They were isolated, purified, and cloned in frame into the expression vector pQE32 as described above. (v) N terminus. peptide epitopes in the fragments and HLA restricting elements that present them. A majority of the T-cell clones recognized an epitope spanning amino acids 149 to 172 within the N terminus, displayed by HLA-DR 15. A minority of the clones, which have been shown to perform a cytolytic function in vitro, recognized an epitope in the MRS1177 tandem repeat displayed by HLA-DPw4, an uncommon restricting element. Tandem repeat epitopes required display by the chain of DPw4 heterodimers. Thus, human T cells with different functions in vitro also recognize distinct regions of WI-1, raising the possibility that HLA restricting elements that present them could modulate immunity during blastomycosis by selection and display of WI-1 peptides. is a dimorphic fungus that causes disease in both healthy and immunodeficient hosts. The fungus is endemic to the Mississippi and Ohio River valleys and northern Wisconsin. The spectrum of infection includes asymptomatic disease, acute or chronic pneumonia, and disseminated disease, especially in immunodeficient patients, who are at higher risk for developing widely disseminated blastomycosis (19, 20). The growing frequency of invasive fungal diseases and the challenge of treating them have stimulated interest in developing ways to prevent fungal infections. The immunodominant and protective antigens for many fungal pathogens have not been elucidated and are actively being investigated (7). For (25), an indication that anti-WI-1 immune responses benefit the host. Thus, WI-1 may serve as a MRS1177 candidate for developing a vaccine against blastomycosis. Studies of mice and humans have established the central importance of delayed-type hypersensitivity in acquired resistance to (5). Since CD4+ T cells are a critical constituent of this response, a deeper understanding of T-cell recognition of WI-1 in people infected MRS1177 with will help elucidate how humans defend against the pathogen and how protective immune responses might be harnessed to prevent infection. In a prior study of a small number of blastomycosis patients, mononuclear cells obtained from their peripheral blood were shown to proliferate in vitro in response to WI-1 (12). These responding T cells were cloned and analyzed functionally: all had a CD4+ phenotype, and a majority of them responded by proliferating in the presence of WI-1, but a small proportion lysed antigen-presenting cells that displayed WI-1 on their surfaces. In the present study, our goals were to (i) investigate peripheral blood mononuclear cell (PBMC) responses to WI-1 in a larger number of patients with blastomycosis, (ii) determine the segments of WI-1 antigen chiefly recognized by T cells, MGC20372 (iii) delineate in these segments the peptide epitopes recognized by cloned T cells, and (iv) determine human leukocyte antigen (HLA) molecules that display these epitopes to T cells. MATERIALS AND METHODS Antigen preparations. (i) Native WI-1. WI-1 was purified from ATCC 60636, a virulent isolate associated with an outbreak of human disease (10). was maintained in the yeast form by growth on Middlebrook 7H10 agar medium containing oleic acid-albumin complex (OADC) (Sigma Chemical Co., St. Louis, Mo.). Liquid cultures of yeasts were grown in macrophage medium (HMM) (24). For large-scale growth of yeast, Roux bottles of 7H10-OADC agar were seeded with 5 108 yeast cells in a final volume of 4 ml of HMM and the yeasts were grown at 37C in a humidified incubator for 7 days. Yeasts were harvested and inoculated into 500 ml of HMM at a final concentration of 2.5 105 yeasts/ml. Cultures were grown for 14 days at 37C with shaking at 250 rpm. Supernatants enriched for secreted WI-1 were collected and frozen at ?20C until WI-1 purification, which was performed using a two-step method (1). (ii) Recombinant WI-1 and its fragments (Fig. ?(Fig.11). Open in a separate window FIG. 1 Full-length WI-1 and its three chief domains (top bar). Below are recombinant fragments and subfragments of WI-1 domains investigated in this study. Amino acid positions are shown above full-length WI-1. Each N-terminal segment extends about 50 residues: N1, amino acids (aa) 8 to 58; N2, aa 58 to 115; N3, aa 115 to 172; N4, aa 29 to 85; N5, aa 85 to 143. EGF, epidermal growth factor; TR, tandem repeat. The full coding sequence of WI-1 was derived by ATCC 26199 (9). The 5.5-kb WI-1 genomic fragment, which is free of introns, was purified and ligated in frame 3 to nucleotides coding for a six-histidine tag for affinity purification in the expression plasmid pQE32 (Qiagen, Valencia, Calif.). Plasmid DNA was electroporated into strain XL1-Blue containing the repressor plasmid pRep4. Transformed cells were grown on Luria-Bertani agar containing.
They were isolated, purified, and cloned in frame into the expression vector pQE32 as described above
