T-cell tolerance induced by repeated antigen stimulation: Selective loss of Foxp3(-) conventional CD4 T cells and induction of CD4 T-cell anergy.

Repeated immunization of mice with bacterial superantigens induces extensive deletion and anergy of reactive CD4 T cells. Here we report that the in vitro proliferation anergy of CD4 T cells from TCR transgenic mice immunized three times with staphylococcal enterotoxin B (SEB) (3x SEB) is partially due to an increased frequency of Foxp3(+) CD4 T cells. Importantly, reduced number of conventional CD25(-) Foxp3(-) cells, rather than conversion of such cells to Foxp3(+) cells, was the cause of that increase and was also seen in mice repeatedly immunized with OVA (3x OVA) and OVA-peptide (OVAp) (3x OVAp). Cell-transfer experiments revealed profound but transient anergy of CD4 T cells isolated from 3x OVAp and 3x SEB mice. However, the in vivo anergy was CD4 T-cell autonomous and independent of Foxp3(+) Treg. Finally, proliferation of transferred CD4 T cells was inhibited in repeatedly immunized mice but inhibition was lost when transfer was delayed, despite the maintenance of elevated frequency of Foxp3(+) cells. These data provide important implications for Foxp3(+) cell-mediated tolerance in situations of repeated antigen exposure such as human persistent infections.