80(hi)Mac-1(low) MΦs induced the differentiation of naive CD4(+) T cells into functional Foxp3(+) regulatory T cells. Additionally, we found that the differentiation of F4/80(hi)Mac-1(low) MΦs was critically regulated by CSF-1, and in vitro-generated bone marrow-derived MΦs induced by CSF-1 suppressed CD4(+) T cell responses and induced the generation of Foxp3(+) regulatory T cells in vivo. These results suggested that splenic CSF-1-dependent F4/80(hi)Mac-1(low) MΦs are a subpopulation of RPMs and regulate peripheral immune homeostasis.

Identification of a third variable lymphocyte receptor in the lamprey.

Jawless vertebrates such as lamprey and hagfish lack T-cell and B-cell receptors; instead, they have unique antigen receptors known as variable lymphocyte receptors (VLRs). VLRs generate diversity by recombining highly diverse leucine-rich repeat modules and are expressed clonally on lymphocyte-like cells (LLCs). Thus far, two types of receptors, VLRA and VLRB, have been identified in lampreys and hagfish. Recent evidence indicates that VLRA and VLRB are expressed on distinct populations of LLCs that resemble T cells and B cells of jawed vertebrates, respectively. Here we identified a third VLR, designated VLRC, in the lamprey. None of the approximately 100 VLRC cDNA clones subjected to sequencing had an identical sequence, indicating that VLRC can generate sufficient diversity to function as antigen receptors. Notably, the C-terminal cap of VLRC exhibits only limited diversity and has important structural differences relative to VLRA and VLRB. Single-cell PCR analysis identified LLCs that rearranged VLRC but not VLRA or VLRB, suggesting the presence of a unique population of LLCs that express only VLRC.

Immunomodulatory Functions of α-GalCer and a Derivative, α-Carba-GalCer.

Certain glycolipids have immunomodulatory potential by activating natural killer T (NKT) cells, a unique T cell subset. Invariant NKT (iNKT) cells recognize α-galactosylceramide (α-GalCer) and synthetic derivatives presented by CD1d molecules and secrete large amounts of cytokines that modulate immune functions. Some iNKT cell ligands induce distinct reactions biased toward either Th1 or Th2 immune responses, while others show mixed responses. We describe the methods for activating iNKT cells by the ligands as represented by α-GalCer using in vitro assays, such as cell-free or co-culture with antigen-presenting cells. In addition, α-GalCer/CD1d multimer can be used to specifically detect iNKT cells using flow cytometry. α-GalCer is also utilized to activate systemic iNKT cells in vivo, which leads to the production of high levels of cytokines in sera. Collectively, α-GalCer and its derivatives activate iNKT cells that modulate immune balance, and we need to understand the characteristics of these ligands for developing their utility.

MR1 deficiency enhances IL-17-mediated allergic contact dermatitis.

Major histocompatibility complex (MHC) class Ib molecules present antigens to subsets of T cells primarily involved in host defense against pathogenic microbes and influence the development of immune-mediated diseases. The MHC class Ib molecule MHC-related protein 1 (MR1) functions as a platform to select MR1-restricted T cells, including mucosal-associated invariant T (MAIT) cells in the thymus, and presents ligands to them in the periphery. MAIT cells constitute an innate-like T-cell subset that recognizes microbial vitamin B2 metabolites and plays a defensive role against microbes. In this study, we investigated the function of MR1 in allergic contact dermatitis (ACD) by examining wild-type (WT) and MR1-deficient (MR1-/-) mice in which ACD was induced with 2,4-dinitrofluorobenzene (DNFB). MR1-/- mice exhibited exaggerated ACD lesions compared with WT mice. More neutrophils were recruited in the lesions in MR1-/- mice than in WT mice. WT mice contained fewer MAIT cells in their skin lesions following elicitation with DNFB, and MR1-/- mice lacking MAIT cells exhibited a significant increase in IL-17-producing αß and γδ T cells in the skin. Collectively, MR1-/- mice displayed exacerbated ACD from an early phase with an enhanced type 3 immune response, although the precise mechanism of this enhancement remains elusive.