Potential of MAIT cells to modulate asthma.

Despite recent advances in asthma treatments, the search for novel therapies remains necessary because there are still patients with recurrent asthma exacerbations and poor responses to the existing treatments. Since group 2 innate lymphoid cells (ILC2) play a pivotal role in asthma by triggering and exacerbating type 2 inflammation, controlling ILC2s function is key to combating severe asthma. Mucosal-associated invariant T (MAIT) cells are innate-like T cells abundant in humans and are activated both in a T cell receptor-dependent and -independent manner. MAIT cells are composed of MAIT1 and MAIT17 based on the expression of transcription factors T-bet and RORγt, respectively. MAIT cells play pivotal roles in host defense against pathogens and in tissue repair and are essential for the maintenance of immunity and hemostasis. Our recent studies revealed that MAIT cells inhibit both ILC2 proliferation and functions in a mouse model of airway inflammation. MAIT cells may alleviate airway inflammation in two ways, by promoting airway epithelial cell barrier repair and by repressing ILC2s. Therefore, reagents that promote MAIT cell-mediated suppression of ILC2 proliferation and function, or designer MAIT cells (genetically engineered to suppress ILC2s or promote repair of airway damage), may be effective therapeutic agents for severe asthma.

Critical Role for Monocytes/Macrophages in Rapid Progression to AIDS in Pediatric Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Infant humans and rhesus macaques infected with the human or simian immunodeficiency virus (HIV or SIV), respectively, express higher viral loads and progress more rapidly to AIDS than infected adults. Activated memory CD4+ T cells in intestinal tissues are major primary target cells for SIV/HIV infection, and massive depletion of these cells is considered a major cause of immunodeficiency. Monocytes and macrophages are important cells of innate immunity and also are targets of HIV/SIV infection. We reported previously that a high peripheral blood monocyte turnover rate was predictive for the onset of disease progression to AIDS in SIV-infected adult macaques. The purpose of this study was to determine if earlier or higher infection of monocytes/macrophages contributes to the more rapid progression to AIDS in infants. We observed that uninfected infant rhesus macaques exhibited higher physiologic baseline monocyte turnover than adults. Early after SIV infection, the monocyte turnover further increased, and it remained high during progression to AIDS. A high percentage of terminal deoxynucleotidyltransferase dUTP nick end label (TUNEL)-positive macrophages in the lymph nodes (LNs) and intestine corresponded with an increasing number of macrophages derived from circulating monocytes (bromodeoxyuridine positive [BrdU+] CD163+), suggesting that the increased blood monocyte turnover was required to rapidly replenish destroyed tissue macrophages. Immunofluorescence analysis further demonstrated that macrophages were a significant portion of the virus-producing cells found in LNs, intestinal tissues, and lungs. The higher baseline monocyte turnover in infant macaques and subsequent macrophage damage by SIV infection may help explain the basis of more rapid disease progression to AIDS in infants.IMPORTANCE HIV infection progresses much more rapidly in pediatric cases than in adults; however, the mechanism for this difference is unclear. Using the rhesus macaque model, this work was performed to address why infants infected with SIV progress more quickly to AIDS than do adults. Earlier we reported that in adult rhesus macaques, increasing monocyte turnover reflected tissue macrophage damage by SIV and was predictive of terminal disease progression to AIDS. Here we report that uninfected infant rhesus macaques exhibited a higher physiological baseline monocyte turnover rate than adults. Furthermore, once infected with SIV, infants displayed further increased monocyte turnover that may have facilitated the accelerated progression to AIDS. These results support a role for monocytes and macrophages in the pathogenesis of SIV/HIV and begin to explain why infants are more prone to rapid disease progression.

Prevention and treatment for intractable infectious diseases through the iPSC-based technology: status quo and perspective.

Nowadays, antibiotic resistance is a serious global health concern, for it is observed every- where on the earth. While antibiotic is effective for controlling pathogens, an inappropriate use of the antibiotic leads to antibiotic resistance. Given that the ability to develop novel anti- biotics is quite limited, a new strategy must be developed to fight against it. Mucosal- associated invariant T cells (MAITs) belong to a family of the innate-like T cells that bridges the gap between the innate and the adaptive immunity. We have generated iPSCs from MAITs and redifferentiated MAITs from the iPSCs. These MAITs exerted anti- mycobacterial activity in mice. Advent of such cells will pave the way to exploit a novel arsen- al against antibiotic resistance.

Mice Generated with Induced Pluripotent Stem Cells Derived from Mucosal-Associated Invariant T Cells.

The function of mucosal-associated invariant T (MAIT) cells, a burgeoning member of innate-like T cells abundant in humans and implicated in many diseases, remains obscure. To explore this, mice with a rearranged T cell receptor (TCR) α or ß locus, specific for MAIT cells, were generated via induced pluripotent stem cells derived from MAIT cells and were designated Vα19 and Vß8 mice, respectively. Both groups of mice expressed large numbers of MAIT cells. The MAIT cells from these mice were activated by cytokines and an agonist to produce IFN-γ and IL-17. While Vß8 mice showed resistance in a cancer metastasis model, Vα19 mice did not. Adoptive transfer of MAIT cells from the latter into the control mice, however, recapitulated the resistance. These mice present an implication for understanding the role of MAIT cells in health and disease and in developing treatments for the plethora of diseases in which MAIT cells are implicated.

A flow-cytometry-based assay to assess the cytolytic activity against tumor cells by combination of mouse MAIT cells and natural killer cells.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells responsible for mucosal immunity in the respiratory and intestinal tracts. Here we present a flow-cytometry-based assay to measure the cytolytic activity of murine MAIT cells and natural killer (NK) cells. We describe steps for differentiating MAIT-like cells from the induced pluripotent stem cells prepared from MAIT cells (reMAIT cells), NK cell isolation, co-culture with target tumor cells, and staining to distinguish dead cells from live cells. For complete details on the use and execution of this protocol, please refer to Sugimoto et al. (2022).1.

Harnessing the Power of Mucosal-Associated Invariant T (MAIT) Cells in Cancer Cell Therapy.

Mucosal-associated invariant T (MAIT) cells, a burgeoning type of the innate-like T cells, play a crucial role in maintaining immune homeostasis, particularly in host defense. Although many studies have implied the use of MAIT cells in tumor immunity, whether MAIT cells are pro-tumor or anti-tumor has remained elusive, as in the case for other innate-like T cells that possess dichotomous roles in tumor immunity. Although this difficulty persists where endogenous MAIT cells are the target for therapeutic intervention, the advent of induced pluripotent stem-cell-derived MAIT cells (reMAIT cells) will make it possible to harness these cells for immune cell therapy. In this review, we will discuss possible roles of MAIT cells in tumor immunity and the potential of reMAIT cells to treat tumors.

Mucosal-associated invariant T cells repress group 2 innate lymphoid cells in Alternaria alternata-induced model of allergic airway inflammation.

Mucosal-associated invariant T (MAIT) cells, a blossoming member of the innate-like T cells, play a pivotal role in host defense through engaging the mucosal immunity. Although it has been suggested that MAIT cells are somehow implicated in the allergic airway inflammation mediated by group 2 innate lymphoid cells (ILC2s) such as asthma, the precise role(s) of MAIT cells in such inflammation has remained elusive. To explore the possible roles of MAIT cells in the inflammation, we examined whether MAIT cells suppressed the production of T helper (Th) 2 and inflammatory cytokines from ILC2s, and constrained the proliferation of ILC2s, both of which are prerequisite for airway inflammation. Given that laboratory mice are poor at MAIT cells, a novel mouse line rich in MAIT cells was used. We found that mice rich in MAIT cells showed alleviated airway inflammation as evidenced by reduced infiltration of the immune cells and hyperplasia in goblet cells in the lung concomitant with compromised production of Th2 and inflammatory cytokines, while wild type mice exhibited severe inflammation upon challenge with the fungal extracts. In vitro coculture experiments using purified ILC2s and MAIT cells unrevealed that cytokine-stimulated MAIT cells suppressed ILC2s to produce the cytokines as well as to proliferate most likely via production of IFN-γ. Furthermore, reconstitution of the allergic airway inflammation in the highly immunocompromised mice showed that ILC2-mediated inflammation was alleviated in mice that received MAIT cells along with ILC2s. We concluded that MAIT cells played a crucial role in suppressing the cytokine-producing capacity of ILC2s and ILC2 proliferation, that ultimately led to decrease in the allergic airway inflammation. The results open up a novel therapeutic horizon in ILC2-mediated inflammatory diseases by modulating MAIT cell activity.

Reprogramming and redifferentiation of mucosal-associated invariant T cells reveal tumor inhibitory activity.

Mucosal-associated invariant T (MAIT) cells belong to a family of innate-like T cells that bridge innate and adaptive immunities. Although MAIT cells have been implicated in tumor immunity, it currently remains unclear whether they function as tumor-promoting or inhibitory cells. Therefore, we herein used induced pluripotent stem cell (iPSC) technology to investigate this issue. Murine MAIT cells were reprogrammed into iPSCs and redifferentiated towards MAIT-like cells (m-reMAIT cells). m-reMAIT cells were activated by an agonist in the presence and absence of antigen-presenting cells and MR1-tetramer, a reagent to detect MAIT cells. This activation accompanied protein tyrosine phosphorylation and the production of T helper (Th)1, Th2, and Th17 cytokines and inflammatory chemokines. Upon adoptive transfer, m-reMAIT cells migrated to different organs with maturation in mice. Furthermore, m-reMAIT cells inhibited tumor growth in the lung metastasis model and prolonged mouse survival upon tumor inoculation through the NK cell-mediated reinforcement of cytolytic activity. Collectively, the present results demonstrated the utility and role of m-reMAIT cells in tumor immunity and provide insights into the function of MAIT cells in immunity.

Regulation of ghrelin signaling by a leptin-induced gene, negative regulatory element-binding protein, in the hypothalamic neurons.

Leptin, the product of the ob gene, plays important roles in the regulation of food intake and body weight through its receptor in the hypothalamus. To identify novel transcripts induced by leptin, we performed cDNA subtraction based on selective suppression of the polymerase chain reaction by using mRNA prepared from the forebrain of leptin-injected ob/ob mice. One of the genes isolated was a mouse homolog of human negative regulatory element-binding protein (NREBP). Its expression was markedly increased by leptin in the growth hormone secretagogue-receptor (GHS-R)-positive neurons of the arcuate nucleus and ventromedial hypothalamic nucleus. The promoter region of GHS-R contains one NREBP binding sequence, suggesting that NREBP regulates GHS-R transcription. Luciferase reporter assays showed that NREBP repressed GHS-R promoter activity in a hypothalamic neuronal cell line, GT1-7, and its repressive activity was abolished by the replacement of negative regulatory element in GHS-R promoter. Overexpression of NREBP reduced the protein expression of endogenous GHS-R without affecting the expression of ob-Rb in GT1-7 cells. To determine the functional importance of NREBP in the hypothalamus, we assessed the effects of NREBP on ghrelin action. Although phosphorylation of AMP-activated protein kinase α (AMPKα) was induced by ghrelin in GT1-7 cells, NREBP repressed ghrelin-induced AMPKα phosphorylation. These results suggest that leptin-induced NREBP is an important regulator of GHS-R expression in the hypothalamus and provides a novel molecular link between leptin and ghrelin signaling.

Constitutively active Stat5A and Stat5B promote adipogenesis.

OBJECTIVE: The metabolic syndrome is an important social problem affecting many people in developed countries. Obesity is a leading cause of this syndrome, hence understanding molecular mechanisms underlying obesity is of prime importance for preventive medicine to develop novel methods to alleviate the corresponding social cost as well as for pharmaceutical companies to develop antimetabolic drugs. METHODS: Since adipocytes play an important role in obesity, we explored the signaling pathways leading to differentiation of adipocytes. We used a preadipocyte cell line to monitor the differentiation of adipocytes, and virus-mediated gene transfer to assess the role of the transcription factor Stat5 in adipogenesis. Adipocyte differentiation was assessed by Northern blot and Western blot analyses as well as accumulation of fat droplets in cells. Promoter activity of the proadipogenic transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ) was evaluated by luciferase assay. RESULTS: Virus-mediated gene transfer of the constitutively active form of both Stat5A and Stat5B resulted in enhanced adipocyte differentiation in the absence of fetal bovine serum (FBS) as judged by expression of proadipogenic factors as well as accumulation of fat droplets in cells. Such a proadipogenic effect of Stat5 is, in part, mediated by its ability to enhance transcription of PPARγ, a master transcriptional regulator in adipogenesis. CONCLUSION: The constitutively active form of Stat5A and Stat5B promoted adipocyte differentiation in the absence of FBS via induction of PPARγ.

Bone marrow allograft rejection mediated by a novel murine NK receptor, NKG2I.

Natural killer (NK) cells mediate bone marrow allograft rejection. However, the molecular mechanisms underlying such a rejection remain elusive. In previous analyses, it has been shown that NK cells recognize allogeneic target cells through Ly-49s and CD94/NKG2 heterodimers. Here, we describe identification and characterization of a novel murine NK receptor, NKG2I, belonging to the NKG2 family. NKG2I, which was composed of 226 amino acids, showed approximately 40% homology to the murine NKG2D and CD94 in the C-type lectin domain. Flow cytometric analysis with anti-NKG2I monoclonal antibody (mAb) revealed that expression of NKG2I was largely confined to NK and NKT cells, but was not seen in T cells. Furthermore, anti-NKG2I mAb inhibited NK cell-mediated cytotoxicity, whereas cross-linking of NKG2I enhanced interleukin 2- and interleukin 12-dependent interferon-gamma production. Similarly, the injection of anti-NKG2I mAb before the allogeneic bone marrow transfer in vivo impinged on the function of NKG2I, resulting in the enhanced colony formation in the spleen. NKG2I is a novel activating receptor mediating recognition and rejection of allogeneic target cells.

Reprogramming of MAIT Cells to Pluripotency and Redifferentiation.

Reprogramming differentiated cells into induced pluripotent stem cells (iPSCs) consists in dedifferentiation of the cells into the pluripotent state, i.e., stem cells. Since T cells play a pivotal role in our immune system, T cell reprogramming into iPSCs and subsequent redifferentiation of iPSCs toward the original cells hold a great promise for future cell therapy and for further exploring the biology of such T cells. Mucosal-associated invariant T (MAIT) cells are an innate-like T cells linking innate immunity to adaptive immunity, and believed to be implicated in host protection to infection, in inflammation, and in immune homeostasis, which makes them an attractive target for the clinical intervention. In this chapter, we will outline the protocol for reprogramming MAIT cells to pluripotency with Sendai virus vector and redifferentiation. This technique will allow expansion of MAIT cells for cell therapy against the intractable infectious diseases such as HIV/Tuberculosis or cancer.

In vitro induction of natural killer T cells from embryonic stem cells prepared using somatic cell nuclear transfer.

The ectopic expression of the Notch receptor ligand delta-like 1 on stromal cells allows the induction of T cells from embryonic stem cells (ESCs). However, these in vitro-generated T cells are not transplantable because they are too immature to mount an immune response in an immunocompromised animal. We efficiently generated a subset of T cells called invariant natural killer T (iNKT) cells from ESCs derived from peripheral iNKT cells using somatic cell nuclear transfer (ntESCs). These iNKT cells matured autonomously in vivo and exhibited an adjuvant effect accompanying the production of interferon-gamma in an antigen-specific manner. This adjuvant effect culminated in the inhibition of inoculated tumor cell growth. Our results indicate that ntESC-derived iNKT cells are transplantable lymphocytes that will be beneficial for the induction of immune tolerance and the treatment of autoimmune diseases, tumors, and infections.

Generation of cloned mice by direct nuclear transfer from natural killer T cells.

Cloning mammals by nuclear transfer (NT) remains inefficient. One fundamental question is whether clones have really been derived from differentiated cells rather than from rare stem cells present in donor-cell samples. To date, cells, such as mature lymphocytes, with genetic differentiation markers have been cloned to generate mice only via a two-step NT involving embryonic stem (ES) cell generation and tetraploid complementation [1, 2 and 3]. Here, we show that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT. The pups and their placentas possessed the rearranged TCR loci specific for NKT cells. The NKT-cell-cloned embryos had a high developmental potential in vitro: Most (71%) developed to the morula/blastocyst stage, in marked contrast to embryos from peripheral blood T cells (12%; p < 1 x 10(-25)). Furthermore, ES cell lines were efficiently established from these NKT-cell blastocysts. These findings clearly indicate a high level of plasticity in the NKT-cell genome. Thus, differentiation of the genome is not always a barrier to NT cloning for either reproductive or therapeutic purposes, so we can now postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.

Mucosal-Associated Invariant T Cells in Regenerative Medicine.

Although antibiotics to inhibit bacterial growth and small compounds to interfere with the productive life cycle of human immunodeficiency virus (HIV) have successfully been used to control HIV infection, the recent emergence of the drug-resistant bacteria and viruses poses a serious concern for worldwide public health. Despite intensive scrutiny in developing novel antibiotics and drugs to overcome these problems, there is a dilemma such that once novel antibiotics are launched in markets, sooner or later antibiotic-resistant strains emerge. Thus, it is imperative to develop novel methods to avoid this vicious circle. Here, we discuss the possibility of using induced pluripotent stem cell (iPSC)-derived, innate-like T cells to control infection and potential application of these cells for cancer treatment. Mucosal-associated invariant T (MAIT) cells belong to an emerging family of innate-like T cells that link innate immunity to adaptive immunity. MAIT cells exert effector functions without priming and clonal expansion like innate immune cells and relay the immune response to adaptive immune cells through production of relevant cytokines. With these characteristics, MAIT cells are implicated in a wide range of human diseases such as autoimmune, infectious, and metabolic diseases, and cancer. Circulating MAIT cells are often depleted by these diseases and often remain depleted even after appropriate remedy because MAIT cells are susceptible to activation-induced cell death and poor at proliferation in vivo, which threatens the integrity of the immune system. Because MAIT cells have a pivotal role in human immunity, supplementation of MAIT cells into immunocompromised patients suffering from severe depletion of these cells may help recapitulate or recover immunocompetence. The generation of MAIT cells from human iPSCs has made it possible to procure MAIT cells lost from disease. Such technology creates new avenues for cell therapy and regenerative medicine for difficult-to-cure infectious diseases and cancer and contributes to improvement of our welfare.

Epigenetic silencing of V(D)J recombination is a major determinant for selective differentiation of mucosal-associated invariant t cells from induced pluripotent stem cells.

Mucosal-associated invariant T cells (MAITs) are innate-like T cells that play a pivotal role in the host defense against infectious diseases, and are also implicated in autoimmune diseases, metabolic diseases, and cancer. Recent studies have shown that induced pluripotent stem cells (iPSCs) derived from MAITs selectively redifferentiate into MAITs without altering their antigen specificity. Such a selective differentiation is a prerequisite for the use of MAITs in cell therapy and/or regenerative medicine. However, the molecular mechanisms underlying this phenomenon remain unclear. Here, we performed methylome and transcriptome analyses of MAITs during the course of differentiation from iPSCs. Our multi-omics analyses revealed that recombination-activating genes (RAG1 and RAG2) and DNA nucleotidylexotransferase (DNTT) were highly methylated with their expression being repressed throughout differentiation. Since these genes are essential for V(D)J recombination of the T cell receptor (TCR) locus, this indicates that nascent MAITs are kept from further rearrangement that may alter their antigen specificity. Importantly, we found that the repression of RAGs was assured in two layers: one by the modulation of transcription factors for RAGs, and the other by DNA methylation at the RAG loci. Together, our study provides a possible explanation for the unaltered antigen specificity in the selective differentiation of MAITs from iPSCs.

Mucosal-associated invariant T cells from induced pluripotent stem cells: A novel approach for modeling human diseases.

Mice have frequently been used to model human diseases involving immune dysregulation such as autoimmune and inflammatory diseases. These models help elucidate the mechanisms underlying the disease and in the development of novel therapies. However, if mice are deficient in certain cells and/or effectors associated with human diseases, how can their functions be investigated in this species? Mucosal-associated invariant T (MAIT) cells, a novel innate-like T cell family member, are a good example. MAIT cells are abundant in humans but scarce in laboratory mice. MAIT cells harbor an invariant T cell receptor and recognize nonpeptidic antigens vitamin B2 metabolites from bacteria and yeasts. Recent studies have shown that MAIT cells play a pivotal role in human diseases such as bacterial infections and autoimmune and inflammatory diseases. MAIT cells possess granulysin, a human-specific effector molecule, but granulysin and its homologue are absent in mice. Furthermore, MAIT cells show poor proliferation in vitro. To overcome these problems and further our knowledge of MAIT cells, we have established a method to expand MAIT cells via induced pluripotent stem cells (iPSCs). In this review, we describe recent advances in the field of MAIT cell research and our approach for human disease modeling with iPSC-derived MAIT cells.

The dynamics of mucosal-associated invariant T cells in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination, gliosis and axonal loss in the Central Nervous System. Although the etiology of the disease has remained enigmatic, recent studies have suggested a role of the innate-like T cells, called Mucosal Associated Invariant T cells (MAITs) in the pathophysiology. In the present study, we have analyzed the relative frequency of MAITs and the expression of the cell surface antigens in MAITs to seek a possible link to the disease. RESULTS: There was little difference in the frequency of total MAITs between healthy donors (HDs) and untreated MS patients, whereas the latter harbored more CD8(lo/neg) (DN) MAITs concomitant with a decrease in CD8(high) MAITs and in CD4 MAITs compared with those in HDs. While the expression of CCR5, CCR6, CD95, CD127, and CD150 has increased in untreated subjects compared with that in HDs, CD45RO has declined in untreated subjects in both DN MAITs and CD8(hi) MAITs. FTY720 therapy has increased the relative frequency of total MAITs in a time-dependent fashion up to 2 years. Intriguingly, FTY720 therapy for 3 years reversed the above phenotype, engendering more CD8(high) MAITs accompanied with decreased DN MAITs. FTY720 therapy affected the cytokine production from CD4 T cells and also enhanced the relative frequency of cells producing both TNF-α and IFN-γ from MAITs, CD8 T cells, and CD4 T cells compared with that in untreated subjects. CONCLUSIONS: FTY 720 therapy enhanced the relative frequency of MAITs in MS patients in a time-dependent manner. Although the expression of CD8 in MAITs has been affected early by FTY720, longer treatment has reversed the phenotypic change. These data demonstrated that FTY720 induced dynamic change in the relative frequency and in the phenotype of MAITs in MS.