Apoptosis by Low-Dose Methotrexate Occurs in Fully Activated Proliferating CD4+ and CD8+ Lymphocytes and in Leukemic LGL after Mitogenic Stimulation using a FADD-Dependent Pathway.
Authors: Epling-Burnette P.K. , Painter Jeffrey S. , Bai Fanqi , Mohaptra Subra , Loughran Thomas P. 
Published: April 2020

Abstract
Low-dose methotrexate (MTX) is used as an immunosuppressive agent for the treatment of rheumatoid arthritis (RA), Large Granular Lymphocyte (LGL) leukemia, Cutaneous T Cell Lymphoma (CTCL), autoimmune diseases, and prevention of GvHD during bone marrow transplants. The mechanism for immunosuppression is not clearly understood but most data suggests that apoptosis of activated lymphocytes plays a critical role. In this study, we wanted to define the MTX-sensitive population and to determine the apoptotic pathway activated by MTX. Using a clinically relevant dosage range (8 nM- 1 μM), MTX-mediated apoptosis was first examined in a T lymphoblastic leukemia cell line (CEM). The apoptotic pathway induced by MTX included phosphotidylinositol externalization and caspase-3 activation along with a slight increase in mitochondrial membrane depolarization. We next examined a series of tumor cell lines and normal cells for evidence of MTX-induced apoptosis. Using the same clinically relevant dosage range, we found that MTX-induced apoptosis was primarily observed in the four T cell leukemia cell lines including CEM, Jurkat, MT-2, and HUT78 and in normal PBMCs activated with mitogens and IL-2. Less MTX-induced apoptosis was observed in two myeloid leukemia cell lines including HL-60 and K562 and in a B cell leukemia cell line Raji, and the multiple myeloma cell line 8226. Unactivated PBMCs were resistant to MTX-mediated apoptosis. T cells that are clonally expanded in patients with T-LGL leukemia have a CD8+ cytotoxic phenotype, whereas other diseases that are treated with low-dose MTX, such as CTCL and RA, are characterized by the expansion of CD4+ T cells. We found that both freshly sorted CD4+ and CD8+ cells were MTX resistant. In contrast, PHA plus IL-2 treatment induced MTX sensitivity in T cell with both immunophenotypes. We also examined clinical samples from patients with LGL leukemia. We found that freshly isolated PBMCs from T-LGL leukemia patients were resistant to MTX. Clonal cells from the peripheral blood of LGL leukemia patients are in G0/G1 phase of the cell cycle. Interestingly, we found that PHA plus IL-2 treatment induced the cells to enter S-phase and to become MTX sensitive. These results suggest that only fully activated, proliferating T cells from patients with LGL leukemia undergo apoptosis in response to low-dose MTX.
Because there was only minor depolarization of mitochondria after MTX treatment in both CEM cells and normal activated PBMCs, we wanted to examine upstream apoptotic events after MTX treatment. We found that caspase-8 cleavage and enzymatic activity was induced by MTX in both CD95 Type I (HUT78) and Type II (CEM and Jurkat) cells but that there was a differential requirement for caspase-8 activity for apoptosis. We found that caspase-8 activation was independent of the Fas receptor as shown by immunoprecipitation experiments and MTX apoptotic assays in the JM3A5 Fas-receptor mutant Jurkat cell line. Using a Jurkat cell line with a homozygous deletion of the FADD gene, we found that caspase-8 activation, caspase-3 activation, and apoptosis in response to MTX were dependent on the adaptor protein FADD. These findings have important implications for understanding the mechanism of MTX for immunosuppressive therapy.