Wassim Elyaman

IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells.

Transcription factor Foxp3 is critical for generating regulatory T cells (Treg cells). Transforming growth factor-β (TGF-β) induces Foxp3 and suppressive Treg cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible Treg cells. Here we show that IL-4 blocked the generation of TGF-β-induced Foxp3+ Treg cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9+IL-10+ T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9+IL-10+ T cells into recombination-activating gene 1–deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9+IL-10+ T cells were transferred with CD45RBhi CD4+ effector T cells. Thus IL-9+IL-10+ T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.

IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells

The development of T helper (TH)17 and regulatory T (Treg) cells is reciprocally regulated by cytokines. Transforming growth factor (TGF)-β alone induces FoxP3+ Treg cells, but together with IL-6 or IL-21 induces TH17 cells. Here we demonstrate that IL-9 is a key molecule that affects differentiation of TH17 cells and Treg function. IL-9 predominantly produced by TH17 cells, synergizes with TGF-β1 to differentiate naïve CD4+ T cells into TH17 cells, while IL-9 secretion by TH17 cells is regulated by IL-23. Interestingly, IL-9 enhances the suppressive functions of FoxP3+ CD4+ Treg cells in vitro, and absence of IL-9 signaling weakens the suppressive activity of nTregs in vivo, leading to an increase in effector cells and worsening of experimental autoimmune encephalomyelitis. The mechanism of IL-9 effects on TH17 and Tregs is through activation of STAT3 and STAT5 signaling. Our findings highlight a role of IL-9 as a regulator of pathogenic versus protective mechanisms of immune responses.

IL-4 inhibits TGF-β-induced Foxp3 + T cells and, together with TGF-β, generates IL-9 + IL-10 + Foxp3 − effector T cells

Transcription factor Foxp3 is critical for generating regulatory T cells (Treg cells). Transforming growth factor-β (TGF-β) induces Foxp3 and suppressive Treg cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible Treg cells. Here we show that IL-4 blocked the generation of TGF-β-induced Foxp3+ Treg cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9+IL-10+ T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9+IL-10+ T cells into recombination-activating gene 1–deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9+IL-10+ T cells were transferred with CD45RBhi CD4+ effector T cells. Thus IL-9+IL-10+ T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.

Localizing central nervous system immune surveillance: Meningeal antigen-presenting cells activate T cells during experimental autoimmune encephalomyelitis

The onset of neurological signs in experimental autoimmune encephalomyelitis is tightly associated with infiltration and reactivation of T cells in the central nervous system. The anatomic localization of the initial T cell‐antigen‐presenting cell (APC) interactions leading to reactivation of T cells in the central nervous system is, however, still unclear. We hypothesized that activated CD4+ T cells gain direct access to the subarachnoid space and become reactivated on encounter with cognate antigen in this compartment.

Robust tumor immunity to melanoma mediated by interleukin-9–producing T cells

Interleukin-9 (IL-9) is a T cell cytokine that acts through a γC-family receptor on target cells and is associated with inflammation and allergy. We determined that T cells from mice deficient in the T helper type 17 (TH17) pathway genes encoding retinoid-related orphan receptor γ (ROR-γ) and IL-23 receptor (IL-23R) produced abundant IL-9, and we found substantial growth inhibition of B16F10 melanoma in these mice. IL-9–blocking antibodies reversed this tumor growth inhibition and enhanced tumor growth in wild-type (WT) mice. Il9r−/− mice showed accelerated tumor growth, and administration of recombinant IL-9 (rIL-9) to tumor-bearing WT and Rag1−/− mice inhibited melanoma as well as lung carcinoma growth. Adoptive transfer of tumor-antigen–specific TH9 cells into both WT and Rag1−/− mice suppressed melanoma growth; this effect was abrogated by treatment with neutralizing antibodies to IL-9. Exogenous rIL-9 inhibited tumor growth in Rag1−/− mice but not in mast-cell–deficient mice, suggesting that the targets of IL-9 in this setting include mast cells but not T or B cells. In addition, we found higher numbers of TH9 cells in normal human skin and blood compared to metastatic lesions of subjects with progressive stage IV melanoma. These results suggest a role for IL-9 in tumor immunity and offer insight into potential therapeutic strategies.

Monocytes from Patients with Type 1 Diabetes Spontaneously Secrete Proinflammatory Cytokines Inducing Th17 Cells

Autoimmune diseases including type 1 diabetes (T1D) are thought to have a Th1/Th17 bias. The underlying mechanisms driving the activation and differentiation of these proinflammatory T cells are unknown. We examined the monocytes isolated directly from the blood of T1D patients and found they spontaneously secreted the proinflammatory cytokines IL-1β and IL-6, which are known to induce and expand Th17 cells. Moreover, these in vivo-activated monocytes from T1D subjects induced more IL-17-secreting cells from memory T cells compared with monocytes from healthy control subjects. The induction of IL-17-secreting T cells by monocytes from T1D subjects was reduced in vitro with a combination of an IL-6-blocking Ab and IL-1R antagonist. In this study, we report a significant although modest increase in the frequency of IL-17-secreting cells in lymphocytes from long-term patients with T1D compared with healthy controls. These data suggest that the innate immune system in T1D may drive the adaptive immune system by expanding the Th17 population of effector T cells.

TGF-β Induces IL-9 Production from Human Th17 Cells

The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-β and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-β during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-β to the Th17-inducing cytokines (IL-1β, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-β–dependent coexpression of IL-9 and IL-17 was identified to be IL-1β. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1β. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9+IL-17+ cells. These data demonstrate the presence of IL-17+IL-9+ CD4 cells induced by IL-1β that may play a role in human autoimmune disease.

Notch Receptors and Smad3 Signaling Cooperate in the Induction of Interleukin-9-Producing T Cells

Interleukin 9 (IL-9) is a pleiotropic cytokine that can regulate autoimmune responses by enhancing regulatory CD4(+)FoxP3(+) T regulatory (Treg) cell survival and T helper 17 (Th17) cell proliferation. Here, we analyzed the costimulatory requirements for the induction of Th9 cells, and demonstrated that Notch pathway cooperated with TGF-β signaling to induce IL-9. Conditional ablation of Notch1 and Notch2 receptors inhibited the development of Th9 cells. Notch1 intracellular domain (NICD1) recruited Smad3, downstream of TGF-β cytokine signaling, and together with recombining binding protein (RBP)-Jκ bound the Il9 promoter and induced its transactivation. In experimental autoimmune encephalomyelitis (EAE), Jagged2 ligation regulated clinical disease in an IL-9-dependent fashion. Signaling through Jagged2 expanded Treg cells and suppressed EAE when administered before antigen immunization, but worsened EAE when administered concurrently with immunization by favoring Th17 cell expansion. We propose that Notch and Smad3 cooperate to induce IL-9 and participate in regulating the immune response.

Jagged1 and Delta1 Differentially Regulate the Outcome of Experimental Autoimmune Encephalomyelitis

Notch signaling plays an important role during T cell development in the thymus and in T cell activation but the role of Notch in autoimmunity is not clear. We investigated the role of Jagged1 and Delta1 in experimental autoimmune encephalomyelitis. During experimental autoimmune encephalomyelitis, Delta1 expression is up-regulated on dendritic cells and B cells after priming while Jagged1 is up-regulated only on dendritic cells. Administration of anti-Jagged1 Ab exacerbated clinical disease while that of anti-Delta1 Ab reduced the severity of the clinical disease. In contrast, administration of Jagged1-Fc protected from disease, that of Delta1-Fc exacerbated disease. Treatment with Jagged1-Fc was associated with increased IL-10-producing Ag-specific cells in the CNS, while anti-Jagged1 decreased the frequency of IL-10-producing cells. Treatment with Delta1-Fc increased Th1 cells in the CNS, while anti-Delta-1 decreased the frequency of Th1 cells. Manipulation of Delta1 or Jagged1 had no effect on the frequency of Th17 cells or FoxP3+ cells. Moreover, Jagged1 may play a role in CNS homeostasis because murine astrocytes specifically express Jagged1 that is up-regulated by TGF-β, whereas IFN-γ, TNF-α, and IL-17 decrease Jagged1 expression. Our study provides novel data about differential roles of Notch ligands in regulating inflammation in the periphery as well as in the CNS.

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood–derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2–DQ8 heterodimer–restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.