Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration

CD25(+)CD4(+) T cells are naturally occurring regulatory T cells that are anergic and have suppressive properties. Although they can be isolated from the spleens of normal mice, there are limited studies on how they can be activated or expanded in vivo. We found that oral administration of OVA to OVA TCR transgenic mice resulted in a modification of the ratio of CD25(+)CD4(+) to CD25(-)CD4(+) cells with an increase of CD25(+)CD4(+) T cells accompanied by a decrease of CD25(-)CD4(+) T cells. The relative increase in CD25(+)CD4(+) T cells persisted for as long as 4 wk post feeding. We also found that CTLA-4 was dominantly expressed in CD25(+)CD4(+) T cells and there was an increase in the percentage of CD25(+)CD4(+) T cells expressing CTLA-4 in OVA-fed mice. In contrast to CD25(-)CD4(+) cells, CD25(+)CD4(+) cells from fed mice proliferated only minimally to OVA or anti-CD3 and secreted IL-10 and elevated levels of TGF-beta(1) following anti-CD3 stimulation. CD25(+)CD4(+) cells from fed mice suppressed the proliferation of CD25(-)CD4(+) T cells in vitro more potently than CD25(+)CD4(+) T cells isolated from unfed mice, and this suppression was partially reversible by IL-10 soluble receptor or TGF-beta soluble receptor and high concentration of anti-CTLA-4. With anti-CD3 stimulation, CD25(+)CD4(+) cells from unfed mice secreted IFN-gamma, whereas CD25(+)CD4(+) cells from fed mice did not. Adoptive transfer of CD25(+)CD4(+) T cells from fed mice suppressed in vivo delayed-type hypersensitivity responses in BALB/c mice. These results demonstrate an Ag-specific in vivo method to activate CD25(+)CD4(+) regulatory T cells and suggest that they may be involved in oral tolerance.     

Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4(+)CD25(+) T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4(+)CD25(+) regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA x DO11.10 mice had increased numbers of CD4(+)CD25(+) regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA x DO11.10 mice, T cells expressing endogenous TCR alpha beta chains were CD4(+)CD25(-) T cells, whereas T cells expressing autoreactive TCR were selected as CD4(+)CD25(+) T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA x DO11.10 mice. In contrast, in DO11.10 mice, CD4(+)CD25(+) T cells expressed endogenous TCR alpha beta chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4(+)CD25(+) T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4(+)CD25(+) T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4(+)CD25(-) T cells from autoreactive T cell repertoire.     

Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells

The capacity of airway eosinophils, potentially pertinent to allergic diseases of the upper and lower airways, to function as professional APCs, those specifically able to elicit responses from unprimed, Ag-naive CD4(+) T cells has been uncertain. We investigated whether airway eosinophils are capable of initiating naive T cell responses in vivo. Eosinophils, isolated free of other APCs from the spleens of IL-5 transgenic mice, following culture with GM-CSF expressed MHC class II and the costimulatory proteins, CD40, CD80, and CD86. Eosinophils, incubated with OVA Ag in vitro, were instilled intratracheally into wild-type recipient mice that adoptively received i.v. infusions of OVA Ag-specific CD4(+) T cells from OVA TCR transgenic mice. OVA-exposed eosinophils elicited activation (CD69 expression), proliferation (BrdU incorporation), and IL-4, but not IFN-gamma, cytokine production by OVA-specific CD4(+) T cells in paratracheal lymph nodes (LN). Exposure of eosinophils to lysosomotropic NH(4)Cl, which inhibits Ag processing, blocked each of these eosinophil-mediated activation responses of CD4(+) T cells. By three-color fluorescence microscopy, OVA Ag-loaded eosinophil APCs were physically interacting with naive OVA-specific CD4(+) T cells in paratracheal LN after eosinophil airway instillation. Thus, recruited luminal airway eosinophils are distinct allergic "inflammatory" professional APCs able to activate primary CD4(+) T cell responses in regional LNs.     

CD4+CD25+ T cells regulate airway eosinophilic inflammation by modulating the Th2 cell phenotype

We used a TCR-transgenic mouse to investigate whether Th2-mediated airway inflammation is influenced by Ag-specific CD4+CD25+ regulatory T cells. CD4+CD25+ T cells from DO11.10 mice expressed the transgenic TCR and mediated regulatory activity. Unexpectedly, depletion of CD4+CD25+ T cells before Th2 differentiation markedly reduced the expression of IL-4, IL-5, and IL-13 mRNA and protein when compared with unfractionated (total) CD4+ Th2 cells. The CD4+CD25--derived Th2 cells also expressed decreased levels of IL-10 but were clearly Th2 polarized since they did not produce any IFN-gamma. Paradoxically, adoptive transfer of CD4+CD25--derived Th2 cells into BALB/c mice induced an elevated airway eosinophilic inflammation in response to OVA inhalation compared with recipients of total CD4+ Th2 cells. The pronounced eosinophilia was associated with reduced levels of IL-10 and increased amounts of eotaxin in the bronchoalveolar lavage fluid. This Th2 phenotype characterized by reduced Th2 cytokine expression appeared to remain stable in vivo, even after repeated exposure of the animals to OVA aerosols. Our results demonstrate that the immunoregulatory properties of CD4+CD25+ T cells do extend to Th2 responses. Specifically, CD4+CD25+ T cells play a key role in modulating Th2-mediated pulmonary inflammation by suppressing the development of a Th2 phenotype that is highly effective in vivo at promoting airway eosinophilia. Conceivably, this is partly a consequence of regulatory T cells facilitating the production of IL-10.     

Listeriolysin O expressed in a bacterial vaccine suppresses CD4+CD25high regulatory T cell function in vivo

CD4(+)CD25(high) regulatory T cells (Treg) protect the host from autoimmune diseases but are also obstacles against cancer therapies. An ideal cancer vaccine would stimulate specific cytotoxic responses and reduce/suppress Treg function. In this study, we showed that Escherichia coli expressing listeriolysin O and OVA (E. coli LLO/OVA) demonstrated remarkable levels of protection against OVA-expressing tumor cells. By contrast, E. coli expressing OVA only (E. coli OVA) showed poor protection. High-avidity OVA-specific CTL were induced in E. coli LLO/OVA-vaccinated mice, and CD8(+) depletion--but not NK cell depletion, abolished the antitumor activity of the E. coli LLO/OVA vaccine. Phenotypic analysis of T cells following vaccination with either vaccine revealed preferential generation of CD44(high)CD62L(low) CD8(+) effector memory T cells over CD44(high)CD62L(high) central memory T cells. Unexpectedly, CD4(+) depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4(+) cells suppressed the CD8(+) T cell-mediated antitumor response. Further depletion experiments demonstrated that these suppressive cells consisted of CD4(+)CD25(high) regulatory T cells. We therefore assessed these vaccines for Treg function and found that although CD4(+)CD25(high) expansion and Foxp3 expression within this population was similar in all groups of mice, Treg cells from E. coli LLO/OVA-vaccinated animals were unable to suppress conventional T cells proliferation. These findings provide the first evidence that LLO expression affects Treg cell function and may have important implications for enhancing antitumor vaccination strategies in humans.     

Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8(+) T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8(+) T cells in the development of these allergen-induced responses. CD8-deficient (CD8(-/-)) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8(-/-) mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8(-/-) mice with allergen-sensitized CD8(+) T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8(+) T cells or allergen-sensitized CD8(+) T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8(+) T cells were a source of IL-13. These data suggest that Ag-primed CD8(+) T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.     

Dendritic cells genetically engineered to express IL-10 induce long-lasting antigen-specific tolerance in experimental asthma

Dendritic cells (DCs) are professional APCs that have a unique capacity to initiate primary immune responses, including tolerogenic responses. We have genetically engineered bone marrow-derived DCs to express the immunosuppressive cytokine IL-10 and tested the ability of these cells to control experimental asthma. A single intratracheal injection of OVA-pulsed IL-10-transduced DCs (OVA-IL-10-DCs) to naive mice before OVA sensitization and challenge prevented all of the cardinal features of airway allergy, namely, eosinophilic airway inflammation, airway hyperreactivity, and production of mucus, Ag-specific Igs, and IL-4. OVA-IL-10-DCs also reversed established experimental asthma and had long-lasting and Ag-specific effects. We furthermore showed, by using IL-10-deficient mice, that host IL-10 is required for mediating the immunomodulatory effects of OVA-IL-10-DCs and demonstrated a significant increase in the percentage of OVA-specific CD4(+)CD25(+)Foxp3(+)IL-10(+) regulatory T cells in the mediastinal lymph nodes of OVA-IL-10-DC-injected mice. Finally, adoptive transfer of CD4(+) mediastinal lymph node T cells from mice injected with OVA-IL-10-DCs protected OVA-sensitized recipients from airway eosinophilia upon OVA provocation. Our study describes a promising strategy to induce long-lasting Ag-specific tolerance in airway allergy.     

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.     

Visualizing the role of Cbl-b in control of islet-reactive CD4 T cells and susceptibility to type 1 diabetes

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4(+) T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4(+) T cells in prediabetic 3A9-TCR × insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4(+) cells or the numbers of islet-specific CD4(+) or CD4(+)Foxp3(+) T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-β-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4(+) cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4(+) cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity.     

Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+ and Foxp3-CD25- CD4+ regulatory T cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (T(reg)). We found that total T(reg), KJ1-26+ T(reg) and CTLA-4+ T(reg) were all increased in Peyer's patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-beta in serum. This could be abolished by co-administering cholera toxin or by treatment with anti-TGF-beta mAb. CD25+ T(reg), but also CD25-CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ T(reg) from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ T(reg). Furthermore, in Rag1(-/-) mice that had adoptively received highly purified Foxp3-CD25-CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ T(reg) cells, which expressed Foxp3 more strongly than naturally developing T(reg) and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25- T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-beta and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ T(reg) together with the generation of both Foxp3+ and Foxp3-CD25- CD4+ T(reg).     

ICOS Mediates the Generation and Function of CD4+CD25+Foxp3+ Regulatory T Cells Conveying Respiratory Tolerance

Costimulatory molecules like ICOS are crucial in mediating T cell differentiation and function after allergen contact and thereby strongly affect the immunologic decision between tolerance or allergy development. In this study, we show in two independent approaches that interruption of the ICOS signaling pathway by application of a blocking anti-ICOSL mAb in wild-type (WT) mice and in ICOS(-/-) mice inhibited respiratory tolerance development leading to eosinophilic airway inflammation, mucus hypersecretion, and Th2 cytokine production in response to OVA sensitization. Respiratory Ag application almost doubled the number of CD4(+)Foxp3(+) regulatory T cells (Tregs) in the lung of WT mice with 77% of lung-derived Tregs expressing ICOS. In contrast, in ICOS(-/-) mice the number of CD4(+)CD25(+)Foxp3(+) Tregs did not increase after respiratory Ag application, and ICOS(-/-) Tregs produced significantly lower amounts of IL-10 than those of WT Tregs. Most importantly, in contrast to WT Tregs, ICOS(-/-) Tregs did not convey protection when transferred to "asthmatic" recipients demonstrating a strongly impaired Treg function in the absence of ICOS signaling. Our findings demonstrate a crucial role of ICOS for the generation and suppressive function of Tregs conveying respiratory tolerance and support the importance of ICOS as a target for primary prevention strategies.     

CD4(+) T cell-dependent airway mucus production occurs in response to IL-5 expression in lung

The potential role of airway interleukin-5 (IL-5) expression in eliciting mucus production was demonstrated in a pulmonary IL-5 transgenic mouse model (NJ.1726) in which naive transgenic mice display comparable levels of airway mucus relative to allergen-sensitized and -challenged wild-type mice. The intrinsic mucus accumulation of NJ.1726 was abolished in compound transgenic-gene knockout mice deficient of either CD4(+) cells [NJ.1726/CD4(-/-)] or alphabeta T cell receptor-positive (TCR(+)) cells [NJ.1726/alphabeta TCR(-/-)]. In addition, mucus production in naive NJ.1726 was inhibited by >90% after administration of the soluble anti-IL-4 receptor alpha-subunit antagonist. The loss of mucus production in NJ.1726/CD4(-/-), NJ.1726/alphabeta TCR(-/-), and anti-IL-4 receptor alpha-subunit antagonist-treated mice occurred notwithstanding the significant pulmonary eosinophilia and expansion of airway B cells induced by ectopic IL-5 expression. Furthermore, the loss of mucus accumulation occurred in these mice despite elevated levels of airway and peripheral IL-5, indicating that IL-5 does not directly induce goblet cell metaplasia and mucus production. Thus pulmonary expression of IL-5 alone is capable of inducing CD4(+) T cell-dependent goblet cell metaplasia, apparently mediated by IL-4 receptor alpha-subunit-ligand interactions, and represents a previously unrecognized novel pathway for augmenting allergen-induced mucus production.     

Naturally occurring lung CD4(+)CD25(+) T cell regulation of airway allergic responses depends on IL-10 induction of TGF-beta

Peripheral tolerance to allergens is mediated in large part by the naturally occurring lung CD4(+)CD25(+) T cells, but their effects on allergen-induced airway responsiveness have not been well defined. Intratracheal, but not i.v., administration of naive lung CD4(+)CD25(+) T cells before allergen challenge of sensitized mice, similar to the administration of the combination of rIL-10 and rTGF-beta, resulted in reduced airway hyperresponsiveness (AHR) and inflammation, lower levels of Th2 cytokines, higher levels of IL-10 and TGF-beta, and less severe lung histopathology. Significantly, CD4(+)CD25(+) T cells isolated from IL-10(-/-) mice had no effect on AHR and inflammation, but when incubated with rIL-10 before transfer, suppressed AHR, and inflammation, and was associated with elevated levels of bronchoalveolar lavage TGF-beta levels. By analogy, anti-TGF-beta treatment reduced regulatory T cell activity. These data identify naturally occurring lung CD4(+)CD25(+) T cells as capable of regulating lung allergic responses in an IL-10- and TGF-beta-dependent manner.     

Listeria monocytogenes infection overcomes the requirement for CD40 ligand in exogenous antigen presentation to CD8(+) T cells.

In vivo priming of CD8(+) T lymphocytes against exogenously processed model Ags requires CD4(+) T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4(+) T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8(+) T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8(+) T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8(+) T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8(+) T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8(+) T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-gamma was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8(+) T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8(+) T cell responses against exogenous Ags during infection.     

TRAIL deficiency does not rescue impaired CD8+ T cell memory generated in the absence of CD4+ T cell help

Ag-specific CD8(+) T cells immunized in the absence of CD4(+) T cell help, so-called "unhelped" CD8(+) T cells, are defective in function and survival. We investigated the role of the proapoptotic molecule TRAIL in this defect. We first demonstrate that TRAIL does not contribute to the CD8(+) T cell response to Listeria monocytogenes strain expressing OVA (LmOVA) in the presence of CD4(+) T cells. Secondly, we generated mice doubly deficient in CD4(+) T cells and TRAIL and analyzed their CD8(+) T cell response to LmOVA. Memory CD8(+) T cells in double-deficient mice waned over time and were not protective against rechallenge, similar to their TRAIL-sufficient unhelped counterparts. To avoid the effects of CD4(+) T cell deficiency during memory maintenance, and to address whether TRAIL plays a role in the early programming of the CD8(+) T cell response, we performed experiments using heterologous prime and early boost immunizations. We did not observe activation-induced cell death of unhelped CD8(+) T cells when mice were infected with followed vaccinia virus expressing OVA 9 days later by LmOVA infection. Furthermore, primary immunization of CD4(+) T cell-deficient mice with cell-associated Ag followed by LmOVA infection did not reveal a role for TRAIL-mediated activation-induced cell death. Overall, our results suggest that CD4(+) T cell help for the CD8(+) T cell response is not contingent on the silencing of TRAIL expression and prevention of TRAIL-mediated apoptosis.     

Dose-dependent recruitment of CD25+ and CD26+ T cells in a novel F344 rat model of asthma

The ovalbumin (OVA)-induced airway inflammation in rats is a commonly used model to explore the pathobiology of asthma. However, its susceptibility varies greatly between rat strains, and presently Brown Norway (BN) rats are preferentially used. Since recruitment of T cells to the lungs depends on the CD26 (dipeptidyl peptidase IV, DPPIV) expression, Fischer 344 strain (F344) rats are a highly relevant rat strain, in particular because CD26-deficient substrains are available. To establish a F344 rat model of asthma, we challenged F344 rats using different doses of aerosolized antigen (0%, 1%, 2.5%, 5%, and 7.5% OVA) and compared these effects with intratracheal instillation of OVA (1.5 mg/0.3 ml). Asthmoid responsiveness was determined by analysis of early airway responsiveness (EAR), antigen-specific IgE levels, as well as airway inflammation including the composition of T cell subpopulations in the bronchoalveolar lavage (BAL) and lung tissue with special respect to the T cell activation markers CD25 and CD26. Even low allergen doses caused allergen-specific EAR and increases of antigen-specific IgE levels. However, EAR and IgE levels did not increase dose dependently. Higher concentrations of OVA led to a dose-dependent increase of several immunological markers of allergic asthma including an influx of eosinophils, T cells, and dendritic cells. Interestingly, a dose-dependent increase of CD4(+)/CD25(+)/CD26(+) T cells was found in the lungs. Summarizing, we established a novel F344 rat model of aerosolized OVA-induced asthma. Thereby, we found a dose-dependent recruitment of cellular markers of allergic asthma including the activated CD4(+)/CD25(+)/CD26(+) T cell subpopulation, which has not been described in asthma yet.     

Regulatory T cell properties of chicken CD4+CD25+ cells

Chicken CD4(+)CD25(+) cells were characterized for mammalian regulatory T cells' suppressive and cytokine production properties. Anti-chicken CD25 mAb was produced in mice and conjugated with a fluorescent tag. The specificity of the Ab against chicken CD25 was confirmed by evaluating Con A-induced CD25 upregulation in thymocytes and by quantifying the CD25 mRNA content of positive and negative cells identified by anti-chicken CD25 Ab. The percentage of CD4(+)CD25(+) cells, expressed as a percentage of CD4(+) cells, in thymus and blood was ～3-7%, in spleen was 10%, and in cecal tonsil, lung, and bone marrow was ～15%. Bursa had no detectable CD4(+)CD25(+) cells. CD25(+) cells were mostly CD4(+) in the thymus, whereas in every other organ studied, CD25(+) cells were distributed between CD4(+) and CD4(-) cells. Chicken thymic CD4(+)CD25(+) cells did not proliferate in vitro in the absence of recombinant chicken IL-2 (rCIL-2). In the presence of rCIL-2, PMA plus ionomycin or Con A stimulated CD4(+)CD25(+) cell proliferation, whereas anti-CD3 plus CD28 did not stimulate CD4(+)CD25(+) cell proliferation. Naive CD4(+)CD25(+) cells had 29-fold more IL-10 mRNA and 15-fold more TGF-β mRNA than the naive CD4(+)CD25(-) cells. Naive CD4(+)CD25(+) had no detectable IL-2 mRNA. Both naive and PMA plus ionomycin-stimulated thymic CD4(+)CD25(+) cells suppressed naive T cell proliferation. The suppressive properties were partially contact dependent. Supplementing CD4(+)CD25(+) cell coculture with rCIL-2 reversed the suppressive properties of CD4(+)CD25(+) cells. Chicken CD4(+)CD25(+) cells have suppressive properties similar to that of mammalian regulatory T cells.     

Antigen specificity acquisition of adoptive CD4+ regulatory T cells via acquired peptide-MHC class I complexes

The Ag-specific CD4(+) regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4(+) Tr cells is unclear. In this study, we generated IL-10- and IFN-gamma-expressing type 1 CD4(+) Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4(+) T cells with IL-10-expressing adenovirus (AdV(IL-10))-transfected and OVA-pulsed dendritic cells (DC(OVA/IL-10)). We demonstrated that both in vitro and in vivo DC(OVA/IL-10)-stimulated CD4(+) Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4(+) Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8(+) T cells, leading to an enhanced suppression of DC(OVA)-induced CD8(+) T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by approximately 700% relative to analogous CD4(+) Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4(+)25(+) Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8(+) T cell responses and antitumor immunity after uptake of DC(OVA)-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4(+) Tr cells via acquiring DC's pMHC I may be an important mean in augmenting CD4(+) Tr cell suppression.     

Whole protein and defined CD8(+) and CD4(+) peptides linked to penetratin targets both MHC class I and II antigen presentation pathways

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.     

Early events in peripheral regulatory T cell induction via the nasal mucosa

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4(+) regulatory T cells (T(R) cells) from the spleen, but little is known about the induction of mucosal T(R) cells in vivo. To investigate the induction of T(R) cells in the nose-draining cervical lymph node (CLN), CD4(+) T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4(+) DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4(+) DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4(+) DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4(+) DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4(+) DO11.10 T cells from CLN were phenotypically similar to CD4(+) DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25(+) proliferating CD4(+) DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal T(R) cells because both CD25(+) and CD25(-) CD4(+) DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of T(R) and effector T cells, this does not include their function. We are the first to demonstrate that functional T(R) cells, which reside within both CD25(+) and CD25(-) subsets, can be isolated from CLN as early as 3 days after nasal OVA application.