T1/ST2 expression is enhanced on CD4+ T cells from schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo

Th cells are categorized into subsets based on the cytokine production of in vitro-differentiated Th populations. For in vivo-differentiated Th subsets, little is known about the heterogeneity of cytokine production in single cells. We recently described a molecule, T1/ST2, that is preferentially expressed on the surface of Th2 cells. Here we combined high-gradient magnetic cell separation with four-color single-cell cytometry to analyze simultaneously three intracellular cytokines and T1/ST2 surface expression on CD4+ cells from lungs containing granulomas induced by Schistosoma mansoni eggs. T1/ST2 was highly up-regulated on CD4+ T cells from hepatic granulomas and granulomatous lungs. T1/ST2+ cells from granulomatous lungs preferentially produced type 2 cytokines ex vivo. In the total CD4+ population, coexpression of type 1 and type 2 cytokines occurred frequently. However, such coproduction was drastically reduced in T1/ST2+ cells compared with T1/ST2- cells. Coexpression of type 1 and type 2 cytokines was also rare in cells simultaneously producing two cytokines of one type. These findings indicate that individual CD4+ T cells in vivo have different levels of commitment to a certain Th phenotype. Coexpression of two type 2 cytokines or production of one type 2 cytokine together with surface expression of T1/ST2 indicate advanced commitment to the Th2 phenotype.     

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.     

Chronic morphine treatment promotes specific Th2 cytokine production by murine T cells in vitro via a Fas/Fas ligand-dependent mechanism

Improper homeostasis of Th1 and Th2 cell differentiation can promote pathological immune responses such as autoimmunity and asthma. A number of factors govern the development of these cells including TCR ligation, costimulation, death effector expression, and activation-induced cell death (AICD). Although chronic morphine administration has been shown to selectively promote Th2 development in unpurified T cell populations, the direct effects of chronic morphine on Th cell skewing and cytokine production by CD4(+) T cells have not been elucidated. We previously showed that morphine enhances Fas death receptor expression in a T cell hybridoma and human PBL. In addition, we have demonstrated a role for Fas, Fas ligand (FasL), and TRAIL in promoting Th2 development via killing of Th1 cells. Therefore, we analyzed whether the ability of morphine to affect Th2 cytokine production was mediated by regulation of Fas, FasL, and TRAIL expression and AICD directly in purified Th cells. We found that morphine significantly promoted IL-4 and IL-13 production but did not alter IL-5 or IFN-gamma. Furthermore, morphine enhanced the mRNA expression of Fas, FasL and TRAIL and promoted Fas-mediated AICD of CD4(+) T cells. Additionally, blockade of Fas/FasL interaction by anti-FasL inhibited the morphine-induced production of IL-4 and IL-13 and AICD of CD4(+) T cells. These results suggest that morphine preferentially enhances Th2 cell differentiation via killing of Th1 cells in a Fas/FasL-dependent manner.     

A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression

ST2/ST2L, a member of the IL-1R gene family, is expressed by fibroblasts, mast cells, and Th2, but not Th1, cells. It exists in both membrane-bound (ST2L) and soluble forms (ST2). Although ST2L has immunoregulatory properties, its ligand, cellular targets, and mode of action remain unclear. Using a soluble ST2-human IgG fusion protein, we demonstrated that ST2 bound to primary bone marrow-derived macrophages (BMM) and that this binding was enhanced by treatment with LPS. The sST2 treatment of BMMs inhibited production of the LPS-induced proinflammatory cytokines IL-6, IL-12, and TNF-alpha but did not alter IL-10 or NO production. Treatment of BMMs with sST2 down-regulated expression of Toll-like receptors-4 and -1 but induced nuclear translocation of NF-kappaB. Administration of sST2 in vivo after LPS challenge significantly reduced LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-alpha. Conversely, blockade of endogenous ST2 through administration of anti-ST2 Ab exacerbated the toxic effects of LPS. Thus, ST2 has anti-inflammatory properties that act directly on macrophages. We demonstrate here a novel regulatory pathway for LPS-induced shock via the ST2-Toll-like receptor 4 route. This may be of considerable therapeutic potential for reducing the severity and pathology of inflammatory diseases.     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

Human IL-18 receptor and ST2L are stable and selective markers for the respective type 1 and type 2 circulating lymphocytes

CD4(+) (Th) and CD8(+) (Tc) T and NK lymphocytes can be divided into type 1 and 2 subsets according to their cytokine secretion profile. Studies on the role of lymphocyte subsets in human diseases have been hampered by the lack of stable surface markers to define them. Recently, we reported that ST2L and IL-18R are stably expressed on murine Th2 and Th1 cells, respectively. In this study, we generated Abs to human homologues of ST2L and IL-18R and tested them against Th1/Th2, Tc1/Tc2, and NK1/NK2 lines and PBMCs from healthy individuals. We show for the first time that ST2L and IL-18R are stable selective cell surface markers for human Th2/Tc2/NK2 and Th1/Tc1/NK1 lymphocytes, respectively. We then investigated PBMCs from HIV-infected patients and HIV-negative individuals, to test whether Abs to these two surface markers could be used directly to monitor lymphocyte subset distribution in human diseases. We found a clear Th1 to Th2 shift in the HIV-infected individuals, thus settling a long-standing controversy and include, for the first time, Tc and NK cells as well. Therefore, these cell surface molecules could serve as important determinants of the immune status of human diseases in general, and thereby could be useful for therapeutic monitoring and intervention.     

T1/ST2--an IL-1 receptor-like modulator of immune responses

Th2-associated factors such as IL-4 are involved in both the development of Th2 responses (via modulating Th2 cell differentiation) and in the effector phase of Th2 responses (via modulating macrophage activation). The IL-1 receptor-like protein ST2 (T1, Fit-1, or DER4) is expressed as a membrane-bound (ST2L) or secreted form (sST2), and has been clearly implicated as a regulator of both the development and effector phases of Th2-type responses. Here we analyze the mechanisms and therapeutic implications of the unique ability of ST2 to promote development and function of type 2 helper T cells through a positive feedback loop, as well as to act as a negative feedback modulator of macrophage pro-inflammatory function.     

IL-1beta-driven ST2L expression promotes maturation resistance in rapamycin-conditioned dendritic cells

Maturation resistance and tolerogenic properties can be conferred on human and murine dendritic cells (DC), crucial regulators of T cell responses, by exposure to rapamycin (RAPA), a "tolerance-sparing" immunosuppressive agent. Mechanisms underlying this acquired unresponsiveness, typified by diminished functional responses to TLR or CD40 ligation, have not been identified. We report that in vitro and in vivo conditioning of murine myeloid DC with RAPA elicits the de novo production of IL-1beta by otherwise phenotypically immature DC. Interestingly, IL-1beta production promotes overexpression of the transmembrane form of the IL-1R family member, IL-1R-like 1, also know as ST2 on RAPA-conditioned DC (RAPA-DC). ST2 is the recently identified receptor for IL-33, a cytokine favoring Th2 responses. In addition, transmembrane ST2, or ST2L, has been implicated as a potent negative regulator of TLR signaling. RAPA-DC generated from ST2-/- mice exhibited higher levels of costimulatory molecules (CD86) than wild-type RAPA-DC. Consistent with its regulatory function, IL-1beta-induced ST2L expression suppressed the responsiveness of RAPA-DC to TLR or CD40 ligation. Thus, as a result of their de novo production of IL-1beta, RAPA-DC up-regulate ST2L and become refractory to proinflammatory, maturation-inducing stimuli. This work identifies a novel mechanism through which a clinically important immunosuppressant impedes the capacity of DC to mature and consequently stimulate effector/adaptive T cell responses.     

CD8+ T lymphocytes regulating Th2 pathology escape neonatal tolerization

Transplantation tolerance induced by neonatal injection of semiallogeneic spleen cells is associated in several strain combinations with a pathological syndrome caused by Th2 differentiation of donor-specific CD4(+) T lymphocytes. We investigated the role of host CD8(+) T cells in the regulation of this Th2 pathology. IgE serum levels and eosinophilia significantly increased in BALB/c mice neonatally injected with (A/J x BALB/c)F(1) spleen cells when CD8(+) T cells were depleted by administration of anti-CD8 mAb or when beta(2)-microglobulin-deficient mice were used as recipients. In parallel, increased serum levels of IL-5 and IL-13 were measured in blood of tolerant CD8(+) T cell-deficient mice. Whereas neonatally injected mice were unable to generate anti-donor cytotoxic effectors, their CD8(+) T cells were as efficient as control CD8(+) T cells in reducing the severity of Th2 pathology and in restoring donor-specific cytotoxicity in vitro after in vivo transfer in beta(2)-microglobulin-deficient mice. Likewise, CD8(+) T cells from control and tolerant mice equally down-regulated the production of Th2 cytokines by donor-specific CD4(+) T cells in vitro. The regulatory activity of CD8(+) T cells depended on their secretion of IFN-gamma for the control of IL-5 production but not for IL-4 or IL-13. Finally, we found that CD8(+) T cells from 3-day-old mice were already able to down-regulate IL-4, IL-5, and IL-13 production by CD4(+) T cells. We conclude that regulatory CD8(+) T cells controlling Th2 responses are functional in early life and escape neonatal tolerization.     

Regulatory roles of IL-2 and IL-4 in H4/inducible costimulator expression on activated CD4+ T cells during Th cell development

We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4(+) T cells obtained from six different murine strains. BALB/c-activated CD4(+) T cells expressed approximately 10-fold more H4/ICOS on their surfaces and produced approximately 10-fold more IL-4 upon restimulation than C57BL/6-activated CD4(+) T cells. BALB/c naive CD4(+) T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4(+) T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4(+) T cells strongly down-regulated both H4/ICOS expression on activated CD4(+) T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4(+) T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4(+) T cells from C57BL/6 or IL-4(-/-) mice increased H4/ICOS expression on activated CD4(+) T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4(+) T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4(+) T cells.     

Role of IL-12 receptor beta 1 in regulation of T cell response by APC in experimental autoimmune encephalomyelitis

IL-12 was thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. However, we have recently found that IL-12 responsiveness, via IL-12Rbeta2, is not required in the induction of EAE. To determine the role of IL-12Rbeta1, a key subunit for the responsiveness to both IL-12 and IL-23, in the development of autoimmune diseases, we studied EAE in mice deficient in this subunit of IL-12R. IL-12Rbeta1(-/-) mice are completely resistant to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with an autoantigen-specific Th2 response. To study the mechanism underlying this Th2 bias, we cocultured purified CD4(+) T cells and APCs of MOG-immunized mice. We demonstrate that IL-12Rbeta1(-/-) APCs drive CD4(+) T cells of both wild-type and IL-12Rbeta1(-/-) mice to an Ag-induced Th2 phenotype, whereas wild-type APCs drive these CD4(+) T cells toward a Th1 type. IL-12Rbeta1(-/-) CD4(+) T cells, in turn, appear to exert an immunoregulatory effect on the capacity of wild-type APCs to produce IFN-gamma and TNF-alpha. Furthermore, decreased levels of IL-12p40, p35, and IL-23p19 mRNA expression were found in IL-12Rbeta1(-/-) APCs, indicating an autocrine pathway of IL-12/IL-23 via IL-12Rbeta1. IL-18 production and IL-18Ralpha expression are also significantly decreased in IL-12Rbeta1(-/-) mice immunized with MOG. We conclude that in the absence of IL-12Rbeta1, APCs play a prominent regulatory role in the induction of autoantigen-specific Th2 cells.     

IL-12, but not IFN-alpha, promotes STAT4 activation and Th1 development in murine CD4+ T cells expressing a chimeric murine/human Stat2 gene

Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4(+) Th1 development in both murine and human T cells, IFN-alphabeta drives Th1 development only in human cells. This IFN-alphabeta-dependent pathway is not conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T cells would provide the opportunity to more closely model specific human disease states that rely on CD4(+) T cell responses to IFN-alphabeta. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2 sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in murine CD4(+) T cells and was activated by murine IFN-alpha signaling. However, the murine/human STAT2 protein was insufficient to restore full IFN-alpha-driven Th1 development as defined by IFN-gamma expression. Furthermore, IL-12, but not IFN-alpha, promoted acute IFN-gamma secretion in collaboration with IL-18 stimulation in both CD4(+) and CD8(+) T cells. The inability of T cells to commit to Th1 development correlated with the lack of STAT4 phosphorylation in response to IFN-alpha. This finding suggests that, although the C terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-alphabetaR), it is not sufficient to restore this process through the murine IFNAR complex.     

An iminosugar N-pentafluorobenzyl-1-deoxynojirimycin as a novel potential immunosuppressant for the treatment of Th2-related diseases

Increased levels of Th2 cytokine interleukin-4 (IL-4) have been reported to be involved in the pathogenesis of the parasite Schistosoma japonicum (S. japonicum) infection or detected in the serum of the causative agent of acquired immunodeficiency syndrome (AIDS) patients. This correlates with a worsened outcome of AIDS. The inhibition of a Th2 type response might aid in the treatment of these Th2-related diseases. Previously, we found that N-pentafluorobenzyl-1-deoxynojirimycin (5F-DNM), a new derivative of 1-deoxynojirimycin (DNM) (an inhibitor of the glycoprotein processing enzymes, glucosidase I and II), had specific inhibition effects on human CD4(+) T cells. In this study, we further found that 5F-DNM not only markedly inhibited in vitro IL-4 production from human PBMCs, CD4(+) T cells and mouse splenocytes but also strongly inhibited the production of IL-4 in splenocytes from a mouse model of S. japonicum infection. The numbers of S. japonicum worms were significantly decreased in vivo upon the treatment of mice with 5F-DNM. We demonstrated the mechanism of 5F-DNM effects on CD4(+) T cells acts via the inhibition of the IL-4/JAK1/STAT6 signaling pathway. Moreover, 5F-DNM was found to induce CD4 internalization (transfer from the cellular surface to the cytoplasm) in CD4(+) T cells and had no significant effects on the overall expression levels of CD4. These findings indicate that 5F-DNM might be used as a potential candidate for the treatment of S. japonicum parasitic infection, AIDS and other Th2-related diseases.     

Induction of a CD4+ T regulatory type 1 response by cyclooxygenase-2-overexpressing glioma

PGE(2), synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4(+) T regulatory type 1 (Tr1) induction by CD11c(+) mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Ralpha expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-beta with negligible IL-4 secretion by CD4(+) T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4(+) T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE(2). Taken together, robust levels of PGE(2) from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.     

Analysis of IL-12 receptor beta 2 chain expression of circulating T lymphocytes in patients with atopic asthma

Th2 cell predominance relative to Th1 cells contributes to pathological immune responses in patients with atopic asthma. IL-12 is a key cytokine in the induction of Th1 cells, and downregulation of IL-12 production is reported in these patients. However, IL-12 receptor expression of their T lymphocytes has not been clarified. In this study, expression of IL-12 receptor beta 2 on T cells and secretion of cytokines which affect IL-12 receptor beta 2 expression by their PBMC were examined. We found that IL-12 receptor beta 2 expression of the T cells is reduced. This is partly due to the diminished production of IL-12 and enhanced secretion of IL-4 by their PBMC. IL-18 production is not significantly modulated in these patients. Furthermore, intrinsic defects of the CD4(+) T cells, which reduce their IL-12 receptor beta 2 expression in response to IL-12 and/or IL-18 stimulation, are evident and are importantly involved in the Th1/Th2 imbalance of patients with atopic asthma.