Clinical studies have demonstrated that HLA-DPB1–mismatched allogeneic stem cell transplantation (allo-SCT) is associated with a decreased risk of disease relapse and an increased risk of graft-versus-host disease (GVHD) compared with HLA-DPB1–matched SCT. In T cell–depleted allo-SCT, mismatching of HLA-DPB1 was not associated with an increased risk of severe GVHD, but a significant decreased risk of disease relapse was still observed. To investigate whether patient HLA-DP–specific CD4⁺ T cell responses were frequently induced after T cell–depleted HLA-DPB1–mismatched allo-SCT and donor lymphocyte infusion (DLI), we developed a method to screen for the presence of HLA-DP–specific CD4⁺ T cells using CD137 as an activation marker and analyzed 24 patient–donor combinations. The patients suffered from various B cell malignancies, multiple myeloma, and myeloid leukemias. Patient HLA-DP–specific CD4⁺ T cells were detected after DLI in 13 of 18 patients who exhibited a clinical response to DLI, compared with only 1 of 6 patients without a clinical response to DLI. Eight patients developed significant GVHD. These data show that patient HLA-DP–specific CD4⁺ T cells frequently occur after HLA-DPB1–mismatched T cell–depleted allo-SCT and DLI, and are associated with graft-versus-leukemia reactivity both in the presence and absence of GVHD.