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Predicted HLA-E binding Mtb peptide specific T-cell lines are cytotoxic.

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posted on 21.02.2013, 02:31 by Simone A. Joosten, Krista E. van Meijgaarden, Pascale C. van Weeren, Fatima Kazi, Annemieke Geluk, Nigel D. L. Savage, Jan W. Drijfhout, Darren R. Flower, Willem A. Hanekom, Michèl R. Klein, Tom H. M. Ottenhoff

(A) HLA-E expression on K562 cell lines after peptide loading. Upper graph, untransfected K562 cells do not stain with anti-HLA-E (3D12) antibodies, whereas HLA-E transfected K562 cells expressing the natural HLA-E ligand (HLA-B7) or cells loaded with a Mtb derived HLA-E binding peptide (#55, see Table 1) express similar levels of HLA-E. Middle graph, similar staining is observed when an antibody against HLA class I (W6/32) is used, with equal expression levels of HLA-E in the presence of a natural ligand or an Mtb derived peptide. Bottom graph, the isotype control staining for 3D12 and W6/32 was negative on all cell lines tested. HLA-E expression was always confirmed before functional experiments. (B) Peptide specific T-cell lines were generated by stimulation with peptide in the presence of IL-7 and further expansion using IL-2. K562 cells expressing HLA-E were loaded with specific and control peptides, labeled with 1 µCi 51Cr and co-cultured with T-cell lines for 5 hours in different ratios before 51Cr release was measured. Data are expressed as % specific lysis. Left graph: T-cell line derived from donor 4 directed against peptide 55 (using the high affinity HLA-E binding peptide 68 or the natural B7 ligand as irrelevant control), right graph: T-cell line derived from donor 2 directed against peptide 62 (using peptide 68 on transfected K562, or peptide 62 on untransfected K562 as irrelevant controls). Representative of 4 lines derived from 3 donors. (C) Fully HLA-A,B,C mismatched adherent monocytes were infected with live BCG (MOI 5, overnight) before labeling with 1 µCi 51Cr. Cells were co-cultured with T-cell lines for 5 hours in 6-replicate cultures. Grey bars represent BCG infected monocytes whereas white bars represent uninfected monocytes. All HLA-E peptide restricted T-cell lines recognized BCG infected target cells. In contrast a CD8+ T-cell clone restricted to the male HY antigen presented in HLA-A2 did not lyse infected monocytes, whereas the natural ligand (B-cells expressing HY in the context of HLA-A2) was specifically lysed, resulting in up to 100% target cell lysis. Data are depicted as mean+standard error of the mean and represent multiple experiments.

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