Erin E. West

T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells.

Innate immune crosstalk in T cells The classical view of immune activation is that innate immune cells, such as macrophages and dendritic cells, recognize invading microbes and then alert adaptive immune cells, such as T cells, to respond. Arbore et al. now show that innate and adaptive immunity converge in human and mouse T cells. Activated T cells express components of the complement cascade, which in turn leads to the assembly of NLRP3 inflammasomes—both critical components of innate immunity that help hosts detect and eliminate microbes. In T cells, complement and inflammasomes work together to push T cells to differentiate into a specialized subset of T cells important for eliminating intracellular bacteria. Science, this issue p. 10.1126/science.aad1210 Complement and NLRP3 inflammasomes work together to promote T helper 1 cell differentiation. INTRODUCTION The inflammasomes and the complement system are traditionally viewed as quintessential components of innate immunity required for the detection and elimination of pathogens. Assembly of the NLRP3 inflammasome in innate immune cells controls the maturation of interleukin (IL)–1β, a proinflammatory cytokine critical to host defense, whereas activation of the liver-derived complement key components C3 and C5 in serum leads to opsonization and removal of microbes and induction of the inflammatory reaction. Recent studies, however, have highlighted an unanticipated direct role for complement C3 also in human T cell immunity: The anaphylatoxin C3a receptor (C3aR) and the complement regulator CD46 (which binds C3b) are critical checkpoints in human T cell lineage commitment, and they control initiation and resolution of T helper 1 (TH1) responses in an autocrine fashion via T cell–derived and intracellularly activated C3. We explored a novel functional cross-talk of complement with the NLRP3 inflammasome within CD4+ T cells and determined how the cooperation between these two “classically” innate systems directly affects interferon-γ (IFN-γ) production by adaptive immune cells. RATIONALE Given the critical role of intracellular C3 activation in human TH1 responses and the importance of C5 activation products in inflammation, we investigated whether human CD4+ T cells also harbor an “intracellular C5 activation system” and by what means this system may contribute to effector responses by using C5aR1 and C5aR2 agonists and antagonists, T cells from patients with cryopyrin-associated periodic syndromes (CAPS), and mouse models of infection and autoimmunity. RESULTS Human CD4+ T cells expressed C5 and generated increased intracellular C5a upon T cell receptor activation and CD46 autocrine costimulation. Subsequent engagement of the intracellular C5aR1 by C5a induced the generation of reactive oxygen species (ROS) and the unexpected assembly of a functional NLRP3 inflammasome in CD4+ T cells, whereas the surface-expressed C5aR2 negatively controlled this process. NLRP3 inflammasome–dependent autocrine IL-1β secretion and activity were required for optimal IFN-γ production by T cells; consequently, dysregulation of NLRP3 function in these cells affected their normal effector responses. For example, mutated, constitutively active NLRP3 in T cells from patients with CAPS induced hyperactive TH1 responses that could be normalized with a NLRP3 inhibitor. The in vivo importance of a T cell–intrinsic NLRP3 inflammasome was further supported by the finding that IFN-γ production by Nlrp3–/– CD4+ T cells was significantly reduced during viral infections in mice and that diminished TH1 induction due to lack of NLRP3 function in a CD4+ T cell transfer model of colitis led to uncontrolled TH17 infiltration and/or expansion in the intestine and aggravated disease. CONCLUSION Our results demonstrate that the regulated cross-talk between intracellularly activated complement components (the “complosome”) and the NLRP3 inflammasome is fundamental to human TH1 induction and regulation. The finding that established innate immune pathways are also operative in adaptive immune cells and orchestrate immunological responses contributes to our understanding of immunobiology and immune system evolution. In addition, the results suggest that the complement-NLRP3 axis in T cells represents a novel therapeutic target for the modulation of TH1 activity in autoimmunity and infection. An intrinsic complement-NLRP3 axis regulates human TH1 responses. T cell receptor activation and CD46 costimulation trigger NLRP3 expression and intracellular C5a generation. Subsequent intracellular C5aR1 engagement induces ROS production (and possibly IL1B gene transcription) and NLRP3 assembly, which in turn mediates IL-1β maturation. Autocrine IL-1β promotes TH1 induction (IFN-γ production) but restricts TH1 contraction (IL-10 coexpression). C5aR2 cell surface activation by secreted C5a negatively controls these events via undefined mechanisms. Dysfunction of this system contributes to impaired TH1 responses in infection or increased TH17 responses during intestinal inflammation. The NLRP3 inflammasome controls interleukin-1β maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4+ T cells and initiates caspase-1–dependent interleukin-1β secretion, thereby promoting interferon-γ production and T helper 1 (TH1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to “innate immune cells” but is an integral component of normal adaptive TH1 responses.

Opposing actions of IL-2 and IL-21 on Th9 differentiation correlate with their differential regulation of BCL6 expression

Significance Interleukin-9 (IL-9) is a γc-family cytokine produced by Th9 cells that regulates a range of immune responses, including allergic inflammation. We show that IL-2 via STAT5 is required for Th9 differentiation. IL-2 inhibits B cell lymphoma 6 (BCL6), which inhibits Th9 differentiation, whereas IL-21 induces BCL6. BCL6 bound near STAT5 and STAT6 binding sites, including at the Il9 (gene encoding IL-9) promoter, and BCL6 binding increased and STAT binding decreased after treatment with anti–IL-2/IL-2R. Thus, IL-2 and IL-21 have opposing actions on BCL6 expression, which inversely correlates with Th9 differentiation and IL-9 production, with implications for controlling Th9 differentiation and potentially allergic inflammation. Interleukin 9 (IL-9) is a γc-family cytokine that is highly produced by T-helper 9 (Th9) cells and regulates a range of immune responses, including allergic inflammation. Here we show that IL-2–JAK3–STAT5 signaling is required for Th9 differentiation, with critical STAT5 binding sites in the Il9 (the gene encoding IL-9) promoter. IL-2 also inhibited B cell lymphoma 6 (BCL6) expression, and overexpression of BCL6 impaired Th9 differentiation. In contrast, IL-21 induced BCL6 and diminished IL-9 expression in wild-type but not Bcl6−/− cells, and Th9 differentiation was increased in Il21−/− and Il21r−/− T cells. Interestingly, BCL6 bound in proximity to many STAT5 and STAT6 binding sites, including at the Il9 promoter. Moreover, there was increased BCL6 and decreased STAT binding at this site in cells treated with blocking antibodies to IL-2 and the IL-2 receptor, suggesting a possible BCL6–STAT5 binding competition that influences IL-9 production. BCL6 binding was also increased when cells were Th9-differentiated in the presence of IL-21. Thus, our data reveal not only direct IL-2 effects via STAT5 at the Il9 gene, but also opposing actions of IL-2 and IL-21 on BCL6 expression, with increased BCL6 expression inhibiting IL-9 production. These data suggest a model in which increasing BCL6 expression decreases efficient Th9 differentiation, indicating possible distinctive approaches for controlling this process.

The Cytokines IL-21 and GM-CSF Have Opposing Regulatory Roles in the Apoptosis of Conventional Dendritic Cells

Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.

Persistent Cytomegalovirus-Specific Memory Responses in the Lung Allograft and Blood following Primary Infection in Lung Transplant Recipients

Primary CMV infection in lung transplant recipients (LTRs) is associated with increased mortality. We studied 22 donor CMV-positive, recipient-negative (D+R−) LTRs for the development of posttransplant CMV-specific immunity. We found that 13 of 22 D+R− LTRs (59.1%) seroconverted (CMV IgG Ab+). Using pooled peptides of the immunodominant CMV Ags pp65 and IE1, we detected CMV-specific CD8+IFN-γ+ T cells in the PBMC of 90% of seroconverted individuals following primary infection by intracellular cytokine staining. In contrast, few seroconverters had detectable CMV-specific CD4+IFN-γ+ T cells during viral latency. However, the majority of IgG+ LTRs demonstrated CMV-specific CD4+ and CD8+ T cell proliferative responses from PBMC, with CD4+IFN-γ+ T cells detectable upon re-expansion. Examination of lung allograft mononuclear cells obtained by bronchoalveolar lavage revealed both CMV-specific CD4+ and CD8+IFN-γ+ T cells, including patients from whom CD4+IFN-γ+ T cells were simultaneously undetectable in the PBMC, suggesting differential effector memory populations between these compartments. Moreover, both responses in the PBMC and lung allograft were found to persist, despite substantial immunosuppression, long after primary infection. Clinical correlation in this cohort demonstrated that the acquisition of CMV immunity was associated with freedom from CMV disease (p ≤ 0.009) and preservation of allograft function (p ≤ 0.02) compared with those who failed to develop CMV immunity. Together, our data reveal immunologic heterogeneity in D+R− LTRs, with the development and persistence of primary CMV responses that may provide clinical benefit.

Differential CMV-Specific CD8+ Effector T Cell Responses in the Lung Allograft Predominate over the Blood during Human Primary Infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient− (D+R−) individuals. In 15 D+R− LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA− during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA− phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.

TSLP: A Key Regulator of Asthma Pathogenesis.

Asthma is a complex disorder of the airways that is characterized by T helper type 2 (Th2) inflammation. The pleiotrophic cytokine TSLP has emerged as an important player involved in orchestrating the inflammation seen in asthma and other atopic diseases. Early research elucidated the role of TSLP on CD4+ T cells, and recent work has revealed the impact of TSLP on multiple cell types. Furthermore, TSLP plays an important role in the sequential progression of atopic dermatitis to asthma, clarifying the key role of TSLP in the pathogenesis of asthma, a finding with therapeutic implications.

Thymic Stromal Lymphopoietin and Interleukin-4 Mediate the Pathogenesis of Halothane-Induced Liver Injury in Mice

Liver eosinophilia has been associated with incidences of drug‐induced liver injury (DILI) for more than 50 years, although its role in this disease has remained largely unknown. In this regard, it was recently shown that eosinophils played a pathogenic role in a mouse model of halothane‐induced liver injury (HILI). However, the signaling events that drove hepatic expression of eosinophil‐associated chemokines, eotaxins, eosinophil infiltration, and subsequent HILI were unclear. We now provide evidence implicating hepatic epithelial‐derived cytokine thymic stromal lymphopoietin (TSLP) and type 2 immunity, in particular, interleukin‐4 (IL‐4) production, in mediating hepatic eosinophilia and injury during HILI. TSLP was constitutively expressed by mouse hepatocytes and increased during HILI. Moreover, the severity of HILI was reduced in mice deficient in either the TSLP receptor (TSLPR) or IL‐4 and was accompanied by decreases in serum levels of eotaxins and hepatic eosinophilia. Similarly, concanavalin A–induced liver injury, where type 2 cytokines and eosinophils play a significant role in its pathogenesis, was also reduced in TSLPR‐deficient mice. Studies in vitro revealed that mouse and human hepatocytes produce TSLP and eotaxins in response to treatment with combinations of IL‐4 and proinflammatory cytokines IL‐1β and tumor necrosis factor alpha. Conclusion: This report provides the first evidence implicating roles for hepatic TSLP signaling, type 2 immunity, and eosinophilia in mediating liver injury caused by a drug. (Hepatology 2014;60:1741‐1752)

A TSLP-complement axis mediates neutrophil killing of methicillin-resistant Staphylococcus aureus

Thymic stromal lymphopoietin increases MRSA killing in a neutrophil- and complement-dependent manner. “Complement”ary MRSA fight Thymic stromal lymphopoietin (TSLP) is a cytokine thought to promote allergic responses; however, its role in fighting infectious diseases is less clear. Now, West et al. report that TSLP in the skin can enhance killing of methicillin-resistant Staphylococcus aureus (MRSA). TSLP enhances killing in both mouse and human neutrophils, in part through interactions with the complement system that induce reactive oxygen species in neutrophils. This enhanced killing is not limited to MRSA because TSLP also boosts killing of Streptococcus pyogenes. These data suggest that TSLP may augment innate immune cells and complement to fight bacterial infection. Community-acquired Staphylococcus aureus infections often present as serious skin infections in otherwise healthy individuals and have become a worldwide epidemic problem fueled by the emergence of strains with antibiotic resistance, such as methicillin-resistant S. aureus (MRSA). The cytokine thymic stromal lymphopoietin (TSLP) is highly expressed in the skin and in other barrier surfaces and plays a deleterious role by promoting T helper cell type 2 (TH2) responses during allergic diseases; however, its role in host defense against bacterial infections has not been well elucidated. We describe a previously unrecognized non-TH2 role for TSLP in enhancing neutrophil killing of MRSA during an in vivo skin infection. Specifically, we demonstrate that TSLP acts directly on both mouse and human neutrophils to augment control of MRSA. Additionally, we show that TSLP also enhances killing of Streptococcus pyogenes, another clinically important cause of human skin infections. Unexpectedly, TSLP mechanistically mediates its antibacterial effect by directly engaging the complement C5 system to modulate production of reactive oxygen species by neutrophils. Thus, TSLP increases MRSA killing in a neutrophil- and complement-dependent manner, revealing a key connection between TSLP and the innate complement system, with potentially important therapeutic implications for control of MRSA infection.

STAT5-mediated chromatin interactions in superenhancers activate IL-2 highly inducible genes: Functional dissection of the Il2ra gene locus

Significance Superenhancers regulate the expression of genes that specify cell type-specific development, but little is known regarding their function and regulation in vivo. Here, we study the cytokines IL-2 and IL-21, which critically control the immune response. These cytokines induce the binding of transcription factors STAT5 and STAT3, respectively, at superenhancers in a cytokine- and gene-specific manner. STAT5-bound superenhancers regulate genes highly induced by IL-2, with STAT5 mediating chromatin looping within such genes, including Il2ra, which mediates responsiveness to IL-2. By deleting three STAT5 binding sites that mediate IL-2–induced chromatin looping at the Il2ra locus, we demonstrate that superenhancer elements cooperatively control gene expression. Overall, we elucidate cytokine-dependent superenhancer function in general and provide detailed analysis of the Il2ra superenhancer. Cytokines critically control immune responses, but how regulatory programs are altered to allow T cells to differentially respond to distinct cytokine stimuli remains poorly understood. Here, we have globally analyzed enhancer elements bound by IL-2–activated STAT5 and IL-21–activated STAT3 in T cells and identified Il2ra as the top-ranked gene regulated by an IL-2–activated STAT5-bound superenhancer and one of the top genes regulated by STAT3-bound superenhancers. Moreover, we found that STAT5 binding was rapidly superenriched at genes highly induced by IL-2 and that IL-2–activated STAT5 binding induces new and augmented chromatin interactions within superenhancer-containing genes. Based on chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing data, we used CRISPR-Cas9 gene editing to target three of the STAT5 binding sites within the Il2ra superenhancer in mice. Each mutation decreased STAT5 binding and altered IL-2–induced Il2ra gene expression, revealing that individual elements within the superenhancer were not functionally redundant and that all were required for normal gene expression. Thus, we demonstrate cooperative utilization of superenhancer elements to optimize gene expression and show that STAT5 mediates IL-2–induced chromatin looping at superenhancers to preferentially regulate highly inducible genes, thereby providing new insights into the mechanisms underlying cytokine-dependent superenhancer function.

Complement and the Regulation of T Cell Responses

The complement system is an evolutionarily ancient key component of innate immunity required for the detection and removal of invading pathogens. It was discovered more than 100 years ago and was originally defined as a liver-derived, blood-circulating sentinel system that classically mediates the opsonization and lytic killing of dangerous microbes and the initiation of the general inflammatory reaction. More recently, complement has also emerged as a critical player in adaptive immunity via its ability to instruct both B and T cell responses. In particular, work on the impact of complement on T cell responses led to the surprising discoveries that the complement system also functions within cells and is involved in regulating basic cellular processes, predominantly those of metabolic nature. Here, we review current knowledge about complements role in T cell biology, with a focus on the novel intracellular and noncanonical activities of this ancient system.