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T he folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.