Selective depletion of FOXP3(high) cells by Fas-Fas-L-induced apoptosis occurs in CD4(+)CD25(+)-enriched populations during repeated expansion.

Abstract

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. 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. 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. Taken together, the data show that Fas-Fas-L-mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.