Selective depletion of FOXP3high cells by Fas–Fas-L–induced apoptosis occurs in CD4+CD25+-enriched populations during repeated expansion

Abstract

Background aimsExpansion 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. MethodsMagnetic-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. ResultsRepeated 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+FOXP3high cells and rapid cell-cycling of CD8+ T cells were collectively responsible for the reduced proportion of CD4+FOXP3high cells in expanded cultures. The depletion of CD4+FOXP3high cells and activation of Caspase 8 in CD4+FOXP3high cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4+FOXP3high cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies. ConclusionsTaken together, the data show that Fas–Fas-L–mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.