Figure 3

TSN-exposed M2-like macrophages significantly induce aTreg cells from CD4⁺CD25⁻ T cells. CD14⁺ monocytes were cultured with TSNs from a laryngeal carcinoma cell line (Mϕ_SNU899), followed by coculture with autologous CD4⁺CD25⁻ T cells for 4 days with or without neutralising mAbs against TGFβ-1, IL-10, or TGFβ-1/IL-10 in the presence of 2 μg ml^−l anti-CD3 mAb plus 1 μg ml⁻¹ anti-CD28 mAb. (A) Foxp3⁺CD25⁺ Treg cells were actively induced by Mϕ_SNU899 from CD25-depleted cells (a and b). Flow cytometry of each subset of induced Treg cells (c). (B) The frequency of the induced Treg cells from CD4⁺CD25⁻ T cells was compared with that of the pre-existing naturally occurring Treg cells in autologous CD4⁺ T cells. Representative plots of the frequency of Fr. I and Fr. II Treg cells among CD4⁺ T cells and in the co-culture of Mϕ_SNU899 with CD4⁺CD25⁻ T cells. (C) Statistical analysis showed that the percentage of Fr. II aTreg cells was markedly increased in the co-culture of Mϕ_SNU899 with CD4⁺CD25⁻ T cells (mean±s.d., n=3; **P<0.01 compared with CD4⁺ T cells). (D) Representative plots of the frequency of Fr. II aTreg cells assessed after 4 days of co-culture with or without the indicated neutralising mAbs. (E) A histogram revealed that the increase of Fr. II aTreg cells was partially blocked by an anti-IL-10 antibody (mean±s.d., n=3; *P<0.05). (A–E) I, CD45RA⁺Foxp3^low resting Treg (rTreg) cells; II, CD45RA⁻Foxp3^high activated Treg (aTreg) cells; III, CD45RA⁻Foxp3^lowCD4⁺ T cells. Both of Fr. I and Fr. II were immunosuppressive, and Fr. III was non-suppressive. TSNs, tumour culture supernatants.