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Invariant natural killer T (iNKT) cells mediate rapid immune responses which bridge the gap between innate and adaptive responses to pathogens while also providing key regulation to maintain immune homeostasis. Both types of important iNKT immune responses are mediated through interactions with innate and adaptive B cells. As such, iNKT cells sit at the decision-making fulcrum between regulating inflammatory or autoreactive B cells and supporting protective or regulatory B cell populations. iNKT cells interpret the signals in their environment to set the tone for subsequent adaptive responses, with outcomes ranging from getting licensed to maintain homeostasis as an iNKT regulatory cell (iNKT(reg)) or being activated to become an iNKT follicular helper (iNKT(FH)) cell supporting pathogen-specific effector B cells. Here we review iNKT and B cell cooperation across the spectrum of immune outcomes, including during allergy and autoimmune disease, tumor surveillance and immunotherapy, or pathogen defense and vaccine responses. Because of their key role as influencers, iNKT cells provide a valuable target for therapeutic interventions. Understanding the nature of the interactions between iNKT and B cells will enable the development of clinical interventions to strategically target regulatory iNKT and B cell populations or inflammatory ones, depending on the circumstance.

CD4+ T cells enable the critical B cell humoral immune protection afforded by most effective vaccines. We and others have recently identified an alternative source of help for B cells in mice, invariant NK T (iNKT) cells. iNKT cells are innate glycolipid-specific T cells restricted to the nonpolymorphic Ag-presenting molecule CD1d. As such, iNKT cells respond to glycolipids equally well in all people, making them an appealing adjuvant for universal vaccines. We tested the potential for the iNKT glycolipid agonist, α-galactosylceramide (αGC), to serve as an adjuvant for a known human protective epitope by creating a nanoparticle that delivers αGC plus antigenic polysaccharides from Streptococcus pneumoniae. αGC-embedded nanoparticles activate murine iNKT cells and B cells in vitro and in vivo, facilitate significant dose sparing, and avoid iNKT anergy. Nanoparticles containing αGC plus S. pneumoniae polysaccharides elicits robust IgM and IgG in vivo and protect mice against lethal systemic S. pneumoniae. However, codelivery of αGC via nanoparticles actually eliminated Ab protection elicited by a T-independent S. pneumoniae vaccine. This is consistent with previous studies demonstrating iNKT cell help for B cells following acute activation, but negative regulation of B cells during chronic inflammation. αGC-containing nanoparticles represent a viable platform for broadly efficacious vaccines against deadly human pathogens, but their potential for eliminating B cells under certain conditions suggests further clarity on iNKT cell interactions with B cells is warranted.

Rapid immune responses regulated by invariant Natural Killer T (iNKT) cells bridge the gap between innate and adaptive responses to pathogens, while also providing key regulation to maintain immune homeostasis. iNKT immune protection and immune regulation are both mediated through interactions with innate and adaptive B cell populations that express CD1d. Recent studies have expanded our understanding of the position of iNKT cells at the fulcrum between regulating inflammatory and autoreactive B cells. Environmental signals influence iNKT cells to set the tone for subsequent adaptive responses, ranging from maintaining homeostasis as an iNKT regulatory cell (iNKT(reg)) or supporting pathogen-specific effector B cells as an iNKT follicular helper (iNKT(FH)). Here we review recent advances in iNKT and B cell cooperation during autoimmunity and sterile inflammation. Understanding the nature of the interactions between iNKT and B cells will enable the development of clinical interventions to strategically target regulatory iNKT and B cell populations or inflammatory ones, across a range of indications.

Epidemiological data and animal studies suggest that helminth infection exerts potent immunomodulatory effects that dampen host immunity against unrelated pathogens. Despite this notion, we unexpectedly discovered that prior helminth infection resulted in enhanced protection against subsequent systemic and enteric bacterial infection. A population of virtual memory CD8 T (CD8 TVM) cells underwent marked expansion upon infection with the helminth Heligmosomoides polygurus by an IL-4-regulated, antigen-independent mechanism. CD8 TVM cells disseminated to secondary lymphoid organs and established a major population of the systemic CD8 T cell pool. IL-4 production elicited by protein immunization or selective activation of natural killer T cells also results in the expansion of CD8 TVM cells. Notably, CD8 TVM cells expanded by helminth infection are sufficient to transfer innate non-cognate protection against bacteria to naive animals. This innate non-cognate "collateral protection" mediated by CD8 TVM might provide parasitized animals an advantage against subsequent unrelated infections, and represents a potential novel strategy for vaccination.

Significance iNKT cells can both provide help and inhibit B cell responses. Our data show that when iNKT cells are activated with the glycolipid agonist αGalCer together with the inflammatory cytokine IL-18, they switch from regulating autoreactive B cells to promoting their expansion. As a consequence, autoreactive B cell responses remain unchecked by iNKT cells. The glycolipid αGalCer has been shown to have promising effects when administered as an adjuvant to achieve a better response to vaccines, as an antitumor agent, as well as in the regulation of autoimmunity. Our results highlight a facet of αGalCer-mediated iNKT cell activation in the context of inflammation and have broad implications for understanding the regulation of autoimmunity and use of αGalCer in therapy., Invariant natural killer T (iNKT) cells serve as early rapid responders in the innate immune response to self-derived autoantigens and pathogen-derived danger signals and antigens. iNKT cells can serve both as helpers for effector B cells and negatively regulate autoreactive B cells. Specifically, iNKT cells drive B cell proliferation, class switch, and antibody production to induce primary antigen-specific immune responses. On the other hand, inflammasome-mediated activation drives accumulation of neutrophils, which license iNKT cells to negatively regulate autoreactive B cells via Fas ligand (FasL). This positions iNKT cells at an apex to support or inhibit B cell responses in inflammation. However, it is unknown which effector mechanism dominates in the face of cognate glycolipid activation during chronic inflammation, as might result from glycolipid vaccination or infection during chronic autoimmune disease. We stimulated iNKT cells by cognate glycolipid antigen α-galactosylceramide (αGalCer) and measured B cell activation during interleukin 18 (IL-18)-induced chronic inflammation. Moreover, glycolipid-activated iNKT cells increased the serum concentration of autoantibodies, frequency of germinal center (GC) B cells, and antigen-specific plasma cells induced during chronic IL-18–mediated inflammation, as compared with IL-18 alone. Further, activation of iNKT cells via cognate glycolipid during IL-18–mediated inflammation overrides the licensing function of neutrophils, instead inducing iNKT follicular helper (iNKTfh) cells that in turn promote autoimmunity. Thus, our data demonstrate that glycolipids which engage iNKT cells support antigen-specific B cell help during inflammasome-mediated inflammation.

Immune cells are increasingly appreciated to play a role in inflammation in adipose and hepatic tissue during obesity and may contribute to the development of non-alcoholic fatty liver disease (NAFLD). Both invariant Natural Killer T (iNKT) cells and B cells are enriched in the liver and adipose tissues of humans and mice, but the degree and nature of their influence in the development of NAFLD remains unclear. iNKT cells interact with B cells in many contexts, including during the chronic inflammation associated with autoimmune disease and infection, so it is likely they interact during obesity. Emerging evidence implicates intrahepatic B cells in the progression of human NAFLD, and proinflammatory B cells increase in the steatotic livers of HFD-fed mice. Our preliminary studies demonstrate an iNKT cell-dependent expansion of inflammatory CD11c+ T-bet+ B cells in the subcutaneous adipose tissue of obese humans and perigonadal adipose tissue of obese mice. We now extend those findings to other iNKT cell-rich fatty depots. Flow cytometry identified increased frequencies of T-bet+ B cells in the livers, but not the omenta or mesenteric adipose tissue, of obese mice. In parallel to the in vivo studies, in vitro co-cultures revealed that iNKT cells are sufficient to mediate expansion of T-bet+ B cells. Ongoing experiments will examine the potential role and mechanism of iNKT cells in mediating hepatic Tbet+ B cell expansion. Characterization of the specific contribution of immune inflammation in the progression NAFLD to NASH will allow for the design of successful therapeutic interventions for this increasingly frequent public health problem. Funding support from NIH K12GM111726 (BTE), NIH TL1 TR002647 (TS), Swedish Research Council (TH), and NIH R01 AI32798-01A1 (EAL).

Invariant natural killer T cells (iNKT) have a well-documented role in direct and indirect killing of tumor cells through their release of proinflammatory cytokines and cytotoxic compounds. To improve outcomes in poorly immunogenic cancers while avoiding NKT cell anergy, we induced anti-tumor responses by activating NKT cells with polymeric nanoparticles containing a low dose of a glycolipid adjuvant, α-Galactosyl-Ceramide (aGalCer), and a model tumor neoantigen, ovalbumin (OVA). Prophylactic vaccination of mice with aGalCer-OVA nanoP before inoculation with B16F10-OVA melanoma cells reduced tumor growth rate and increased overall survival compared to mice immunized with control nanoparticles. In mice with an established B16 tumor, therapeutic administration of aGalCer-OVA nanoP provided significantly greater anti-tumor effects, reduced tumor growth rate and increased survival, compared to control nanoparticles. Therapeutic treatment with aGalCer-OVA nanoP, but not control nanoPs, also induced significant infiltration of iNKT, NK, and antigen-specific CD8 T cells in the tumor, and enhanced production of granzyme B and perforin by CD8 T cells. Furthermore, immune checkpoint blockade treatment (anti-CTLA-4 + anti-PD-1) enhanced the effectiveness of aGalCer-OVA nanoP, leading to clearance of established tumors nearly 50% of the time. These results provide strong evidence that activation of iNKT cells via nanoparticles containing a glycolipid adjuvant and tumor antigen is a viable anti-cancer strategy that partners well with other clinically used immunotherapies. Supported by grants from NIH (T32 AI138944) and the CTSA (TL1 TR002467)

Alpha-galactosylceramide (αGalCer) is a glycolipid derived from a marine sponge that is a potent activator of both mouse and human invariant natural killer T (iNKT) cells. For that reason, αGalCer is a promising vaccine adjuvant that has been shown to improve both humoral and cellular immunity when co-administered with various vaccines, including candidate vaccines for biodefense. In the current study, we tested the effectiveness of αGalCer as an adjuvant for the clinically-relevant ricin toxin subunit vaccine, RiVax. αGalCer had a potent adjuvant effect, as shown by a rapid onset of anti-ricin IgG titers, accelerated development of serum toxin-neutralizing activity, and enhanced protection from lethal ricin challenge in a mouse model. These results underscore the potential of αGalCer to augment the protective immune response to a vaccine designed to counteract ricin toxin, a fast-acting biothreat agent.

Homeostasis is a fundamental principle of biological systems. A paradigm of immune homeostasis is the remarkably constant number of naive T and B lymphocytes in the body that continuously circulate through the secondary lymphoid organs to maximize immune surveillance. Whether the dynamics and distribution of the systemic naive lymphocyte pool is affected following organ-specific infection is not known. Here we show that, following infection of mice with an enteric helminth, naive T and B lymphocytes accumulate in the T helper type 2-reactive mesenteric lymph node while they are concurrently depleted from non-draining peripheral lymph nodes. This systemic redistribution of naive lymphocytes is sustained into the chronic phase of the infection, requires lymphotoxin beta receptor-dependent signals and is associated with a reduced ability of parasitized animals to mount antigen-specific cellular and humoral immune responses to heterologous immunization or infection at peripheral sites. Our data suggest that the function of the homeostatic naive lymphocyte pool can be modulated by its systemic distribution following infection and may provide a novel concept underlying compromised immune responsiveness at peripheral sites in helminth-infected individuals.

Obesity is a burgeoning public health crisis, responsible for increasing chronic systemic inflammation which leads to metabolic disease in humans and mice. Understanding the contribution of immune inflammation to metabolic disease is critical for designing effective therapeutic interventions. Invariant Natural Killer T (iNKT) cells are enriched in the adipose tissue, liver, and omenta of both humans and mice, but their frequency is reduced in adipose during obesity. iNKT cells negatively regulate or positively activate other immune cells, including B cells, depending on the immunologic context. Adipose B regulatory cells protect against inflammation, but they are not the only subset of B cells in adipose tissue. Our preliminary studies find increased frequencies of inflammatory Tbet+ B cells in adipose tissue of obese humans and mice which correlates with weight gain in mice and increasing BMI in humans. We now extend those findings to consider iNKT cell interaction with B cells during obesity in the adipose, liver, and omental tissues. Flow cytometry confirmed iNKT cell-dependent expansion of Tbet+ CD11c+ B cells in adipose tissue of obese mice. Interestingly, similar expansion was not observed in liver or omental tissue from the same obese mice. Instead, the livers and omenta of obese mice demonstrated significant increases in iNKT cell frequency compared with lean mice. In contrast to the adipose tissue, the increase in liver and omental iNKT cells during obesity was missing in mice genetically deficient in Tbet+ B cells. Thus, we find that iNKT cell-dependent expansion of Tbet+ CD11c+ B cells within adipose tissue of obese mice is counter-balanced by a reciprocal Tbet+ B cell-dependent expansion of iNKT cells in liver and omentum.

Mutations in RIP kinases underlie the recently described human autoimmune syndrome, CRIA, characterized by lymphadenopathy, splenomegaly, and autoantibody production. While disease mechanisms for CRIA remain undescribed, RIP kinases work together with Caspase-8 to regulate cell death, which is critical for normal differentiation of many cell types. Here, we describe a key role for RIPK1 in facilitating innate B cell differentiation and subsequent activation. By comparing RIPK1, RIPK3, and Caspase8 triple deficient and RIPK3, Caspase 8 double deficient mice, we identified selective contributions of RIPK1 to an accumulation of murine splenic Marginal Zone (MZ) B cells and B1-b cells. We used mixed bone-marrow chimeras to determine that innate B cell commitment required B cell-intrinsic RIPK1, RIPK3, and Casp8 sufficiency. RIPK1 regulated MZ B cell differentiation rather than development and RIPK1 mediates its innate immune effects independent of the RIPK1 kinase domain. NP-KLH/alum and NP-Ficoll vaccination of RIPK3, Casp8, and RIPK1 triple deficient mice revealed uniquely delayed T-dependent and T-independent IgG responses, abnormal splenic germinal center architecture, and reduced extrafollicular plasmablast formation compared to double deficient and WT mice. RIPK1 deficiency was beneficial for survival following lethal, systemic Streptococcus pneumoniae infection, confirming the functionality of innate B cell populations which increased in the absence of RIPK1. Thus, RIPK1 orchestrates B cell fate, and delayed effector function, through a B cell-intrinsic mechanism.

The innate responsiveness of the immune system is important not only for quick responses to pathogens but also for the initiation and shaping of the subsequent adaptive immune response. Activation via the cytokine IL-18, a product of inflammasomes, gives rise to a rapid response that includes the production of self-reactive antibodies. As increased concentrations of this cytokine are found in inflammatory diseases, we investigated the origin of the B cell response and its regulation. We identified an accumulation of B cell-helper neutrophils in the spleen that interacted with innate-type invariant natural killer T cells (iNKT cells) to regulate B cell responses. We found that neutrophil-dependent expression of the death-receptor ligand FasL by iNKT cells was needed to restrict autoantibody production. Neutrophils can thus license iNKT cells to regulate potentially harmful autoreactive B cell responses during inflammasome-driven inflammation.

Receptor-interacting protein kinase (RIPK) 1, RIPK3, and Caspase-8, are essential adapter proteins in death receptor-induced apoptosis and programmed necrosis. Apoptosis and necroptosis are crucial for normal cell development, homeostasis, and immune responses. RIP kinases and Caspase-8 form parts of multiprotein complexes like the Death-Inducing Signaling Complex (DISC) and the necrosome, thus mediating signaling events in these cell death pathways. We compared RipK3−/−, RipK1−/−RipK3−/−Caspase-8−/−, RipK3−/−Caspase-8−/−, and RIPK1 kinase-dead (RIPK1kd) mice to define the role of RIP kinase inflammatory pathways and Caspase-8 in immune responses and lymphocyte development. Preliminary studies show that lack of RIPK1 and Caspase-8, but not RIPK3, leads to the accumulation of Marginal Zone (MZ) B cells. Evidence from mixed-bone marrow chimera experiments reveals an intrinsic requirement for RIPK1 and caspase 8 in normal B cell differentiation post-T2 B cell status. Immunofluorescence showed splenic architecture disorganization in the T cell zone and MZB populations in RIPK-deficient mice. Surprisingly, humoral immune responses to T-dependent and T-independent antigens were largely normal, but immunization-induced splenic GC formation was diminished in mice lacking both Caspase-8 and RIPK3. Elucidation of cell development/death in the context of RIPK1 (whose ubiquitination status influences cellular homeostasis) could lead to better priming of B cells for chemotherapy-induced apoptosis. Previous studies also suggest human RIP kinases are potential therapeutic targets for treating a wide range of autoimmune, inflammatory, and neurodegenerative diseases.

Obesity is accompanied by inflammation in adipose tissue, impaired glucose tolerance, and changes in adipose leukocyte populations. These studies of adipose tissue from humans and mice revealed that increased frequencies of T-bet

Invariant natural killer T cells (iNKT cells) are lipid-sensing innate T cells that are restricted by the antigen-presenting molecule CD1d and express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, as well as their targets in adipose tissue, are unknown. Here we found that iNKT cells in adipose tissue had a unique transcriptional program and produced interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lacked PLZF but expressed the transcription factor E4BP4, which controlled their IL-10 production. The adipose iNKT cells were a tissue-resident population that induced an anti-inflammatory phenotype in macrophages and, through the production of IL-2, controlled the number, proliferation and suppressor function of regulatory T cells (Treg cells) in adipose tissue. Thus, iNKT cells in adipose tissue are unique regulators of immunological homeostasis in this tissue.

TLR-mediated signaling and the production of IL-12 by APCs, rather than recognition of microbial antigens, enables rapid iNKT cell responses to diverse microbial infections., Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor–driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12–induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.

A new haptenated derivative of alpha-galactosyl ceramide (alpha-GalCer) has been synthesized to assist in the study of the mechanism of T cell help for the production of B cell antibodies. Our synthetic route provides access to an amine intermediate which can easily be extended to generate an array of compounds, useful in various ongoing studies. Herein, we also describe the biological evaluation of the nitrophenyl (NP) haptenated alpha-GalCer and demonstrate its importance in such mechanistic studies. For instance, in vitro studies showed that NP-alpha-GalCer stimulates both T and B cell proliferation while in vivo studies in immunized mice showed the production of IgG anti-NP antibodies after exposure to NP-alpha-GalCer. The interpretation of these results helps toward a better understanding of T cell help for the production of antibodies.

The mechanisms of T cell help for production of antilipid antibodies are largely unknown. This study shows that invariant NK T cells (iNK T cells) and B cells cooperate in a model of antilipid antigen-specific antibody responses. We use a model haptenated lipid molecule, 4-hydroxy-3-nitrophenyl-αGalactosylCeramide (NP-αGalCer), to demonstrate that iNK T cells provide cognate help to lipid-antigen-presenting B cells. B cells proliferate and IgG anti-NP is produced fromin vivo-immunized mice andin vitrococultures of B and NK T cells after exposure to NP-αGalCer, but not closely related control glycolipids. This B cell response is absent in CD1d−/−and Jα18−/−mice but not CD4−/−mice. The antibody response to NP-αGalCer is dominated by the IgM, IgG3, and IgG2c isotypes, and marginal zone B cells stimulate betterin vitrolipid antigen-driven proliferation than follicular B cells, suggesting an important role for this B cell subset. iNK T cell help for B cells is shown to involve cognate help from CD1d-instructed lipid-specific iNK T cells, with help provided via CD40L, B7–1/B7–2, and IFN-γ, but not IL-4. This model provides evidence of iNK T cell help for antilipid antibody production, an important aspect of infections, autoimmune diseases, and vaccine development. Our findings also now allow prediction of those microbial antigens that would be expected to elicit cognate iNKT cell help for antibody production, namely those that can stimulate iNKT cells and at the same time have a polar moiety that can be recognized by antibodies.

Successful induction of B-cell activation and memory depends on help from CD4+ T cells. Invariant natural killer T (iNKT) cells (glycolipid-specific, CD1d-restricted innate lymphocytes) provide both cognate (direct) and noncognate (indirect) helper signals to enhance B-cell responses. Both forms of iNKT-cell help induce primary humoral immune responses, but only noncognate iNKT-cell help drives humoral memory and plasma cells. Here, we show that iNKT cognate help for B cells is fundamentally different from the help provided by conventional CD4+ T cells. Cognate iNKT-cell help drives an early, unsustained germinal center B-cell expansion, less reduction of T follicular regulatory cells, an expansion of marginal zone B cells, and early increases in regulatory IL-10-producing B-cell numbers compared with noncognate activation. These results are consistent with a mechanism whereby iNKT cells preferentially provide an innate form of help that does not generate humoral memory and has important implications for the application of glycolipid molecules as vaccine adjuvants.

The onset of systemic autoimmunity is variable, making it difficult to identify early events. In this study, we show in rheumatoid factor (RF) Ig-transgenic autoimmune-prone mice that the appearance of RF B cells in blood signifies the onset of RF B cell activation in spleen, providing a novel window into the initiation of an autoantibody response. This allowed us to study the early and late phases of spontaneous induction of the B cell autoimmune response. Using this approach we showed that extensive Ab-forming cell generation in spleen, accompanied by somatic hypermutation, occurred despite the lack of an early germinal center response. The onset of the RF response correlated with the levels of IgG2a-containing immune complexes but not total IgG2a. By identifying the time of onset in individual mice, we were able to track progression of disease. We found remissions of RF Ab-forming cell production in some mice, suggesting that at the clonal level, chronic autoantibody responses are dynamic and episodic, much like acute pathogen responses. Surprisingly, there was little accumulation of long-lived plasma cells in bone marrow of mice with long-standing RF responses in spleen. These studies are among the first to define the early events of a spontaneous B cell autoimmune response.

Synthetic single-stranded oligodeoxynucleotides (15—30 bp) containing CpG motifs and phosphorothioate backbones (CpG s-ODN), immune complexes consisting of anti-nucleosome mAbs and mammalian chromatin (chromatin IC), and immune complexes consisting of anti-hapten mAbs and haptenated-double stranded DNA fragments (~600 bp) can all effectively stimulate transgenic B cells expressing a rheumatoid factor receptor by a TLR9-dependent process. However, differential sensitivity to both s-ODN and small molecule inhibitors suggests that stimulatory CpG sODN and chromatin IC may either access TLR9 via different routes or depend on discrete activation parameters. These data have important implications regarding the therapeutic application of TLR9 inhibitors to the treatment of systemic autoimmune diseases.

Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but typically remain quiescent. When this homeostasis is perturbed, the formation of self-reactive antibodies can have serious pathological consequences. B cells expressing an antigen receptor specific for self-immunoglobulin-gamma (IgG) make a class of autoantibodies known as rheumatoid factor (RF). Here we show that effective activation of RF+ B cells is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the antigen receptor and a member of the MyD88-dependent Toll-like receptor (TLR) family. Inhibitor studies implicate TLR9. These data establish a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease and explain the preponderance of autoantibodies reactive with nucleic acid-protein particles. The unique features of this dual-engagement pathway should facilitate the development of therapies that specifically target autoreactive B cells.

Background/Aims: Inflammation is a major and critical component of the lung pathology in the hereditary disease cystic fibrosis. The molecular mechanisms of chronic inflammation in cystic fibrosis require definition. Methods: We used several genetic mouse models to test a role of iNKT cells and ceramide in pulmonary inflammation of cystic fibrosis mice. Inflammation was determined by the pulmonary cytokine profil and the abundance of inflammatory cells in the lung. Results: Here we provide a new concept how inflammation in the lung of individuals with cystic fibrosis is initiated. We show that in cystic fibrosis mice the mutation in the Cftr gene provokes a significant up-regulation of iNKT cells in the lung. Accumulation of iNKT cells serves to control autoimmune disease, which is triggered by a ceramide-mediated induction of cell death in CF organs. Autoimmunity becomes in particular overt in cystic fibrosis mice lacking iNKT cells and although suppression of the autoimmune response by iNKT cells is beneficial, IL-17+ iNKT cells attract macrophages and neutrophils to CF lungs resulting in chronic inflammation. Genetic deletion of iNKT cells in cystic fibrosis mice prevents inflammation in CF lungs. Conclusion: Our data demonstrate an important function of iNKT cells in the chronic inflammation affecting cystic fibrosis lungs. iNKT cells suppress the auto-immune response induced by ceramide-mediated death of epithelial cells in CF lungs, but also induce a chronic pulmonary inflammation.

Multiple sclerosis is an autoimmune disease of the central nervous system in which T cell reactivity to several myelin proteins, including myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG), has been implicated in the perpetuation of the disease state. Experimental autoimmune encephalomyelitis (EAE) is used commonly as a model in which potential therapies for multiple sclerosis are evaluated. The ability of T cell epitope-containing peptides to down-regulate the disease course is well documented for both MBP- and proteolipid protein-induced EAE, and recently has been shown for MOG-induced EAE. In this study, we describe a novel EAE model, in which development of severe disease symptoms in (PL/J × SJL)F1 mice is dependent on reactivity to two different immunizing Ags, MBP and MOG. The disease is often fatal, with a relapsing/progressive course in survivors, and is more severe than would be predicted by immunization with either Ag alone. The MOG plus MBP disease can be treated postinduction with a combination of the MOG 41–60 peptide (identified as the major therapeutic MOG epitope for this strain) and the MBP Ac1–11[4Y] peptide. A significant treatment effect can also be obtained by administration of the MBP peptide alone, but this effect is strictly dose dependent. This MBP peptide does not treat the disease induced only with MOG. These results suggest that peptide immunotherapy can provide an effective means of mitigating disease in this model, even when the treatment is targeted to only one component epitope or one component protein Ag of a diverse autoimmune response.

Inflammation is an essential protective part of the body’s response to infection, yet many diseases are the product of inflammation. For example, inflammation can lead to autoimmune disease and tissue damage, and is a key element in chronic health conditions such as heart disease, diabetes, rheumatoid arthritis, and also drives changes associated with aging. Animal models of infectious and chronic disease are important tools with which to dissect the pathways whereby inflammatory responses are initiated and controlled. Animal models therefore provide a prism through which the role of inflammation in health and disease can be viewed, and are important means by which to dissect mechanisms and identify potential therapies to be tested in the clinic. A meeting, “The Yin and Yang of Inflammation” was organized by Trudeau Institute and was held between April 4-6, 2014. The main goal was to bring together experts from biotechnology and academic organizations to examine and describe critical pathways in inflammation and place these pathways within the context of human disease. A group of ~80 scientists met for three days of intense formal and informal exchanges. A key focus was to stimulate interactions between basic research and industry.

B cells rely on CD4(+) T cells helper signals to optimize their responses to T-dependent antigens. Recently another subset of T cells has been identified which provides help for B cells, invariant natural killer T (iNKT) cells. iNKT cells are unique because they provide both innate and adaptive forms of help to B cells, with divergent outcomes. iNKT cells are widely distributed throughout the spleen at rest, consolidate in the marginal zone of the spleen early after activation, and are later found in germinal centers. Understanding the activation requirements for iNKT cells has led to the development of glycolipid containing nanoparticles which efficiently activate iNKT cells, enhance their cooperation with B cells, and which hold promise for vaccine development.

Invariant NKT (iNKT) cells are glycolipid-specific innate lymphocytes emerging as critical players in the immune response to diverse infections and disease. iNKT cells are activated through cognate interactions with lipid-loaded APCs, by Ag-independent cytokine-mediated signaling pathways, or a combination of both. Although each of these modes of iNKT cell activation plays an important role in directing the humoral and cell-mediated immune response, the spatio-temporal nature of these interactions and the cellular requirements for activation are largely undefined. Combining novel in situ confocal imaging of αGalactosylceramide-loaded CD1d tetramer labeling to localize the endogenous iNKT cell population with cytokine reporter mice, we reveal the choreography of early murine splenic iNKT cell activation across diverse settings of glycolipid immunization and systemic infection with Streptococcus pneumoniae. We find that iNKT cells consolidate in the marginal zone and require dendritic cells lining the splenic marginal zone for activation following administration of cognate glycolipids and during systemic infection but not following exogenous cytokine administration. Although further establishing the importance of cognate iNKT cell interactions with APCs, we also show that noncognate iNKT-dependent mechanisms are sufficient to mediate effector outcomes, such as STAT signaling and dendritic cell licensing throughout the splenic parenchyma. Collectively, these data provide new insight into how iNKT cells may serve as a natural adjuvant in facilitating adaptive immune responses, irrespective of their tissue localization.

One of the manifestations of X-linked lymphoproliferative disease (XLP) is progressive agammaglobulinemia, caused by the absence of a functional signaling lymphocyte activation molecule (SLAM)–associated protein (SAP) in T, invariant natural killer T (NKT) cells and NK cells. Here we report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. Whereas NKT cell–dependent B cell responses were absent in SAP−/−.B6 mice that completely lack NKT cells, the small number of SAP-deficient NKT cells in SAP−/−.BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted after development in SAPfl/fl.tgCreERT2.B6 mice. We find that NKT cell intrinsic expression of SAP is dispensable for noncognate helper functions, but is critical for providing cognate help to antigen-specific B cells. These results demonstrate that SLAM-family receptor-regulated cell-cell interactions are not limited to T-B cell conjugates. We conclude that in the absence of SAP, several routes of NKT cell–mediated antibody production are still accessible. The latter suggests that residual NKT cells in XLP patients might contribute to variations in dysgammaglobulinemia.

Progesterone's (P) actions in the ventral medial hypothalamus (VMH) and the ventral tegmental area (VTA) are essential for sexual receptivity in hamsters. This study investigates the notion that P works in the VTA in the absence of intracellular P receptors (PRs) by 5 alpha-reduction to progestins, which would subsequently bind to membrane gamma-Aminobutyric Acid receptor complexes (GBRs). To test the importance of P metabolism, a 5 alpha-reductase inhibitor, 17 beta-N, N-diethylcarbamoyl-4-methyl-4aza-5 alpha-androstan-3-one (4MA) was administered SC (0, 3, 9, 15, 24, or 30 mg/kg) to ovx estradiol benzoate(EB)-primed hamsters, followed by one of six doses of SC P (0, 25, 50, 100, 200, or 500 micrograms) and sexual receptivity testing. 200 micrograms P-treated animals administered higher (24 and 30 mg/kg) doses of 4MA had significantly decreased total lordosis durations (TLDs) compared to 0 mg/kg 4MA controls (exp 1). In exp 2, 4MA was aimed bilaterally at the VTA prior to SC P. After 200 micrograms P, animals had significantly lower TLDs than after 500 micrograms P, 3 hours following bilateral VTA implantation of 4MA, but not cholesterol. In exp 3, cycling female hamsters were infused with 1.0 microgram 4MA or vehicle unilaterally into the VTA on diestrus, proestrus, and estrus. 4MA, but not vehicle, infusions into the VTA interrupted receptivity in estrus and proestrus animals, but had no effect on diestrus animals. 4MA's reduction of receptivity when given systemically and intracranially strongly supports the hypothesis that 5 alpha-reduced P metabolites, possibly interacting with GBRs in the VTA, are essential for sexual receptivity in hamsters.

Mouse invariant natural killer T cells (iNKT cells) provide cognate and noncognate help for lipid and protein-specific B cells, respectively. However, the long-term outcome for B cells after cognate help is provided by iNKT cells is unknown at present. Here we found that cognate iNKT cell help resulted in a B cell differentiation program characterized by extrafollicular plasmablasts, germinal-center formation, affinity maturation and a robust primary immunoglobulin G (IgG) antibody response that was uniquely dependent on iNKT cell-derived interleukin 21 (IL-21). However, cognate help from iNKT cells did not generate an enhanced humoral memory response. Thus, cognate iNKT cell help for lipid-specific B cells induces a unique signature that is a hybrid of classic T cell-dependent and T cell-independent type 2 B cell responses.

An unexpected link between immunity and lipid metabolism has been found. Apolipoproteins critical in lipid trafficking during normal energy delivery and in atherosclerosis have been found to have a role in the immune system. Here they serve a special function in CD1-mediated presentation of lipid antigens for T-cell activation. This has implications for microbial immunity, autoimmunity and atherosclerosis. Peptide antigens are presented to T cells by major histocompatibility complex (MHC) molecules, with endogenous peptides presented by MHC class I and exogenous peptides presented by MHC class II. In contrast to the MHC system, CD1 molecules bind lipid antigens that are presented at the antigen-presenting cell (APC) surface to lipid antigen-reactive T cells1. Because CD1 molecules survey endocytic compartments2, it is self-evident that they encounter antigens from extracellular sources. However, the mechanisms of exogenous lipid antigen delivery to CD1-antigen-loading compartments are not known. Serum apolipoproteins are mediators of extracellular lipid transport for metabolic needs3. Here we define the pathways mediating markedly efficient exogenous lipid antigen delivery by apolipoproteins to achieve T-cell activation. Apolipoprotein E binds lipid antigens and delivers them by receptor-mediated uptake into endosomal compartments containing CD1 in APCs. Apolipoprotein E mediates the presentation of serum-borne lipid antigens and can be secreted by APCs as a mechanism to survey the local environment to capture antigens or to transfer microbial lipids from infected cells to bystander APCs. Thus, the immune system has co-opted a component of lipid metabolism to develop immunological responses to lipid antigens.

The critical role of Toll-like receptors (TLRs) as mediators of pathogen recognition by the innate immune system is now firmly established. Such recognition results in the initiation of an inflammatory immune response and subsequent instruction of the adaptive immune system, both of which are designed to rid the host of the invading pathogen. More controversial is the potential role of TLRs in the recognition of endogenous ligands and what effect this might have on the consequent development of autoimmune or other chronic sterile inflammatory disorders. An increasing number of studies implicate TLRs as being involved in the immune response to self-molecules that have in some way been altered from their native state or accumulate in non-physiologic sites or amounts, although questions have been raised about possible contaminants in certain of these studies. In this review, we discuss the evidence for endogenous ligand-TLR interactions with particular emphasis on mammalian chromatin, systemic lupus erythematosus, and atherosclerosis. Overall, the data support the general concept of a role for TLRs in the recognition of endogenous ligands. However, the precise details of the interactions and the extent to which they may contribute to the pathogenesis of human disease remain to be clarified.

The fate of an autoreactive B cell is determined in part by the nature of the interaction of the B cell receptor with its autoantigen. In the lpr model of systemic autoimmunity, as well as in certain human diseases, autoreactive B cells expressing rheumatoid factor (RF) binding activity are prominent. A murine B cell transgenic model in which the B cell receptor is a RF that recognizes IgG2a of the j allotype (IgG2aj), but not the b allotype, was used in this study to investigate how the form of the autoantigen influences its ability to activate B cells. We found that sera from autoimmune mice, but not from nonautoimmune mice, were able to induce the proliferation of these RF+ B cells but did not stimulate B cells from RF− littermate controls. The stimulatory factor in serum was found to be IgG2aj, but the IgG2aj was stimulatory only when in the form of immune complexes. Monomeric IgG2aj failed to stimulate. Immune complexes containing lupus-associated nuclear and cytoplasmic autoantigens were particularly potent B cell activators in this system. Appropriate manipulation of such autoantibody/autoantigen complexes may eventually provide a means for therapeutic intervention in patients with certain systemic autoimmune disorders.