Wilms tumor gene 1 product (WT1) has been recognized as an attractive target antigen of immunotherapy for various malignancies including leukemia. Because tumor-associated antigen-specific CD4+ T lymphocytes undoubtedly play an important role in the induction of an antitumor immune response, we attempted to generate WT1-specific CD4+ T lymphocytes in vitro and examined their antileukemia functions. A CD4+ T-cell line, designated NIK-1, which proliferated and produced Th1 cytokines specifically in response to stimulation with the WT1-derived peptide, WT1(337-347) LSHLQMHSRKH, in an HLA-DP5-restricted manner was established. NIK-1 exhibited cytotoxicity against HLA-DP5-positive, WT1-expressing leukemia cells but did not lyse HLA-DP5-negative, WT1-expressing leukemia cells or HLA-DP5-positive, WT1-negative cells. NIK-1 did not inhibit colony formation by normal bone marrow cells of HLA-DP5-positive individuals. This is the first report to describe WT1-specific and HLA class II-restricted CD4+ T lymphocytes possessing direct cytotoxic activity against leukemia cells.