Selective depletion of FOXP3^high cells by Fas-Fas-L-induced apoptosisoccurs in CD4⁺CD25⁺-enriched populations during repeated expansion

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⁺ Tcells 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 invitro.