Cutting Edge: OX40 agonists can drive regulatory T cell expansion if the cytokine milieu is right

Abstract

We report that OX40 stimulation drives all lineages of CD4 T cell development, including regulatory T cells (Tregs), and the plasticity of the response is dependant on local cytokines. In TGF-beta1-treated cultures, an OX40 agonist increased IFN-gamma and IL-4 production and diverted T cells from the Treg lineage. However, cytokine blockade in the context of OX40 stimulation promoted enhanced Treg accumulation. This observation was evident in naive mice, as OX40 engagement enhanced Treg proliferation and accumulation in vivo. Lastly, OX40 agonist administration influenced experimental autoimmune encephalomyelitis disease severity in opposing directions, depending on the timing of administration. Given during Ag priming, the OX40 agonist drove Treg expansion and inhibited disease, whereas given later it enhanced T cell effector cytokine production in the CNS and exacerbated disease. Hence, OX40 signaling can augment the accumulation of all CD4 T cell lineages; however, its accentuation of immune responses may have vastly different biologic outcomes depending upon the local cytokine milieu.

FIGURE 1

The cytokines IFNγ and IL-4 determine the effect of OX40 stimulation on activated T cells in the presence of TGFβ-1. Isolated CD25⁻FoxP3⁻ T cells were stimulated by αCD3 and αCD28 in the presence of IL-2. (A) Cultures were then treated with TGFβ-1 and/or agonist OX40 antibody (αOX40) and incubated for 72 hrs. (B) Blocking Abs specific for IL-4, IL-6, and IFNγ or (C) combinations of IL-4, IL-6, and IFNγ Abs were added to cultures. (D) Levels of IFNγ and IL-4 from cultures (72 hrs). (E) Dose

FIGURE 2

Agonist OX40 augments the accumulation and cycling of functionally suppressive CD4⁺FoxP3⁺ T cells in vivo. (A) Naïve wt mice were injected with rat IgG or αOX40 (100 or 500 µg) i.p. and splenic CD4⁺FoxP3⁺ T cells were enumerated six days later. (B) Splenocytes from mice injected with 500 µg αOX40 were analyzed for CD4⁺FoxP3⁺ T cells at various times. (C) TGFβ-1-generated Tregs (10⁶) (Thy1.2) were injected into Thy1.1 congenic hosts and treated with rat IgG or αOX40 (500 µg) one day later. Ki-67 expression was analyzed six days after treatment in Thy1.1⁺CD4⁺FoxP3⁺ and Thy1.1⁻CD4⁺FoxP3⁺ T cells. (D) FoxP3⁺ and FoxP3⁻ CD4 T cells were isolated by FACs sorting from FoxP3eGFP spleens by gating for CD4⁺GFP⁺ and CD4⁺GFP⁻ respectively. FoxP3⁺ T cells (10⁶) or FoxP3⁻ T cells (5 X 10⁶) were transferred to CD45.1 congenic mice and treated with rat IgG or αOX40 (500 µg). Spleens were analyzed for CD45.2⁺FoxP3⁺ T cell expansion (d6). (E) Isolated CD4⁺FoxP3⁺ T cells (50,000) from the spleens of mice six days after rat IgG or αOX40 treatment were mixed with naïve CFSE-labeled CD8 T cells and stimulated with αCD3. Two-three days later CFSE dilution was measured.

FIGURE 3

The timing of OX40-engagement in a model of EAE influences disease severity by alterations in Tregs. (A) EAE disease progression and severity is decreased following αOX40 administration (250 µg) compared to rat IgG at Ag-priming (days 0 and 4), but mediated an increase in severity when administered at disease onset (day of onset, day +2 and +4). (B) αOX40 treatment at disease onset increases IL-2, IL-6, IL-17, and IFNγ produced by isolated CNS cells. (C) αOX40 treatment at PLP/CFA-priming alters the frequency of FoxP3⁺ CD4 cells in the spleen and LNs of EAE mice after seven days. Values of p≤0.05 were considered significant and expressed as */# p≤0.05 and **/## p≤0.001.