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. 2004 Dec 1;114(11):1612–1623. doi: 10.1172/JCI22787

Figure 3.

Figure 3

MHC class II_restricted interactions between ANK and CD4+ T cells. (A) OFR3 CD4+ T cells were stained with PE-labeled DR0401 tetramer loaded with HA (DR0401-HA) or control peptide (DR0401-ospA) (gray histograms). Staining of freshly isolated CD4+ T cells with the same tetramers was used as a background setting (white histograms). (B and C) All APC_T cell incubations applied in the following experiments were maintained for 48 hours followed by addition of thymidine for an additional 24 hours. (B) Irradiated cells (50 × 103) were incubated with either HA306–318 (HA pep.) or control peptide (con pep.) (150 ng/ml) and added to the OFR3 T cell clone (50 × 103 cells per well). (C) Antigen-processing capability was tested by measurement of OFR3 clone proliferation in the presence of 150 ng/ml recombinant HA or BSA proteins (prot.). The contribution of CD80 and CD86 to OFR3 T cell activation was evaluated by pretreatment of APCs with 25 ng/ml CTLA-4Ig. (D) CD4+ T cells isolated from HLA-DR4+ donors were incubated for 10 days with UaNK or ANK cells pulsed with HA protein or BSA (indicated on the top of each panel), or only with recombinant HA protein. Cells were stained for CD4 and DR0401-HA or control DR0401-ospA tetramers. The percentage of tetramer-positive fraction out of total CD4+ T cells is indicated. Staining of PBLs from the same donor is shown as a control. One representative staining obtained from a single donor out of 3 donors examined is shown. (E) Naive umbilical cord_derived CD4+ T cells were cultured with HA- or BSA-pulsed ANK cells for 14 days and stained with DR4 peptide_loaded tetramers. (F) DR0401-HA_positive umbilical cord_derived CD4+ T cells were characterized for CD45RO and antigen-specific effector responses. At day 25 of coculturing, 150 × 103 cells from each donor were incubated for 96 hours with 150 × 103 irradiated autologous ANK cells either unpulsed or pulsed with HA or control peptide, in the presence of 2 μCi of [3H]thymidine per well. Prior to harvesting, supernatant was taken for measurement of IL-2 and IFN-γ. One representative characterization from a single donor out of 3 is shown. Values are mean ± SD for triplicate samples. **P < 0.01 by Student’s t test.