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.     

Chloroquine‐treated dendritic cells require STAT1 signaling for their tolerogenic activity

MS and EAE are T cell‐driven autoimmune diseases of the CNS where IL‐17‐producing Th17 cells promote damage and are pathogenic. Conversely, tolerogenic DCs induce Treg cells and suppress Th17 cells. Chloroquine (CQ) suppresses EAE through the modulation of DCs by unknown mechanisms. Here, we show that STAT 1 is necessary for CQ‐induced tolerogenic DCs (tolDCs) to efficiently suppress EAE. We observed that CQ induces phosphorylation of STAT1 in DCs in vivo and in vitro. Genetic blockage of STAT1 abrogated the suppressive activity of CQ‐treated DCs. Opposed to its WT counterparts, CQ‐treated STAT1^?/? BMDCs were unable to suppress Th17 cells and increased EAE severity. Our findings show that STAT1 is a major signaling pathway in CQ‐induced tolDCs and may shed light on new therapeutic avenues for the induction of tolDCs in autoimmune diseases such as MS.     

Structure-guided design of an invariant natural killer T cell agonist for optimum protection from type 1 diabetes in non-obese diabetic mice

Because invariant natural killer T (iNK T) cells link innate and adaptive immunity, the structure‐dependent design of iNK T cell agonists may have therapeutic value as vaccines for many indications, including autoimmune disease. Previously, we showed that treatment of non‐obese diabetic (NOD) mice with the iNK T cell activating prototypic glycolipid α‐galactosylceramide (α‐GalCer) protects them from type 1 diabetes (T1D). However, α‐GalCer is a strong agonist that can hyperactivate iNK T cells, elicit several side effects and has shown only limited success in clinical trials. Here, we used a structure‐guided design approach to identify an iNK T cell agonist that optimally protects from T1D with minimal side effects. Analyses of the kinetics and function of a panel of synthetic α‐GalCer fatty acyl chain derivatives (C8:0‐C16:0) were performed in NOD mice. C16:0 elicited the highest protection from insulitis and T1D, which was associated with a higher frequency and survival of iNK T cells and enhanced activity of tolerogenic dendritic cells (DCs) in draining pancreatic lymph nodes (PLN), inability to transactivate NK cells and a more rapid kinetics of induction and recovery of iNK T cells from anergy. We conclude that the length and structure of the acyl chain of α‐GalCer regulates the level of protection against T1D in mice, and propose that the extent of this protection depends on the relative capacity of the acyl chain to accommodate an endogenous spacer lipid of appropriate length and structure. Thus, our findings with the α‐GalCer C16:0 derivative suggest strongly that it be considered as a lead glycolipid candidate in clinical trials of T1D.     

Activation of murine invariant NKT cells promotes susceptibility to candidiasis by IL‐10 induced modulation of phagocyte antifungal activity

Invariant NKT (iNKT) cells play an important role in a variety of antimicrobial immune responses due to their ability to produce high levels of immune‐modulating cytokines. Here, we investigated the role of iNKT cells in host defense against candidiasis using Jα18‐deficient mice (Jα18^?/?), which lack iNKT cells. Jα18^?/? mice were more resistant to the development of lethal candidiasis than wild‐type (WT) mice. In contrast, treatment of WT mice with the iNKT cell activating ligand α‐galactosylceramide markedly enhanced their mortality after infection with Candida albicans. Serum IL‐10 levels were significantly elevated in WT mice in response to infection with C. albicans. Futhermore, IL‐10 production increased after in vitro coculture of peritoneal macrophages with iNKT cells and C. albicans. The numbers of peritoneal macrophages, the production of IL‐1β and IL‐18, and caspase‐1 activity were also significantly elevated in Jα18^?/? mice after infection with C. albicans. The adoptive transfer of iNKT cells or exogenous administration of IL‐10 into Jα18^?/? reversed susceptibility to candidiasis to the level of WT mice. These results suggest that activation of iNKT cells increases the initial severity of C. albicans infection, most likely mediated by IL‐10 induced modulation of macrophage antifungal activity.     

Gal‐3 regulates the capacity of dendritic cells to promote NKT‐cell‐induced liver injury

Galectin‐3 (Gal‐3), an endogenous lectin, exhibits pro‐ and anti‐inflammatory effects in various disease conditions. In order to explore the role of Gal‐3 in NKT‐cell‐dependent pathology, we induced hepatitis in C57BL/6 WT and Gal‐3‐deficient mice by using specific ligand for NKT cells: α‐galactosylceramide, glycolipid Ag presented by CD1d. The injection of α‐galactosylceramide significantly enhanced expression of Gal‐3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal‐3 (induced by Gal‐3‐inhibitor TD139) abrogated the susceptibility to NKT‐cell‐dependent hepatitis. Blood levels of pro‐inflammatory cytokines (TNF‐α, IFN‐γ, IL‐12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal‐3 alleviated influx of inflammatory CD11c⁺CD11b⁺ DCs in the liver and favored tolerogenic phenotype and IL‐10 production of liver NKT and DCs. Deletion of Gal‐3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro‐inflammatory cytokines in vitro. Gal‐3‐deficient DCs failed to optimally stimulate production of pro‐inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal‐3 regulates the capacity of DCs to support NKT‐cell‐mediated liver injury, playing an important pro‐inflammatory role in acute liver injury.     

Mast cells control insulitis and increase Treg cells to confer protection against STZ‐induced type 1 diabetes in mice

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low‐dose streptozotocin (MLD‐STZ) treatments, using two strains of mast cell‐deficient mice (W/W^v or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL‐10, TGF‐β, and IL‐6 expression in the pancreatic tissue. Interestingly, IL‐6‐deficient mice are more susceptible to T1D associated with reduced Treg‐cell numbers in the PLNs, but mast cell transfer from wild‐type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD‐STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL‐17‐producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ‐induced T1D via an immunological tolerance mechanism mediated by Treg cells.     

Identification of C‐glycoside analogues that display a potent biological activity against murine and human invariant natural killer T cells

We have recently shown that α‐C‐galactosylceramide (α‐C‐GalCer) stimulates invariant natural killer T (iNKT) cells and preferentially induces a T helper 1 (Th1)‐type response in mice. However, α‐C‐GalCer was found to be a rather weak ligand against human iNKT cells in vitro. Therefore, in this study, we sought to identify a compound that displays a strong stimulatory activity against human iNKT cells, by determining the biological activities of several C‐glycoside analogues. From the in vitro screening assays, we found that almost all C‐glycoside analogues, which have an E‐alkene linker between sugar and lipid moieties, are able to activate human iNKT cells and to induce the maturation and activation of human dendritic cells through iNKT‐cell activation. In summary, although α‐galactosylceramide (α‐GalCer) remains the strongest iNKT‐cell ligand, our study identified E‐alkene‐linked C‐glycoside analogues as potent human iNKT‐cell stimulants, and indicated that these analogues could be used as a therapeutic agent in the future for diseases resolved by Th1‐type responses.     

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.     

In vivo activation of invariant natural killer T cells induces systemic and local alterations in T‐cell subsets prior to preterm birth

Preterm birth, the leading cause of neonatal morbidity and mortality worldwide, is frequently preceded by spontaneous preterm labour, a syndrome of multiple aetiologies. Pathological inflammation is causally linked to spontaneous preterm labour. Indeed, direct activation of invariant natural killer T (iNKT) cells via α‐galactosylceramide induces preterm labour/birth largely by initiating systemic and local (i.e. decidua and myometrium) innate immune responses. Herein, we investigated whether iNKT‐cell activation altered local and systemic T‐cell subsets. Administration of α‐galactosylceramide induced an expansion of activated CD1d‐restricted iNKT cells in the decidua and a reduction in the number of: (1) total T cells (conventional CD4⁺ and CD8⁺ T cells) through the down‐regulation of the CD3? molecule in the peripheral circulation, spleen, uterine‐draining lymph nodes (ULNs), decidua and/or myometrium; (2) CD4⁺ regulatory T cells in the spleen, ULNs and decidua; (3) T helper type 17 (Th17) cells in the ULNs but an increase in the number of decidual Th17 cells; (4) CD8⁺ regulatory T cells in the spleen and ULNs; and (5) CD4⁺ and CD8⁺ forkhead box protein 3 negative (Foxp3^–) responder T cells in the spleen and ULNs. As treatment with rosiglitazone prevents iNKT‐cell activation‐induced preterm labour/birth, we also explored whether the administration of this peroxisome proliferator‐activated receptor gamma (PPARγ) agonist would restore the number of T cells. Treating α‐galactosylceramide‐injected mice with rosiglitazone partially restored the number of T cells in the spleen but not in the decidua. In summary, iNKT‐cell activation altered the systemic and local T‐cell subsets prior to preterm labour/birth; however, treatment with rosiglitazone partially reversed such effects.     

Pathogenic T helper type 17 cells contribute to type 1 diabetes independently of interleukin‐22

We have shown that pathogenic T helper type 17 (Th17) cells differentiated from naive CD4⁺ T cells of BDC2·5 T cell receptor transgenic non‐obese diabetic (NOD) mice by interleukin (IL)‐23 plus IL‐6 produce IL‐17, IL‐22 and induce type 1 diabetes (T1D). Neutralizing interferon (IFN)‐γ during the polarization process leads to a significant increase in IL‐22 production by these Th17 cells. We also isolated IL‐22‐producing Th17 cells from the pancreas of wild‐type diabetic NOD mice. IL‐27 also blocked IL‐22 production from diabetogenic Th17 cells. To determine the functional role of IL‐22 produced by pathogenic Th17 cells in T1D we neutralized IL‐22 in vivo by using anti‐IL‐22 monoclonal antibody. We found that blocking IL‐22 did not alter significantly adoptive transfer of disease by pathogenic Th17 cells. Therefore, IL‐22 is not required for T1D pathogenesis. The IL‐22Rα receptor for IL‐22 however, increased in the pancreas of NOD mice during disease progression and based upon our and other studies we suggest that IL‐22 may have a regenerative and protective role in the pancreatic islets.     

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.     

RELT negatively regulates the early phase of the T‐cell response in mice

RELT (tumor necrosis factor receptor superfamily member 19‐like, TNFRSF19L) is a TNFR superfamily member that is primarily expressed in immune cells and lymphoid tissues, but whose immunological function is not well‐defined. Here, we show that RELT is expressed by naive T cells and DCs, and their activation or maturation decreases RELT expression. Using RELT knockout (RELT^?/?) mice, we demonstrate that RELT deficiency selectively promotes the homeostatic proliferation of CD4⁺ T cells but not CD8⁺ T cells, and enhances anti‐tumor CD8⁺ T‐cell responses. We also demonstrate, using an adoptive transfer model in which RELT is knocked‐out in either the transferred transgenic CD8⁺ T cells or the recipient melanoma‐bearing mice, that RELT on multiple immune cells limits the hyper‐response of tumor‐specific CD8⁺ T cells. Hyper‐responsiveness of RELT‐deficient T cells was induced by promoting their proliferation. Taken together, our findings suggest that RELT acts as a negative regulator that controls the early phase of T‐cell activation probably by promoting T‐cell apoptosis.     

Abrogation of ICOS/ICOS ligand costimulation in NOD mice results in autoimmune deviation toward the neuromuscular system

NOD mice spontaneously develop insulin‐dependent diabetes around 10–40 wk of age. Numerous immune gene variants contribute to the autoimmune process. However, genes that direct the autoimmune response toward β cells remain ill defined. In this study, we provide evidence that the Icos and Icosl genes contribute to the diabetes process. Protection from diabetes in ICOS^?/? and ICOSL^?/? NOD mice was unexpectedly associated with the development of an autoimmune disorder of the neuro‐muscular system, characterized by myositis, sensory ganglionitis and, to a reduced extent, inflammatory infiltrates in the CNS. This syndrome was reproduced upon adoptive transfer of CD4⁺ and CD8⁺ T cells from diseased donors to naïve NOD.scid recipients. Our data further show that protection from diabetes results from defective activation of autoimmune diabetogenic effector T cells in ICOS^?/? NOD mice, whereas acceleration of diabetes in BDC2.5 ICOS^?/? NOD mice is induced by a dominant defect in Treg. Taken together, our findings indicate that costimulation signals play a key role in regulating immune tolerance in peripheral tissues and that the ICOS/ICOSL costimulatory pathway influences the balance between Treg and diabetogenic effector T cells.     

T cells generated in the absence of a thoracic thymus fail to establish homeostasis

Cervical thymus mimics the thoracic thymus in supporting T‐cell development and exists in a subset of mice and humans. Importantly, it remains unknown whether the cervical thymus can generate T cells that are self‐tolerant in the complete absence of signals from the thoracic thymus. Using a fetal liver reconstitution model in thoracic thymectomized RAG^?/? mice, we found that T cells could be generated without contribution from the thoracic thymus. However, these mice had decreased T cells, increased proportions of effector memory T cells and Treg phenotype cells, increased serum IgG1/2b, and increased frequency of T cells expressing IFN‐γ, IL‐17 or IL‐10. Half of the mice that received a thoracic thymectomy and fetal liver cells, unlike sham surgery controls, developed substantial morbidity with age. Disease was associated with lymphopenia‐driven activation rather than inherent defects in the cervical thymus, as both thoracic and cervical thymocytes could generate disease in lymphopenic recipients. Administration of the homeostatic cytokine IL‐7 caused a rapid, transient increase in T‐cell numbers and reduced the time to disease onset. Together the data suggests that the cervical thymus can function in the complete absence of the thoracic thymus; however, the T cells generated do not establish homeostasis.     

Modification of anti-tumor immunity by tolerogenic dendritic cells

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag^?/? mice, expressing a transgenic TCR in CD8⁺ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8⁺OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1⁺CD27^? NK cells as well as Foxp3⁺ and IL-10 secreting CD8⁺OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8⁺ Tregs.     

B6.g7 mice reconstituted with BDC2·5 non‐obese diabetic (BDC2·5NOD) stem cells do not develop autoimmune diabetes

In BDC2·5 non‐obese diabetic (BDC2·5NOD) mice, a spontaneous model of type 1 diabetes, CD4⁺ T cells express a transgene‐encoded T cell receptor (TCR) with reactivity against a pancreatic antigen, chromogranin. This leads to massive infiltration and destruction of the pancreatic islets and subsequent diabetes. When we reconstituted lethally irradiated, lymphocyte‐deficient B6.g7 (I‐A^g7+) Rag^–/– mice with BDC2·5NOD haematopoietic stem and progenitor cells (HSPC; ckit⁺Lin^–Sca‐1^hi), the recipients exhibited hyperglycaemia and succumbed to diabetes. Surprisingly, lymphocyte‐sufficient B6.g7 mice reconstituted with BDC2·5NOD HSPCs were protected from diabetes. In this study, we investigated the factors responsible for attenuation of diabetes in the B6.g7 recipients. Analysis of chimerism in the B6.g7 recipients showed that, although B cells and myeloid cells were 98% donor‐derived, the CD4⁺ T cell compartment contained ～50% host‐derived cells. These host‐derived CD4⁺ T cells were enriched for conventional regulatory T cells (T_regs) (CD25⁺forkhead box protein 3 (FoxP3)⁺] and also for host‐ derived CD4⁺CD25^–FoxP3^– T cells that express markers of suppressive function, CD73, FR4 and CD39. Although negative selection did not eliminate donor‐derived CD4⁺ T cells in the B6.g7 recipients, these cells were functionally suppressed. Thus, host‐derived CD4⁺ T cells that emerge in mice following myeloablation exhibit a regulatory phenoytpe and probably attenuate autoimmune diabetes. These cells may provide new therapeutic strategies to suppress autoimmunity.     

Antigen presenting cell‐targeted proinsulin expression converts insulin‐specific CD8+ T‐cell priming to tolerance in autoimmune‐prone NOD mice

Type 1 diabetes (T1D) results from autoimmune destruction of insulin‐producing pancreatic β cells. Therapies need to incorporate strategies to overcome the genetic defects that impair induction or maintenance of peripheral T‐cell tolerance and contribute to disease development. We tested whether the enforced expression of an islet autoantigen in antigen‐presenting cells (APC) counteracted peripheral T‐cell tolerance defects in autoimmune‐prone NOD mice. We observed that insulin‐specific CD8⁺ T cells transferred to mice in which proinsulin was transgenically expressed in APCs underwent several rounds of division and the majority were deleted. Residual insulin‐specific CD8⁺ T cells were rendered unresponsive and this was associated with TCR downregulation, loss of tetramer binding and expression of a range of co‐inhibitory molecules. Notably, accumulation and effector differentiation of insulin‐specific CD8⁺ T cells in pancreatic lymph nodes was prominent in non‐transgenic recipients but blocked by transgenic proinsulin expression. This shift from T‐cell priming to T‐cell tolerance exemplifies the tolerogenic capacity of autoantigen expression by APC and the capacity to overcome genetic tolerance defects.