Figure 3.

Belatacept does not induce indoleamine 2,3‐dioxygenase (IDO) activity in vitro in human monocyte‐derived dendritic cells (DCs). (a) Immature DCs (iDCs) were either left unstimulated or were stimulated with lipopolysaccharide (LPS) (50 ng/ml) for 24 h and treated subsequently with or without graded doses of belatacept (20 µg/ml, 100 µg/ml, 200 µg/ml). Treatment of unstimulated DCs with increasing doses of belatacept did not induce IDO protein expression. Activation of iDCs with LPS (50 ng/ml) resulted in IDO protein expression which was, however, not increased in the presence of belatacept. (b) DCs were recovered and recultured in fresh medium for another 48 h. Supernatants were analysed for kynurenine accumulation after stimulation and after reculture. DCs without LPS prestimulation did not release kynurenine independent of further belatacept treatment. LPS prestimulation induced kynurenine release, which was not increased upon addition of increasing doses of belatacept. (c) Differently stimulated DCs were harvested and co‐cultured in the presence of allogeneic T cells (DC : T cell ratio 1 : 10) for 7 days, each condition in the presence or absence of belatacept (100 µg/ml). Neither LPS‐stimulated nor LPS/belatacept‐stimulated DCs dampened proliferation of CD3⁺ T cells (mean proliferation 75 and 66%, respectively, P = not significant). In contrast, the co‐culture of both DC populations and CD3⁺ T cells in the presence of belatacept resulted in a significantly lower proliferative response (mean proliferation 25 and 26%, respectively, *P < 0·001). (d) Kynurenine release was equally low during the first 48 h of co‐culture, independent of DC‐type or presence or absence of belatacept.