Intestinal epithelial antigen induces mucosal CD8 T cell tolerance, activation, and inflammatory response

Intestinal autoimmune diseases are thought to be associated with a breakdown in tolerance, leading to mucosal lymphocyte activation perhaps as a result of encounter with bacterium-derived Ag. To study mucosal CD8(+) T cell activation, tolerance, and polarization of autoimmune reactivity to self-Ag, we developed a novel (Fabpl(4x at -132)-OVA) transgenic mouse model expressing a truncated form of OVA in intestinal epithelia of the terminal ileum and colon. We found that OVA-specific CD8(+) T cells were partially tolerant to intestinal epithelium-derived OVA, because oral infection with Listeria monocytogenes-encoding OVA did not elicit an endogenous OVA-specific MHC class I tetramer(+)CD8(+) T cell response and IFN-gamma-, IL-4-, and IL-5-secreting T cells were decreased in the Peyer's patches, mesenteric lymph nodes, and intestinal mucosa of transgenic mice. Adoptive transfer of OVA-specific CD8(+) (OT-I) T cells resulted in their preferential expansion in the Peyer's patches and mesenteric lymph nodes and subsequently in the epithelia and lamina propria but failed to cause mucosal inflammation. Thus, CFSE-labeled OT-I cells greatly proliferated in these tissues by 5 days posttransfer. Strikingly, OT-I cell-transferred Fabpl(4x at -132)-OVA transgenic mice underwent a transient weight loss and developed a CD8(+) T cell-mediated acute enterocolitis 5 days after oral L. monocytogenes-encoding OVA infection. These findings indicate that intestinal epithelium-derived "self-Ag" gains access to the mucosal immune system, leading to Ag-specific T cell activation and clonal deletion. However, when Ag is presented in the context of bacterial infection, the associated inflammatory signals drive Ag-specific CD8(+) T cells to mediate intestinal immunopathology.     

Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.     

In vivo adjuvant-induced mobilization and maturation of gut dendritic cells after oral administration of cholera toxin

Although dendritic cells (DCs) regulate immune responses, they exhibit functional heterogeneity depending on their anatomical location. We examined the functional properties of intestinal DCs after oral administration of cholera toxin (CT), the most potent mucosal adjuvant. Two CD11c+ DC subsets were identified both in Peyer's patches and mesenteric lymph nodes (MLN) based on the expression of CD8alpha (CD8+ and CD8- DCs, respectively). A third subset of CD11c+CD8int was found exclusively in MLN. Feeding mice with CT induced a rapid and transient mobilization of a new CD11c+CD8- DC subset near the intestinal epithelium. This recruitment was associated with an increased production of the chemokine CCL20 in the small intestine and was followed by a massive accumulation of CD8int DCs in MLN. MLN DCs from CT-treated mice were more potent activators of naive T cells than DCs from control mice and induced a Th2 response. This increase in immunostimulating properties was accounted for by CD8int and CD8- DCs, whereas CD8+ DCs remained insensitive to CT treatment. Consistently, the CD8int and CD8- subsets expressed higher levels of costimulatory molecules than CD8+ and corresponding control DCs. Adoptive transfer experiments showed that these two DC subsets, unlike CD8+ DCs, were able to present Ags orally coadministered with CT in an immunostimulating manner. The ability of CT to mobilize immature DCs in the intestinal epithelium and to promote their emigration and differentiation in draining lymph nodes may explain the exceptional adjuvant properties of this toxin on mucosal immune responses.     

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.     

Fc gamma receptor IIB on dendritic cells enforces peripheral tolerance by inhibiting effector T cell responses

The uptake of immune complexes by FcRs on APCs augments humoral and cellular responses to exogenous Ag. In this study, CD11c+ dendritic cells are shown to be responsible in vivo for immune complex-triggered priming of T cells. We examine the consequence of Ab-mediated uptake of self Ag by dendritic cells in the rat insulin promoter-membrane OVA model and identify a role for the inhibitory FcgammaRIIB in the maintenance of peripheral CD8 T cell tolerance. Effector differentiation of diabetogenic OT-I CD8+ T cells is enhanced in rat insulin promoter-membrane OVA mice lacking FcgammaRIIB, resulting in a high incidence of diabetes. FcgammaRIIB-mediated inhibition of CD8 T cell priming results from suppression of both DC activation and cross-presentation through activating FcgammaRs. Further FcgammaRIIB on DCs inhibited the induction of OVA-specific Th1 effectors, limiting Th1-type differentiation and memory T cell accumulation. In these MHC II-restricted responses, the presence of FcgammaRIIB only modestly affected initial CD4 T cell proliferative responses, suggesting that FcgammaRIIB limited effector cell differentiation primarily by inhibiting DC activation. Thus, FcgammaRIIB can contribute to peripheral tolerance maintenance by inhibiting DC activation alone or by also limiting processing of exogenously acquired Ag.     

Injection of soluble antigen into the anterior chamber of the eye induces expansion and functional unresponsiveness of antigen-specific CD8+ T cells

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8(+) CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8(+) TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8(+) T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8(+) T cells.     

Breakdown of mucosal immunity in the gut and resultant systemic sensitization by oral antigens in a murine model for systemic lupus erythematosus

Secreted IgA plays a pivotal role in the mucosal immunity to maintain the front line of body defense. We found that the level of fecal IgA was dramatically decreased in aged (NZB x NZW)F(1) (BWF(1)) mice developing lupus nephritis, whereas levels in similarly aged New Zealand Black (NZB) and New Zealand White (NZW) mice remained unchanged compared with young mice. The number of cells obtained from Peyer's patches was markedly decreased in aged BWF(1) mice. Aged BWF(1) mice showed increased susceptibility to pathogenic bacterial infection. Furthermore, oral administration of OVA failed to inhibit secondary IgG response induced by systemic immunization, suggesting defective oral tolerance in aged BWF(1) mice. A significant amount of orally administered OVA was incorporated directly into the intestinal lamina propria in aged BWF(1) mice whereas it was mainly localized in subepithelial domes and interfollicular region in Peyer's patches in young mice. T cells obtained from renal and pulmonary lymph nodes of aged BWF(1) mice that had been orally administered with OVA showed an Ag-specific T cell proliferation, whereas those from young BWF(1), aged NZB, and aged NZW mice did not. Interestingly, aerosol exposure to OVA of aged BWF(1) mice, which had been orally administered with the same Ag, provoked an eosinophil infiltration in the lung. These results demonstrate that mucosal immunity in the gut is impaired and oral Ags induce systemic sensitization instead of oral tolerance in the development of murine lupus.     

Urinary bladder epithelium antigen induces CD8+ T cell tolerance, activation, and autoimmune response

The effort to explore the specific autoimmune mechanisms of urinary bladder has long been hindered due to a lack of proper animal models. To better elucidate this issue, we developed a novel line of transgenic (Tg) mice, designated as URO-OVA mice, that express the model Ag OVA as a "self"-Ag on the bladder epithelium. URO-OVA mice are naturally tolerant to OVA and show no response to OVA stimulation. Adoptive transfer of naive OVA-specific T cells showed cell proliferation, activation, and infiltration but no bladder histopathology. In contrast, adoptive transfer of activated OVA-specific T cells induced OVA-mediated histological bladder inflammation. Increased mast cells and up-regulated mRNA expressions of TNF-alpha, nerve growth factor, and substance P precursor were also observed in the inflamed bladder. To further facilitate bladder autoimmunity study, we crossbred URO-OVA mice with OVA-specific CD8(+) TCR Tg mice (OT-I mice) to generate a dual Tg line URO-OVA/OT-I mice. The latter mice naturally acquire clonal deletion for autoreactive OT-I CD8(+) T cells (partial deletion in the thymus and severe deletion in the periphery). Despite this clonal deletion, URO-OVA/OT-I mice spontaneously develop autoimmune cystitis at 10 wk of age. Further studies demonstrated that the inflamed bladder contained infiltrating OT-I CD8(+) T cells that had escaped clonal deletion and gained effector functions before developing histological bladder inflammation. Taken together, we demonstrate for the first time that the bladder epithelium actively presents self-Ag to the immune system and induces CD8(+) T cell tolerance, activation, and autoimmune response.     

Abrogation of functional selectin-ligand expression reduces migration of pathogenic CD8+ T cells into heart

CD8+ T cells are involved in autoimmune and infectious myocarditis and cardiac allograft rejection. The role of selectins in cardiac recruitment of CD8+ T cells is not understood. In this study, the contribution of T cell selectin ligands to effector CD8+ T cell recruitment into the heart was examined using a model of myocarditis, which depends on transfer of OVA peptide-specific CD8+ T cells (OT-I) into mice (CMy-mOva) that express OVA in the heart. alpha-(1,3)-Fucosyltransferase (FucT)-VII-deficient OT-I cells displayed over a 95% reduction in their ability to interact with P-selectin under flow conditions in vitro, compared with wild-type OT-I cells. Interaction of FucT-VII-deficient OT-I cells with E-selectin was reduced approximately 50%. FucT-VII-deficient OT-I cells were also less efficiently recruited into a dermal site of Ag and adjuvant injection. Significantly, FucT-VII-deficient OT-I cells were also impaired in their ability to migrate into CMy-mOva hearts, compared with wild-type OT-I cells. Transfer of FucT-VII-deficient T cells caused less severe early myocarditis and myocyte damage than transfer of wild-type T cells. Combined FucT-IV/VII-deficient OT-I cells displayed a more profound reduction in E-selectin interactions in vitro compared with FucT-VII-deficient T cells, and the FucT-IV/VII-deficient T cells also showed less early recruitment and pathogenicity in the CMy-mOva myocarditis model. These results identify a prominent role for selectin ligands in contributing to effector CD8+ T cell recruitment into the myocardium and indicate that selectin-dependent T cell recruitment is relevant to other tissues besides the skin.     

Control of syngeneic tumor growth by activation of CD8+ T cells: efficacy is limited by migration away from the site and induction of nonresponsiveness

Activation of Ag-specific CD8+ T cells in response to syngeneic tumor has been visualized by adoptive transfer of CD8+ T cells from OT-I mice, with a transgenic TCR specific for H-2Kb and an OVA peptide, into Thy-1 congenic recipients. Intraperitoneal challenge with E.G7, the EL-4 thymoma transfected with OVA, results in activation and clonal expansion of the OT-I cells in the peritoneal cavity and transient control of tumor growth. However, within 2 days after becoming activated, the OT-I cells migrate out of the peritoneal cavity into the spleen and lymph nodes, and tumor growth resumes in the peritoneal cavity. The OT-I cells in lymph nodes and spleen have lytic effector activity, but exhibit split anergy in that they cannot proliferate in response to Ag unless exogenous IL-2 is provided. The failure to remain at the tumor site and continue to control tumor growth is not due to selection of Ag loss variants or development of suppression. These results suggest that effective CD8-targeted immunotherapy may depend less on enhancing the initial activation and more on sustaining the response at the appropriate location and/or reactivating cells that have left the site of tumor growth and become nonresponsive.     

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).     

The state of CD4+ T cell activation is a major factor for determining the kinetics and location of T cell responses to oral antigen

Current models suggest that inductive immune responses to enteric Ag are initiated in Peyer's patches (PP) and mesenteric lymph nodes (MLN) followed by migration of activated, memory-like CD4(+) T cells to extralymphoid sites in the intestinal lamina propria (LP). The resultant immune system contains both naive and activated T cells. To examine the differential responses of naive and memory-like T cells to oral Ag, bone marrow chimeras (BMC) were generated. Irradiated BALB/c hosts were reconstituted with a mix of DO11.10 x RAG-1(-/-) and BALB/c bone marrow. In unprimed DO11.10 and BMC models, LP and PP DO11.10 T cells responded to oral Ag with similar kinetics. Responses of activated, memory-like T cells to oral Ag were examined in thymectomized BMC 60 days after i.p. immunization with OVA peptide in Freund's adjuvant (OVA(323-339)/CFA). Results indicate that i.p. OVA(323-339)/CFA generated a high proportion of memory-like CD45RB(low) DO11.10 T cells in peripheral lymphoid (40%) and intestinal LP (70%) tissue. Previously activated DO11.10 T cells in the LP responded to oral Ag earlier and at 50% higher levels compared with memory CD4(+) T cells localized to PP tissue. These data indicate that responses to oral Ag in antigenically naive animals are initiated in PP whereas in Ag-experienced animals LP T cells respond earlier and more vigorously than cells in PP. Taken together, these data suggest that previous activation alters the hierarchy of T cell responses to oral Ag by enhancing the efficiency of LP T cell activation.     

The mannose receptor mediates uptake of soluble but not of cell-associated antigen for cross-presentation

The mannose receptor (MR) has been implicated in the recognition and clearance of microorganisms and serum glycoproteins. Its endocytic function has been studied extensively using macrophages, although it is expressed by a variety of cell types, including dendritic cells (DC). In this study, we investigated its role in Ag presentation by DC using MR-/- mice. Uptake of the model Ag, soluble OVA, by bone marrow-derived DC and in vitro activation of OVA-specific CD8 T cells (OT-I cells) strictly depended on the MR. In vivo, MR deficiency impaired endocytosis of soluble OVA by DC and concomitant OT-I cell activation. No alterations in the DC subtype composition in MR-/- mice were accountable. Uptake of cell-associated OVA was unaffected by MR deficiency, resulting in unchanged activation of OT-I cells. These findings demonstrate that DC use the MR for endocytosis of a particular Ag type intended for cross-presentation.     

In vivo augmentation of tumor-specific CTL responses by class I/peptide antigen complexes on microspheres (large multivalent immunogen)

Tumor membrane Ag immobilized on cell size microspheres (large multivalent immunogen (LMI)) was previously shown to augment tumor-specific CTL activity and reduce tumor growth, and a clinical trial examining this approach is in progress. In the current study, LMI treatment has been examined using adoptive transfer of TCR-transgenic CD8 T cells to visualize Ag-specific cells during the response. OT-I T cells specific for H-2K(b)/OVA(257-264) were transferred into mice that were then challenged with LMI made by immobilizing H-2K(b)/OVA(257-264) on microspheres (K(b)/OVA(257-264)-LMI) alone, or along with i.p. challenge with OVA-expressing E.G7 tumor. K(b)/OVA(257-264)-LMI caused significant reduction of tumor growth when administered to E.G7-bearing mice. When administered alone, the K(b)/OVA(257-264)-LMI caused only weak clonal expansion of OT-I cells in the spleen and lymph nodes, although most of the OT-I cells up-regulated expression of CD44 and VLA-4. In contrast, K(b)/OVA(257-264)-LMI administration to E.G7-bearing mice stimulated no detectable expansion of OT-I cells in the spleen and lymph nodes but caused a rapid increase in the number of OT-I cells in the peritoneal cavity, the site of the growing tumor. These results demonstrate the potential for using class I/tumor peptide complexes for immunotherapy. In addition, they suggest a model for the mechanism of CTL augmentation in which recognition of the LMI Ag results in altered trafficking of the tumor-specific CD8 T cells so that they reach the site of a growing tumor more rapidly and in greater numbers, where they may further expand and acquire effector function.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

PD-1 regulates self-reactive CD8+ T cell responses to antigen in lymph nodes and tissues

PD-1, an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. We tested the role of PD-1:PD-1 ligand (PD-L) interactions in cross-presentation and the generation and control of CD8(+) responses against self-Ag. Ag-naive PD-1(-/-) OVA-specific OT-I CD8(+) T cells exhibited exacerbated responses to cross-presented Ag in mice expressing soluble OVA under the control of the rat insulin promoter (RIP-ova(high)). Following adoptive transfer into RIP-ova(high) recipients, PD-1(-/-) OT-I T cells expanded in the pancreatic lymph node. In contrast to wild-type OT-I cells, PD-1(-/-) OT-I T cells secreted IFN-gamma and migrated into the pancreas, ultimately causing diabetes. Loss of PD-1 affected CD8(+) cells intrinsically, and did not significantly alter the responses of wild-type OT-I T cells adoptively transferred into the same RIP-ova(high) recipient mouse. PD-1:PD-L interactions also limited CD8(+) effector cells, and PD-L1 expression on parenchymal tissues protected against effector OT-I T cell attack. Finally, we found that the loss of PD-1 on effector OT-I cells lowers the threshold for Ag recognition in peripheral tissues. These findings indicate two checkpoints where PD-1 attenuates self-reactive T cell responses: presentation of self-Ag to naive self-reactive T cells by dendritic cells in the draining lymph node and reactivation of pathogenic self-reactive T cells in the target organ.     

The role of antigen in the localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza pneumonia.

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2(+)CD8(+) OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1(+) recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVA(I)) containing the K(b) restricted OVA(257-264) epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant K(b) epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8(+) T cells reactive to a murine gamma-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.     

Chronic allergy to dietary ovalbumin induces lymphocyte migration to rat small intestinal mucosa that is inhibited by MAdCAM-1

Few models have described a chronic food allergy with morphological changes in the intestinal mucosa. Here we established an ovalbumin (OVA)-induced, cell-mediated, allergic rat model and examined lymphocyte migration in the gut. Brown Norway rats were intraperitoneally sensitized to OVA and then given 10 mg OVA/day by gastric intubation for 6 wk. Lymphocyte subsets and adhesion molecules were examined immunohistochemically, and the migration of T lymphocytes to microvessels of Peyer's patches and villus mucosa was observed by using an intravital microscope. Serum OVA-specific IgG and IgE levels were increased in animals repeatedly exposed to OVA. Significant villus atrophy and increased crypt depth was accompanied by increased infiltration of T lymphocytes in the small intestinal mucosa of the group given OVA. Expression of rat mast cell protease II and of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was also increased in these groups. The administration of anti-MAdCAM-1 antibody significantly attenuated the OVA-induced changes in the mucosal architecture and in CD4 T lymphocyte infiltration. Intravital observation demonstrated that in rats with a chronic allergy, T lymphocytes significantly accumulated in villus microvessels as well as in Peyer's patches via a MAdCAM-1-dependent process. Our model of chronic food allergy revealed that lymphocyte migration was increased with MAdCAM-1 upregulation.     

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

Functional tolerance of CD8+ T cells induced by muscle-specific antigen expression

Skeletal muscles account for more than 30% of the human body, yet mechanisms of immunological tolerance to this tissue remain mainly unexplored. To investigate the mechanisms of tolerance to muscle-specific proteins, we generated transgenic mice expressing the neo-autoantigen OVA exclusively in skeletal muscle (SM-OVA mice). SM-OVA mice were bred with OT-I or OT-II mice that possess a transgenic TCR specific for OVA peptides presented by MHC class I or class II, respectively. Tolerance to OVA did not involve clonal deletion, anergy or an increased regulatory T cell compartment. Rather, CD4+ T cell tolerance resulted from a mechanism of ignorance revealed by their response following OVA immunization. In marked contrast, CD8+ T cells exhibited a loss of OVA-specific cytotoxic activity associated with up-regulation of the immunoregulatory programmed death-1 molecule. Adoptive transfer experiments further showed that OVA expression in skeletal muscle was required to maintain this functional tolerance. These results establish a novel asymmetric model of immunological tolerance to muscle autoantigens involving Ag ignorance for CD4+ T cells, whereas muscle autoantigens recognized by CD8+ T cells results in blockade of their cytotoxic function. These observations may be helpful for understanding the breakage of tolerance in autoimmune muscle diseases.