Variation in cytotoxic T-lymphocyte responses to peptides derived from tyrosinase-related protein-2.

In this study, we developed three optimized peptide ligands (OPL) that demonstrate increased affinities for HLA-A*0201 compared with wild-type tyrosinase-related protein-2 (TRP-2) peptide. The OPL contain amino acids from TRP-2((180-188)) and preferred primary and auxiliary HLA-A*0201 anchor residues. Cytotoxic T lymphocyte (CTL) lines were generated against wild-type TRP-2 peptide and OPL by multiple rounds of peptide stimulation of peripheral blood mononuclear cells from HLA-A2*0201(+) healthy individuals. CTL reactivity profiles to three different OPL were donor-dependent. Among donors, at least one OPL was particularly stimulatory and elicited high levels of CTL that cross-reacted with wild-type TRP-2 peptide. Cytotoxicity assays using CTL raised on wild-type TRP-2 peptide or OPL demonstrated lysis of HLA-A2-positive glioblastoma cells. Molecular models of TRP-2 and OPL peptides docked with HLA-A*0201 demonstrated that substitution of F for S at position 1 (P1) oriented the peptides favoring a pi-pi aromatic interaction with W 167 of HLA-A*0201. This in turn positions P5 and P8 aromatic rings to face solvent that may promote binding to the T-cell receptor, leading to a robust T-cell activation. The results of this study further substantiate the concept that rational design and testing of multiple peptides for the same T-cell epitope should elicit a broader response among different individuals than single peptide immunization. Our results may partially explain why some patients have better clinical responses to peptide-based immunotherapy, whereas others respond poorly.

Mannose-binding lectin serum levels are low in persons with clinically active coccidioidomycosis.

BACKGROUND: Mannose-binding lectin (MBL) is a circulating collectin that is part of the innate immune response. We explored the serum levels of MBL in persons with different forms of coccidioidomycosis. METHODS: Serum MBL was measured by ELISA from samples obtained from healthy donors with immunity to Coccidioides, and those with various forms of active coccidioidomycosis. Blood cell specimens from a subgroup of subjects with active coccidioidomycosis were examined for single nucleotide polymorphisms of the MBL gene and promoter regions. RESULTS: The control group comprised 29 healthy immune subjects. Patient groups with active coccidioidomycosis consisted of 20 patients with symptomatic primary pulmonary coccidioidomycosis, 26 with non-meningeal disseminated coccidioidomycosis, and nine with coccidioidal meningitis. The group with active coccidioidomycosis was significantly older and more likely to be male than the control group (for both, P < 0.001). The mean +/- SEM level of serum MBL in the healthy controls was 169.4 +/- 28.6 ng/ml, significantly higher than the 79.2 +/- 10.9 ng/ml for all active groups (P < 0.001). Moreover, the active coccidioidomycosis group was significantly more likely to have serum MBL level

Spherules derived from Coccidioides posadasii promote human dendritic cell maturation and activation.

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 E2. 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.

The mannose receptor mediates the cellular immune response in human coccidioidomycosis.

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.

Functional characterization of CTL against gp100 altered peptide ligands.

In this study, four modified gp100 peptides were designed by combining amino acids from the melanoma peptide antigen gp100((209-217)) with preferred primary and auxiliary HLA-A *0201 anchor residues previously identified from combinatorial peptide library screening with recombinant HLA-A*0201. These modified peptides demonstrated stronger binding affinity for the HLA-A*0201 molecule compared to wild-type gp100 peptide. Nine CTL lines generated from patients immunized with the g209-2 M peptide and one CTL line from a non-immunized patient were tested for the ability to respond to these modified gp100 peptides. Stimulation of CTL by two of four modified peptides induced higher levels of IFN-gamma secretion than the wild-type gp100 peptide, demonstrating that higher peptide binding affinity for HLA molecules does not necessarily equate to functional activity of CTL. Two major and one minor CTL recognition pattern were observed, irrespective of previous peptide immunization, suggesting that multiple, rationally designed modified tumor peptides for the same epitope stimulate a broad CTL response by activating multiple CTL capable of cross-reacting with the natural antigenic peptide.

Her-2/ neu altered peptide ligand-induced CTL responses: implications for peptides with increased HLA affinity and T-cell-receptor interaction.

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.

Identification of HLA-Cw6.02 and HLA-Cw7.01 allele-specific binding motifs by screening synthetic peptide libraries.

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.