Bhanu P. Pappu

T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORα and RORγ

T cell functional differentiation is mediated by lineage-specific transcription factors. T helper 17 (Th17) has been recently identified as a distinct Th lineage mediating tissue inflammation. Retinoic acid receptor-related orphan receptor gamma (ROR gamma) was shown to regulate Th17 differentiation; ROR gamma deficiency, however, did not completely abolish Th17 cytokine expression. Here, we report Th17 cells highly expressed another related nuclear receptor, ROR alpha, induced by transforming growth factor-beta and interleukin-6 (IL-6), which is dependent on signal transducer and activator of transcription 3. Overexpression of ROR alpha promoted Th17 differentiation, possibly through the conserved noncoding sequence 2 in Il17-Il17f locus. ROR alpha deficiency resulted in reduced IL-17 expression in vitro and in vivo. Furthermore, ROR alpha and ROR gamma coexpression synergistically led to greater Th17 differentiation. Double deficiencies in ROR alpha and ROR gamma globally impaired Th17 generation and completely protected mice against experimental autoimmune encephalomyelitis. Therefore, Th17 differentiation is directed by two lineage-specific nuclear receptors, ROR alpha and ROR gamma.

Molecular Antagonism and Plasticity of Regulatory and Inflammatory T Cell Programs

Regulatory T (Treg) and T helper 17 (Th17) cells were recently proposed to be reciprocally regulated during differentiation. To understand the underlying mechanisms, we utilized a Th17 reporter mouse with a red fluorescent protein (RFP) sequence inserted into the interleukin-17F (IL-17F) gene. Using IL-17F-RFP together with a Foxp3 reporter, we found that the development of Th17 and Foxp3(+) Treg cells was associated in immune responses. Although TGF-beta receptor I signaling was required for both Foxp3 and IL-17 induction, SMAD4 was only involved in Foxp3 upregulation. Foxp3 inhibited Th17 differentiation by antagonizing the function of the transcription factors RORgammat and ROR*. In contrast, IL-6 overcame this suppressive effect of Foxp3 and, together with IL-1, induced genetic reprogramming in Foxp3(+) Treg cells. STAT3 regulated Foxp3 downregulation, whereas STAT3, RORgamma, and ROR* were required for IL-17 expression in Treg cells. Our data demonstrate molecular antagonism and plasticity of Treg and Th17 cell programs.

CCR6 Regulates the Migration of Inflammatory and Regulatory T Cells

Th17 and regulatory T (Treg) cells play opposite roles in autoimmune diseases. However, the mechanisms underlying their proper migration to inflammatory tissues are unclear. In this study, we report that these two T cell subsets both express CCR6. CCR6 expression in Th17 cells is regulated by TGF-β and requires two nuclear receptors, RORα and RORγ. Th17 cells also express the CCR6 ligand CCL20, which is induced synergistically by TGF-β and IL-6, which requires STAT3, RORγ and IL-21. Th17 cells, by producing CCL20, promote migration of Th17 and Treg cells in vitro in a CCR6-dependent manner. Lack of CCR6 in Th17 cells reduces the severity of experimental autoimmune encephalomyelitis and Th17 and Treg recruitment into inflammatory tissues. Similarly, CCR6 on Treg cells is also important for their recruitment into inflammatory tissues. Our data indicate an important role of CCR6 in Treg and Th17 cell migration.

Toll-like Receptor 2 Signaling in CD4+ T Lymphocytes Promotes T Helper 17 Responses and Regulates the Pathogenesis of Autoimmune Disease

Toll-like receptors (TLRs) have previously been shown to play critical roles in the activation of innate immunity. Here, we describe that T cell expression of TLR2 regulates T helper 17 (Th17) cell responses. Stimulation with TLR2 agonists promoted Th17 differentiation in vitro and led to more robust proliferation and Th17 cytokine production. Using the experimental autoimmune encephalomyelitis (EAE) model, we found that TLR2 regulated Th17 cell-mediated autoimmunity in vivo and that loss of TLR2 in CD4(+) T cells dramatically ameliorated EAE. This study thus reveals a critical role of a TLR in the direct regulation of adaptive immune response and pathogenesis of autoimmune diseases.

TL1A–DR3 interaction regulates Th17 cell function and Th17-mediated autoimmune disease

T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A–DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A−/− dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A−/− animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A–DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.

Phosphorylation and ubiquitination of the IκB kinase complex by two distinct signaling pathways

The IκB kinase (IKK) complex serves as the master regulator for the activation of NF‐κB by various stimuli. It contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKα/β at its activation loop and the K63‐linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T‐cell receptor (TCR) stimulation. However, the phosphorylation of IKKα/β activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1‐independent manner and mediate IKKα/β phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKα/β and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.

Regulatory mechanisms of helper T cell differentiation: New lessons learned from interleukin 17 family cytokines

Interleukin 17 (IL-17) family consists of six cytokines in mammals. Among them, IL-17 and IL-17F are expressed by a novel subset of CD4+ helper T (Th) cells and play critical function in inflammation and autoimmunity. On the other hand, IL-17E, also called IL-25, has been associated with allergic responses. Here we summarize recent work by us as well as other investigators in understanding the regulation and function of these three cytokines. From these studies, IL-17 family cytokines may serve as novel targets for pharmaceutical intervention of immune and inflammatory diseases.

B Cell Lymphoma 10 Is Essential for FcεR-Mediated Degranulation and IL-6 Production in Mast Cells

The adaptor protein B cell lymphoma 10 (Bcl10) plays an essential role in the functions of the AgRs in T and B cells. In this study, we report that Bcl10 also plays an important role in mast cells. Bcl10 is expressed in mast cells. Although Bcl10-deficient mast cells undergo normal development, we demonstrate that Bcl10 is essential for specific functions of FcεR. Although Bcl10-deficient mast cells have normal de novo synthesis and release of the lipid mediator arachidonic acid, the mutant cells possess impaired FcεR-mediated degranulation, indicated by decreased serotonin release, and impaired cytokine production, measured by release of IL-6. In addition, Bcl10-deficient mice display impaired IgE-mediated passive cutaneous anaphylaxis. Moreover, although Bcl10-deficient mast cells have normal FcεR-mediated Ca2+ flux, activation of PI3K, and activation of the three types of MAPKs (ERKs, JNK, and p38), the mutant cells have markedly diminished FcεR-mediated activation of NF-κB and decreased activation of AP-1. Thus, Bcl10 is essential for FcεR-induced activation of AP-1, NF-κB, degranulation, and cytokine production in mast cells.

Potential role of CARMA1 in CD40-induced splenic B cell proliferation and marginal zone B cell maturation

NF‐κB activation through B cell receptor (BCR) ligation is critical for B cell development, survival and antigen‐mediated activation of B cells. CARD domain and MAGUK‐domain containing protein‐1 (CARMA1), recently identified adaptor molecule, has been shown to play an essential role in BCR‐induced NF‐κB activation. CARMA1‐deficient B cells fail to proliferate upon BCR stimulation, leading to defective humoral responses. Surprisingly, CARMA1‐deficient B cells are also defective in CD40‐induced proliferation. The mechanisms responsible for CD40‐induced proliferation defect have not yet been characterized. In this study, we show that signaling cascades activated by CD40 stimulation are largely unaffected in CARMA1‐deficient B cells. Instead, we have found that the defective proliferation of CARMA1‐deficient B cells is due to two events. First, CARMA1‐deficient B cells show defective cell‐cycle progression. Secondly, the numbers of marginal zone (MZ) B cells, which are the main responders upon CD40 stimulation, are greatly diminished in CARMA1‐deficient mice. Since B cell maturation requires basal signaling through BCR and NF‐κB activation, we propose that impaired BCR signaling in CARMA1‐deficient mice leads to defective maturation of MZ B cell population, which in turn, contributes to impaired proliferation upon CD40 stimulation.

Measurement of Interleukin‐17

Upon antigenic stimulation, naive CD4+ T cells undergo proliferation and differentiate into cytokine‐producing T helper (TH) effector cells. TH1 cells secrete effector cytokine IFN‐γ and regulate cell‐mediated immunity, whereas TH2 cells produce IL‐4, IL‐5, and IL‐13 cytokines, and mediate immunity against extracellular pathogens and allergic reactions. Recent studies have identified a novel TH subset, called TH17, THIL‐17, or inflammatory TH (THi) cells, characterized by the production of a proinflammatory cytokine, IL‐17, and regulating inflammatory responses. In this unit, we describe the protocols for the differentiation of mouse IL‐17‐expressing T cells in vitro, detection of IL‐17‐expressing T cells by intracellular cytokine staining, and measurement of IL‐17 secretion in culture supernatants by ELISA. Generation of IL‐17‐expressing T cells in vitro under defined culture conditions allows investigation of their differentiation regulation. Detection of IL‐17 in cell culture and tissue samples helps in monitoring inflammatory diseases and determining efficacy of therapeutic interventions. Curr. Protoc. Immunol. 79:6.25.1‐6.25.8.