Oral tolerance induced by continuous feeding: enhanced up-regulation of transforming growth factor-beta/interleukin-10 and suppression of experimental autoimmune encephalomyelitis

Oral administration of antigen leads to specific immune hyporesponsiveness termed as oral tolerance. Different doses and feeding regimens have been demonstrated to induce different types of tolerance and degrees of immune suppression. Herein, we compare distinct different regimens of feeding using equivalent final doses of antigen in order to investigate the role of frequency of antigen uptake in the induction of oral tolerance. We demonstrate that continuous feeding of antigen in the drinking water, as compared to a single feeding or feeding once per day over several days enhances suppression to both Th1 and Th2 type responses in B6D2F1 and BALB/c mice. Continuous feeding suppresses antibody responses in aged B6D2F1 mice, which are otherwise refractory to oral tolerance induction. Continuous feeding of ovalbumin (OVA) in high or low doses, as compared to control or single daily feeding over several days, up-regulates interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) production in both OVA TCR transgenic and BALB/c mice. In all regimens tested in wild type mice, low doses were more efficacious than high doses in inducing IL-10 and TGF-beta. Serial feeding (multiple low dose daily gavages) using OVA or myelin basic protein (MBP), also led to up-regulation of TGF-beta and IL-10 production in OVA TCR and MBP TCR transgenic mice, as well as enhanced inhibition of MBP-induced experimental autoimmune encephalomyelitis (EAE) in (PLxSJL) F1 mice. We did not find differences in the cytokine profile between serial (multiple low dose daily gavages) and continuous feeding regimens, suggesting that repetitive discrete delivery of oral antigen provides a sustained signal for the induction of oral tolerance. Thus, using different regimens of feeding that resemble natural feeding with equivalent final doses of antigen, we found enhancement of oral tolerance utilizing regimens that resemble natural feeding. Such feeding regimens may be advantageous in the application of oral tolerance for clinical purposes in the treatment of autoimmune and other inflammatory conditions.     

Suppression of Th1 and Th17, but not Th2, responses in a CD8+ T cell-mediated model of oral tolerance

The role of CD8⁺ T cells in oral tolerance remains unclear. To address this, we developed a model to induce CD8⁺ Tregs by feeding the major histocompatibility complex class I immunodominant epitope of OVA, OVA_(257–264). OVA_(257–264) feeding induced tolerance similar to that observed in OVA protein-fed mice, capable of suppressing the production of Th1 and Th17 cytokines and inhibiting a Th1-driven delayed-type hypersensitivity response following immunization with whole OVA (wOVA) protein. OVA_(257–264) peptide-induced suppression could be transferred to naive mice with CD8⁺ cells, but not CD8-depleted cells, isolated from mesenteric lymph nodes of peptide-fed mice. Interestingly, while capable of inhibiting Th1 and Th17 responses, OVA_(257–264) feeding could not suppress any feature of a Th2 inflammatory response, though OVA protein feeding could, suggesting that these cells function through a different mechanism than their CD4⁺ counterparts generated in response to feeding with wOVA. Thus, CD8⁺ T cells are functionally capable of mediating tolerance to Th1 and Th17 responses.     

Ag and IL‐2 immune complexes efficiently expand Ag‐specific Treg cells that migrate in response to chemokines and reduce localized immune responses

An intravenous administration of a high‐dose antigen (Ag) can induce immune tolerance and suppress the immune response, but the mechanism remains unclear. We recently proved that a combined i.v. administration of OVA and IL‐2‐anti‐IL‐2 Ab immune complexes (IL‐2 ICs) efficiently expands OVA‐specific Treg cells in the thymus and induces their migration into peripheral blood, by using OVA‐specific TCR Tg‐expressing DO11.10 mice. Here, we demonstrate that the expanded OVA‐specific Treg cells rapidly move into the air pouch after OVA injection in DO11.10 mice. The migration was inhibited by blocking the axis of a chemokine receptor, CCR2. Moreover, prior treatment with OVA and IL‐2 ICs enhanced OVA‐specific Treg‐cell migration and inhibited OVA‐induced delayed‐type hypersensitivity (DTH) reactions in the skin of BM chimeric mice with 15% of T cells expressing OVA‐specific TCR. Blocking the CCR2 axis reversed this suppression of DTH in these mice. Furthermore, prior treatment with OVA and IL‐2 ICs effectively reduced DTH reactions even in WT mice possessing only a very small population of OVA‐specific T cells. Thus, the treatment with Ag and IL‐2 ICs can efficiently expand Ag‐specific Treg cells with the capacity to migrate and reduce localized immune responses.     

Ontogeny of Th1 memory responses against a Brucella abortus conjugate

Protective immune responses to intracellular pathogens such as Brucella abortus are characteristically Th1-like. Recently we demonstrated that heat-killed B. abortus (HKBa), a strong Th1 stimulus, conjugated to ovalbumin (HKBA-OVA), but not B. abortus alone, can alter the antigen-specific cytokine profile from Th2- to Th1-like. In this report we study the ability of a single injection of B. abortus to switch a Th2 to a Th1 response in immature mice. One-day- and 1-week-old mice were given a single injection of B. abortus in the absence or presence of OVA, and at maturity mice were challenged with an allergenic preparation, OVA with alum (OVA-A). B. abortus given without OVA did not diminish the subsequent Th2 response in either age group. In contrast, mice receiving a single injection of B. abortus-OVA at the age of 1 week, but not those injected at the age of 1 day, had reversal of the ratio of OVA-specific Th1 to Th2 cells and decreased immunoglobulin E levels after allergen challenge as adults. Within 6 h both 1-day- and 1-week-old mice expressed interleukin-12 p40 mRNA following either B. abortus or B. abortus-OVA administration. However, only the 1-week-old mice exhibited increased expression of gamma interferon (IFN-gamma) mRNA. The absence of the early IFN-gamma response in 1-day-old mice may explain their inability to generate a Th1 memory response. These results suggest that at early stages of immune development, responses to intracellular bacteria may be Th2- rather than Th1-like. Furthermore, they suggest that the first encounter with antigen evokes either a Th1- or a Th2-like response which becomes imprinted, so that subsequent memory responses conform to the original Th bias. This has implications for protection against infectious agents and development of allergic responses.     

Prolonged oral treatment with low doses of allergen conjugated to cholera toxin B subunit suppresses immunoglobulin E antibody responses in sensitized mice

BACKGROUND: Oral tolerance is a long recognized method for inducing systemic immunological tolerance. However, large doses of antigen and frequent administrations are often required. By linking the antigen to the nontoxic mucosa-binding B subunit of cholera toxin (CTB), the required amount can be dramatically reduced. We have previously shown that mucosal administration of small amounts of antigens coupled to CTB can suppress peripheral Th1 cell-reactivity and associated inflammatory immunopathology in both naive and systemically-immunized animals. Induction of oral tolerance by repeated feeding of relatively small doses of antigen has, in some cases been shown to involve the generation of regulatory Th2-like CD4+ T cells, and hence could promote rather than suppress type I immunoglobulin (Ig) E-mediated allergic responses. OBJECTIVES: We examined whether oral prophylactic or therapeutic administration of a model allergen coupled to CTB would modulate allergen-specific IgE responses in high IgE responder Balb/c mice. METHODS: Ovalbumin (OVA) was used as a model allergen. Mice were treated perorally with free or CTB-coupled OVA before or after systemic priming with alum-adsorbed OVA. Allergen-specific IgE levels in serum were measured with the passive cutaneous anaphylaxis test at various time-points. RESULTS: Oral administration of a single low dose of CTB-linked OVA, prior to systemic sensitization and challenge with OVA, suppressed allergen-specific serum IgE antibody responses. Treatment with comparable doses of free OVA was much less effective. Most importantly, oral treatment with CTB-OVA conjugate could also suppress an already initiated IgE antibody response, but to achieve such a 'therapeutic effect', administration of multiple low doses of conjugate over a long time was required. Oral treatment with CTB-OVA conjugate could also effectively suppress antigen-specific Th1-mediated delayed-type hypersensitivity. Thus treatment with a CTB-conjugated model allergen can affect a broad range of T-cell-driven immune responses, even in antigen-experienced animals. CONCLUSION: These results may impact on the development of therapeutic vaccines against type I allergies.     

NKT cells are dispensable in the induction of oral tolerance but are indispensable in the abrogation of oral tolerance by prostaglandin E

NK1.1(+) alpha beta T cells (NKT cells) regulate the Th1/Th2 balance in response to dietary Ag, which may be involved in regulation of oral tolerance. OVA-specific IgE and IgG(1) Ab levels were significantly lower following an i.p. injection of OVA (in CFA) in C57BL/6 mice orally given a single, high dose (25 mg) of OVA than in those orally given PBS. The oral tolerance was normally induced in Jalpha281(-/-) mice which lack Valpha14(+) NKT cells, suggesting that NKT cells are dispensable for induction of oral tolerance. Treatment with PGE(1) or PGE(2 )abrogated the oral tolerance in Jalpha281(+/+) mice; this abrogation was accompanied by an OVA-specific Th2-dominant response. The abrogation of oral tolerance by PGE(1 )was not evident in Jalpha281(-/-) mice. Treatment with PGE(1) induced an early increase in IL-4 production by liver NKT cells in normal mice and neutralization of the early IL-4 by administration of anti-IL-4 mAb abolished PGE(1)-induced abrogation of oral tolerance. These results suggest that liver NKT cells producing IL-4 are responsible for the down-regulation of oral tolerance that is caused by the PGE molecules.     

IgE-dependent enhancement of Th2 cell-mediated allergic inflammation in the airways

T helper 2 (Th2) cell-derived cytokines, including interleukin (IL)-4, IL-5 and IL-13, play important roles in causing allergic airway inflammation. In contrast to Th2 cells, however, the role of IgE and mast cells in inducing allergic airway inflammation is not understood fully. In the present study, we addressed this point using transgenic mice expressing trinitrophenyl (TNP)-specific IgE (TNP-IgE mice), which enable us to investigate the role of IgE without the influence of antigen-specific T cell activation and other immunoglobulins. When the corresponding antigen, TNP-BSA, was administered intranasally to TNP-IgE mice, a large number of CD4+ T cells were recruited into the airways. In contrast, TNP-BSA administration did not induce eosinophil recruitment into the airways or airway hyperreactivity. Furthermore, when ovalbumin (OVA)-specific Th2 cells were transferred to TNP-IgE mice and the mice were challenged with inhaled OVA, TNP-BSA administration increased OVA-specific T cell recruitment and then enhanced Th2 cell-mediated eosinophil recruitment into the airways. These results indicate that IgE-induced mast cell activation principally induces CD4+ T cell recruitment into the airways and thus plays an important role in enhancing Th2 cell-mediated eosinophilic airway inflammation by recruiting Th2 cells into the site of allergic inflammation.     

Mucosal tolerance is associated with, but independent of, up-regulation Th2 responses.

Intranasal administration of protein antigen is an efficient way to induce mucosal tolerance. Suppressive mechanisms that might be involved in this phenomenon include down-regulation of T-helper type-1 (Th1)-mediated processes by Th2 cells. However, since Th2 responses can also be subjected to mucosal tolerance, we wanted to investigate whether suppression of a typical Th1 response, such as a delayed-type hypersensitivity (DTH) reaction by intranasal tolerance induction, was causally related to up-regulation of Th2 responses. We therefore treated mice either systemically or locally with anti-interleukin-4 (IL-4) or anti-IL-10 antibodies before intranasal tolerance induction or before sensitization for DTH to see whether we could prevent or abrogate tolerance. Although the up-regulation of antigen-specific IgE levels in tolerant mice could be prevented by anti-IL-4 treatment, the extent of tolerance as measured by suppression of DTH was not affected. We therefore conclude that up-regulation of Th2 responses observed after intranasal tolerance induction is an additional or consequential rather than a necessary reaction.     

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice

Background and aim: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice.

Material and methods: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-γ as well as Th2 cytokines such as IL-4 and IL-5 were measured.

Results: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-γ. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells.

Conclusion: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.     

Antigen dose defines T helper 1 and T helper 2 responses in the lungs of C57BL/6 and BALB/c mice independently of splenic responses

To investigate the effect of antigen dose on immune response, C57BL/6 and BALB/c mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) then challenged with aerosolized OVA. Low-dose sensitization (less than 8 microg of OVA) elicited T helper 2 (Th2)-type immunoglobulins (Igs) secretion from C57BL/6 mice, including high levels of serum IgE, IgG1 and low levels of IgG2a, while BALB/c mice secreted T helper 1 (Th1)-type Igs, including low levels of IgE, IgG1 and high levels of IgG2a. In contrast, high-dose sensitization (more than 50 microgram) elicited Th1-type Igs secretion in C57BL/6mice, while BALB/c mice exhibited Th2-type Igs secretion. Furthermore, the number of eosinophils infiltrating into the lungs of low-dose OVA-sensitized C57BL/6 mice was significantly greater than in BALB/c mice sensitized with the same amount of OVA. Only a very high dose of OVA (1 mg) could induce greater eosinophil infiltration into the lungs of BALB/c mice compared with C57BL/6 mice. Additionally, low-dose sensitization generated Th2-type cytokines, including high levels of interleukin (IL) -4, IL-5 and a low level of interferon-gamma (IFN-gamma) in the lungs of C57BL/6 mice, while BALB/c mice generated Th1-type cytokines in their lungs, including low levels of IL-4, IL-5 and a high level of IFN-gamma. In contrast, high-dose sensitization elicited Th1-type cytokines production in the lungs of C57BL/6 mice, while BALB/c mice generated Th2-type cytokines in their lungs. Interestingly, splenocyte cultures from C57BL/6 mice produced Th1-type cytokines, while cultures from BALB/c mice produced Th2-type cytokines regardless of OVA sensitization dose (100 ng-1 mg). These results indicate that C57BL/6 and BALB/c mice have different susceptibilities to OVA-sensitization and OVA-induced pulmonary eosinophilia regulated by Th1- and Th2-type cytokines, independent of splenic Th1- and Th2-type cytokines production.     

Suppression of specific IgE antibody responses by liposome-conjugated ovalbumin in mice sensitized with ovalbumin via the respiratory tract

BACKGROUND: Previously we have shown that intranasal administration of ovalbumin (OVA) together with cholera toxin (CT) abrogates nasal tolerance to OVA, resulting in the induction of specific IgE antibody (Ab) responses, and that intraperitoneal injection of OVA coupled with liposomes (OVA-liposomes) induces a selective suppression of IgE Ab responses to OVA. Whether OVA-liposomes suppress anti-OVA IgE Ab responses in mice sensitized with CT-combined OVA via the respiratory tract remains to be clarified. METHODS: In some experiments, mice were given OVA, liposomes or OVA-liposomes with or without CT intranasally three times, at 2-week intervals (weeks 0, 2 and 4). In other experiments, mice were given OVA-liposomes intranasally 2 days before or 1 and 3 weeks after CT-combined OVA (week 0), which was administered intranasally three times, at 2-week intervals (weeks 0, 2 and 4). Two weeks after the third administration of CT-combined OVA (week 0), nasal wash and serum IgA, IgG and IgE Ab responses were assayed. RESULTS: Pretreatment with OVA-liposomes suppressed IgE Ab responses to CT-combined OVA, with a significantly high production of both nasal IgA and serum IgG Abs. Moreover, treatment with OVA-liposomes 1 and 3 weeks after CT-combined OVA administration also suppressed IgE Ab responses. The suppression of anti-OVA IgE Ab production by OVA-liposomes was accompanied by a simultaneous enhancement of specific IgA and IgG (IgG1, and especially IgG2a) Ab production. CONCLUSIONS: Postimmunization treatment with OVA-liposomes, as well as preimmunization treatment, suppressed specific IgE Ab responses in mice sensitized intranasally with CT-combined OVA. Allergens conjugated to liposomes may be appropriate for preventing the development of allergies to inhaled or dietary antigens in humans.     

Suppression of Th2 immune responses by mekabu fucoidan from Undaria pinnatifida sporophylls

BACKGROUND: We demonstrated that mekabu fucoidan obtained from Undaria pinnatifida (Up) sporophylls augments the type 1 T-helper (Th1) cell response in normal BALB/c mice. In this study, we examined the effects of the fucoidan of mekabu on the type 2 T-helper (Th2) response in bronchoalveolar lavage fluid (BALF) after ovalbumin (OVA) aerosol challenge. METHODS: Mekabu fucoidan (50 mg/kg) was injected intraperitoneally into BALB/c mice for 4 days, and then the mice were sensitized with 50 microg/mouse of OVA plus alum (1 mg/mouse) 1 and 8 days later. The mice were challenged with OVA delivered using a nebulizer 7, 8 and 9 days after the second challenge with OVA plus alum. After 24 h, we assessed T cell responses in BALF by measuring the amount of Th2 cytokines (IL-4, IL-5, IL-13) and gamma-interferon (IFN-gamma) produced by Th1 cells. RESULTS: The production of Th2 cytokines was suppressed (p < 0.05), and the amount of IFN-gamma was not increased in the mice treated with mekabu fucoidan. Anti-OVA immunoglobulin E (IgE) and IgE levels in serum determined after challenge with aerosolized OVA at the end of the experiment were lower (p < 0.05) in the treated than in the control mice. CONCLUSIONS: The pulmonary inflammation was relieved by mekabu fucoidan, which also downregulated Th2-dominated responses. These results indicate that mekabu fucoidan modulates Th2 responses and might be useful for treating allergic inflammation.     

Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization

It has been postulated that low-level interleukin (IL)-12 production of antigen-presenting cells is associated with the risk of developing atopic asthma. To study the relationship between IL-12 production capacity of dendritic cells (DCs) and development of T helper type 2 (Th2) responses in the lung, we genetically engineered DCs to constutively overexpress bioactive IL-12. Retrovirally mediated overexpression of IL-12 in DCs strongly polarized naive ovalbumin (OVA)-specific CD4+ T cells toward Th1 effector cells in vitro. After intratracheal injection, OVA-pulsed IL-12-overexpressing DCs failed to induce Th2 responses in vivo and no longer primed mice for Th2-dependent eosinophilic airway inflammation upon OVA aerosol challenge, readily observed in mice immunized with sham-transfected, OVA-pulsed DCs. Analysis of a panel of cytokines and chemokines in the lung demonstrated that the lack of Th2 sensitization was accompanied by increased production of the Th1 cytokine interferon-gamma (IFN-gamma), chemokines induced by IFN-gamma, and the immunoregulatory cytokine IL-10. When Th2 priming was induced using OVA/alum prior to intratracheal DC administration, DCs constitutively expressing IL-12 were no longer capable of preventing eosinophilic airway inflammation and even enhanced it. These data show directly that high-level expression of IL-12 in DCs prevents the development of Th2 sensitization. Enhancing IL-12 production in DCs should be seen as a primary prevention strategy for atopic disorders. Enhancing IL-12 production in DCs is less likely to be of benefit in already Th2-sensitized individuals.     

Selective down-regulation of Th2 immune responses following treatment with antigen-coupled splenocytes

Intravenous injection of antigen-coupled splenocytes has been widely used to induce specific tolerance to a variety of antigens. In this study, we investigated the effects of such a treatment on Th1 and Th2 antigen-specific immune responses. Using both well-characterized model antigens and crude homogenates from Leishmania major promastigotes, we found that intravenous injection of antigen-coupled splenocytes strongly down-regulated antigen-specific Th2 responses but had no or only moderate effects on Th1 responses. Because the susceptibility of inbred strains of mice to murine leishmaniasis has been found to be correlated with a strong Th2 response against parasite antigens, we investigated whether administration of splenocytes chemically coupled to parasite antigens could protect susceptible mice from murine leishmaniasis. We found that this was indeed the case and further demonstrated that protection was associated with a strong decrease in the number of parasite-specific Th2-like cells. Because administration of antigen-coupled splenocytes is believed to induce ligation of the T cell receptor complex without inducing a co-stimulatory signal, our results further suggest that priming of Th1 cells is less dependent on co-stimulatory signals than the priming of Th2 cells.     

Environmental pollutant tributyltin promotes Th2 polarization and exacerbates airway inflammation

It has been shown that a relatively high dose of tributyltin (TBT), which is recognized as a particularly notable environmental pollutant, exerts immunotoxic effects such as thymic atrophy via induction of T cell apoptosis. However, the effect of low doses of TBT on the immune responses remains unknown. Here we show that environmentally relevant doses of TBT promoted strong Th2 polarization via suppression and augmentation of Th1 and Th2 development, respectively, from naive CD4(+) T cells primed with anti-CD3 and splenic antigen-presenting cells (APC). TBT-induced Th2 polarization was indirect, working through APC via suppression of IL-12 production by macrophages/DC and the augmentation of IL-10 production by B cells. Th2 polarization was also induced in mice treated with TBT and immunized with OVA or infected with Nippostrongylus brasiliensis. Furthermore, airway inflammation in mice sensitized and challenged with OVA was exacerbated by the administration of TBT with concomitant augmentation of Th2-type immunity. Our results highlight the fact that an important environmental pollutant TBT may present significant risk for the induction of allergic diseases via promotion of Th2 polarization.     

Selective suppression of antigen-specific Th2 cells by continuous micro-dose oral tolerance

The effect on antigen (Ag)-specific Th2 response as well as IgE production of continuous oral administration of micro-doses of Ag was investigated. Transgenic (Tg) mice carrying the α β-T cell receptor (TCR) genes specific for ovalbumin (OVA) peptide fragment 323 – 339 were continuously fed with micro-doses of OVA (100 μg/day) for 14 days. Mice were first immunized by OVA in alum and pertussis toxin 7 days before the oral feeding and given a second immunization 1 day after the oral treatment. This feeding regimen tolerized Th2 but not Th1 responses as shown by decrease of Ag-driven cell proliferation and cytokine secretion of IL- 4 but not of IL-2 or IFN-γ as well as by the absence of Ag-specific antibody production of IgE and IgG1, but not of IgG2a or total IgG. Numbers of clonotype-specific TCR-high CD4-positive T cells in peripheral lymphoid tissues markedly decreased in the orally treated group but not in the control group. However, total numbers of CD4-positive T cells in thymus, spleen and lymph nodes were not affected by the oral treatment, indicating that tolerance induction in Th2 cells was mainly due to the down-regulation of TCR and not clonal deletion. The population of antigen-presenting cells expressing B7-2 (CD86) Ag on the surface was decreased in the spleen of the mice which underwent the feeding regimen. The present results suggest that Ag-specific low responsiveness in Th2 cells, which resulted in suppres sion of the Ag-specific IgE production, can be achieved by continuous feeding with microdoses of Ag.     

Oral administration of Enterococcus faecalis FK-23 suppresses Th17 cell development and attenuates allergic airway responses in mice

Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4+ T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25, 26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Ιnterleukin (IL)-17-expressing CD4+ lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-β mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4+ cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.     

A Distinct Role of CD4<Superscript>+</Superscript> Th17- and Th17-Stimulated CD8<Superscript>+</Superscript> CTL in the Pathogenesis of Type 1 Diabetes and Experimental Autoimmune Encephalomyelitis

Both CD4(+) Th17-cells and CD8(+) cytotoxic T lymphocytes (CTLs) are involved in type 1 diabetes and experimental autoimmune encephalomyelitis (EAE). However, their relationship in pathogenesis of these autoimmune diseases is still elusive. We generated ovalbumin (OVA)- or myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells expressing RORγt and IL-17 by in vitro co-culturing OVA-pulsed and MOG(35-55) peptide-pulsed dendritic cells (DC(OVA) and DC(MOG)) with CD4(+) T cells derived from transgenic OTII and MOG-T cell receptor mice, respectively. We found that these Th17 cells when transferred into C57BL/6 mice stimulated OVA- and MOG-specific CTL responses, respectively. To assess the above question, we adoptively transferred OVA-specific Th17 cells into transgenic rat insulin promoter (RIP)-mOVA mice or RIP-mOVA mice treated with anti-CD8 antibody to deplete Th17-stimulated CD8(+) T cells. We demonstrated that OVA-specific Th17-stimulated CTLs, but not Th17 cells themselves, induced diabetes in RIP-mOVA. We also transferred MOG-specific Th17 cells into C57BL/6 mice and H-2K(b-/-) mice lacking of the ability to generate Th17-stimulated CTLs. We further found that MOG-specific Th17 cells, but not Th17-activated CTLs induced EAE in C57BL/6 mice. Taken together, our data indicate a distinct role of Th17 cells and Th17-stimulated CTLs in the pathogenesis of TID and EAE, which may have great impact on the overall understanding of Th17 cells in the pathogenesis of autoimmune diseases.     

Th1 and Th2 cells help CD8 T-cell responses

Help from CD4 T cells is often important for the establishment of primary and memory CD8 T-cell responses. However, it has yet to be determined whether T helper polarization affects the delivery of help and/or whether responding CD8 T cells helped by Th1 or Th2 cells express distinct effector properties. To address these issues, we compared CD8 T-cell responses in the context of Th1 or Th2 help by injecting dendritic cells copulsed with the major histocompatibility complex class I-restricted OVA peptide plus, respectively, bacterial or helminth antigens. We found that Th2 cells, like Th1 cells, can help primary and long-lived memory CD8 T-cell responses. Experiments in interleukin-12 (IL-12)-/- and IL-4-/- mice, in which polarized Th1 or Th2 responses, respectively, fail to develop, indicate that the underlying basis of CD4 help is independent of attributes acquired as a response to polarization.