A cyclic peptide accelerates the loading of peptide antigens in major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC)-loading enhancers (MLE) have recently attracted attention because of their ability to enhance the efficacy of peptide immunotherapeutics. As small molecular weight compounds, they influence the loading of peptides in MHC molecules by converting them from a non-receptive to a receptive state. Herein, we report a 14-mer cyclic peptide 1 (CP-1) as a new class of MLE-peptide. This peptide was used to investigate its loading on human leukocyte antigen (HLA)-DR molecules. It was found that CP-1 strongly accelerates peptide-loading on both soluble and cell surface HLA-DR molecules in a dose-dependent manner. The effect was evident for all subsets of HLA-DR tested, including HLA-DRB1*1501, indicating that it acts independently of P1-pocket size, which is the canonical MLE-binding site. Importantly, increased peptide-loading by CP-1 was correlated with improved CD4(+) T cell responses in vitro, while propidium iodide staining indicated low peptide-induced cytotoxicity. Thus, this study revealed a new class of peptide-based enhancers that catalyze peptide-loading by allosteric interactions with MHC molecules. Because of its low cellular cytotoxicity and high MLE activity, it may be useful in stimulating antigen-specific T cell responses for therapeutic purposes.

A xenon-129 biosensor for monitoring MHC-peptide interactions.

Caged in: The formation of a complex between a peptide ligand and a major histocompatibility complex (MHC) class II protein is detected by a (129)Xe biosensor. Cryptophane molecules that trap Xe atoms are modified with a hemagglutinin (HA) peptide, which binds to the MHC protein. The interaction can be monitored by an NMR chemical shift change of cage-HA bound (129)Xe.

Enhancement of tumour-specific immune responses in vivo by 'MHC loading-enhancer' (MLE).

BACKGROUND: Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered. PRINCIPAL FINDINGS: Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with 'MHC-loading enhancer' (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE. CONCLUSION: MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy.

Interferon-gamma regulates cathepsin G activity in microglia-derived lysosomes and controls the proteolytic processing of myelin basic protein in vitro.

The serine protease cathepsin (Cat) G dominates the proteolytic processing of the multiple sclerosis (MS)-associated autoantigen myelin basic protein (MBP) in lysosomes from primary human B cells and dendritic cells. This is in contrast to B-lymphoblastoid cell lines, where the asparagine endopeptidase (AEP) is responsible for this task. We have analysed microglia-derived lysosomal proteases for their ability to process MBP in vitro. In lysosomes derived from primary murine microglia, CatD, CatS, AEP and CatG were involved in the processing of MBP. Interestingly, when microglia were treated with interferon-gamma to mimic a T helper type 1-biased cytokine milieu in MS, CatG was drastically down-regulated, in contrast to CatS, CatB, CatL, CatD or AEP. This resulted in significantly increased stability of MBP and a selective lack of CatG-derived proteolytic fragments; however, it did not affect the gross pattern of MBP processing. Inhibition of serine proteases eliminated the processing differences between lysosomal extracts from resting microglia compared to interferon-stimulated microglia. Thus, the cytokine environment modulates lysosomal proteases in microglia by a selective down-regulation of CatG, leading to decreased MBP-processing by microglia-derived lysosomal proteases in vitro.