Mutant mouse lysozyme carrying a minimal T cell epitope of hen egg lysozyme evokes high autoantibody response

Self proteins including foreign T cell epitope induce autoantibodies. We evaluated the relationship between the size of foreign Ag introduced into self protein and the magnitude of autoantibody production. Mouse lysozyme (ML) was used as a model self protein, and we prepared three different ML derivatives carrying T cell epitope of hen egg white lysozyme (HEL) 107-116, i.e, heterodimer of ML and HEL (ML-HEL), chimeric lysozyme that has residue 1-82 of ML and residue 83-130 of HEL in its sequence (chiMH), and mutant ML that has triple mutations rendering the most potent T cell epitope of HEL (sequence 107-116). Immunization of BALB/c mice with these three ML derivatives induced anti-ML autoantibody responses, whereas native ML induced no detectable response. In particular, mutML generated a 10(4) times higher autoantibody titer than did ML-HEL. Anti-HEL107-116 T cell-priming activities were almost similar among the ML derivatives. The heterodimerization of mutant ML and HEL led to significant reduction of the autoantibody response, whereas the mixture did not. These results show that size of the nonself region in modified self Ag has an important role in determining the magnitude of the autoantibody response, and that decrease in the foreign region in a modified self protein may cause high-titered autoantibody response.     

Breaking T cell tolerance with foreign and self co-immunogens. A study of autoimmune B and T cell epitopes of cytochrome c.

The initiation of autoimmune B cell and T cell responses by self Ag or by foreign pathogens (molecular mimics) is not well understood. In the present study, cytochrome c (cyt c) was used as a model autoantigen to investigate how self-proteins are involved in the priming of autoimmune T cell responses. Immunization with foreign cyt c has been extensively analyzed in previous studies as a model for both humoral and cellular immune responses. Mice do not, however, make antibody or T cell responses to immunization with self (mouse) cyt c. In addition, T cell tolerance can be broken by autoreactive B cells that are readily elicited by immunization with cross-reactive foreign cyt c. These immune B cells presumably bind self cyt c and process and present the self Ag to stimulate an autoreactive T cell response. Autoreactive T cell clones derived by this mechanism are all specific for determinants within amino acids 1-80 of the cyt c protein presented by I-Ek. No T cell responses were observed to the carboxyl terminal 81-104 fragment that dominates the response to foreign cyt c. All clones derived in this study are stimulated by a polypeptide encompassing amino acids 54-68 and utilized the V beta 8.2 TCR gene. In contrast, T cells stimulated by foreign cyt c did indeed respond to fragment 81-104 and appear to utilize alternate TCR genes. Our data demonstrate that B cells specific for linear determinants distributed along the entire length of the foreign cyt c molecule can provide the stimulus required for breaking T cell tolerance to self cyt c. The applications of this work to understanding the mechanisms of autoimmune disease are discussed.     

Role of B7 in T cell tolerance

The induction of effective immune responses requires costimulation by B7 molecules, and Ag recognition without B7 is thought to result in no response or tolerance. We compared T cell responses in vivo to the same Ag presented either by mature dendritic cells (DCs) or as self, in the presence or absence of B7. We show that Ag presentation by mature B7-1/2-deficient DCs fails to elicit an effector T cell response but does not induce tolerance. In contrast, using a newly developed adoptive transfer system, we show that naive OVA-specific DO11 CD4+ T cells become anergic upon encounter with a soluble form of OVA, in the presence or absence of B7. However, tolerance in DO11 cells transferred into soluble OVA transgenic recipients can be broken by immunization with Ag-pulsed DCs only in B7-deficient mice and not in wild-type mice, suggesting a role of B7 in maintaining tolerance in the presence of strong immunogenic signals. Comparing two double-transgenic models--expressing either a soluble or a tissue Ag--we further show that B7 is not only essential for the active induction of regulatory T cells in the thymus, but also for their maintenance in the periphery. Thus, the obligatory role of B7 molecules paradoxically is to promote effective T cell priming and contain effector responses when self-Ags are presented as foreign.     

Blockade of CTLA-4 signals inhibits Th2-mediated murine chronic graft-versus-host disease by an enhanced expansion of regulatory CD8+ T cells

CTLA-4 (CD152) is thought to be a negative regulator of T cell activation. Little is known about the function of CTLA-4 in Th2-type immune responses. We have investigated the effect of initial treatment with anti-CTLA-4 mAb on murine chronic graft-vs-host disease. Transfer of parental BALB/c splenocytes into C57BL/6 x BALB/c F1 mice induced serum IgE production, IL-4 expression by donor CD4+ T cells, and host allo-Ag-specific IgG1 production at 6-9 wk after transfer. Treatment with anti-CTLA-4 mAb for the initial 2 wk significantly reduced IgE and IgG1 production and IL-4 expression. Analysis of the splenic phenotype revealed the enhancement of donor T cell expansion, especially within the CD8 subset, and the elimination of host cells early after anti-CTLA-4 mAb treatment. This treatment did not affect early IFN-gamma expression by CD4+ and CD8+ T cells and anti-host cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced CD8+ T cell expansion, and this may result in the regulation of consequent Th2-mediated humoral immune responses. These findings suggest a new approach for regulating IgE-mediated allergic immune responses by blockade of CTLA-4 during a critical period of Ag sensitization.     

Absence of CTLA-4 lowers the activation threshold of primed CD8+ TCR-transgenic T cells: lack of correlation with Src homology domain 2-containing protein tyrosine phosphatase

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.     

OX40-mediated memory T cell generation is TNF receptor-associated factor 2 dependent

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.     

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.     

T cell studies in a peptide-induced model of systemic lupus erythematosus

We have previously reported that immunization with a peptide mimetope of dsDNA on a branched polylysine backbone (DWEYSVWLSN-MAP) induces a systemic lupus erythematosus-like syndrome in the nonautoimmune BALB/c mouse strain. To understand the mechanism underlying this breakdown in self tolerance, we examined the role of T cells in the response. Our results show that the anti-foreign and anti-self response induced by immunization is T cell dependent and is mediated by I-E(d)-restricted CD4(+) T cells of the Th1 subset. In addition, generation of the critical T cell epitope requires processing by APCs and depends on the presence of both DWEYSVWLSN and the MAP backbone. The breakdown in self tolerance does not occur through cross-reactivity between the T cell epitope of DWEYSVWLSN-MAP and epitopes derived from nuclear Ags. In this induced-model of SLE, therefore, autoreactivity results from the activation of T cells specific for foreign Ag and of cross-reactive anti-foreign, anti-self B cells. Despite the fact that tissue injury is mediated by Ab, the critical initiating T cell response is Th1.     

Blockade of CTLA-4 decreases the generation of multifunctional memory CD4+ T cells in vivo

CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti-CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ-producing CD4(+) T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized "multifunctional" memory CD4(+) T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ-producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4-competent and -deficient cells unmask, which these CTLA-4-driven mechanisms are mediated CD4(+) T cell nonautonomously. In addition, the depletion of CD25(+) T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4-expressing natural regulatory T cells determine the quality of memory CD4(+) T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4(+) T cells capable of mounting a robust response to Ag rechallenge.     

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.     

Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli.

CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells. CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells. Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells. A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo. Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success. Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure. Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay.     

Janus kinase 2 is associated with a box 1-like motif and phosphorylates a critical tyrosine residue in the cytoplasmic region of cytotoxic T lymphocyte associated molecule-4

It is a consensus that a cytotoxic T lymphocyte associated molecule-4 (CTLA-4) transduces inhibitory signal for T cell activation under physiological condition, indicating that this molecule is an important regulator of T cell homeostasis in vivo. It has been reported that phosphorylation and dephosphorylation of tyrosine residue Y-165 in the cytoplasmic region of CTLA-4 play an important role in its negative signaling and cell surface expression. Some signaling molecules such as Src homology 2 protein tyrosine phosphatase 2 (SHP-2) and the p85 subunit of phosphatidylinositol 3 kinase (PI3 kinase) associate with phosphorylated tyrosine residue Y-165, through Src homology 2 (SH2) domains. On the other hand, the adapter complex proteins, AP-2 and AP-50 interact with the same tyrosine residue when unphosphorylated, resulting in clathrin-mediated endocytosis of CTLA-4 molecules. The objective of this study is to identify a tyrosine kinase that can directly bind and phosphorylate the critical tyrosine residue, Y-165 in the cytoplasmic domain of CTLA-4. Here, we demonstrated that 1) Janus Kinase 2 (Jak2) was directly associated with a box 1-like motif in the cytoplasmic tail of CTLA-4 molecule, 2) Jak2 phosphorylated Y-165 residue in the cytoplasmic region of CTLA-4 molecule, and 3) Jak2 was associated with CTLA-4 in HUT 78 T cell lines.     

Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25- TGF-beta 1+ adaptive regulatory T cells

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.     

A new role of CTLA-4 on B cells in thymus-dependent immune responses in vivo

The expression of CTLA-4 (CD152) on the cell surface of B cells and its consequences for the humoral immune response in vivo are unknown. We investigated the expression of CTLA-4 mRNA and protein in B cells in T cell-independent or -dependent ways. B cells in the presence of Ag-stimulated Th2 cells expressed mRNA of CTLA-4 and up-regulated intracellular CTLA-4 protein. Using a liposome-enhanced staining technique, we show for the first time, that surface CTLA-4 protein is expressed by 11-15% of B cells in a T cell-dependent culture system. To dissect the role of CTLA-4 on B cells in vivo, we used bone marrow chimeric mice in which only B cells were CTLA-4 deficient. These mice showed that early B cell development and homeostasis is not influenced by CTLA-4 deficiency of B cells. Ag-specific responses after immunization of the chimeric mice revealed elevated levels of IgM Abs in mice deficient for B cell CTLA-4. We propose that CTLA-4 signals on B cells determine the early fate of B cells in thymus-dependent immune responses.     

Established T cell-driven germinal center B cell proliferation is independent of CD28 signaling but is tightly regulated through CTLA-4

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.     

CTLA-4. FasL induces alloantigen-specific hyporesponsiveness

The APC:T cell interface can be effectively targeted with immunotherapeutic proteins. We previously described a unique trans signal converter protein, CTLA-4. Fas ligand (FasL), that has the inherent capacities to tether the T cell inhibitor FasL (CD95 ligand) to the surfaces of B7 (CD80 and CD86)-positive APC (via CTLA-4:B7 interaction), and in so doing, to simultaneously interfere with B7-to-CD28 T cell activation signals. Given the continuing need for agents capable of inducing allograft tolerance without generalized immunosuppression, we have explored in depth the functional activity of CTLA-4. FasL in human allogeneic MLR. CTLA-4. FasL inhibits 1 degrees MLR and induces specific hyporesponsiveness in 2 degrees MLR, with both effects only partially reversible with exogenous IL-2. Moreover, the presence of exogenous IL-2 during the 1 degrees MLR does not affect the induction of hyporesponsiveness upon restimulation. Furthermore, CTLA-4. FasL enables partial activation of allostimulated T cells, reduces the fraction of actively dividing cells, and increases the percentage of dead cells among dividing T cells. Taken together, these findings suggest that CTLA-4. FasL-mediated inhibition of secondary alloantigenic responses involves both anergy induction and clonal deletion. Thus, CTLA-4. FasL, a paradigmatic trans signal converter protein, manifests unique functional properties and emerges as a potentially useful immunotherapeutic for modulating alloresponsiveness.     

An Exhaustion-Like Phenotype Constrains the Activity of CD4+ T Cells Specific for a Self and Melanoma Antigen

While the immune system has the capacity to recognize and destroy melanoma, tolerance mechanisms often hinder the development of effective anti-tumor immune responses. Since many melanoma antigens are self proteins expressed in normal melanocytes, self antigen exposure before tumor development can negatively impact the function of T cells specific for these self/tumor antigens. However, the contribution of self tolerance to anti-melanoma T cell dysfunction remains largely unexplored. We have previously described a TCR transgenic (Tg) mouse model in which T cells specific for the self/melanoma antigen, tyrosinase-related protein 1 (TRP1), develop in the presence of endogenous TRP1 expression (Ag+) and diminished antigen presentation due to the absence of gamma-interferon-inducible lysosomal thiol reductase (GILT-/-). We show that TRP1-specific T cells from these Ag+GILT-/-Tg mice do not protect from melanoma tumor growth, fail to induce autoimmune vitiligo, and undergo diminished proliferation compared to T cells from Ag-GILT+/+Tg mice. Despite an increased frequency of TRP1-specific Treg cells in Ag+GILT-/-Tg mice compared to Ag-GILT+/+Tg animals, Treg cell depletion only partially rescues the proliferative capacity of T cells from TRP1-expressing mice, suggesting the involvement of additional suppressive mechanisms. An increased percentage of melanoma-specific T cells from Ag+GILT-/-Tg animals express PD-1, an inhibitory receptor associated with the maintenance of T cell exhaustion. Antibody blockade of PD-1 partially improves the ability of TRP1-specific T cells from Ag+GILT-/-Tg mice to produce IL-2. These findings demonstrate that melanoma-specific T cells exposed to a self/melanoma antigen in healthy tissue develop an exhaustion-like phenotype characterized by PD-1-mediated immunosuppression prior to encounter with tumor.     

CTLA-4 signaling regulates the intensity of hypersensitivity responses to food antigens, but is not decisive in the induction of sensitization

Although food allergy has emerged as a major health problem, the mechanisms that are decisive in the development of sensitization to dietary Ag remain largely unknown. CTLA-4 signaling negatively regulates immune activation, and may play a crucial role in preventing induction and/or progression of sensitization to food Ag. To elucidate the role of CTLA-4 signaling in responses to food allergens, a murine model of peanut allergy was used. During oral exposure to peanut protein extract (PPE) together with the mucosal adjuvant cholera toxin (CT), which induces peanut allergy, CTLA-4 ligation was prevented using a CTLA-4 mAb. Additionally, the effect of inhibition of the CTLA-4 pathway on oral exposure to PPE in the absence of CT, which leads to unresponsiveness to peanut Ag, was explored. During sensitization, anti-CTLA-4 treatment considerably enhanced IgE responses to PPE and the peanut allergens, Ara h 1, Ara h 3, and Ara h 6, resulting in elevated mast cell degranulation upon an oral challenge. Remarkably, antagonizing CTLA-4 during exposure to PPE in the absence of CT resulted in significant induction of Th2 cytokines and an elevation in total serum IgE levels, but failed to induce allergen-specific IgE responses and mast cell degranulation upon a PPE challenge. These results indicate that CTLA-4 signaling is not the crucial factor in preventing sensitization to food allergens, but plays a pivotal role in regulating the intensity of a food allergic sensitization response. Furthermore, these data indicate that a profoundly Th2-biased cytokine environment is insufficient to induce allergic responses against dietary Ag.     

The CD28/CTLA-4-B7 signaling pathway is involved in both allergic sensitization and tolerance induction to orally administered peanut proteins

Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.