Gerhard Wingender

Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO

CpG‐rich oligonucleotides (CpG‐ODN) bind to Toll‐like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG‐ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG‐ODN resulted in antigen‐specific T cell activation in local lymph nodes. In contrast, systemic application of CpG‐ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3‐dioxygenase (IDO) as indicated by the observation that CpG‐ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1‐methyl‐tryptophan (1‐MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG‐ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG‐ODN‐induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG‐ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down‐modulation of immune responses by CpG‐ODN may be possible in certain pathological conditions.

IL-10–producing NKT10 cells are a distinct regulatory invariant NKT cell subset

Invariant natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after activation, thereby impacting a wide variety of different immune reactions. However, strong activation of iNKT cells with α-galactosylceramide (αGalCer) reportedly induces a hyporeactive state that resembles anergy. In contrast, we determined here that iNKT cells from mice pretreated with αGalCer retain cytotoxic activity and maintain the ability to respond to TCR-dependent as well as TCR-independent cytokine-mediated stimulation. Additionally, αGalCer-pretreated iNKT cells acquired characteristics of regulatory cells, including production and secretion of the immunomodulatory cytokine IL-10. Through the production of IL-10, αGalCer-pretreated iNKT cells impaired antitumor responses and reduced disease in experimental autoimmune encephalomyelitis, a mouse model of autoimmune disease. Furthermore, a subset of iNKT cells with a similar inhibitory phenotype and function were present in mice not exposed to αGalCer and were enriched in mouse adipose tissue and detectable in human PBMCs. These data demonstrate that IL-10-producing iNKT cells with regulatory potential (NKT10 cells) represent a distinct iNKT cell subset.

Intestinal Microbes Affect Phenotypes and Functions of Invariant Natural Killer T Cells in Mice

BACKGROUND & AIMSnInvariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells.nnnMETHODSnWe analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free environments.nnnRESULTSnSpecific pathogen-free mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vβ7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hyporesponsive to activation with the antigen α-galactosylceramide. Intragastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require Toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation.nnnCONCLUSIONSnIntestinal microbes can affect iNKT cell phenotypes and functions in mice.

Cross-presentation of oral antigens by liver sinusoidal endothelial cells leads to CD8 T cell tolerance.

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood‐borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross‐presented on H‐2Kb by LSEC to naive CD8 T cells. Cross‐presentation efficiency in LSEC but not dendritic cells was increased by antigen‐exposure to heat or low pH. Mechanistically, cross‐presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H‐2Kb‐restricted cross‐presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2–/– knockout mice, previously reconstituted with naive ovalbumin‐specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN‐γ in CD8 T cells. Using a new transgenic mouse line expressing H‐2Kb only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H‐2Kb‐restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.

Mechanisms for Glycolipid Antigen-Driven Cytokine Polarization by Vα14i NKT Cells

Certain glycolipid Ags for Vα14i NKT cells can direct the overall cytokine balance of the immune response. Th2-biasing OCH has a lower TCR avidity than the most potent agonist known, α-galactosylceramide. Although the CD1d-exposed portions of OCH and α-galactosylceramide are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. Th1-biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFN-γ, accounting for its promotion of Th1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer in vivo. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating invariant NKT cell responses.

Invariant NKT cells are required for airway inflammation induced by environmental antigens

House dust contains antigens capable of activating mouse and human iNKT cells, contributing to allergen-induced airway inflammation.

Commensal Microbiota and CD8+ T Cells Shape the Formation of Invariant NKT Cells

Commensal bacteria play an important role in formation of the immune system, but the mechanisms involved are incompletely understood. In this study, we analyze CD1d-restricted invariant NKT (iNKT) cells in germfree mice and in two colonies of C57BL/6 mice termed conventional flora and restricted flora (RF), stably bearing commensal microbial communities of diverse but distinct composition. In germfree mice, iNKT cells were moderately reduced, suggesting that commensal microbiota were partially required for the antigenic drive in maintaining systemic iNKT cells. Surprisingly, even greater depletion of iNKT cell population occurred in RF mice. This was in part attributable to reduced RF levels of intestinal microbial taxa (Sphingomonas spp.) known to express antigenic glycosphingolipid products. However, memory and activated CD8+ T cells were also expanded in RF mice, prompting us to test whether CD8+ T cell activity might be further depleting iNKT cells. Indeed, iNKT cell numbers were restored in RF mice bearing the CD8α−/− genotype or in adult wild-type RF mice acutely depleted with anti-CD8 Ab. Moreover, iNKT cells were restored in RF mice bearing the Prf1−/− phenotype, a key component of cytolytic function. These findings indicate that commensal microbiota, through positive (antigenic drive) and negative (cytolytic depletion by CD8+ T cells) mechanisms, profoundly shape the iNKT cell compartment. Because individuals greatly vary in the composition of their microbial communities, enteric microbiota may play an important epigenetic role in the striking differences in iNKT cell abundance in humans and therefore in their potential contribution to host immune status.

Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency.

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag α-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.

Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance

Development of tumor‐specific T cell tolerance contributes to the failure of the immune system to eliminate tumor cells. Here we report that hematogenous dissemination of tumor cells followed by their elimination and local removal of apoptotic tumor cells in the liver leads to subsequent development of T cell tolerance towards antigens associated with apoptotic tumor cells. We provide evidence that liver sinusoidal endothelial cells (LSEC) remove apoptotic cell fragments generated by induction of tumor cell apoptosis through hepatic NK1.1+ cells. Antigen associated with apoptotic cell material is processed and cross‐presented by LSEC to CD8+ T cells, leading to induction of CD8+ T cell tolerance. Adoptive transfer of LSEC isolated from mice challenged previously with tumor cells promotes development of CD8+ T cell tolerance towards tumor‐associated antigen in vivo. Our results indicate that hematogenous dissemination of tumor cells, followed by hepatic tumor cell elimination and local cross‐presentation of apoptotic tumor cells by LSEC and subsequent CD8+ T cell tolerance induction, represents a novel mechanism operative in tumor immune escape.

Fibrocyte-like cells recruited to the spleen support innate and adaptive immune responses to acute injury or infection

Bone marrow (BM)-derived fibrocytes are a population of CD45+ and collagen Type I-expressing cells that migrate to the spleen and to target injured organs, such as skin, lungs, kidneys, and liver. While CD45+Col+ fibrocytes contribute to collagen deposition at the site of injury, the role of CD45+Col+ cells in spleen has not been elucidated. Here, we demonstrate that hepatotoxic injury (CCl4), TGF-β1, lipopolysaccharide, or infection with Listeria monocytogenes induce rapid recruitment of CD45+Col+ fibrocyte-like cells to the spleen. These cells have a gene expression pattern that includes antimicrobial factors (myleoperoxidase, cathelicidin, and defensins) and MHC II at higher levels than found on quiescent or activated macrophages. The immune functions of these splenic CD45+Col+ fibrocyte-like cells include entrapment of bacteria into extracellular DNA-based structures containing cathelicidin and presentation of antigens to naïve CD8+ T cells to induce their proliferation. Stimulation of these splenic fibrocyte-like cells with granulocyte macrophage-colony stimulating factor or macrophage-colony stimulating factor induces downregulation of collagen expression and terminal differentiation into the dendritic cells or macrophage. Thus, splenic CD45+Col+ cells are a population of rapidly mobilized BM-derived fibrocyte-like cells that respond to inflammation or infection to participate in innate and adaptive immune responses.