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BACKGROUND AIMS: Expansion of anti-CD25 bead-isolated human Tregs culture has paradoxically resulted in reduced suppressive activity, but the mechanism(s) responsible for these observations are poorly defined. METHODS: Magnetic-bead isolated human CD25(+) cells were expanded with anti-CD3/CD28 beads and high doses of rhIL-2. Detection of Fas and Fas ligand (Fas-L) expression, activation of Caspase 8, cell proliferation and cytokine production was evaluated by multi-color fluorescence-activated cell sorting analysis. The role of Fas-Fas-L-mediated cell death was dissected through the use of agonist or antagonist monoclonal antibodies directed at Fas and Fas-L. RESULTS: Repeated expansion of bead-enriched CD4(+)CD25(+) cells generated a cellular product with markedly reduced suppressive activity and with significantly increased CD8(+) T cells and CD4(+) T cells producing interferon-γ and/or interleukin-2. We showed that Fas-Fas-L-mediated apoptosis of CD4(+)FOXP3(high) cells and rapid cell-cycling of CD8(+) T cells were collectively responsible for the reduced proportion of CD4(+)FOXP3(high) cells in expanded cultures. The depletion of CD4(+)FOXP3(high) cells and activation of Caspase 8 in CD4(+)FOXP3(high) cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4(+)FOXP3(high) cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies. CONCLUSIONS: Taken together, the data show that Fas-Fas-L-mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.

Original publication




Journal article



Publication Date





1286 - 1296


FOXP3, Fas, Treg, apoptosis, cell therapy, expansion, Apoptosis, CD4 Antigens, Caspase 8, Cell Proliferation, Cells, Cultured, Cytokines, Fas Ligand Protein, Flow Cytometry, Forkhead Transcription Factors, Humans, Immunomagnetic Separation, Immunotherapy, Interleukin-2, Interleukin-2 Receptor alpha Subunit, Lymphocyte Activation, Lymphocyte Depletion, T-Lymphocytes, Regulatory, fas Receptor