Recognition of skin-derived fibroblasts by allo-reactive HLA-DPB1–specific CD4 T-cell clones. (A) Expression of HLA-DR, -DQ, and -DP on skin fibroblasts from patient 1 (upper) and patient 2 (lower) cultured for 5 days with and without IFN-γ (200 IU/mL) was analyzed by flow cytometry. Histograms show the mean fluorescence intensity after staining with HLA-DR– (solid lines), HLA-DQ– (dashed lines), and HLA-DP– (dotted lines) specific moAb (filled histograms). Nonfilled histograms represent the MFI of unstained cells. (B) Reactivity of all allo-reactive HLA-DPB1–specific CD4+ T-cell clones from patient 1 (upper) and patient 2 (lower) was tested for cytokine production against skin-derived fibroblasts from the patients cultured with and without IFN-γ in ELISA. Each dot represents the release of IFN-γ (pg/mL) in 10 µL supernatants by a single CD4+ T-cell clone. None of the HLA-DPB1–specific CD4+ T-cell clones recognized skin fibroblasts in the absence of IFN-γ pretreatment, in line with the observed lack of HLA class II expression. From patient 1, 7 CD4+ T-cell clones specific for HLA-DPB1*0101 and 2 CD4+ T-cell clones specific for HLA-DPB1*0301 recognized skin-derived fibroblasts cultured with IFN-γ. From patient 2, 10 CD4+ T-cell clones specific for HLA-DPB1*0301 recognized skin-derived fibroblasts cultured with IFN-γ.