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