Permissive and nonpermissive HLA-DPB1 mismatches induced HLA-DP specific immune responses in vivo and in vitro. Two patients transplanted with an HLA-A, -B, -C, -DRB1, -DQB1 matched, HLA-DPB1 mismatched SCT were analyzed for the presence of HLA-DP specific CD4⁺ T cells after DLI. Patient 1 (HLA-DPB1*0201,0301) was transplanted for a chronic B-cell leukemia with a nonpermissive HLA-DPB1 mismatched donor (HLA-DPB1*0402,0501). Patient 2 (HLA-DPB1*0401) was transplanted for multiple myeloma with a permissive HLA-DPB1 mismatched donor (HLA-DPB1*0301,0402) in the GVH direction. (A) Purified CD4⁺ T cells obtained during the clinical immune response to DLI were stimulated with HLA class II negative HeLa cells transduced with either donor or patient HLA-DP molecules. After 48 hours, CD137 expression on CD4⁺ T cells was determined using flow cytometry. Percentages of CD137 positive CD4⁺ T cells in response to stimulation with different HLA-DP molecules are shown. (B) CD137 expressing CD4⁺ T cells shown in panel A were clonally isolated and tested for recognition of different target cells. IFN-γ production (pg/mL) was determined in 50 μL of supernatant. Mean results ± SD of a selection of 11 HLA-DPB1*0201 specific CD4⁺ T-cell clones, 15 HLA-DPB1*0301 specific CD4⁺ T-cell clones, and 15 HLA-DPB1*0401 specific CD4⁺ T-cell clones are shown. (C) Purified CD4⁺ T cells derived from 4 different healthy persons were stimulated with HeLa cells transduced with permissive mismatched HLA-DPB1 molecules (n = 2) or nonpermissive mismatched HLA-DPB1 molecules (n = 2). At day 14, 25 000 CD4⁺ T cells from each cell line were restimulated with 50 000 HLA-DP transduced HeLa cells used for stimulation (■) or HeLa cells transduced with control donor HLA-DP molecules (□). IFN-γ release (pg/mL) measured in 50 μL of supernatant upon restimulation is shown.