OX40 ligand shuts down IL-10-producing regulatory T cells

Abstract

IL-10-producing CD4(+) type 1 regulatory T (Tr1) cells play a critical role in the maintenance of peripheral tolerance. Although immunosuppressive drugs, cytokines, costimulatory molecules, and immature dendritic cells are implicated in the induction of Tr1 cells, the signals that negatively regulate the generation and function of Tr1 cells have been elusive. We report that OX40 ligand (OX40L) completely inhibited the generation of IL-10-producing Tr1 cells from naïve and memory CD4(+) T cells induced by the immunosuppressive drugs dexamethasone and vitamin D3. This unique function of OX40L was not shared by two costimulatory TNF family members, GITR ligand and 4-1BB ligand. OX40L strongly inhibited the generation of IL-10-producing Tr1 cells induced by two physiologic stimuli, the inducible costimulatory ligand and immature dendritic cells. In addition, OX40L strongly inhibited IL-10 production and suppressive function of differentiated IL-10-producing Tr1 cells. These two novel functions of OX40L shed light on the mechanism by which OX40/OX40L regulates immunity and tolerance.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

OX40L inhibits the generation and function of IL-10-producing Tr1 cells from naïve CD4+ T cells induced by different polarizing signals. CD4+ naïve T cells were cultured with anti-CD3 and anti-CD28 mAbs in the presence of IL-2 on parental L cells or OX40L-L cells with the indicated recombinant cytokines or reagents for 7 days. (A) Cytokine production by CD4+ T cells was analyzed intracellularly by flow cytometry. Percentages of the respective cytokine-producing T cells are indicated. (B) Cytokine production was measured in supernatants after restimulation with anti-CD3 and anti-CD28 mAbs for 24 h by ELISA. Data are means ± SEM of four independent experiments. (C) Mixtures of indicated T cell populations were restimulated for 5 days by anti-CD3 and anti-CD28 mAbs. Suppressive function was assessed by [3H]thymidine incorporation. Error bars represent the SEM of triplicate wells. Data are representative of three independent experiments.

Fig. 2.

OX40L inhibits the generation of IL-10-producing Tr1 cells from memory CD4+ T cells under a condition with Dex + Vit D3. CD4+CD45RO+ memory T cells were cultured with anti-CD3 mAb, anti-CD28 mAb, and IL-2 on parental L cells or OX40L-L cells in the presence or absence of Dex + Vit D3 for 7 days. (A) Cytokine production by CD4+ T cells was analyzed intracellularly by flow cytometry. Percentages of the respective cytokine-producing T cells are indicated. (B) IL-10 production was measured in supernatants after restimulation with anti-CD3 and anti-CD28 mAbs for 24 h by ELISA. Data are means ± SEM of four independent experiments.

Fig. 3.

OX40L but not GITRL or 4-1BBL inhibits the generation of IL-10-producing Tr1 cells. CD4+ naïve T cells were cultured with anti-CD3 mAb, anti-CD28 mAb, and IL-2 on parental L cells, OX40L-L cells, GITRL-L cells, or 4-1BBL-L cells in the presence of Dex + Vit D3 for 7 days. (A) IL-10 and TNF-α production by CD4+ T cells was analyzed intracellularly by flow cytometry. Percentages of the respective cytokine-producing T cells are indicated. (B) IL-10 production was measured in supernatants after restimulation with anti-CD3 and anti-CD28 mAbs for 24 h by ELISA. (C) The number of viable T cells was counted. In B and C, data are means ± SEM of four independent experiments.

Fig. 4.

OX40L inhibits the generation of IL-10-producing Tr1 cells from CD4+ T cells induced by ICOSL or immature DCs. (A–D) CD4+ naïve and memory T cells were cultured for 7 days on parental L cells, a mixture of ICOSL-L cells and L cells, or a mixture of ICOSL-L cells and OX40L-L cells, which were precoated with anti-CD3 mAb. (E and F) CD4+ naïve T cells were cocultured with immature DCs or DCs precultured with IFN-α, IL-10, or CD40L in round-bottom 96-well culture plates in the presence or absence of the soluble recombinant OX40L for 7 days. (A, C, and E) IL-10 and IFN-γ production by CD4+ T cells was analyzed intracellularly by flow cytometry. Percentages of the respective cytokine-producing T cells are indicated. (B, D, and F) IL-10 production was measured in supernatants after restimulation with anti-CD3 and anti-CD28 mAbs for 24 h by ELISA. Statistical significance was determined by using a paired Student t test. Data are means ± SEM of three independent experiments. sOX40L, soluble recombinant OX40L. ∗, P < 0.05.

Fig. 5.

OX40L inhibits IL-10 production and function of already differentiated Tr1 cells. CD4+CD45RO+ memory T cells were first cultured for 7 days with anti-CD3 mAb, anti-CD28 mAb, and IL-2 on parental L cells in the presence of Dex + Vit D3. Cells were washed and then restimulated for 24 h with anti-CD3 mAb and anti-CD28 mAb in the presence of parental L cells or OX40L-L cells. (A) Cytokine production by CD4+ T cells was analyzed intracellularly by flow cytometry. Percentages of the respective cytokine-producing T cells are indicated. (B) Cytokine production was measured in supernatants after restimulation with anti-CD3 and anti-CD28 mAbs for 24 h by ELISA. Data are means ± SEM of three independent experiments. Statistical significance was determined by using a paired Student t test. ∗, P < 0.05. (C) Indicated T cell population alone or mixtures of indicated T cell populations were restimulated for 5 days by anti-CD3 and anti-CD28 mAbs. Suppressive function was assessed by [3H]thymidine incorporation. Error bars represent the SEM of triplicate wells. Data are representative of three independent experiments.