Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.     

B7H1-Ig fusion protein activates the CD4+ IFN-gamma receptor+ type 1 T regulatory subset through IFN-gamma-secreting Th1 cells

It has been demonstrated in our previous work that, in the human skin-grafting model, the expression of costimulatory molecule B7H1 (PD-L1) by keratinocytes plays an essential role in inducing local tolerance via activation of IL-10-secreting T cells. This study further analyzes the role of B7H1 in differentiation of type 1 T regulatory (Tr1) cells and explores underlying mechanisms. Mouse fusion protein B7H1-Ig is used, together with immobilized anti-CD3 mAb, to costimulate the purified naive CD4+ T cells. B7H1-Ig-treated CD4+ T cells were found to activate a characteristic Tr1 population possessing a CD4+ CD25- Foxp3- CD45RBlow phenotype. These regulatory T cells strongly inhibited the Th1-dominated MLR by secretion of IL-10 and TGF-beta. Moreover, B7H1-treated Tr1 cells also resulted in suppressed clinical scores and demyelination when adoptively transferred into mice with experimental allergic encephalomyelitis. Furthermore, analysis of the cytokine profile indicated that there were two differential reaction patterns during the B7H1-Ig-induced Tr1 development. These two patterns were characterized by activation of IFN-gammaR+ IL-10R- Th1 and IFN-gammaR+ IL-10R+ Tr1 cells, respectively. Secretion of IFN-gamma by Th1 and the expression of IFN-gammaR on Tr1 were critical for further Tr1 differentiation, as demonstrated by mAb blocking and by analysis in IFN-gamma(-/-) mice. In conclusion, B7H1 is capable of inducing Tr1 differentiation from naive CD4+ T cells by coactivation in an IFN-gamma- or Th1-dependent manner. Our study may shed some light upon the clinical usage of B7H1 as a therapeutic reagent for induction of tolerance.     

T regulatory cells 1 inhibit a Th2-specific response in vivo

We recently described a new population of CD4(+) regulatory T cells (Tr1) that inhibits proliferative responses of bystander T cells and prevents colitis induction in vivo through the secretion of IL-10. IL-10, which had been primarily described as a Th2-specific cytokine inhibiting Th1 responses, has displayed in several models a more general immune suppression on both types of effector T cell responses. Using an immediate hypersensitivity model in which BALB/c mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of OVA-specific Tr1 T cell clones to inhibit OVA-specific cytokines and Ab responses. In contrast to Th2 or Th1 T cell clones, transfer of Tr1 T cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses, whereas IgG1 and IgG2a synthesis were not affected. This specific inhibition was mediated in part through IL-10 secretion as anti-IL-10 receptor Abs treatment reverted the inhibitory effect of Tr1 T cell clones. Although specifically targeted to IgE responses, Tr1 clones' inhibitory effects were more profound as they affected Ag-specific Th2 cell priming both in term of proliferative responses and cytokine secretion. These results suggest that regulatory T cells may play a fundamental role in maintaining the balance of the immune system to prevent allergic disorders.     

Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora

We showed previously that cecal bacterial Ag (CBA)-specific CD4(+) T cells induce colitis when transferred into SCID mice. The purpose of this study was to generate and characterize CBA-specific regulatory T cells in C3H/HeJBir (Bir) mice. CD4(+) T cells were stimulated with CBA-pulsed APC in the presence of IL-10 every 10-14 days. After four or more cycles, these T cells produced high levels of IL-10, low levels of IL-4 and IFN-gamma, and no IL-2, consistent with the phenotype of T regulatory-1 (Tr1) cells. Bir Tr1 cells proliferated poorly, but their proliferation was dependent on CD28-B7 interactions and was MHC class II-restricted. Transfer of Bir Tr1 cells into SCID mice did not result in colitis, and cotransfer of Bir Tr1 T cells with pathogenic Bir CD4(+) Th1 cells prevented colitis. Bir Tr1 cells inhibited proliferation and IFN-gamma production of a CBA-specific Th1 cell line in vitro. Such inhibition was partly due to IL-10 and TGFbeta1, but cognate interactions with either APCs or Th1 cells were also involved. Normal intestinal lamina propria CD4(+) T cells had Tr1-like activity when stimulated with CBA-pulsed APCs. We conclude that CD4(+) T cells with the properties of Tr1 cells are present in the intestinal lamina propria and hypothesize that these cells maintain intestinal immune homeostasis to the enteric flora.     

Restricted T cell expression of IL-2/IFN-gamma mRNA in human inflammatory disease

It has been difficult to demonstrate functionally distinct T cell populations in humans on the basis of cytokine secretion. As previous investigators have examined the T cell cytokine profile from immunized animals, we examined whether Th1 or Th2 type T cells could be identified in the peripheral blood or cerebrospinal fluid (CSF) immune compartments from subjects with or without inflammatory diseases. Using limiting dilution analysis and growth with PHA and IL-2/IL-4, we directly cloned a total of 177 T cells from the peripheral blood and CSF of seven subjects, four with inflammatory disease and three control subjects, and examined the cytokine message profile after stimulation with ionomycin and PMA. We found that most clones from both the peripheral blood and CSF express IL-1, IL-2, IL-4, IFN-gamma, or TNF-alpha cytokine mRNA after activation with ionomycin and PMA. All T cell clones tested produced TNF-alpha mRNA, and all but 14 produced IFN-gamma mRNA. As reported previously, Th0 cells, which produced IFN-gamma, IL-2, IL-4, and IL-5 mRNA, were found in most subjects. In striking contrast, Th1 cells, which expressed IL-2 and IFN-gamma but not IL-4 or IL-5 mRNA, were present in both peripheral blood and CSF of subjects with inflammatory disease but not found in peripheral blood or CSF of subjects without systemic inflammation. Th2 cells, expressing IL-4 and IL-5 but not IFN-gamma or IL-2 mRNA, were not found in any subject. These data present the first evidence for Th1 T cell clones in humans that may be associated with systemic inflammation.     

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.     

IFN-gamma is necessary but not sufficient for anti-CD40 antibody-mediated inhibition of the Th2 response to Schistosoma mansoni eggs

The injection of Schistosoma mansoni eggs into the footpads of mice results in a localized Th2 cytokine response and tissue eosinophilia. We examined whether treatment with CD40-activating Abs would block the development of Th2 cytokine responses and eosinophilic tissue pathology in this model. Seven days after C57BL/6 mice were injected with eggs and the FGK45 anti-CD40 Ab, Ag-specific synthesis of IL-4, IL-5, and IL-13 in lymph node culture was reduced (>10-fold) relative to control mice treated with eggs and rat IgG. In contrast, IFN-gamma and IL-12 were increased in both culture supernatants and in the serum. Similar changes in lymph node cytokine mRNA were observed in vivo, and tissue eosinophilia was reduced nearly 20-fold. Th2 cytokine responses in anti-CD40-treated IFN-gamma-/- and IL-12 p40-/- C57BL/6 mice were unaffected, although anti-CD40 induced high levels of systemic and local IFN-gamma production in both wild-type and IL-12 p40-/- mice. We conclude that CD40-activating treatments strongly reverse the immune phenotype generated in response to a classic, Th2-biasing stimulus and stimulate IFN-gamma through a novel IL-12-independent pathway. This model for Th1-deviating immune therapy may have relevance to the treatment of Th2-dependent diseases in general.     

Adenosine - cyclic AMP pathways and cytokine expression.

Adenosine and cAMP are potent modulators of immune-triggered cytokine production. Their effects overlap with regard to the inhibition of the pro-inflammatory cytokines TNF-alpha, IFN-gamma, IL-12, and the stimulation of production of the major anti-inflammatory cytokine IL-10. They may tentatively be considered to be upregulators of the production of Th2 cytokines (IL-10, IL-6), but downregulators of the production of Th1 cytokines (IL-2 and IFN-gamma). Cytokines produced in common by Th0, Th1 and Th2 cells are affected as well, although the low quantity and heterogeneity of the contemporary experimental data do not allow unambiguous conclusions to be drawn. Nevertheless, IL-3, IL-4, MIP-1alpha/beta and GM-CSF have usually been found to be inhibited, IL-5 stimulated, while IL-1 remains largely unaffected by adenosine or cAMP. These effects, and in particular the inhibition of TNF-alpha and stimulation of IL-10 expression, might be of therapeutic value in a variety of pathophysiological conditions.     

Proinflammatory properties of IL-4 in the intestinal microenvironment

IL-4 is involved in type 2 T helper cell (Th)2-type immune responses and, in some cases, can promote Th1 responses. However, the proinflammatory potential of IL-4 alone is unclear. In this study, we examined the ability of IL-4 to induce colitis after its overexpression in the colon using an adenoviral vector (Ad5) and compared results with those obtained after overexpression of IL-12, a cytokine implicated in several models of colitis. Overexpression of IL-4 or IL-12 caused a fatal colitis within 24 h in 60% of animals and was dose and strain dependent. IL-12-induced colitis was accompanied by the local expression of IFN-gamma and TNF-alpha but not IL-4 mRNA and protein. Conversely, IL-4-induced colitis was accompanied by the local expression of IL-4 and TNF-alpha but not IFN-gamma mRNA and protein. The Ad5-IL4-induced colitis did not persist beyond 3 days and was present in recombinase activation gene-2 (RAG-2)-/- mice but not in STAT6-/- mice. Acute lethal colitis induced by Ad5IL12 was T cell mediated and IFN-gamma receptor (IFN-gamma R) dependent. Furthermore, TNF-alpha was found to be important in the pathogenesis of Ad5IL-4 and Ad5IL-12-induced colitis. Results of this study indicate that IL-4 alone can act as a proinflammatory cytokine in the gut of normal mice, inducing a rapid onset and short-lived colonic injury while maintaining a Th2-type cytokine profile that functions via a local T cell-independent mechanism involving TNF-alpha.     

Dendritic cells transduced with SOCS-3 exhibit a tolerogenic/DC2 phenotype that directs type 2 Th cell differentiation in vitro and in vivo

Dendritic cells (DCs) have been suggested to direct a type of Th differentiation through their cytokine profile, e.g., high IL-12/IL-23 for Th1 (named DC1/immunogenic DCs) and IL-10 for Th2 (DC2/tolerogenic DCs). Suppressor of cytokine signaling (SOCS)-3 is a potent inhibitor of Stat3 and Stat4 transduction pathways for IL-23 and IL-12, respectively. We thus hypothesize that an enhanced SOCS-3 expression in DCs may block the autocrine response of IL-12/IL-23 in these cells, causing them to become a DC2-type phenotype that will subsequently promote Th2 polarization of naive T cells. Indeed, in the present study we found that bone marrow-derived DCs transduced with SOCS-3 significantly inhibited IL-12-induced activation of Stat4 and IL-23-induced activation of Stat3. These SOCS-3-transduced DCs expressed a low level of MHC class II and CD86 on their surface, produced a high level of IL-10 but low levels of IL-12 and IFN-gamma, and expressed a low level of IL-23 p19 mRNA. Functionally, SOCS-3-transduced DCs drove naive myelin oligodendrocyte glycoprotein-specific T cells to a strong Th2 differentiation in vitro and in vivo. Injection of SOCS-3-transduced DCs significantly suppressed experimental autoimmune encephalomyelitis, a Th1 cell-mediated autoimmune disorder of the CNS and an animal model of multiple sclerosis. These results indicate that transduction of SOCS-3 in DCs is an effective approach to generating tolerogenic/DC2 cells that then skew immune response toward Th2, thus possessing therapeutic potential in Th1-dominant autoimmune disorders such as multiple sclerosis.     

Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice

To elucidate the pathogenesis of Helicobacter pylori-associated gastritis, we studied immune responses of C57BL/6J wild-type (WT), SCID, and gene deficient (IFN-gamma-/- and IL-4-/-) mice following infection with a pathogenic isolate of H. pylori (SPM326). During early infection in WT mice, mononuclear and polymorphonuclear cells accumulated in the gastric lamina propria, and the numbers of cells in the inflamed mucosa expressing IFN-gamma, but not IL-4, mRNA rose significantly (p < 0.005), consistent with a local Th1 response. Splenic T cells from the same infected WT mice produced high levels of IFN-gamma, no detectable IL-4, and low amounts of IL-10 following in vitro H. pylori urease stimulation, reflecting a systemic Th1 response. Infected C57BL/6J SCID mice did not develop gastric inflammation despite colonization by many bacteria. Infected C57BL/10J and BALB/c mice also did not develop gastric inflammation and displayed a mixed Th1/Th2 splenic cytokine profile. These data imply a major role for the Th1 cytokine IFN-gamma in H. pylori-associated gastric inflammation in C57BL/6J mice. Compared with WT animals, infected IL-4-/- animals had more severe gastritis and higher levels of IFN-gamma production by urease-stimulated splenocytes (p < 0.01), whereas IFN-gamma-/- mice exhibited no gastric inflammation and higher levels of IL-4 production by stimulated splenocytes. These findings establish C57BL/6J mice as an important model for H. pylori infection and demonstrate that up-regulated production of IFN-gamma, in the absence of the opposing effects of IL-4 (and possibly IL-10), plays a pivotal role in promoting H. pylori-induced mucosal inflammation.     

The role of interferon-gamma on immune and allergic responses

Allergic diseases have been closely related to Th2 immune responses, which are characterized by high levels of interleukin (IL) IL-4, IL-5, IL-9 and IL-13. These cytokines orchestrate the recruitment and activation of different effector cells, such as eosinophils and mast cells. These cells along with Th2 cytokines are key players on the development of chronic allergic inflammatory disorders, usually characterized by airway hyperresponsiveness, reversible airway obstruction, and airway inflammation. Accumulating evidences have shown that altering cytokine-producing profile of Th2 cells by inducing Th1 responses may be protective against Th2-related diseases such as asthma and allergy. Interferon-gamma (IFN-gamma), the principal Th1 effector cytokine, has shown to be crucial for the resolution of allergic-related immunopathologies. In fact, reduced production of this cytokine has been correlated with severe asthma. In this review, we will discuss the role of IFN-gamma during the generation of immune responses and its influence on allergic inflammation models, emphasizing its biologic properties during the different aspects of allergic responses.     

Preferential messenger RNA expression of Th1-type cells (IFN-gamma+, IL-2+) in classical delayed-type (tuberculin) hypersensitivity reactions in human skin.

We recently established that the allergen-induced late-phase cutaneous reaction in atopic subjects was associated with high mRNA expression for the cytokine gene cluster IL-3, IL-4, IL-5, and granulocyte/macrophage-CSF (GM-CSF), compared with IFN-gamma and IL-2, suggesting that allergic skin reactions contained the equivalent of murine Th2 cells. We now show that, in humans, classical delayed-type hypersensitivity is associated with cells preferentially expressing a Th1-type cytokine profile. Cryostat sections from skin biopsies from 24-h tuberculin reactions in 10 nonatopic subjects were hybridized with 35S-labeled RNA probes and processed by using in situ hybridization. On the whole, tuberculin biopsies showed preferential expression of mRNA encoding IFN-gamma and IL-2, although in some cases mRNA expression for IL-3, IL-4, IL-5, and GM-CSF was also observed. Biopsies from diluent control sites gave only occasional signals. The difference in the number of cells expressing mRNA in the diluent compared with tuberculin sites was statistically significant for IL-2 and IFN-gamma (p less than 0.01) but not for IL-3, IL-4, IL-5, and GM-CSF. These results suggest that cells infiltrating the site of the 24-h tuberculin reaction preferentially transcribe mRNA encoding IFN-gamma and IL-2, supporting the hypothesis that delayed-type hypersensitivity is associated with preferential activation of cells having a cytokine profile similar to the murine Th1 subset.     

Lower expression of mRNA for interferon-gamma in T helper cells of children with newly diagnosed lymphomas

The complex interactions between cancer and host cells are far from being fully elucidated. Assessment of Th1/Th2/Th3/Tr1 balance is an interesting approach to explain immunological disturbances in lymphomas. The aim of our study was to assess mRNA for pro- and anti-inflammatory cytokines in T-cells in 20 children with Hodgkin- and non-Hodgkin lymphomas. CD4+ and CD8+ cells were isolated from whole peripheral blood and four different cytokine mRNA levels (IFN-gamma, IL-10, IL-4, TGF-beta) were determined by real-time PCR technique. Comparing to the control group, we found lower expression of mRNA for IFN-gamma in CD4+ cells at the time of lymphoma diagnosis. It may be one of the pathogenetic mechanisms of impaired immunity in these patients.     

Role of Th1 and Th2 cytokines in BCG-induced IFN-gamma production: cytokine promotion and simulation of BCG effect

Induction of a T-helper-type 1 (Th1) immune response is indispensable for successful treatment of superficial bladder cancer with BCG. In this study possible involvement of various cytokines in BCG action as well as their potential roles in enhancing and mimicking BCG effect were explored. In immunocompetent cell cultures, IFN-gamma, a major Th1 cytokine, appears to be a late responsive cytokine to BCG stimulation. Its induction requires involvement of various endogenously produced Th1 and Th2 cytokines. Functional abolishment of any one of these cytokines (IL-2, IL-6, IL-12, IL-18, GMCSF, TNF-alpha, or IFN-alpha, except IL-10) by neutralizing antibodies leads to reduced IFN-gamma production (19-82% inhibition in mouse and 44-77% inhibition in human systems, respectively). In mice cytokines IL-2, IL-12, IL-18, and GMCSF are observed to synergize with BCG for IFN-gamma production, whereas in human cytokines IL-2, IL-12, TNF-alpha, and IFN-alpha exhibit similar synergistic effects. Rational combinations of these Th1-stimulating cytokines (IL-12 plus IL-18 in mice and IL-2 plus IL-12 in humans, respectively) dramatically up-regulate IFN-gamma production that is incomparably superior to BCG for induction of this cytokine. These results suggest that combined Th1-stimulating cytokines and combinations of BCG plus selected Th1-stimulating cytokines are rational candidates for further study in the treatment of bladder cancer patients.