The ever-expanding role of cytokine receptor DR3 in T cells.

Death Receptor 3 (DR3) is a cytokine receptor of the Tumor Necrosis Factor receptor superfamily that plays a multifaceted role in both innate and adaptive immunity. Based on the death domain motif in its cytosolic tail, DR3 had been proposed and functionally affirmed as a trigger of apoptosis. Further studies, however, also revealed roles of DR3 in other cellular pathways, including inflammation, survival, and proliferation. DR3 is expressed in various cell types, including T cells, B cells, innate lymphocytes, myeloid cells, fibroblasts, and even outside the immune system. Because DR3 is mainly expressed on T cells, DR3-mediated immune perturbations leading to autoimmunity and other diseases were mostly attributed to DR3 activation of T cells. However, which T cell subset and what T effector functions are controlled by DR3 to drive these processes remain incompletely understood. DR3 engagement was previously found to alter CD4 T helper subset differentiation, expand the Foxp3+ Treg cell pool, and maintain intraepithelial γδ T cells in the gut. Recent studies further unveiled a previously unacknowledged aspect of DR3 in regulating innate-like invariant NKT (iNKT) cell activation, expanding the scope of DR3-mediated immunity in T lineage cells. Importantly, in the context of iNKT cells, DR3 ligation exerted costimulatory effects in agonistic TCR signaling, unveiling a new regulatory framework in T cell activation and proliferation. The current review is aimed at summarizing such recent findings on the role of DR3 on conventional T cells and innate-like T cells and discussing them in the context of immunopathogenesis.

The cytokine receptor DR3 identifies and promotes the activation of thymic NKT17 cells.

Invariant natural killer T (iNKT) cells correspond to a population of thymus-generated T cells with innate-like characteristics and effector functions. Among the various iNKT subsets, NKT17 is the only subset that produces the proinflammatory cytokine IL-17. But, how NKT17 cells acquire this ability and what would selectively trigger their activation remain incompletely understood. Here, we identified the cytokine receptor DR3 being specifically expressed on thymic NKT17 cells and mostly absent on other thymic iNKT subsets. Moreover, DR3 ligation promoted the in vivo activation of thymic NKT17 cells and provided costimulatory effects upon agonistic α-GalCer stimulation. Thus, we identified a specific surface marker for thymic NKT17 cells that triggers their activation and augments their effector functions both in vivo and in vitro. These findings provide new insights for deciphering the role and function of murine NKT17 cells and for understanding the development and activation mechanisms of iNKT cells in general.

JAK-STAT signaling in human disease: From genetic syndromes to clinical inhibition.

Since its discovery, the Janus kinase-signal transduction and activation of transcription (JAK-STAT) pathway has become recognized as a central mediator of widespread and varied human physiological processes. The field of JAK-STAT biology, particularly its clinical relevance, continues to be shaped by 2 important advances. First, the increased use of genomic sequencing has led to the discovery of novel clinical syndromes caused by mutations in JAK and STAT genes. This has provided insights regarding the consequences of aberrant JAK-STAT signaling for immunity, lymphoproliferation, and malignancy. In addition, since the approval of ruxolitinib and tofacitinib, the therapeutic use of JAK inhibitors (jakinibs) has expanded to include a large spectrum of diseases. Efficacy and safety data from over a decade of clinical studies have provided additional mechanistic insights while improving the care of patients with inflammatory and neoplastic conditions. This review discusses major advances in the field, focusing on updates in genetic diseases and in studies of clinical jakinibs in human disease.

JAK inhibitors: Ten years after.

The European Journal of Immunology was launched 50 years ago, coinciding with the discovery of many cytokines and growth factors and the emergence of an entirely new field of research. Ultimately, our knowledge about the biological activity of these factors allowed us to better understand how the immune system functions in the context of inflammatory and autoimmune diseases leading to the development of targeted biologic therapies. The study of cytokine signal transduction led to the discovery of Janus kinases (JAK), and the consideration of therapeutically targeting JAKs to treat immune and inflammatory diseases. This year also marks the tenth anniversary of the approval of the first JAK inhibitor (jakinib) and now there are a total of nine approved jakinibs for treatment of rheumatologic, dermatologic, gastrointestinal, and neoplastic indications and most recently COVID-19. Here, we summarized the discoveries that led to development of first-generation jakinibs, discussed some of the newer, possibly more selective jakinibs, as well as jakinibs that also target other kinases. We also illustrated the rationale behind the application of these drugs in the treatment of COVID-19 cytokine storm. In this review, we will discuss the clinical success of jakinibs, the gaps in our understanding of their biological activities as well as challenges in regard to their clinical application.

MicroRNA-221 and -222 modulate intestinal inflammatory Th17 cell response as negative feedback regulators downstream of interleukin-23.

MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.

Circulating TNF-like protein 1A (TL1A) is elevated early in rheumatoid arthritis and depends on TNF.

BACKGROUND: The tumor necrosis factor (TNF) superfamily cytokine TNF-like protein 1A (TL1A) and its receptor DR3 are essential for diverse animal models of autoimmune disease and may be pathogenic in rheumatoid arthritis (RA). However, the relationship of TL1A to disease duration, activity, and response to anti-TNF and other therapies in RA is not clear. METHODS: We measured soluble TL1A in synovial fluid (SF), serum, or plasma from RA first-degree relatives (FDRs) and in early RA and established disease. We measured the effects of anti-TNF and methotrexate (MTX) therapy on circulating TL1A from multiple independent RA treatment trials. We also determined the ability of a blocking anti-TL1A antibody to inhibit clinical disease and articular bone destruction in the murine collagen-induced arthritis (CIA) model of human RA. RESULTS: Soluble TL1A was specifically elevated in the blood and SF of patients with RA compared to patients with other diseases and was elevated early in disease and in at-risk anti-cyclic citrullinated peptide (CCP) (+) first-degree relatives (FDRs). Therapeutic TNF inhibition reduced serum TL1A in both responders and non-responders, whereas TL1A declined following MTX treatment only in responders. In murine CIA, TL1A blockade was clinically efficacious and reduced bone erosions. CONCLUSIONS: TL1A is specifically elevated in RA from early in the disease course and in at-risk FDRs. The decline in TL1A after TNF blockade suggests that TL1A levels may be a useful biomarker for TNF activity in RA. These results support the further investigation of the relationship between TL1A and TNF and TL1A blockade as a potential therapeutic strategy in RA.

Reduced monocyte and macrophage TNFSF15/TL1A expression is associated with susceptibility to inflammatory bowel disease.

Chronic inflammation in inflammatory bowel disease (IBD) results from a breakdown of intestinal immune homeostasis and compromise of the intestinal barrier. Genome-wide association studies have identified over 200 genetic loci associated with risk for IBD, but the functional mechanisms of most of these genetic variants remain unknown. Polymorphisms at the TNFSF15 locus, which encodes the TNF superfamily cytokine commonly known as TL1A, are associated with susceptibility to IBD in multiple ethnic groups. In a wide variety of murine models of inflammation including models of IBD, TNFSF15 promotes immunopathology by signaling through its receptor DR3. Such evidence has led to the hypothesis that expression of this lymphocyte costimulatory cytokine increases risk for IBD. In contrast, here we show that the IBD-risk haplotype at TNFSF15 is associated with decreased expression of the gene by peripheral blood monocytes in both healthy volunteers and IBD patients. This association persists under various stimulation conditions at both the RNA and protein levels and is maintained after macrophage differentiation. Utilizing a "recall-by-genotype" bioresource for allele-specific expression measurements in a functional fine-mapping assay, we localize the polymorphism controlling TNFSF15 expression to the regulatory region upstream of the gene. Through a T cell costimulation assay, we demonstrate that genetically regulated TNFSF15 has functional relevance. These findings indicate that genetically enhanced expression of TNFSF15 in specific cell types may confer protection against the development of IBD.

Somatic activating mutations in MAP2K1 cause melorheostosis.

Melorheostosis is a sporadic disease of uncertain etiology characterized by asymmetric bone overgrowth and functional impairment. Using whole exome sequencing, we identify somatic mosaic MAP2K1 mutations in affected, but not unaffected, bone of eight unrelated patients with melorheostosis. The activating mutations (Q56P, K57E and K57N) cluster tightly in the MEK1 negative regulatory domain. Affected bone displays a mosaic pattern of increased p-ERK1/2 in osteoblast immunohistochemistry. Osteoblasts cultured from affected bone comprise two populations with distinct p-ERK1/2 levels by flow cytometry, enhanced ERK1/2 activation, and increased cell proliferation. However, these MAP2K1 mutations inhibit BMP2-mediated osteoblast mineralization and differentiation in vitro, underlying the markedly increased osteoid detected in affected bone histology. Mosaicism is also detected in the skin overlying bone lesions in four of five patients tested. Our data show that the MAP2K1 oncogene is important in human bone formation and implicate MEK1 inhibition as a potential treatment avenue for melorheostosis.

RORα-expressing T regulatory cells restrain allergic skin inflammation.

Atopic dermatitis is an allergic inflammatory skin disease characterized by the production of the type 2 cytokines in the skin by type 2 innate lymphoid cells (ILC2s) and T helper 2 (TH2) cells, and tissue eosinophilia. Using two distinct mouse models of atopic dermatitis, we show that expression of retinoid-related orphan receptor α (RORα) in skin-resident T regulatory cells (Tregs) is important for restraining allergic skin inflammation. In both models, targeted deletion of RORα in mouse Tregs led to exaggerated eosinophilia driven by interleukin-5 (IL-5) production by ILC2s and TH2 cells. Expression of RORα in skin-resident Tregs suppressed IL-4 expression and enhanced expression of death receptor 3 (DR3), which is the receptor for tumor necrosis factor (TNF) family cytokine, TNF ligand-related molecule 1 (TL1A), which promotes Treg functions. DR3 is expressed on both ILC2s and skin-resident Tregs Upon deletion of RORα in skin-resident Tregs, we found that Tregs were no longer able to sequester TL1A, resulting in enhanced ILC2 activation. We also documented higher expression of RORα in skin-resident Tregs than in peripheral blood circulating Tregs in humans, suggesting that RORα and the TL1A-DR3 circuit could be therapeutically targeted in atopic dermatitis.

Cleavage of TL1A Differentially Regulates Its Effects on Innate and Adaptive Immune Cells.

TNF superfamily cytokines play major roles in the regulation of adaptive and innate immunity. The TNF superfamily cytokine TL1A (TNFSF15), through its cognate receptor DR3 (TNFRSF25), promotes T cell immunity to pathogens and directly costimulates group 2 and 3 innate lymphoid cells. Polymorphisms in the TNFSF15 gene are associated with the risk for various human diseases, including inflammatory bowel disease. Like other cytokines in the TNF superfamily, TL1A is synthesized as a type II transmembrane protein and cleaved from the plasma membrane by metalloproteinases. Membrane cleavage has been shown to alter or abrogate certain activities of other TNF family cytokines; however, the functional capabilities of membrane-bound and soluble forms TL1A are not known. Constitutive expression of TL1A in transgenic mice results in expansion of activated T cells and promotes intestinal hyperplasia and inflammation through stimulation of group 2 innate lymphoid cells. Through the generation of membrane-restricted TL1A-transgenic mice, we demonstrate that membrane TL1A promotes expression of inflammatory cytokines in the lung, dependent upon DR3 expression on T cells. Soluble TL1A alone was unable to produce this phenotype but was still able to induce intestinal type 2 inflammation independently of T cells. These data suggest differential roles for membrane and soluble TL1A on adaptive and innate immune cells and have implications for the consequences of blocking these two forms of TL1A.

TNF superfamily cytokines in the promotion of Th9 differentiation and immunopathology.

The tumor necrosis factor (TNF) receptors and their corresponding cytokine ligands have been implicated in many aspects of the biology of immune functions. TNF receptors have key roles during various stages of T cell homeostasis. Many of them can co-stimulate lymphocyte proliferation and cytokine production. Additionally, several TNF cytokines can regulate T cell differentiation, including promoting Th1, Th2, Th17, and more recently the newly described Th9 subset. Four TNF family cytokines have been identified as regulators for IL-9 production by T cells. OX40L, TL1A, and GITRL can promote Th9 formation but can also divert iTreg into Th9, while 4-1BBL seems to inhibit IL-9 production from iTreg and has not been studied for its ability to promote Th9 generation. Regulation of IL-9 production by TNF family cytokines has repercussions in vivo, including enhancement of anti-tumor immunity and immunopathology in allergic lung and ocular inflammation. Regulating T cell production of IL-9 through blockade or agonism of TNF family cytokine receptors may be a therapeutic strategy for autoimmune and allergic diseases and in tumor.

Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction.

Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.

The TNF-family cytokine TL1A: from lymphocyte costimulator to disease co-conspirator.

Originally described in 2002 as a T cell-costimulatory cytokine, the tumor necrosis factor family member TNF-like factor 1A (TL1A), encoded by the TNFSF15 gene, has since been found to affect multiple cell lineages through its receptor, death receptor 3 (DR3, encoded by TNFRSF25) with distinct cell-type effects. Genetic deficiency or blockade of TL1A-DR3 has defined a number of disease states that depend on this cytokine-receptor pair, whereas excess TL1A leads to allergic gastrointestinal inflammation through stimulation of group 2 innate lymphoid cells. Noncoding variants in the TL1A locus are associated with susceptibility to inflammatory bowel disease and leprosy, predicting that the level of TL1A expression may influence host defense and the development of autoimmune and inflammatory diseases.

The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells.

A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk(-/-) CD4(+) T cells develop higher percentages of functional FoxP3(+) cells, associated with increased sensitivity to IL-2. Itk(-/-) CD4(+) T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk(-/-) cells also exhibit reduced IL-2-induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4(+) T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4(+) T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells.

TL1A and DR3, a TNF family ligand-receptor pair that promotes lymphocyte costimulation, mucosal hyperplasia, and autoimmune inflammation.

DR3 (TNFRSF25) is a member of the tumor necrosis factor receptor (TNFR) superfamily expressed primarily on lymphocytes and is a receptor for the TNF family cytokine TL1A (TNFSF15). DR3 costimulates T-cell activation, but it is unique among these receptors in that it signals through an intracytoplasmic death domain and the adapter protein TRADD (TNFR-associated death domain). TL1A costimulates T cells to produce a wide variety of cytokines and can promote expansion of activated and regulatory T cells in vivo. Studies in mice deficient in DR3 or TL1A or in animals treated with antibodies that block the activity of TL1A have revealed a specific role for DR3 in enhancing effector T-cell proliferation at the site of tissue inflammation in autoimmune disease models. DR3 appears to be required in autoimmune disease models dependent on a variety of different T-cell subsets and also invariant natural killer T (iNKT) cells. Chronic expression of TL1A induces a distinct interleukin-13-dependent pathology in the small intestine marked by goblet cell hyperplasia and other features associated with allergic and anti-parasitic responses. These studies suggest that TL1A may be a viable target for therapies designed to inhibit the T-cell-dependent component of diverse autoimmune diseases.

Sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 are vital to recovery from anaphylactic shock in mice.

Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors (S1PR1-S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2-/- mice had a rapid recovery from anaphylaxis. In contrast, Sphk1-/- mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into Sphk1-/- mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of S1pr2-/- mice from anaphylaxis, whereas S1pr2+/- mice showed partial recovery. Unlike Sphk2-/- mice, Sphk1-/- and S1pr2-/- mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.

Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi.

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.

The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.

DR3 (TRAMP, LARD, WSL-1, TNFRSF25) is a death-domain-containing tumor necrosis factor (TNF)-family receptor primarily expressed on T cells. TL1A, the TNF-family ligand for DR3, can costimulate T cells, but the physiological function of TL1A-DR3 interactions in immune responses is not known. Using DR3-deficient mice, we identified DR3 as the receptor responsible for TL1A-induced T cell costimulation and dendritic cells as the likely source for TL1A during T cell activation. Despite its role in costimulation, DR3 was not required for in vivo T cell priming, for polarization into T helper 1 (Th1), Th2, or Th17 effector cell subtypes, or for effective control of infection with Toxoplasma gondii. Instead, DR3 expression was required on T cells for immunopathology, local T cell accumulation, and cytokine production in Experimental Autoimmune Encephalomyelitis (EAE) and allergic lung inflammation, disease models that depend on distinct effector T cell subsets. DR3 could be an attractive therapeutic target for T cell-mediated autoimmune and allergic diseases.