ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement

We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to ga in a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amou nts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks I COS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4's actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costiniulation by interferring with the signals needed to induc e ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induc tion of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting syn ergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL- 2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Final ly, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement fo r exogenous IL-2.