Previous studies have shown that dendritic cells (DC) pulsed with T27K, an antigenic preparation derived from spherules (of Coccidioides posadasii), activate peripheral blood mononuclear cells (PBMC) from nonimmune subjects as well as from patients with disseminated coccidioidomycosis. In this study, we have assessed the interaction between human DC and C. posadasii spherules in order to better understand the initial response between Coccidioides and the human host. Whole autoclaved spherules induced lymphocyte transformation in PBMC obtained from immune but not from nonimmune donors. Immature DC (iDC) bound fluorescein isothiocyanate-labeled spherules in a time-and temperature-dependent manner. This binding was blocked by the addition of mannan, suggesting mannose receptor involvement in the DC-Coccidioides interaction. Binding was subsequently associated with ingestion and intracellular processing of spherules. Coculturing of spherules with iDC was associated with the development of mature DC that were morphologically, phenotypically, and functionally similar to those induced by tumor necrosis factor alpha and prostaglandin E 2 . Finally, spherules incubated with iDC induced activation of PBMC from nonimmune donors. These data indicate that human DC are capable of binding, internalizing, and presenting antigens from Coccidioides spherules and suggest that DC may play a critical early role in the formation of a cellular immune response in human coccidioidomycosis.
Unlike HLA-A and HLA-B, few peptide epitope motifs have been reported for HLA-C molecules. However, a number of cytotoxic T-lymphocyte epitopes derived from tumor antigens that bind to HLA-C molecules have been described. Here we report peptide-binding motifs for both HLA-Cw6.02 and HLA-Cw7.01 molecules. Recombinant human HLA molecules were generated and used to screen combinatorial 9mer peptide libraries. Complexes of HLA molecules properly folded and associated with beta2-microglobulin and peptides were identified using a conformation-specific HLA class I antibody conjugated to alkaline phosphatase. In the presence of substrate, peptide beads can be readily isolated and microsequenced to determine peptide identity. Of the peptides that bound to HLA-Cw6.02 and HLA-Cw7.01, 19 and 18 peptides, respectively, were sequenced, allowing motif identification for each C allele. This is the first report of an HLA-Cw7.01 peptide motif and extends the findings of Falk et al. [(1993) Proc Natl Acad Sci USA 90:12005] for an HLA-Cw6.02 motif. Anchoring amino acids for the HLA-Cw6.02 motif were phenylalanine or tyrosine in position (P)1, arginine in P2, and an aliphatic/aromatic residue at P9. Anchoring residues for HLA-Cw7.01 were positively charged amino acids in P1 and P2. Unlike most other HLA molecules, we were unable to assign P9 an anchoring residue, and we suspect that HLA-Cw7.01 binds peptides in an unconventional manner. Additionally, preferred amino acids were identified for both molecules. Identification of HLA-Cw6.02 and HLA-Cw7.01 peptide-binding motifs makes a significant contribution to the C allele peptide-binding motifs and will allow investigators to predict, design, and test HLA-Cw6.02 and HLA-Cw7.01 engineered peptides for immunotherapy.
Mannose is the predominant monosaccharide in the coccidioidal antigen preparation T27K. Mannan and anti-CD206 antibody significantly decreased the surface expression of mannose receptor (MR) on adherent peripheral blood mononuclear cells and reduced the interleukin-2 (IL-2) release induced by T27K. These data suggest that MR mediates IL-2 release by T27K.The mannose receptor (MR) is found on the surface of macrophages and dendritic cells, can bind terminal mannoses found on fungi and other pathogens (6), and has been shown to mediate the in vitro cellular immune response to fungal antigens (7). We used mannan, an MR ligand (11), and anti-CD206 (␣CD206), a monoclonal antibody directed against MR (4), to block the surface expression of MR on adherent peripheral blood mononuclear cells (PBMC) and examined the effect of this on the cellular immune response induced by T27K, a glycosylated coccidioidal antigen preparation (1-3).PBMC, derived from the blood of healthy human donors of known coccidioidal immunity, were resuspended in RPMI 1640 (GIBCO, Grand Island, Mich.) with 10% autologous serum, added to 35-mm flat-bottom wells (Falcon, Becton Dickinson Labware), and incubated at 37°C in 95% air-5% CO 2 . For the first 30 min, mannan (from Sacchyromyces cerevisiae; Sigma Chemical Company, St. Louis, Mo.) or ␣CD206 (from clone 19.2; BD Biosciences Pharmingen, San Diego, Calif.) was added to wells. In some experiments, immunoglobulin G1 (IgG1) (no. 555748; BD Biosciences Pharmingen), the isotype of ␣CD206, was used. Control wells received nothing. After 30 min, 10-g/ml T27K was added to cells and further incubated for 72 h. Adherent cells were removed and incubated with fluorescein isothiocyanate (FITC)-labeled ␣CD206 or FITC-labeled IgG1 for 30 min at 22°C in the dark. Cell viability just prior to flow cytometry was Ͼ90%, as determined by trypan blue exclusion. A gate was set around viable nonlymphocytes and 4,000 events were collected. MR surface expression was measured as the ratio of the geometric mean fluorescent intensity (MFI) of samples stained with FITC-labeled ␣CD206 divided by the geometric MFI of cells stained with labeled isotype. Interleukin-2 (IL-2) and gamma interferon (IFN-␥) concentrations in harvested supernatant were measured using a flow cytometry-based immunoassay (CBA; BD Immunosciences, San Jose, Calif.) or by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn.).Monosaccharide analysis of T27K was performed by the Glycotechnology Core Resource of the University of California at San Diego by using high-pH anion-exchange chromatography with pulsed amperometric detection after protein denaturization and desalting (A. Datta, personal communication). The Wilcoxon signed-rank test for paired data was employed for statistical analysis. All work was approved by the Human Subjects Protection Program of the University of Arizona.To assess mannan blocking of MR, 3.0 ϫ 10 6 PBMC in 2 ml of RPMI 1640 with 10% autologous serum were incubated for 72 h with or without 3 mg of mannan/ml. Subsequent...
In this study, we developed two Her-2/ neu-derived E75 altered peptide ligands (APLs) that demonstrate increased affinities for the HLA-A*0201 allele compared with wild-type E75 peptide. The APLs contain amino acids from E75(369-377), an immunodominant Her-2/ neu-derived peptide, and preferred primary and auxiliary HLA-A*0201 molecule anchor residues previously identified from combinatorial peptide library screening with the recombinant molecule. CTL lines were generated against wild-type E75 peptide (KIFGSLAFL) and APLs by multiple rounds of peptide stimulation of peripheral blood mononuclear cells (PBMCs) from HLA-A2+ antigen normal individuals. CTL lines raised on wild-type E75 peptide cross-reacted with APLs and similarly, CTL lines raised on APLs cross-reacted with wild-type E75 peptide, as measured by IFN-gamma ELISpot and target cell lysis assays. One of five individuals demonstrated specificity for APL 2 (FLFGSLAFL), whereas APL 5 (FLFESLAFL)-specific responses were observed from all five individuals tested. Molecular models of the E75, APL 2, and APL 5/HLA-A2 complexes indicated that the substitution of glycine with glutamic acid at position four of APL 5 resulted in the presentation of a large, negatively charged side chain that interacts with the outer edge of the HLA-A2 antigen alpha helix and is freely available to interact with cognate T-cell receptors. The results of this study further substantiate the concept that rational design of T-cell epitopes may lead to stronger peptide immunogens than natural, wild-type peptides.
These data suggest that there is an association between low serum MBL levels and symptomatic coccidioidomycosis.
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