Intranasal exposure to protein antigen induces immunological tolerance mediated by functionally disabled CD4+ T cells.

In this study we examined the immunological parameters underlying the natural immunity to inhaled nonpathogenic proteins. We addressed this question by examining the effect of intranasal exposure to OVA in both wild-type mice and mice reconstituted with OVA-TCR transgenic CD4+ T cells. Intranasal administration of OVA induced an initial phase of activation during which CD4+ T cells were capable of proliferating and producing cytokines. Although many of the OVA-specific CD4+ T cells were subsequently depleted from the lymphoid organs, a stable population of such T cells survived but remained refractory to antigenic rechallenge. The unresponsive state was not associated with immune deviation due to selective secretion of Th1- or Th2-type cytokines, and the presence of regulatory CD8+ T cells was not required. Moreover, neutralization of the immunosuppressive cytokines IL-10 and TGF-beta did not abrogate the induction of tolerance. Inhibition of the interaction of T cells with CD86, but not CD80, at the time of exposure to intranasal Ag prevented the development of unresponsiveness, while selective blockade of CTLA-4 had no effect. Our results suggest that intranasal exposure to Ags results in immunological tolerance mediated by functionally impaired CD4+ T cells via a costimulatory pathway that requires CD86.     

Antigen chemically coupled to the surface of liposomes are cross-presented to CD8+ T cells and induce potent antitumor immunity

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4+ and CD8+ T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4+ T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8+ T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8+ T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.     

The IL-4 receptor alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25+CD4+ regulatory T cells from CD25-CD4+ precursors

The mechanisms underlying the extrathymic generation of CD25+CD4 regulatory T cells (Tregs) are largely unknown. In this study the IL-4R alpha-chain-binding cytokines, IL-4 and IL-13, were identified as inducers of CD25+ Tregs from peripheral CD25-CD4 naive T cells. IL-4-induced CD25+ Tregs phenotypically and functionally resemble naturally occurring Tregs in that they are anergic to mitogenic stimulation, inhibit the proliferation of autologous responder T cells, express high levels of the Forkhead box P3 and the surface receptors glucocorticoid-induced TNFR family-related protein and CTLA-4, and inhibit effector T cells in a contact-dependent, but cytokine-independent, manner. The IL-4-induced generation of peripheral Tregs was independent of the presence of TGF-beta or IL-10, but was dependent on Ag-specific stimulation and B7 costimulation. The significance of the IL-4Ralpha-binding cytokines in the generation of Ag-specific Tregs was emphasized in a mouse model of oral tolerance, in which neutralization of IL-4 and IL-13 in mice transgenic for the TCR specific for OVA completely inhibited the expansion of OVA-specific Tregs that can be induced in untreated mice by feeding the nominal Ag. Together, our results demonstrate that IL-4 and IL-13 play an important role in generating Forkhead box P3-expressing CD25+ Tregs extrathymically in an Ag-dependent manner and therefore provide an intriguing link between the well-established immunoregulatory capacity of Th2 cells and the powerful CD25+ Treg population. Moreover, our findings might provide the basis for the design of novel therapeutic approaches for targeted immunotherapy with Tregs to known Ags in autoimmune diseases or graft-vs-host reactions.     

Administration of an antigen at a high dose generates regulatory CD4+ T cells expressing CD95 ligand and secreting IL-4 in the liver

Ags administered orally at a high dose are absorbed in immunogenic forms and perfuse the liver, which raises a question regarding the relevance of hepatic lymphocyte activation to the systemic hyporesponsiveness against the ingested Ag. Oral administration of 100 mg of OVA to the mice led to massive cell death of OVA-specific (KJ1-26+)CD4+ T cells by Fas-Fas ligand (FasL)-mediated apoptosis in the liver, which was associated with the emergence of hepatic KJ1-26+CD4+ T cells expressing FasL. Hepatic CD4+ T cells in OVA-fed mice secreted large amounts of IL-4, IL-10, and TGF-beta(1) upon restimulation in vitro and inhibited T cell proliferation. Adoptive transfer of these hepatic CD4+ T cells to naive mice and subsequent antigenic challenge led to suppression of T cell proliferation as well as IgG Ab responses to OVA; this effect was mostly abrogated by a blocking Ab to FasL. i.p. administration of an Ag at a high dose also generated hepatic CD4+FasL+ T cells with similar cytokine profile as T cells activated by oral administration of Ags at a high dose. Finally, we did not see an increase in FasL+ cells in the hepatic CD4+Vbeta8+ T cell subset of MRL/lpr/lpr mice given staphylococcal enterotoxin B, indicating the requirement for Fas-mediated signals. These hepatic CD4+FasL+ regulatory cells may explain the tolerogenic property of the liver and play roles in systemic hyporesponsiveness induced by an Ag administered at a high dose.     

TGF-beta expression by allogeneic bone marrow stromal cells ameliorates diabetes in NOD mice through modulating the distribution of CD4+ T cell subsets

BMSCs could promote the regeneration of islet beta-cell, but the status of BMSCs under diabetes is still unknown. Our study verified the effect of allogeneic BMSCs (ICR) transferred into NOD mice on blood glucose and CD4+ T cells subsets function. In vivo experiment, BMSCs could decrease blood glucose, weaken lymphocytes proliferation. In vitro experiment, the distribution of CD4+ T cell subsets was changed after co-culture with BMSCs, resulting in a greater frequency of Treg cells and reduced representation of Th17 cells. After TGF-beta blockade, CD4+ T cells differentiated along a route favoring development of Th17, but not Treg cells. Thus, NOD can be treated by BMSCs which changes the distribution of CD4+ T cells, increases the number of Treg cells, and inhibits the differentiation of Th17 cells. And the positive effects of allogeneic BMSCs in the treatment of NOD mice depend on the regulation of TGF-beta secreted by BMSCs.     

Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.     

In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11. 10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.     

Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity

Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-gamma and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.     

Th17 augmentation in OTII TCR plus T cell-selective type 1 sphingosine 1-phosphate receptor double transgenic mice

Sphingosine 1-phosphate (S1P) in blood and lymph controls lymphoid traffic and tissue migration of T cells through signals from the type 1 S1PR (S1P(1)), but less is known of effects of the S1P-S1P(1) axis on nonmigration functions of T cells. CD4 T cells from a double transgenic (DTG) mouse express OTII TCRs specific for OVA peptide 323-339 (OVA) and a high level of transgenic S1P(1), resistant to suppression by T cell activation. OVA-activated DTG CD4 T cells respond as expected to S1P by chemotactic migration and reduction in secretion of IFN-gamma. In addition, DTG CD4 T cells stimulated by OVA secrete a mean of 2.5-fold more IL-17 than those from OTII single transgenic mice with concomitantly higher levels of mRNA encoding IL-17 by real-time PCR and of CD4 T cells with intracellular IL-17 detected by ELISPOT assays. OVA challenge of s.c. air pockets elicited influx of more OTII TCR-positive T cells producing a higher level of IL-17 in DTG mice than OTII control mice. Augmentation of the number and activity of Th17 cells by the S1P-S1P(1) axis may thus enhance host defense against microbes and in other settings increase host susceptibility to autoimmune diseases.     

Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T cells and contraction of inflammatory Th17 cells

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H4(71-94), when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-beta. The induced Treg cells suppress nuclear autoantigen-specific Th and B cells and block renal inflammation. Splenic dendritic cells (DC) captured the s.c.-injected H4(71-94) peptide rapidly and expressed a tolerogenic phenotype. The DC of the tolerized animal, especially plasmacytoid DC, produced increased amounts of TGF-beta, but diminished IL-6 on stimulation via the TLR-9 pathway by nucleosome autoantigen and other ligands; and those plasmacytoid DC blocked lupus autoimmune disease by simultaneously inducing autoantigen-specific Treg and suppressing inflammatory Th17 cells that infiltrated the kidneys of untreated lupus mice. Low-dose tolerance with H4(71-94) was effective even though the lupus immune system is spontaneously preprimed to react to the autoepitope. Thus, H4(71-94) peptide tolerance therapy that preferentially targets pathogenic autoimmune cells could spare lupus patients from chronically receiving toxic agents or global immunosuppressants and maintain remission by restoring autoantigen-specific Treg cells.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

A model vaccine exploiting fungal mannosylation to increase antigen immunogenicity

Ag mannosylation represents a promising strategy to augment vaccine immunogenicity by targeting Ag to mannose receptors (MRs) on dendritic cells. Because fungi naturally mannosylate proteins, we hypothesized that Ags engineered in fungi would have an enhanced capacity to stimulate T cell responses. Using the model Ag OVA, we generated proteins that differentially expressed N- and O-linked mannosylation in the yeast Pichia pastoris and compared them to their unglycosylated counterparts produced in Escherichia coli. We found that yeast-derived OVA proteins containing N-linkages, extensive O-linkages, or both were more potent than the unmannosylated Ags at inducing OVA-specific CD4+ T cell proliferation. This elevated response to fungal Ags was inhibited by mannan, suggesting involvement of MRs. However, the macrophage MR (CD206) was not essential, because macrophage MR-deficient dendritic cells were fully competent in presenting yeast-derived OVA Ags. Thus, the use of fungal glycosylation to provide N-linked and/or extensive O-linked mannosylation increased the capacity of the model Ag OVA to stimulate Ag-specific T cell responses in an MR-dependent manner. These data have implications for vaccine design by providing proof of principle that yeast-derived mannosylation can enhance immunogenicity.     

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.     

Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells

Data regarding the role of TGF-beta for the in vivo function of regulatory CD4(+)CD25(+) T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-beta signaling specifically in T cells was used to assess the role of endogenous TGF-beta for the in vivo function of CD4(+)CD25(+) Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4(+)CD25(+) Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4(+)CD25(+) Treg we could demonstrate that endogenous TGF-beta promotes the expansion of CD4(+)CD25(+) Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4(+)CD25(+) Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-beta signaling in CD4(+)CD25(+) Treg is required for their in vivo expansion and suppressive capacity.     

Heme oxygenase-1-mediated CD4+CD25high regulatory T cells suppress allergic airway inflammation

Heme oxygenase-1 (HO-1) has anti-inflammatory effects in asthma. CD4+CD25(high) regulatory T cells (Treg) are a potent immunoregulator that suppresses the immune response. We studied the effects of HO-1-mediated CD4+CD25(high) Treg on suppression of allergic airway inflammation by comparing mice treated with hemin, OVA, Sn-protoporphyrin (SnPP), and hemin plus SnPP. Airway responsiveness, airway eosinophil infiltration, the level of OVA-specific IgE, and the numbers of cells in general and eosinophils in particular in bronchial alveolar lavage fluid were lower in the hemin group than in the OVA, SnPP, and hemin plus SnPP groups. The expressions of HO-1 mRNA and protein in the lung were increased by repeated administrations of hemin and SnPP. However, the activity of HO-1 was highest in hemin mice. The percentage and suppressive function of CD4+CD25(high) Treg and the expression of Foxp3 mRNA were obviously enhanced after treatment with hemin. This increase was diminished by the administration of SnPP. The concentration of serum IL-10 was higher in the hemin group than in the other groups, whereas the level of serum TGF-beta did not significantly differ across groups. Furthermore, the ratio of IFN-gamma/IL-4 mRNA in the lung was higher in hemin-treated mice than in OVA and SnPP mice. The suppressive capacity of CD4+CD25(high) Treg was not enhanced in the IL-10-deficient mice treated with hemin. In conclusion, our experiments in the animal model demonstrated that HO-1 has anti-inflammatory effects, probably via enhancement of the secretion of IL-10 and promotion of the percentage of CD4+CD25(high) Treg.     

Minimal tolerance to a tumor antigen encoded by a cancer-germline gene

Central tolerance toward tissue-restricted Ags is considered to rely on ectopic expression in the thymus, which was also observed for tumor Ags encoded by cancer-germline genes. It is unknown whether endogenous expression shapes the T cell repertoire against the latter Ags and explains their weak immunogenicity. We addressed this question using mouse cancer-germline gene P1A, which encodes antigenic peptide P1A(35-43) presented by H-2L(d). We made P1A-knockout (P1A-KO) mice and asked whether their anti-P1A(35-43) immune responses were stronger than those of wild-type mice and whether P1A-KO mice responded to other P1A epitopes, against which wild-type mice were tolerized. We observed that both types of mice mounted similar P1A(35-43)-specific CD8 T cell responses, although the frequency of P1A(35-43)-specific CD8 T cells generated in response to P1A-expressing tumors was slightly higher in P1A-KO mice. This higher reactivity allowed naive P1A-KO mice to reject spontaneously P1A-expressing tumors, which progressed in wild-type mice. TCR-Vβ usage of P1A(35-43)-specific CD8 cells was slightly modified in P1A-KO mice. Peptide P1A(35-43) remained the only P1A epitope recognized by CD8 T cells in both types of mice, which also displayed similar thymic selection of a transgenic TCR recognizing P1A(35-43). These results indicate the existence of a minimal tolerance to an Ag encoded by a cancer-germline gene and suggest that its endogenous expression only slightly affects diversification of the T cell repertoire against this Ag.     

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).     

A bone marrow-derived APC in the gut-associated lymphoid tissue captures oral antigens and presents them to both CD4+ and CD8+ T cells

We have previously reported that feeding OVA to C57BL/6 mice can lead to a weak CTL response that is dependent on CD4+ T cell help and is capable of causing autoimmunity. In this study, we investigated the basis of the class I and class II-restricted Ag presentation required for such CTL induction. Two days after feeding OVA, Ag-specific CD4+ and CD8+ T cells were seen to proliferate in the Peyer's patches and mesenteric lymph nodes. Little proliferation was evident in other lymphoid tissues, except at high Ags doses, in which case some dividing CD4+ T cells were observed in the spleen and peripheral lymph nodes. Using chimeric mice, the APC responsible for presenting orally derived Ags was shown to be derived from the bone marrow. Examination of the Ag dose required to activate either CD4+ or CD8+ T cells indicated that a single dose of 6 mg OVA was the minimum dose that consistently stimulated either T cell subset. These data indicate that oral Ags can be transported from the gut into the gut-associated lymphoid tissue, where they are captured by a bone marrow-derived APC and presented to both CD4+ and CD8+ T cells.     

P38 MAP kinase signaling is required for the conversion of CD4+CD25- T cells into iTreg

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.     

Multiple antigen-specific processing pathways for activating naive CD8+ T cells in vivo

Current knowledge of the processing of viral Ags into MHC class I-associated ligands is based almost completely on in vitro studies using nonprofessional APCs (pAPCs). This is two steps removed from real immune responses to pathogens and vaccines, in which pAPCs activate naive CD8(+) T cells in vivo. Rational vaccine design requires answers to numerous questions surrounding the function of pAPCs in vivo, including their abilities to process and present peptides derived from endogenous and exogenous viral Ags. In the present study, we characterize the in vivo dependence of Ag presentation on the expression of TAP by testing the immunogenicity of model Ags synthesized by recombinant vaccinia viruses in TAP1(-/-) mice. We show that the efficiency of TAP-independent presentation in vitro correlates with TAP-independent activation of naive T cells in vivo and provide the first in vivo evidence for proteolytic processing of antigenic peptides in the secretory pathway. There was, however, a clear exception to this correlation; although the presentation of the minimal SIINFEKL determinant from chicken egg OVA in vitro was strictly TAP dependent, it was presented in a TAP-independent manner in vivo. In vivo presentation of the same peptide from a fusion protein retained its TAP dependence. These results show that determinant-specific processing pathways exist in vivo for the generation of antiviral T cell responses. We present additional findings that point to cross-priming as the likely mechanism for these protein-specific differences.