Immunodominant T-cell epitopes of hnRNP-A2 associated with disease activity in patients with rheumatoid arthritis.

The heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) has been described as an important autoantigen in rheumatoid arthritis (RA) since it is targeted by autoantibodies, autoreactive T cells, and is aberrantly expressed in synovial cells in patients. To identify hnRNP-A2-specific T-cell epitopes possibly associated with pathogenicity, we used an innovative approach. We first scanned 280 overlapping hnRNP-A2 peptides for binding to the RA-associated class II molecules HLA-DR4 and HLA-DR1, leading to a comprehensive selection of binders. The selected peptides were tested in IFN-gamma-specific ELISPOT assay: PBMC from 18% of RA patients showed a significant IFN-gamma response to hnRNP-A2 peptides, 15% to the overlapping sequences 117-133 and/or 120-133, whereas PBMC from healthy individuals tested negative. We measured proliferative responses to these two peptides in another cohort of patients with RA or osteoarthritis: positive responses were found in 28% of RA, but also in 11% of osteoarthritis patients and these responses could be blocked by anti-MHC class II Ab. Remarkably, the presence of 117/120-133-specific T cells was significantly associated with active disease in RA patients, and bone erosion appeared to be more common in T-cell positive patients. These data suggest involvement of hnRNP-A2 specific cellular autoimmune responses in RA pathogenesis.

Scientific workflows as productivity tools for drug discovery.

Large pharmaceutical companies annually invest tens to hundreds of millions of US dollars in research informatics to support their early drug discovery processes. Traditionally, most of these investments are designed to increase the efficiency of drug discovery. The introduction of do-it-yourself scientific workflow platforms has enabled research informatics organizations to shift their efforts toward scientific innovation, ultimately resulting in a possible increase in return on their investments. Unlike the handling of most scientific data and application integration approaches, researchers apply scientific workflows to in silico experimentation and exploration, leading to scientific discoveries that lie beyond automation and integration. This review highlights some key requirements for scientific workflow environments in the pharmaceutical industry that are necessary for increasing research productivity. Examples of the application of scientific workflows in research and a summary of recent platform advances are also provided.

Identification of novel consensus CD4 T-cell epitopes from clade B HIV-1 whole genome that are frequently recognized by HIV-1 infected patients.

OBJECTIVE: To identify promiscuous and potentially protective human CD4 T-cell epitopes in most conserved regions within the protein-coding genome of HIV-1 clade B consensus sequence. DESIGN: We used the TEPITOPE algorithm to screen the most conserved regions of the whole genome of the HIV-1 subtype B consensus sequence to identify promiscuous human CD4 T-cell epitopes in HIV-1. The actual promiscuity of HLA binding of the 18 selected peptides was assessed by binding assays to nine prevalent HLA-DR molecules. Synthetic peptides were tested with interferon-gamma ELISPOT assays on peripheral blood mononuclear cells (PBMC) from 38 HIV-1 infected patients and eight uninfected controls. RESULTS: Most peptides bound to multiple HLA-DR molecules. PBMC from 91% of chronically HIV-1 infected patients recognized at least one of the promiscuous peptides, while none of the healthy controls recognized peptides. All 18 peptides were recognized, and each peptide was recognized by at least 18% of patients; 44% of the patients recognized five or more peptides. This response was not associated to particular HLA-DR alleles. Similar responses were obtained in CD8 T-cell-depleted PBMC. CONCLUSION: In silico prediction of promiscuous epitopes led to the identification of naturally immunodominant CD4 T-cell epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to HIV-1. This combination of CD4 T-cell epitopes - 11 of them not described before - may have the potential for inclusion in a vaccine against HIV-1, allowing the immunization of genetically distinct populations.

Identification of HLA DR7-restricted epitopes from human telomerase reverse transcriptase recognized by CD4+ T-helper cells.

CD4+ T cells play critical roles in initiating, regulating, and maintaining antitumor immune responses. One way to improve current tumor vaccines that mainly induce CTLs would be to activate antigen-specific CD4+ T cells that recognize MHC class II restricted tumor associated antigens. Human telomerase reverse transcriptase (hTRT) is preferentially expressed by various tumors and, therefore, could be a universal tumor antigen. In this study, we used a combined approach of using the prediction software TEPITOPE to select class II epitope candidates and in vitro T-cell biological analysis to identify class II-restricted epitope(s) in hTRT. We first identified several HLA-DR7-restricted class-II epitope candidates in hTRT by examining human T-cell responses to synthetic peptides. We then characterized these HLA-DR7-restricted hTRT epitope candidates by establishing and analyzing peptide-specific T-cell clones. It was demonstrated that CD4+ T cells specific for the HLA-DR7-restricted hTRT(672) epitope (RPGLLGASVLGLDDI) can respond to naturally processed hTRT proteins. Furthermore, the hTRT(672)-specific T cells recognized hTRT antigen from various tumors, including prostate cancer, breast cancer, melanoma, and leukemia. Thus, the identification of the naturally processed HLA-DR7-restricted hTRT epitope, together with the previous finding of class I-restricted hTRT epitopes, provide a basis for the combined application of class I- and II-restricted hTRT epitopes to induce potent, long-term CD4+ and CD8+ T-cell responses against a broad spectrum of tumors.

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein.

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.

T-cell recognition and cytokine profile induced by melanocyte epitopes in patients with HLA-DRB1*0405-positive and -negative Vogt-Koyanagi-Harada uveitis.

PURPOSE: Vogt-Koyanagi-Harada disease (VKH), an autoimmune disease targeted against melanocytes, is associated with HLA-DRB1*0405. This study was undertaken to analyze T-cell recognition and the cytokine expression profile induced by melanocyte epitopes in HLA-DRB1*0405-positive and -negative patients with VKH uveitis. METHODS: Peripheral blood mononuclear cells (PBMC) proliferation and Th1 (IFN-gamma) and Th2 (IL-4 and IL-5) cytokine production were analyzed in HLA-DRB1*0405-positive (n = 12) and -negative (n = 22) patients with VKH and HLA-DRB1*0405-positive (n = 9) and -negative (n = 8) control subjects in response to human melanoma cell line lysate (HMCLL) and 28 synthetic peptides derived from the human melanocyte differentiation proteins TYR, TRP1, TRP2, and Pmel17. The peptides were selected using the TEPITOPE algorithm, based on their predicted binding to HLA-DRB1*0405 and to the non-disease-related HLA-DRB1*15. RESULTS: HMCLL was recognized exclusively by the patients' PBMC (44%) but not by those of the control subjects (P < 0.01). PBMC from patients with VKH recognized an increased breadth of melanocyte-derived peptides at lower peptide concentrations than in the control subjects (68% vs. 25%; P < 0.01, at 1 microM) and did not produce the Th2 cytokine IL-4 in response to disease-specific peptides (0% vs. 50%, P < 0.001). Five peptides were exclusively recognized in patients bearing HLA-DRB1*0405. Furthermore, HLA-DRB1*0405-bearing patients, but not those with HLA-DRB1*15, recognized an increased breadth of melanocyte epitopes in comparison to HLA-matched control subjects (60% vs. 28%; P < 0.05). CONCLUSIONS: These data indicate that patients with VKH are sensitized to melanocyte epitopes and display a peptide-specific Th1 cytokine response. In addition, the data indicate that patients bearing HLA-DRB1*0405 recognize a broader melanocyte-derived peptide repertoire, reinforcing the importance of this allele in susceptibility to the development of VKH disease.

Computational dissection of tissue contamination for identification of colon cancer-specific expression profiles.

Microarray profiles of bulk tumor tissues reflect gene expression corresponding to malignant cells as well as to many different types of contaminating normal cells. In this report, we assess the feasibility of querying baseline multitissue transcriptome databases to dissect disease-specific genes. Using colon cancer as a model tumor, we show that the application of Boolean operators (AND, OR, BUTNOT) for database searches leads to genes with expression patterns of interest. The BUTNOT operator for example allows the assignment of "expression signatures" to normal tissue specimens. These expression signatures were then used to computationally identify contaminating cells within conventionally dissected tissue specimens. The combination of several logic operators together with an expression database based on multiple human tissue specimens can resolve the problem of tissue contamination, revealing novel cancer-specific gene expression. Several markers, previously not known to be colon cancer associated, are provided.

Cascades of transcriptional induction during dendritic cell maturation revealed by genome-wide expression analysis.

Dendritic cells (DC) are central regulators of immunity. Signal-induced maturation of DCs is assumed to be the starting point for specific immune responses. To further understand this process, we analyzed the alteration of transcript profiles along the time course of CD40 ligand-induced maturation of human myeloid DCs by Affymetrix GeneChip microarrays covering >6800 genes. Besides rediscovery of genes already described as associated with DC maturation proving reliability of the methods used, we identified clusterin as novel maturation marker. Looking across the time course, we observed synchronized kinetics of distinct functional groups of molecules whose temporal coregulation underscores known cellular events during dendritic cell maturation. For example, an early-peaking wave of inflammatory chemokines was followed by a sustained increase of constitutive chemokines and accompanied by slow but continuous induction of survival proteins. After an immediate but transient induction of cytokine-responsive transcripts, there was an increased expression of a group of genes involved in not only the regulation of cytokine effects, but also of transcription in general. Our results demonstrate that microarray studies along time courses combined with real-time PCR not only discover new marker molecules with functional implications, but also dissect the molecular kinetics of biological processes identifying complex pathways of regulation.

T cell epitope-specific defects in the immune response to cat allergen in patients with atopic dermatitis.

Atopic dermatitis (AD) is often associated with high titer IgE antibodies (ab) to allergens, and IL-10-mediated regulation of IFN-gamma has been proposed to contribute to this IgE ab production. However, the relevance of IL-10 and IFN-gamma to IgE associated with AD has not been examined in the context of an allergen-specific system. Analysis of PBMC responses in vitro showed deficient T cell proliferation to overlapping IL-10- (peptide (P) 2:1) and IFN-gamma- (P2:2) inducing chain 2 major epitopes of cat allergen (Fel d 1) in cultures from sensitized AD patients (mean IgE to cat=20.9 IU/ml). Diminished IFN-gamma induction by Fel d 1 and P2:2, along with elevated peptide-induced IL-10 (except for P2:1) was observed in PBMC cultures from AD subjects compared with non-AD (sensitized and non-sensitized) subjects. Neither T cell proliferation nor IFN-gamma production to chain 2 epitopes could be restored by anti-IL-10 mAb in cultures from sensitized AD subjects. Moreover, allergen avoidance was associated with a paradoxical decrease in both IL-10 and IFN-gamma in peptide-stimulated PBMC from these subjects. Control of IFN-gamma production to chain 2 epitopes by IL-10 may be relevant to sensitization status. Development of high titer IgE ab in AD could reflect a failure of this mechanism.

MCJ/DnaJC15, an endogenous mitochondrial repressor of the respiratory chain that controls metabolic alterations.

Mitochondria are the main engine that generates ATP through oxidative phosphorylation within the respiratory chain. Mitochondrial respiration is regulated according to the metabolic needs of cells and can be modulated in response to metabolic changes. Little is known about the mechanisms that regulate this process. Here, we identify MCJ/DnaJC15 as a distinct cochaperone that localizes at the mitochondrial inner membrane, where it interacts preferentially with complex I of the electron transfer chain. We show that MCJ impairs the formation of supercomplexes and functions as a negative regulator of the respiratory chain. The loss of MCJ leads to increased complex I activity, mitochondrial membrane potential, and ATP production. Although MCJ is dispensable for mitochondrial function under normal physiological conditions, MCJ deficiency affects the pathophysiology resulting from metabolic alterations. Thus, enhanced mitochondrial respiration in the absence of MCJ prevents the pathological accumulation of lipids in the liver in response to both fasting and a high-cholesterol diet. Impaired expression or loss of MCJ expression may therefore result in a "rapid" metabolism that mitigates the consequences of metabolic disorders.

Determination of a HLA II promiscuous peptide cocktail as potential vaccine against EBV latency II malignancies.

The Epstein-Barr virus (EBV) is associated with several malignant diseases, which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the nonimmunodominant antigens EBNA1, LMP1 and LMP2 and is seen in EBV-positive Hodgkin disease, nasopharyngeal carcinomas, and peripheral T/NK-cell lymphomas. CD4 T cells may play a crucial role in controlling these EBV latency II malignancies. In this study, we used the prediction software TEPITOPE to predict promiscuous major histocompatibility complex class II epitopes derived from the latency II antigens EBNA1, LMP1, and LMP2. The predicted peptides were then submitted to peptide-binding assays on HLA II purified molecules, which allowed the selection of 6 peptides (EBNA1: 3; LMP1: 1; and LMP2: 2) with a highly promiscuous capability of binding. This peptide cocktail was immunogenic in a model of HLA-DR1 transgenic mice, leading to a specific cellular and humoral TH1 response. The peptides were also recognized by human CD4 T cells from individuals expressing various HLA II genotypes. This promiscuous peptide cocktail could be immunogenic in the majority of the population and may be used as a peptide-based vaccine in EBV latency II malignancies.

Peptide-binding assays and HLA II transgenic Abeta degrees mice are consistent and complementary tools for identifying HLA II-restricted peptides.

The identification of MHC class II-restricted peptides has become a priority for the development of peptide-based prophylactic and therapeutic vaccines. The aim of this study was to assess the correlations between peptide-binding assays on purified HLA II molecules and immunization of human HLA II transgenic mice deficient in murine class II molecules (Abeta degrees ). We used as models two MHC class II-restricted peptides, one derived from the HIV Nef regulatory protein (Nef (56-68)) and the other from the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST (190-211)). High correlations were found between the two approaches, which showed that the Nef (56-68) and Sm28GST (190-211) peptides may represent promiscuous ligands for HLA-DQ and for HLA-DR molecules, respectively. We suggest a rational method based on the combination of peptide-binding assays and HLA II transgenic mice experiments as consistent and complementary tools for selecting T helper epitopes.

Discovery of promiscuous HLA-II-restricted T cell epitopes with TEPITOPE.

TEPITOPE is a prediction model that has been successfully applied to the in silico identification of T cell epitopes in the context of oncology, allergy, infectious diseases, and autoimmune diseases. Like most epitope prediction models, TEPITOPE's underlying algorithm is based on the prediction of HLA-II peptide binding, which constitutes a major bottleneck in the natural selection of epitopes. An important step in the design of subunit vaccines is the identification of promiscuous HLA-II ligands in sets of disease-specific gene products. TEPITOPE's user interface enables the systematic prediction of promiscuous peptide ligands for a broad range of HLA-binding specificity. We show how to apply the TEPITOPE prediction model to identify T cell epitopes, and provide both a road map and examples of its successful application.

The use of bioinformatics for identifying class II-restricted T-cell epitopes.

An important step in the design of subunit vaccines is the identification of promiscuous T helper cell epitopes in sets of disease-specific gene products. Most of the epitope prediction models are based on HLA-II peptide binding, which constitutes a major bottleneck in the natural selection of epitopes. Here we describe a computer model, TEPITOPE, that enables the systematic prediction of promiscuous peptide ligands for a broad range of HLA binding specificity. We show how to apply the TEPITOPE prediction model to identify T-cell epitopes, and provide examples of its successful application in the context of oncology, allergy, and infectious and autoimmune diseases.

Identification of a MHC class-II restricted epitope in carcinoembryonic antigen.

PURPOSE: The carcinoembryonic antigen (CEA) is extensively expressed on the vast majority of colorectal, gastric, and pancreatic carcinomas, and, therefore, is a good target for tumor immunotherapy. CD4+ T-helper (Th) cells play a critical role in initiation, regulation, and maintenance of immune responses. In this study, we sought to identify Th epitopes derived from CEA which can induce CEA-specific Th responses. The combined application with cytotoxic T lymphocyte (CTL) epitopes would be more potent than tumor vaccines that primarily activate CTL alone. METHODS: We utilized a combined approach of using a computer-based algorithm analysis TEPITOPE and in vitro biological analysis to identify Th epitopes in CEA. RESULTS: Initial screening of healthy donors showed that all five predicted peptides derived from CEA could induce peptide-specific T-cell proliferation in vitro. We characterized these CEA epitopes by establishing and analyzing peptide-specific T-cell clones. It was shown that CD4+ T-cells specific for the CEA(116 )epitope can recognize and respond to naturally processed CEA protein and CEA(116 )epitope can be promiscuously presented by commonly found major histocompatibility complex (MHC) alleles. Furthermore, it was demonstrated that immunization of human leukocyte antigen (HLA)-DR4 transgenic mice with CEA(116) peptide elicited antigen-specific Th responses which can recognize the antigenic peptides derived from CEA protein and CEA-positive tumors. CONCLUSION: The MHC class II-restricted epitope CEA(116) could be used in the design of peptide-based tumor vaccine against several common cancers expressing CEA.

Binding of outer surface protein A and human lymphocyte function-associated antigen 1 peptides to HLA-DR molecules associated with antibiotic treatment-resistant Lyme arthritis.

OBJECTIVE: To assess the binding of outer surface protein A (OspA) and human lymphocyte function-associated antigen 1 (hLFA-1) peptides to 5 major histocompatibility complex (MHC) molecules. METHODS: Peptide binding to the MHC molecules was determined by in vitro binding assays, and binding was correlated with the frequencies of the 5 MHC molecules in patients with treatment-resistant Lyme arthritis. RESULTS: The HLA-DRB1*0401 molecule bound both OspA(163-175) and hLFA-1alpha(L330-342) well. Although the magnitude of the binding was less, the DRB1*0404 molecule also showed binding of both peptides. The DRB1*0101 molecule bound OspA(163-175) well, but hLFA-1alpha(L330-342) only weakly; the DRB1*0801 or *1101 molecule bound both peptides weakly, if at all. The magnitude of OspA(163-175) binding correlated well with the frequencies of the DRB1 alleles in patients with treatment-resistant arthritis, but the binding of hLFA-1alpha(L330-342) showed only an association with the DRB*04 alleles. CONCLUSION: These correlations support the hypothesis that OspA(163-175) is the critical epitope in triggering antibiotic treatment-resistant Lyme arthritis. However, the inability of the DRB*0101 molecule to bind hLFA-1alpha(L330-342) suggests that this peptide may not be a relevant autoantigen, at least in DRB1*0101-positive patients.

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity.

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.

T-cell receptor-mediated cross-allergenicity.

BACKGROUND: Profilins are conserved and ubiquitous plant and animal proteins. We wanted to discover whether the T-cell response to conserved epitopes on birch and grass profilins could account for cross-allergenicity in subjects allergic to these two pollens. METHODS: Thirty-one patients allergic to grass and birch were recruited for the study. Grass and birch reactive T lymphocytes were studied by measuring proliferation to birch and grass allergen, respectively, followed by Vbeta T-cell receptor family-specific polymerase chain reaction and heteroduplex analysis. T-cell clones were derived from patients with cross-proliferating T cells. RESULTS: In 25 of 31 subjects the T-cell response to grass was quite distinct from that to birch. In contrast, in 6 of 31 individuals grass T cells cross-proliferated to birch and this was reproduced in 4 patients by birch profilin. CD4 Th2 cell clones were derived which promiscuously recognized homologously conserved regions on birch and grass profilins. CONCLUSION: We conclude that a functionally relevant T-cell response to conserved regions of panallergens underlie cross-allergenicity in a subset of allergic patients. These results suggest that a reciprocal modulation of the response to one sensitizing allergen can occur following natural exposure to or immunotherapy with another allergen. These results have relevance in the management of patients with multiple allergies.

Identification of immunodominant regions among promiscuous HLA-DR-restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3.

The molecular characterization of the CD4(+) T-cell epitope repertoire on human tumor antigens would contribute to both clinical investigation and cancer immunotherapy. In particular, the identification of promiscuous epitopes would be beneficial to a large number of patients with neoplastic diseases regardless of their HLA-DR type. MAGE-3 is a tumor-specific antigen widely expressed in solid and hematologic malignancies; therefore, is an excellent candidate antigen. We used a major histocompatability complex (MHC) class II epitope prediction algorithm, the TEPITOPE software, to predict 11 sequence segments of MAGE-3 that could form promiscuous CD4(+) T-cell epitopes. In binding assays, the synthetic peptides corresponding to the 11 predicted sequences bound at least 3 different HLA-DR alleles. Nine of the 11 peptides induced proliferation of CD4(+) T cells from both healthy subjects and melanoma patients. Four immunodominant regions (residues 111-125, 146-160, 191-205, and 281-295), containing naturally processed epitopes, were recognized by most of the donors, in association with 3 to 4 different HLA-DR alleles, thus covering up to 94% of the alleles expressed in whites. On the contrary, the other promiscuous regions (residues 161-175 and 171-185) contained epitopes not naturally processed in vitro. The immunodominant epitopes identified will be useful in the design of peptide-based cancer vaccines and in the study of the functional state of tumor-specific CD4(+) T cells in patients bearing tumors expressing MAGE-3.

A role for IL-10-mediated HLA-DR7-restricted T cell-dependent events in development of the modified Th2 response to cat allergen.

Although high dose exposure to inhaled cat allergen (Fel d 1) can cause a form of tolerance (modified Th2 response), the T cell mechanism for this phenomenon has not been studied. T cell responses to Fel d 1 were characterized in both allergic (IgE(pos)) and modified Th2 (IgE(neg)IgG(pos)) responders as well as serum Ab-negative controls (IgE(neg)IgG(neg)). Fel d 1 stimulated high levels of IL-10 in PBMC cultures from all individuals, with evidence of Th2 and Th1 cytokine skewing in allergic and control subjects, respectively. Using overlapping peptides, epitopes at the N terminus of Fel d 1 chain 2 were shown to stimulate strong T cell proliferation and to preferentially induce IL-10 (peptide 2:1 (P2:1)) or IFN-gamma (P2:2) regardless of the allergic status of the donor. Injection of cat extract during conventional immunotherapy stimulated expansion of IL-10- and IFN-gamma-producing chain 2 epitope-specific T cells along with increased Fel d 1-specific serum IgG and IgG4 Ab. Six of 12 modified responders expressed the major HLA-DRB1 allele, *0701, and both P2:1 and P2:2 were predicted ligands for this allele. Cultures from DR7-positive modified responders produced the highest levels of IL-10 to P2:1 in addition to other major and minor epitopes within chains 1 and 2. In the presence of anti-IL-10 mAb, both T cell proliferation and IFN-gamma production were enhanced in a Fel d 1- and epitope-specific manner. We conclude that IL-10-producing T cells specific for chain 2 epitopes are relevant to tolerance induction, and that DR7-restricted recognition of these epitopes favors a modified Th2 response.