Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes

The efficient in vitro expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8(+) cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

CTL recognition of a bulged viral peptide involves biased TCR selection

MHC class I molecules generally present peptides of 8-10 aa long, forming an extended coil in the HLA cleft. Although longer peptides can also bind to class I molecules, they tend to bulge from the cleft and it is not known whether the TCR repertoire has sufficient plasticity to recognize these determinants during the antiviral CTL response. In this study, we show that unrelated individuals infected with EBV generate a significant CTL response directed toward an HLA-B*3501-restricted, 11-mer epitope from the BZLF1 Ag. The 11-mer determinant adopts a highly bulged conformation with seven of the peptide side chains being solvent-exposed and available for TCR interaction. Such a complex potentially creates a structural challenge for TCR corecognition of both HLA-B*3501 and the peptide Ag. Surprisingly, unrelated B*3501 donors recognizing the 11-mer use identical or closely related alpha beta TCR sequences that share particular CDR3 motifs. Within the small number of dominant CTL clonotypes observed, each has discrete fine specificity for the exposed-side chain residues of the peptide. The data show that bulged viral peptides are indeed immunogenic but suggest that the highly constrained TCR repertoire reflects a limit to TCR diversity when responding to some unusual MHC peptide ligands.

Potent cyclic antagonists of the complement C5a receptor on human polymorphonulcear leukocytes. Relationships between structures and activity

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC50, 20 nM(-1) muM). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinity-determining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G protein-coupled receptor.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Versatile peptide dendrimers for nucleic acid delivery

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.

Prediction of HLA-DQ3.2 beta ligands: evidence of multiple registers in class II binding peptides

Motivation: While processing of MHC class II antigens for presentation to helper T-cells is essential for normal immune response, it is also implicated in the pathogenesis of autoimmune disorders and hypersensitivity reactions. Sequence-based computational techniques for predicting HLA-DQ binding peptides have encountered limited success, with few prediction techniques developed using three-dimensional models. Methods: We describe a structure-based prediction model for modeling peptide-DQ3.2 beta complexes. We have developed a rapid and accurate protocol for docking candidate peptides into the DQ3.2 beta receptor and a scoring function to discriminate binders from the background. The scoring function was rigorously trained, tested and validated using experimentally verified DQ3.2 beta binding and non-binding peptides obtained from biochemical and functional studies. Results: Our model predicts DQ3.2 beta binding peptides with high accuracy [area under the receiver operating characteristic (ROC) curve A(ROC) > 0.90], compared with experimental data. We investigated the binding patterns of DQ3.2 beta peptides and illustrate that several registers exist within a candidate binding peptide. Further analysis reveals that peptides with multiple registers occur predominantly for high-affinity binders.

Specific modulation of airway epithelial tight junctions by apical application of an occludin peptide

Tight junctions are directly involved in regulating the passage of ions and macromolecules (gate functions) in epithelial and endothelial cells. The modulation of these gate functions to transiently regulate the paracellular permeability of large solutes and ions could increase the delivery of pharmacological agents or gene transfer vectors. To reduce the inflammatory responses caused by tight junction-regulating agents, alternative strategies directly targeting specific tight junction proteins could prove to be less toxic to airway epithelia. The apical delivery of peptides corresponding to the first extracellular loop of occludin to transiently modulate apical paracellular flux has been demonstrated in intestinal epithelia. We hypothesized that apical application of these occludin peptides could similarly modulate tight junction permeability in airway epithelia. Thus, we investigated the effects of apically applied occludin peptide on the paracellular permeability of molecular tracers and viral vectors in well differentiated human airway epithelial cells. The effects of occludin peptide on cellular toxicity, tight junction protein expression and localization, and membrane integrity were also assessed. Our data showed that apically applied occludin peptide significantly reduced transepithelial resistance in airway epithelia and altered tight junction permeability in a concentration-dependent manner. These alterations enhanced the paracellular flux of dextrans as well as gene transfer vectors. The occludin peptide redistributed occludin but did not alter the expression or distribution of ZO-1, claudin-1, or claudin-4. These data suggest that specific targeting of occludin could be a better-suited alternative strategy for tight junction modulation in airway epithelial cells compared with current agents that modulate tight junctions.