CD8αα⁺ innate-type lymphocytes in the intestinal epithelium mediate mucosal immunity.

Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.

Autophagy-related protein Vps34 controls the homeostasis and function of antigen cross-presenting CD8α+ dendritic cells.

The class III PI3K Vacuolar protein sorting 34 (Vps34) plays a role in both canonical and noncanonical autophagy, key processes that control the presentation of antigens by dendritic cells (DCs) to naive T lymphocytes. We generated DC-specific Vps34-deficient mice to assess the contribution of Vps34 to DC functions. We found that DCs from these animals have a partially activated phenotype, spontaneously produce cytokines, and exhibit enhanced activity of the classic MHC class I and class II antigen-presentation pathways. Surprisingly, these animals displayed a defect in the homeostatic maintenance of splenic CD8α+ DCs and in the capacity of these cells to cross-present cell corpse-associated antigens to MHC class I-restricted T cells, a property that was associated with defective expression of the T-cell Ig mucin (TIM)-4 receptor. Importantly, mice deficient in the Vps34-associated protein Rubicon, which is critical for a noncanonical form of autophagy called "Light-chain 3 (LC3)-associated phagocytosis" (LAP), lacked such defects. Finally, consistent with their defect in the cross-presentation of apoptotic cells, DC-specific Vps34-deficient animals developed increased metastases in response to challenge with B16 melanoma cells. Collectively, our studies have revealed a critical role of Vps34 in the regulation of CD8α+ DC homeostasis and in the capacity of these cells to process and present antigens associated with apoptotic cells to MHC class I-restricted T cells. Our findings also have important implications for the development of small-molecule inhibitors of Vps34 for therapeutic purposes.

Follicular B cell trafficking within the spleen actively restricts humoral immune responses.

Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen, but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naive FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens.

Peripheral tolerance can be modified by altering KLF2-regulated Treg migration.

Tregs are essential for maintaining peripheral tolerance, and thus targeting these cells may aid in the treatment of autoimmunity and cancer by enhancing or reducing suppressive functions, respectively. Before these cells can be harnessed for therapeutic purposes, it is necessary to understand how they maintain tolerance under physiologically relevant conditions. We now report that transcription factor Kruppel-like factor 2 (KLF2) controls naive Treg migration patterns via regulation of homeostatic and inflammatory homing receptors, and that in its absence KLF2-deficient Tregs are unable to migrate efficiently to secondary lymphoid organs (SLOs). Diminished Treg trafficking to SLOs is sufficient to initiate autoimmunity, indicating that SLOs are a primary site for maintaining peripheral tolerance under homeostatic conditions. Disease severity correlates with impaired Treg recruitment to SLOs and, conversely, promotion of Tregs into these tissues can ameliorate autoimmunity. Moreover, stabilizing KLF2 expression within the Treg compartment enhances peripheral tolerance by diverting these suppressive cells from tertiary tissues into SLOs. Taken together, these results demonstrate that peripheral tolerance is enhanced or diminished through modulation of Treg trafficking to SLOs, a process that can be controlled by adjusting KLF2 protein levels.

Spleen supports a pool of innate-like B cells in white adipose tissue that protects against obesity-associated insulin resistance.

Lipid accumulation in obesity triggers a low-grade inflammation that results from an imbalance between pro- and anti-inflammatory components of the immune system and acts as the major underlying mechanism for the development of obesity-associated diseases, notably insulin resistance and type 2 diabetes. Innate-like B cells are a subgroup of B cells that respond to innate signals and modulate inflammatory responses through production of immunomodulatory mediators such as the anti-inflammatory cytokine IL-10. In this study, we examined innate-like B cells in visceral white adipose tissue (VAT) and the relationship of these cells with their counterparts in the peritoneal cavity and spleen during diet-induced obesity (DIO) in mice. We show that a considerable number of innate-like B cells bearing a surface phenotype distinct from the recently identified "adipose natural regulatory B cells" populate VAT of lean animals, and that spleen represents a source for the recruitment of these cells in VAT during DIO. However, demand for these cells in the expanding VAT outpaces their recruitment during DIO, and the obese environment in VAT further impairs their function. We further show that removal of splenic precursors of innate-like B cells through splenectomy exacerbates, whereas supplementation of these cells via adoptive transfer ameliorates, DIO-associated insulin resistance. Additional adoptive transfer experiments pointed toward a dominant role of IL-10 in mediating the protective effects of innate-like B cells against DIO-induced insulin resistance. These findings identify spleen-supplied innate-like B cells in VAT as previously unrecognized players and therapeutic targets for obesity-associated diseases.

KLF2 is a rate-limiting transcription factor that can be targeted to enhance regulatory T-cell production.

Regulatory T cells (Tregs) are a specialized subset of CD4(+) T cells that maintain self-tolerance by functionally suppressing autoreactive lymphocytes. The Treg compartment is composed of thymus-derived Tregs (tTregs) and peripheral Tregs (pTregs) that are generated in secondary lymphoid organs after exposure to antigen and specific cytokines, such as TGF-ß. With regard to this latter lineage, pTregs [and their ex vivo generated counterparts, induced Tregs (iTregs)] offer particular therapeutic potential because these cells can be raised against specific antigens to limit autoimmunity. We now report that transcription factor Krüppel-like factor 2 (KLF2) is necessary for the generation of iTregs but not tTregs. Moreover, drugs that limit KLF2 proteolysis during T-cell activation enhance iTreg development. To the authors' knowledge, this study identifies the first transcription factor to distinguish between i/pTreg and tTreg ontogeny and demonstrates that KLF2 is a therapeutic target for the production of regulatory T cells.

Invariant natural killer T cells as sensors and managers of inflammation.

Invariant natural killer T (iNKT) cells are a subset of innate-like lymphocytes that recognize glycolipid antigens bound by the major histocompatibility complex (MHC)-class-I-related protein CD1d. iNKT cells are activated early during a variety of infections and inflammatory diseases and contribute to the subsequent development of adaptive immune responses. Consequently, iNKT cells play a critical role in the development and resolution of inflammatory diseases and represent attractive targets for the development of immunotherapies. Recent studies have provided important insight into the mechanisms by which iNKT cells become activated in response to diverse inflammatory stimuli. These new findings should be instrumental to promote the immunomodulatory properties of iNKT cells for treatment of inflammatory diseases.

Endothelial JAM-A promotes reovirus viremia and bloodstream dissemination.

Viruses that cause systemic disease often spread through the bloodstream to infect target tissues. Although viremia is an important step in the pathogenesis of many viruses, how viremia is established is not well understood. Reovirus has been used to dissect mechanisms of viral pathogenesis and is being evaluated in clinical trials as an oncolytic agent. After peroral entry into mice, reovirus replicates within the gastrointestinal tract and disseminates systemically via hematogenous or neural routes. Junctional adhesion molecule-A (JAM-A) is a tight junction protein that serves as a receptor for reovirus. JAM-A is required for establishment of viremia and viral spread to sites of secondary replication. JAM-A also is expressed on the surface of circulating hematopoietic cells. To determine contributions of endothelial and hematopoietic JAM-A to reovirus dissemination and pathogenesis, we generated strains of mice with altered JAM-A expression in these cell types and assessed bloodstream spread of reovirus strain type 1 Lang (T1L), which disseminates solely by hematogenous routes. We found that endothelial JAM-A but not hematopoietic JAM-A facilitates reovirus T1L bloodstream entry and egress. Understanding how viruses establish viremia may aid in development of inhibitors of this critical step in viral pathogenesis and foster engineering of improved oncolytic viral vectors.

Natural killer T cells in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory disease that causes demyelination of neurons in the central nervous system. Traditional therapies for MS have involved anti-inflammatory and immunosuppressive drugs with significant side effects that often only provide short-term relief. A more desirable outcome of immunotherapy would be to protect against disease before its clinical manifestation or to halt disease after its initiation. One attractive approach to accomplish this goal would be to restore tolerance by targeting immunoregulatory cell networks. Although much of the work in this area has focused on CD4(+) Foxp3(+) regulatory T cells, other studies have investigated natural killer T (NKT) cells, a subset of T cells that recognizes glycolipid antigens in the context of the CD1d glycoprotein. Studies with human MS patients have revealed alterations in the numbers and functions of NKT cells, which have been partially supported by studies with the experimental autoimmune encephalomyelitis model of MS. Additional studies have shown that activation of NKT cells with synthetic lipid antigens can, at least under certain experimental conditions, protect mice against the development of MS-like disease. Although mechanisms of this protection remain to be fully investigated, current evidence suggests that it involves interactions with other immunoregulatory cell types such as regulatory T cells and immunosuppressive myeloid cells. These studies have provided a strong foundation for the rational design of NKT-cell-based immunotherapies for MS that induce tolerance while sparing overall immune function. Nevertheless, additional pre-clinical and clinical studies will be required to bring this goal to fruition.

Activated invariant NKT cells control central nervous system autoimmunity in a mechanism that involves myeloid-derived suppressor cells.

Invariant NKT (iNKT) cells are a subset of T lymphocytes that recognize glycolipid Ags presented by the MHC class I-related protein CD1d. Activation of iNKT cells with glycolipid Ags, such as the marine sponge-derived reagent α-galactosylceramide (α-GalCer), results in the rapid production of a variety of cytokines and activation of many other immune cell types. These immunomodulatory properties of iNKT cells have been exploited for the development of immunotherapies against a variety of autoimmune and inflammatory diseases, but mechanisms by which activated iNKT cells confer disease protection have remained incompletely understood. In this study, we demonstrate that glycolipid-activated iNKT cells cooperate with myeloid-derived suppressor cells (MDSCs) in protecting mice against the development of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model for multiple sclerosis. We show that α-GalCer induced the expansion and immunosuppressive activities of MDSCs in the spleen of mice induced for development of EAE. Disease protection in these animals also correlated with recruitment of MDSCs to the CNS. Depletion of MDSCs abrogated the protective effects of α-GalCer against EAE and, conversely, adoptive transfer of MDSCs from α-GalCer-treated mice ameliorated passive EAE induced in recipient animals. The cytokines GM-CSF, IL-4, and IFN-γ, produced by activated iNKT cells, and inducible NO synthase, arginase-1, and IL-10 produced by MDSCs, contributed to these effects. Our findings have revealed cooperative immunosuppressive interactions between iNKT cells and MDSCs that might be exploited for the development of improved immunotherapies for multiple sclerosis and other autoimmune and inflammatory diseases.

Impaired autophagy, defective T cell homeostasis, and a wasting syndrome in mice with a T cell-specific deletion of Vps34.

Autophagy plays a critical role in multiple aspects of the immune system, including the development and function of T lymphocytes. In mammalian cells, the class III PI3K vacuolar protein sorting (Vps)34 is thought to play a critical role in autophagy. However, recent studies have cast doubt on the role of Vps34 in autophagy, at least in certain cell types. To study the effects of Vps34 on autophagy in T lymphocytes, we generated mice that selectively lack Vps34 in the T cell lineage. Vps34 ablation in T cells caused profound defects in autophagic flux, resulting in accumulation of cellular organelles and apoptosis. These animals exhibited normal intrathymic development of conventional T cells, but they were profoundly impaired in the intrathymic development of invariant NKT cells. In peripheral organs, T cell-specific ablation of Vps34 had a profound impact on T cell homeostasis and function. Furthermore, aged animals developed an inflammatory wasting syndrome characterized by weight loss, intestinal inflammation, and anemia. Consistent with this phenotype, Vps34 was required for the peripheral maintenance and function of CD4(+)Foxp3(+) regulatory T cells. Collectively, our study reveals a critical role for Vps34 in autophagy and for the peripheral homeostasis and function of T lymphocytes.

Activation of invariant natural killer T cells by lipid excess promotes tissue inflammation, insulin resistance, and hepatic steatosis in obese mice.

Obesity triggers a low-grade systemic inflammation, which plays an important role in the development of obesity-associated metabolic diseases. In searching for links between lipid accumulation and chronic inflammation, we examined invariant natural killer T (iNKT) cells, a subset of T lymphocytes that react with lipids and regulate inflammatory responses. We show that iNKT cells respond to dietary lipid excess and become activated before or at the time of tissue recruitment of inflammatory leukocytes, and that these cells progressively increase proinflammatory cytokine production in obese mice. Such iNKT cells skew other leukocytes toward proinflammatory cytokine production and induce an imbalanced proinflammatory cytokine environment in multiple tissues. Further, iNKT cell deficiency ameliorates tissue inflammation and provides protection against obesity-induced insulin resistance and hepatic steatosis. Conversely, chronic iNKT cell stimulation using a canonical iNKT cell agonist exacerbates tissue inflammation and obesity-associated metabolic disease. These findings place iNKT cells into the complex network linking lipid excess to inflammation in obesity and suggest new therapeutic avenues for obesity-associated metabolic disorders.

Intestinal epithelial cells modulate CD4 T cell responses via the thymus leukemia antigen.

The intestinal epithelium is comprised of a monolayer of intestinal epithelial cells (IEC), which provide, among other functions, a physical barrier between the high Ag content of the intestinal lumen and the sterile environment beyond the epithelium. IEC express a nonclassical MHC class I molecule known as the thymus leukemia (TL) Ag. TL is known to interact with CD8αα-expressing cells, which are abundant in the intestinal intraepithelial lymphocyte compartment. In this report, we provide evidence indicating that expression of TL by IEC modulates the cytokine profile of CD4(+) T cells favoring IL-17 production. We show in an adoptive transfer model of colitis that donor-derived cells become more pathogenic when TL is expressed on IEC in recipient animals. Moreover, TL(+)IEC promote development of IL-17-mediated responses capable of protecting mice from Citrobacter rodentium infection. We also show that modulation of IL-17-mediated responses by TL(+)IEC is controlled by the expression of CD8α on CD4(+) T cells. Overall, our results provide evidence for an important interaction between IEC and CD4(+) T cells via TL, which modulates mucosal immune responses.

In vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.

Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present several self- and foreign antigens to Kb-, Db-, or Qa-1b-restricted CD8+ cytotoxic T cells, presentation of some antigens was unaffected or significantly enhanced. Consistent with these findings, mice generated defective CD8+ T cell responses against class I-presented antigens. These findings reveal an important in vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.

Impact of bacteria on the phenotype, functions, and therapeutic activities of invariant NKT cells in mice.

Invariant NKT (iNKT) cells are innate-like lymphocytes that recognize glycolipid antigens in the context of the MHC class I-like antigen-presenting molecule CD1d. In vivo activation of mouse iNKT cells with the glycolipid alpha-galactosylceramide (alpha-GalCer) results in the acquisition of a hyporesponsive (anergic) phenotype by these cells. Because iNKT cells can become activated in the context of infectious agents, here we evaluated whether iNKT cell activation by microorganisms can influence subsequent responses of these cells to glycolipid antigen stimulation. We found that mouse iNKT cells activated in vivo by multiple bacterial microorganisms, or by bacterial LPS or flagellin, became unresponsive to subsequent activation with alpha-GalCer. This hyporesponsive phenotype of iNKT cells required IL-12 expression and was associated with changes in the surface phenotype of these cells, reduced severity of concanavalin A-induced hepatitis, and alterations in the therapeutic activities of alpha-GalCer. These findings may have important implications for the development of iNKT cell-based therapies.

Invariant natural killer T cells: bridging innate and adaptive immunity.

Cells of the innate immune system interact with pathogens via conserved pattern-recognition receptors, whereas cells of the adaptive immune system recognize pathogens through diverse, antigen-specific receptors that are generated by somatic DNA rearrangement. Invariant natural killer T (iNKT) cells are a subset of lymphocytes that bridge the innate and adaptive immune systems. Although iNKT cells express T cell receptors that are generated by somatic DNA rearrangement, these receptors are semi-invariant and interact with a limited set of lipid and glycolipid antigens, thus resembling the pattern-recognition receptors of the innate immune system. Functionally, iNKT cells most closely resemble cells of the innate immune system, as they rapidly elicit their effector functions following activation, and fail to develop immunological memory. iNKT cells can become activated in response to a variety of stimuli and participate in the regulation of various immune responses. Activated iNKT cells produce several cytokines with the capacity to jump-start and modulate an adaptive immune response. A variety of glycolipid antigens that can differentially elicit distinct effector functions in iNKT cells have been identified. These reagents have been employed to test the hypothesis that iNKT cells can be harnessed for therapeutic purposes in human diseases. Here, we review the innate-like properties and functions of iNKT cells and discuss their interactions with other cell types of the immune system.

PD-1/PD-L blockade prevents anergy induction and enhances the anti-tumor activities of glycolipid-activated invariant NKT cells.

Invariant NKT (iNKT) cells recognize glycolipid Ags, such as the marine sponge-derived glycosphingolipid alpha-galactosylceramide (alphaGalCer) presented by the CD1d protein. In vivo activation of iNKT cells with alphaGalCer results in robust cytokine production, followed by the acquisition of an anergic phenotype. Here we have investigated mechanisms responsible for the establishment of alphaGalCer-induced iNKT cell anergy. We found that alphaGalCer-activated iNKT cells rapidly up-regulated expression of the inhibitory costimulatory receptor programmed death (PD)-1 at their cell surface, and this increased expression was retained for at least one month. Blockade of the interaction between PD-1 and its ligands, PD-L1 and PD-L2, at the time of alphaGalCer treatment prevented the induction iNKT cell anergy, but was unable to reverse established iNKT cell anergy. Consistently, injection of alphaGalCer into PD-1-deficient mice failed to induce iNKT cell anergy. However, blockade of the PD-1/PD-L pathway failed to prevent bacterial- or sulfatide-induced iNKT cell anergy, suggesting additional mechanisms of iNKT cell tolerance. Finally, we showed that blockade of PD-1/PD-L interactions enhanced the antimetastatic activities of alphaGalCer. Collectively, our findings reveal a critical role for the PD-1/PD-L costimulatory pathway in the alphaGalCer-mediated induction of iNKT cell anergy that can be targeted for the development of immunotherapies.

STAT1 negatively regulates lung basophil IL-4 expression induced by respiratory syncytial virus infection.

IL-4 contributes to immunopathology induced in mice by primary respiratory syncytial virus (RSV) infection. However, the cellular source of IL-4 in RSV infection is unknown. We identified CD3(-)CD49b(+) cells as the predominant source of IL-4 in the lungs of RSV-infected BALB/c mice. We ruled out T cells, NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. Using IL4 GFP reporter mice (4get) mice, we identified the IL-4-expressing cells in RSV infection as basophils (CD3(-)CD49b(+)FcepsilonRI(+)c-kit(-)). Because STAT1(-/-) mice have an enhanced Th2-type response to RSV infection, we also sought to determine the cellular source and role of IL-4 in RSV-infected STAT1(-/-) mice. RSV infection resulted in significantly more IL-4-expressing CD3(-)CD49b(+) cells in the lungs of STAT1(-/-) mice than in BALB/c mice. CD49b(+)IL-4(+) cells sorted from the lungs of RSV-infected STAT1(-/-) mice and stained with Wright-Giemsa had basophil characteristics. As in wild-type BALB/c mice, IL-4 contributed to lung histopathology in RSV-infected STAT1(-/-) mice. Depletion of basophils in RSV-infected STAT1(-/-) mice reduced lung IL-4 expression. Thus, we show for the first time that a respiratory virus (RSV) induced basophil accumulation in vivo. Basophils were the primary source of IL-4 in the lung in RSV infection, and STAT1 was a negative regulator of virus-induced basophil IL-4 expression.

Thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease.

Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8alpha alpha homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically-deficient in TL expression. Our findings show that TL expression has a critical role in maintaining IEL effector functions. Also, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.

Role of autophagy in MHC class I-restricted antigen presentation.

Major histocompatibility complex (MHC) class I molecules present peptide antigens to MHC class I-restricted CD8+ T lymphocytes. The peptides loaded onto MHC class I molecules are typically derived from cytosolic antigens, which includes both self and foreign proteins. In addition to this classical MHC class I antigen presentation pathway, some cell types, especially dendritic cells can present antigens from exogenous sources to MHC class I-restricted CD8+ T cells, in a process called cross-presentation. A variety of cellular processes, including endocytosis, vesicle trafficking, and autophagy, play critical roles in these antigen presentation pathways. In this review article, we discuss the role of autophagy, an intracellular degradation system that delivers cytoplasmic constituents to lysosomes, in MHC class I-restricted antigen presentation. A mechanistic understanding of the role of autophagy-related proteins in MHC class I restricted antigen presentation may guide future efforts in manipulating autophagy to prevent or treat human disease.