Ag Dose Research Articles

Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands.

Thymic expression of self-Ags results in the deletion of high-avidity self-specific T cells, but, at least for certain Ags, a residual population of self-specific T cells with low-affinity TCRs remains after negative selection. Such self-specific T cells are thought to play a role in the induction of T cell-mediated autoimmunity, but may also be used for the induction of antitumor immunity against self-Ags. In this study, we examine the functional competence of a polyclonal population of self-specific CD8+ T cells. We show that low-affinity interactions between TCR and peptide are associated with selective loss of critical T cell functions. Triggering of low levels of IFN-gamma production and cytolytic activity through low-affinity TCRs readily occurs provided high Ag doses are used, but IL-2 production and clonal expansion are severely reduced at all Ag doses. Remarkably, a single peptide variant can form an improved ligand for the highly diverse population of low-avidity self-specific T cells and can improve their proliferative capacity. These data provide insight into the inherent limitations of self-specific T cell responses through low-avidity TCR signals and the effect of modified peptide ligands on self-specific T cell immunity.

Monomorphic molecules function as additional recognition structures on haptenated target cells for HLA-A1-restricted, hapten-specific CTL.

Hapten-specific T cells have been shown to recognize haptenated peptides with high avidity and, in some instances, with promiscuous MHC restriction. In this study, the impact of Ag density on MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was investigated. In this study, we demonstrate a novel recognition mechanism used by TNP-specific CD8(+) CTL in the presence of high Ag doses. Although low levels of TNP epitopes on target cells allowed for HLA-A1-restricted CTL activity only, entirely MHC-independent target cell recognition became operative at high TNP loading. In both cases, recognition was mediated by the TCR. This MHC-independent recognition is target cell type restricted and critically involves in our model direct recognition of the ectonucleotidase family surface molecule CD39 by the CTL.

A T cell clone's avidity is a function of its activation state.

At present it is unclear how Ag dose-dependent T cell functions, such as cytokine production, reflect TCR affinity and how the signal strength afforded by the Ag dose affects the kinetics of cytokine production by the individual T cell. We used a computer-assisted ELISPOT approach to address these issues. IFN-gamma release by a clonal population of CD4 T cells was monitored on a clonal population of APC while titrating the nominal peptide. The frequency of cytokine-producing cells, the net per-cell output of cytokine, and the onset of cytokine production were each found to be functions of the signal strength. Sigmoidal dose-response curves were seen at the clonal population level, but the activation thresholds for the individual T cells followed a Gaussian distribution. Moreover, the overall dose-response curve of the T cell clone revealed cyclic changes, becoming increasingly shifted toward lower Ag concentrations with the duration of time that elapsed since the last restimulation with Ag. Therefore, responsiveness to Ag ("functional avidity") is not a constant parameter of a T cell clone but a function of the T cell's history of last Ag encounter. The implications of such shifting activation thresholds are discussed for autoimmune disease.

Generation of anergic and potentially immunoregulatory CD25+CD4 T cells in vivo after induction of peripheral tolerance with intravenous or oral antigen.

Immunoregulatory CD25(+)CD4 T cells are thought to arise from the thymus as a distinct lineage of CD4 T cells specific for self Ags. We used the DO11.10 TCR transgenic adoptive transfer system to show that cells of similar phenotype may also arise in the course of peripheral tolerance induction. Such cells emerged within 1 wk following Ag exposure and correlated negatively with the number of initial cell divisions. Limiting i.v. Ag dose or using an oral tolerance protocol yielded the greatest numbers of Ag-specific CD25(+)CD4 T cells. In contrast, immunogenic Ag exposure in the presence of an adjuvant did not lead to emergence of CD25(+)CD4 T cells. The profound anergic phenotype of these cells and their potential immunoregulatory properties make them an especially desirable population to induce in the course of immunotherapy in numerous clinical settings. This experimental system may be useful in future studies designed to optimize immunologic tolerance induction.

Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens.

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, "cross-priming" can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor's blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual's APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

Visualizing T Cell Competition for Peptide/MHC Complexes: A Specific Mechanism to Minimize the Effect of Precursor Frequency

In vivo antigenic competition of naive CD4 + TCR transgenic T cells was visualized by tracking cell division. Competition reduced both recruitment into cell division and burst size per recruited precursor cell, minimizing the effect of differences in precursor frequency while maintaining the dose-response relationship with antigen. Competition was restricted to T cells of the same specificity, indicating that cells were competing for access to Ag-MHC complexes rather than for Ag nonspecific factors. Moreover, the qualitative distinction between the responses to i.v. peptide and s.c. peptide/CFA was unaffected by precursor frequency. These data explain the paradoxical ability of the immune system to tailor responses to the type and dose of Ag even in individuals with large differences in initial precursor frequency.

American Ginseng Improves Glycemia in Individuals with Normal Glucose Tolerance: Effect of Dose and Time Escalation

Objective: We studied the effect of escalating the dose and administration time of American ginseng (AG, Panax quinquefolius L.) in nondiabetic individuals to achieve further improvements in glucose tolerance seen previously when 3g of AG was taken 40 minutes before a 25g glucose challenge.Methods: Ten nondiabetic individuals (6M:4F; mean ± STD: age = 41 ± 13 years, BMI = 24.8 ± 3.5 kg/m2, FBG = 4.5 ± 0.1mmolL−1) on 12 separate occasions, randomly received 0 (placebo), 3, 6 or 9g of ground AG root at 40, 80, or 120 minutes before a 25g oral glucose challenge. Capillary blood glucose was measured prior to ingestion of AG or placebo capsules and at 0, 15, 30, 45, 60 and 90 minutes from start of challenge.Results: Compared with the placebo, 3, 6 and 9g of AG reduced (p<0.05) postprandial incremental glucose at 30, 45 and 60 minutes; also, 3 and 9g of AG did so at 90 minutes. At 60 minutes, 9g of AG reduced incremental postprandial glucose relative to 3g of AG (p<0.05). All AG doses reduced (p<0.05) area under the incremental glucose curve (3g, 26.6%; 6g, 29.3%; 9g, 38.5%). AG taken at different times did not have an additional influence on postprandial glycemia.Conclusions: In nondiabetic individuals, 3, 6 or 9g of AG taken 40, 80 or 120 minutes before a glucose challenge similarly improved glucose tolerance.

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.

Chronic myeloid lukemia cells: Tyrosine kinase inhibition using ag 957 and arachidonic acid inducing apoptosis

Chronic myelogenous leukemia (CML) is characterized by Philadelphia chromosome that fuses genetic sequences of the BCR gene on chromosome 22. AG 957, a member of the tyrphostin compound produces a selective inhibition of P 210 BCR/ABL tyrosine phosphorylation. A group of ten patients with CML in chronic phase were treated after CD34 separation with 1 to 100 umol/L of AG 957. All patients were 100% Ph1 positive. Eight patients showed a b3a2 BCR/ABL junction and two cases showed a b2a2 junction when analyzed by RT-PCR. The effect of AG 957 on CML and normal CD34 cells in culture assay (CFU/MIX, BFU-E and CFU-GM) is clearly demonstrated. The inhibition on CML untreated and AG957 treated colonies was from 2%–7% for CFU-MIX, 38%–80% for BFU-E and 31%–18–% for CFU-GM. In the group of normal samples the inhibition ranged from 4%–8% for CFU-MIX, 24%–96% for BFU-E and 4%–64% for CFU-GM. The inhibitory effect of AG 957 on CML progenitors is statistically significant at the dose of 1 umol/L for CFU-MIX and BFU-E, 5 umol/L for CFU-GM. For colonies obtained from normal progenitors the inhibiting dose of AG 957 was more consistent: 5 umol/L for CFU-MIX and BFU-E, 10 umol/L for CFU-GM. These data demonstrated the possibility to select non clonal CML progenitors after incubation with the PTK inhibitor AG 957. This selection of “normal” progenitors from CML marrow could be used for autograft in patients without suitable allogeneic bone marrow donors. The research is in progression to explore the potential therapeutic effect of AG 957 in combination with other agents (IFN - ARA-C) and/or inductors of apoptosis as arachidonic acid in cell culture, to briefly translate the “in vitro” experimental approach to clinical therapeutic application.

Correlation of T cell independence of antibody responses with antigen dose reaching secondary lymphoid organs: implications for splenectomized patients and vaccine design.

Many natural viral and bacterial pathogens activate B cells independently of Th cells (TI Ags). This study analyzed the characteristics of the activation of B cells after immunization with various forms of viral Ags using different immunization routes and found a decreasing dependence on T help with increasing amounts of Ag recruited to the spleen. Repetitive antigenic structure facilitated TI B cell responses if Ag was present in lymphoid organs. These results suggest that 1) Ag dose and localization in secondary lymphoid organs are the key for B cell activation in the absence of T help; 2) early TI Ab responses are crucial to protect against systemically spreading acute cytopathic infectious agents; and 3) there may be new rationales for improved vaccine design.

Humoral immune responses in Cr2-/- mice: enhanced affinity maturation but impaired antibody persistence.

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.

An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model.

Immunization of BALB/c mice with alum-adsorbed OVA, followed by three bronchoprovocations with aerosolized OVA, resulted in the development of airway hyperresponsiveness (AHR) and allergic inflammation in the lung accompanied by severe infiltration of eosinophils into airways. In this murine asthma model, administration of monoclonal anti-IL-5 Ab before each Ag challenge markedly inhibited airway eosinophilia, but the treatment did not affect the development of AHR. Immunization and aerosol challenges with OVA following the same protocol failed to induce AHR in the mast cell-deficient W/Wv mice, but induced AHR in their congenic littermates, i.e., WBB6F1 (+/+) mice. No significant difference was found between the W/Wv mice and +/+ mice with respect to the IgE and IgG1 anti-OVA Ab responses and to the airway eosinophilia after Ag provocations. It was also found that reconstitution of W/Wv mice with bone marrow-derived mast cells cultured from normal littermates restored the capacity of developing Ag-induced AHR, indicating that lack of mast cells was responsible for the failure of W/Wv mice to develop Ag-induced AHR under the experimental conditions. However, the OVA-immunized W/Wv mice developed AHR by increasing the frequency and Ag dose of bronchoprovocations. The results suggested that AHR could be developed by two distinct cellular mechanisms. One would go through mast cell activation and the other is IgE/mast cell independent but an eosinophil/IL-5-dependent mechanism.

Molecular mechanisms of IL-2 gene regulation following costimulation through LFA-1

The integrin LFA-1 serves as an accessory molecule in T cell activation. In addition to its well-known role as an adhesion molecule, LFA-1 can contribute to T cell activation and up-regulation of IL-2 gene expression. However, the specific mechanisms by which LFA-1 influences T cell activation have not been elucidated. Therefore, we examined the impact of LFA-1:ICAM-1 interactions on transcriptional and posttranscriptional IL-2 gene regulation, using a costimulation-negative cell line transfected with MHC class II alone, or in combination with ICAM-1 or B7-1. IL-2 transcription was assessed utilizing transgenic mice expressing an IL-2 promoter luciferase reporter construct crossed to DO11.10 TCR-transgenic mice, and IL-2 mRNA stability was evaluated by real-time RT-PCR. Comparison of naive and previously activated T cells demonstrates a dramatic increase in IL-2-luciferase transcription in activated T cells that can, in part, be attributed to downstream signaling events. Costimulation through LFA-1 enhances transcription of the transgenic reporter construct across a wide Ag dose range, but does not affect IL-2 mRNA stability. In contrast, CD28 costimulation is clearly mediated through up-regulation of IL-2 transcription and through enhancement of mRNA stability. These results indicate that the primary pathway whereby engagement of LFA-1 through its ligand ICAM-1 up-regulates IL-2 gene expression is through enhanced IL-2 transcription, in the absence of any effect on IL-2 mRNA stabilization.

CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen.

The involvement of specific accessory/costimulatory molecules in differentiation to Th1 and Th2 phenotypes is controversial. Reports suggest that molecules such as CD4, CD28, and Ox-40 support Th2 differentiation and suppress Th1 differentiation, whereas others such as LFA-1 support Th1 responses and suppress Th2 responses. We have previously defined an in vitro model of differentiation that is absolutely dependent on the initial dose and affinity of peptide presented to a naive CD4 cell. The dose and affinity of Ag regulate autocrine production of IL-2, IL-4, and IFN-gamma, which in turn govern differentiation to Th1 and Th2 phenotypes. We have used this system to confirm that CD4, CD28, and Ox-40 interactions can promote, and LFA-1 interactions can suppress, differentiation of cells secreting the Th2 cytokines IL-5 and IL-13. However, for CD4 and LFA-1, this is only seen over a certain range of peptide doses. In addition, CD28 and Ox-40 interactions also promote Th1 differentiation. In general, agonist Abs to accessory molecules shifted the response curves for IFN-gamma, IL-5, and IL-13 to lower doses, whereas antagonist reagents resulted in similar curves shifted toward the higher doses. We conclude that ligation of cell surface accessory receptors enables low doses of Ag to promote responses normally induced only by higher doses. Individual receptors do not intrinsically regulate one cytokine phenotype or another, suggesting that differentiation is controlled by the level of expression of multiple accessory molecule pairs integrated with the number and affinity of peptide/MHC complexes.

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.

Antiviral B cell memory in the absence of mature follicular dendritic cell networks and classical germinal centers in TNFR1-/- mice.

TNFR1-/- mice have been shown to lack networks of mature follicular dendritic cells (FDCs) and they do not form germinal centers. With nonreplicating Ags, IgG titers were inefficiently induced and not maintained. In this study, the neutralizing Ab response and the establishment of B cell memory in TNFR1-/- mice after infection with vesicular stomatitis virus (VSV) were analyzed histologically and functionally. Immunization with VSV-derived protein Ags without adjuvant induced only IgM but no IgG Abs in TNFR1-/- mice, whereas VSV glycoprotein emulsified in CFA or IFA induced IgM and IgG responses that were short-lived and of moderate titer. However, infection with live VSV induced excellent neutralizing IgM and IgG responses in TNFR1-/- mice, and adoptively transferable B cell memory was generated and persisted for more than 300 days. In contrast, IgG levels and Ab-forming cells in the bone marrow declined within 300 days by 90-95% compared with controls. These findings suggest that 1) increased Ag dose and time of Ag availability can substitute for FDC-stored Ab-complexed Ag in the induction of efficient IgG responses in TNFR1-/- mice devoid of classical germinal centers; 2) the induction and maintenance of adoptively transferable B cell memory can occur in the absence of Ag bound to mature FDCs; and 3) the long-term maintenance of elevated IgG titers is largely dependent on FDC-associated persisting Ag. However, about 5-10% of the Ab production remained in the absence of detectable persisting Ag in TNFR1-/- mice, probably either due to immature FDCs being partially functional and/or due to long-lived plasma cells.

Vaccination with heat-killed Listeria as adjuvant reverses established allergen-induced airway hyperreactivity and inflammation: role of CD8+ T cells and IL-18.

Asthma is a respiratory disorder characterized by airway hyperreactivity (AHR) and inflammation and is associated with high serum IgE and overproduction of IL-4, IL-5, and IL-13 by allergen-specific Th2 cells. Our previous studies demonstrated that heat-killed Listeria monocytogenes (HKL) as an adjuvant in immunotherapy successfully reversed ongoing Ag-specific Th2-dominated responses toward Th1-dominated responses, but it was unclear if such immune modulation could reverse ongoing, established disease in target organs such as the lung. In this paper we show that a single dose of Ag plus HKL as adjuvant significantly reduced AHR in a murine model for asthma and reversed established AHR when given late after allergen sensitization. HKL as adjuvant also dramatically inhibited airway inflammation, eosinophilia, and mucus production, significantly reduced Ag-specific IgE and IL-4 production, and dramatically increased Ag-specific IFN-gamma synthesis. The inhibitory effect of HKL on AHR depended on the presence of IL-12 and CD8+ T cells and was associated with an increase of IL-18 mRNA expression. Thus, our results demonstrate that HKL as an adjuvant for immunotherapy mediates immune deviation from a pathological Th2-dominated response toward a protective immune response in peripheral lymphoid tissues and in the lungs and may be clinically effective in the treatment of patients with established asthma and allergic disease.

Stat6-Dependent and -Independent Pathways for IL-4 Production

Stat6 has been shown to have a crucial role in the IL-4-dependent differentiation of Th2 cells. In this report, we explore whether in vitro Th2 differentiation driven by altered costimulatory signals or Ag dose is Stat6 dependent. We find that blocking B7-1 signaling in vitro promotes the differentiation of IL-4-secreting Th2 cells in wild-type but not Stat6-deficient T cell cultures. Additionally, stimulation with peptide Ag doses that normally result in the production of Th2 cells in vitro fails to do so in cultures of Stat6-deficient cells. We also demonstrate that Stat6 is required for the in vitro differentiation of CD8+ T cells into IL-4-secreting cytotoxic T cell type 2 cells. However, IL-4 expression is not absolutely dependent on Stat6. We demonstrate that populations of T cells that do not require IL-4 for their development, such as NK T cells, are still competent to secrete IL-4 in the absence of Stat6. These results demonstrate that Stat6 is required for the differentiation program leading to the generation of Th2 and cytotoxic T cell type 2 cells but not for IL-4 expression in cells that do not undergo differentiation in response to IL-4.

A Mathematical Model on Germinal Center Kinetics and Termination

We devise a mathematical model to study germinal center (GC) kinetics. Earlier models for GC kinetics are extended by explicitly modeling 1) the cell division history of centroblasts, 2) the Ag uptake by centrocytes, and 3) T cell dynamics. Allowing for T cell kinetics and T-B cell interactions, we study the role of GC T cells in GC kinetics, GC termination, and B cell selection. We find that GC T cells play a major role in GC formation, but that the maintenance of established GC reactions requires very few T cells only. The results therefore suggest that the termination of a GC reaction is largely caused by lack of Ag on the follicular dendritic cells and is hardly influenced by Th cells. Ag consumption by centrocytes is the major factor determining the decay rate of the antigenic stimulus during a GC reaction. Investigating the effect of the Ag dose on GC kinetics, we find that both the total size of the GC and its duration are hardly influenced by the initial amount of Ag. In the model this is due to a buffering effect by competition for limited T cell help and/or competition between proliferating centroblasts.

B7.2 (CD86) But Not B7.1 (CD80) Costimulation Is Required for the Induction of Low Dose Oral Tolerance

Oral administration of Ag leads to systemic unresponsiveness (oral tolerance) to the fed Ag. Oral tolerance is mediated through active suppression by Th2 or TGF-beta-secreting cells or clonal anergy/deletion, depending on the Ag dose used, with low dose favoring active suppression and high dose favoring anergy/deletion. The nature of APC and inductive events leading to the generation of oral tolerance have not been well defined. To determine the role of costimulatory molecules in the induction of oral tolerance, we have tested the effect of anti-B7.1 or anti-B7.2 mAb on the induction of tolerance by both high and low dose Ag feeding regimens. Our results show that the B7.2 molecule is critical for the induction of low-dose oral tolerance. Injection of anti-B7.2 but not anti-B7.1 intact Ab or Fab fragments inhibited the oral tolerance induced by low-dose (0.5 mg) but not high-dose OVA (25 mg) feeding. In addition, anti-B7.2, but not anti-B7.1, inhibited secretion of TGF-beta, one of the primary cytokines that mediates low-dose oral tolerance. Finally, in the in vivo model of experimental allergic encephalomyelitis, anti-B7.2 mAb treatment abrogated protection offered against disease by low-dose myelin basic protein feeding, while anti-B7.1 had no effect. Anti B7.2 had no effect on disease suppression by high-dose oral Ag. These data demonstrate that B7.2 costimulatory molecules play an essential role in the induction of low-dose oral tolerance.