12 research outputs found

    Enhancing sensitivity of detection of immune responses to Mycobacterium leprae peptides in whole-blood assays

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    Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-γ) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-γ than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-γ production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-γ production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-γ in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-γ was mainly mediated by CD4+ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1β (MIP-1β) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-γ-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides

    Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin

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    Celiac disease is a common severe intestinal disease resulting from intolerance to dietary wheat gluten and related proteins. The large majority of patients expresses the HLA-DQ2 and/or DQ8 molecules, and gluten-specific HLA-DQ-restricted T cells have been found at the site of the lesion in the gut. The nature of peptides that are recognized by such T cells, however, has been unclear so far. We now report the identification of a gliadin-derived epitope that dominantly is recognized by intestinal gluten-specific HLA-DQ8-restricted T cells. The characterization of such epitopes is a key step toward the development of strategies to interfere in mechanisms involved in the pathogenesis of celiac disease

    Enhanced Cross-Presentation and Improved CD8<sup>+</sup> T Cell Responses after Mannosylation of Synthetic Long Peptides in Mice

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    <div><p>The use of synthetic long peptides (SLP) has been proven to be a promising approach to induce adaptive immune responses in vaccination strategies. Here, we analyzed whether the efficiency to activate cytotoxic T cells by SLP-based vaccinations can be increased by conjugating SLPs to mannose residues. We could demonstrate that mannosylation of SLPs results in increased internalization by the mannose receptor (MR) on murine antigen-presenting cells. MR-mediated internalization targeted the mannosylated SLPs into early endosomes, from where they were cross-presented very efficiently compared to non-mannosylated SLPs. The influence of SLP mannosylation was specific for cross-presentation, as no influence on MHC II-restricted presentation was observed. Additionally, we showed that vaccination of mice with mannosylated SLPs containing epitopes from either ovalbumin or HPV E7 resulted in enhanced proliferation and activation of antigen-specific CD8<sup>+</sup> T cells. These findings demonstrate that mannosylation of SLPs augments the induction of a cytotoxic T cell response <i>in vitro</i> and <i>in vivo</i> and might be a promising approach to induce cytotoxic T cell responses in e.g. cancer therapy and anti-viral immunity.</p></div

    In vivo T cell activation by mannosylated SLPs.

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    <p>A) Mice were injected i.d. with 75 µg of OVA-specific mannosylated or non-mannosylated SLPs and 20 µg CpG. After 7 days, antigen-specific T cells in the blood were monitored by flow cytometry using epitope-specific tetramers. Cells were gated for CD8. B) Quantitative analysis of epitope-specific T cells in the blood or in the spleen after s.c or i.d. injection of SLPs. Graphs show percentage of tetramer-positive CD8<sup>+</sup> T cells. C) as in A) using HPV-specific SLPs. D) Quantitative analysis of epitope-specific T cells in the blood or in the spleen after s.c or i.d. injection of HPV-specific SLPs. E) Intracellular cytokine staining of splenic CD8<sup>+</sup> T cells after i.d. injection of HPV-specific mannosylated or non-mannosylated peptides and CpG as above. F) Quantitative analysis of intracellular cytokines in T cells isolated from the spleen after s.c or i.d. injection of HPV-specific SLPs. Graphs show percentage of IFNγ<sup>+</sup> TNFα<sup>+</sup> cells amongst all CD8<sup>+</sup> T cells. Dot plots depict representative results of 2 independent experiments. Bar graphs depict pooled results of 2 independent experiments (n = 9–10).</p

    Uptake of mannosylated and non-mannosylated SLPs.

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    <p>A) Wildtype or MR-deficient BM-DCs were incubated with 250 ng/ml fluorochrome-labeled OVA, 200 nM mannosylated SLPs or non-mannosylated SLPs for 15 min, chased with medium for 20 min and analyzed by immunofluorescence microscopy. Nuclei stained with DAPI are depicted in blue. B) Wildtype or MR-deficient BM-DCs were incubated with 250 ng/ml fluorochrome-labeled OVA, 200 nM mannosylated SLPs or non-mannosylated SLPs for 15 min. Antigen uptake was monitored by flow cytometry (gated on all living cells). C) Quantification of B) using different antigen concentrations. Depicted are representative results of at least 3 independent experiments. MFI: mean fluorescence intensity.</p

    Overview of the used SLPs.

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    <p>To study the effect of mannosylation on cross-presentation, we generated mannosylated and non-mannosylated SLPs containing the MHC I-restricted epitope of OVA (OVA<sub>257–264</sub>; peptides 1 and 2). Mannosylation was introduced by a bis-mannosylated Lysin residue. Additionally, we generated mannosylated or non-mannosylated SLPs containing the MHC II-restricted epitope of OVA (OVA<sub>323–339</sub>; peptides 3 and 4). To analyze intracellular trafficking, we synthesized fluorescently-labeled mannosylated or non-mannosylated SLPs (peptides 5 and 6). The Alexa647 fluorochrome was introduced by conjugation to an Alexa647-labeled cysteine. To study T cell responses against the E7 protein of HPV16, we generated mannosylated or non-mannosylated SLPs containing the MHC I restricted epitope of E7 (E7<sub>43–63</sub>; peptides 7 and 8). Epitopes within the SLPs are in bold.</p

    Intracellular localization of mannosylated SLPs.

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    <p>A) Wild-type BM-DCs were incubated simultaneously with Alexa647-labeled SLPs and Alexa488-labeled OVA for 15 min. After medium chase of another 20 min, intracellular localization was determined by immunofluorescence microscopy. B) Wild-type BM-DCs were incubated with fluorochrome-labeled OVA, SLPs, Transferrin and/or Lucifer Yellow for 15 min, chased with medium for another 20 min and stained with antibodies against EEA1, LAMP1 or Lucifer Yellow. Intracellular distribution was analyzed by immunofluorescence microscopy. To analyse co-localization of OVA and SLPs with the indicated markers, the Pearson correlation coefficient (varying between −1 and +1 with −1 for perfect negative correlation, 0 for perfect absence of correlation and 1 for perfect correlation) and the Mander's overlap coefficient (varying between 0 and 1 with 0 for no overlap and +1 for perfect overlap), were calculated. Nuclei stained with DAPI are depicted in blue. PCC: Pearson Correlation Coefficient. MOC: Mander's overlap coefficient.</p
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