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MHC class I molecules bind and present endogenously loaded peptides to immune effector cells, such as natural killer (NK) cells and T cells. Cell stress, including infection and neoplasia, alters the peptides presented to immune effectors by MHC class I. Cell stress additionally results in the induction of heat-shock proteins (HSPs). Based on observations of multiple researchers, we propose that class I presented-peptide epitopes derived from HSPs are a recognition point for innate and adaptive immune responses. HSPs are rich sources of MHC peptides, and the expression of these peptides increases as a result of several cellular stresses, including viral infection. Functionally, several groups have now confirmed the recognition of HSP peptides by cytotoxic T lymphocytes during both viral infection and tumorigenesis. Similarly, NK cells recognize an HSP60-derived peptide presented by HLA-E. These HSP60–HLA-E complexes lead to NK-cell activation, promoting lysis of stressed cells by members of the innate immune response. This peptide-epitope function of HSPs enables these proteins to act as broad sensors of cellular stress to both the innate and adaptive immune response. [Copyright &y& Elsevier]

The article shows that activated dendritic cells fail to cross-prime CD8+ T cell responses. Viruses that infect professional antigen-presenting cells may have contributed to the evolution of cross-priming. Cross-priming of CD8+ T cells involves three specialized features of dendritic cells. In vitro activation of dendritic cells results in inhibition of cross-priming.

The article presents information on hereditary hemochromatosis, a disease resulting from iron overload, is the most common genetic disorder in the United States, afflicting more than a million Americans. Untreated, HH frequently leads to a middle-aged demise due to the failure of an iron-damaged liver, heart, or pancreas. HH was one of the first diseases linked to the major histocompatibility complex. HH results from misfolding of HFE induced by a point mutation that replaces the normal cysteine residue at position 282 with a tyrosine.

Various approaches are currently proposed to successfully develop therapies for the prevention and treatment of infectious diseases and cancer. One of the most promising approaches is the development of vaccines that elicit cytotoxic T lymphocyte (CTL) responses. Consequently, identification and exact definition of molecular parameters involved in peptide-MHC class-I interactions of putative CTL epitopes are of prime importance for the development of immunomodulating compounds. To better facilitate epitope discovery, we developed and validated a novel state-of-the-art biochemical HLA-A*0201 assay, which is comprised of technologically advanced cutting edge reagents. The technique is based on competition and uses a FITC-labeled reference peptide and highly purified soluble HLA-A*0201 molecules to quantitatively measure the binding capacity of nonlabeled peptide candidates. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. During standardization, the problem of assay parameter variation is discussed, showing the dramatic influence of HLA and reference peptide concentrations as well as the choice of the reference peptide itself on IC50 determinations. For validation, a panel of 15 well-defined HLA-A*0201 ligands from various sources covering a broad range of binding affinities was tested. Binding data were used to compare against pre-existing quantitative assay systems. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with recombinant sHLA molecules is highly advantageous for the accurate assessment of peptide binding. Furthermore, the assay also features high-throughput screening capacity, providing uniquely efficient means of identifying and evaluating immune target molecules. [ABSTRACT FROM AUTHOR]

Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the. advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 °C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three β-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate kon values (1 × 104 to 8.7 × 104 M-1 s-1) and slow koff values (1.9 × 10-4 to 4.3 × 10-4 s-1), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies. [ABSTRACT FROM AUTHOR]