The indirect target of belatacept, CD28, can be down regulated by T-cells upon allogeneic stimulation, resulting in IFNγ-producing CD28^NULL T-cells.

<p>FACS-sorted CD28^POS T-cells (A) and FACS-sorted CD28^NULL T-cells (B) from n = 16 kidney-transplant candidates were stimulated for 7 days with allo-antigen (purities ≥95%). CD28-expression was assessed after 7 days of MLR and compared to the CD28-expression of the pure starting populations. Gating strategies to determine proliferated CD4^POS and CD8^POS T-cells are depicted, as well as a typical example for proliferation with and without belatacept (CD). The gating strategy for CD28 expression within proliferated CD4^POS and CD8^POS T-cells, including allogeneic IFNγ production, is depicted in a typical example (C). IFNγ expression is depicted in this typical example in the presence of 100 ng/mL belatacept. The proportion of CD4^POS and CD8^POS T-cells which lost their CD28-molecules are shown for the study population (D). The percentage of IFNγ producing cells within T-cells that remained CD28^POS and within T-cells that differentiated into CD28^NULL are shown, in the presence and absence of belatacept (E). N.B.: Black lines represent the medians. The upper and lower border of the boxes represent the 10^th and 90^th percentile. The error lines represent the minimal and maximal value within 1.5 quartile distances of the box. Values above 1.5 quartile distances of the box are considered outliers and are represented by a dot. Levels of significance in (B) were given for the difference between proliferation in the presence of belatacept and the IgG control. * = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001, **** = <i>p</i><0.0001, T_N = naïve T-cells, T_CM = central-memory T-cells, T_EM = effector-memory T-cells, T_EMRA = terminally differentiated EMRA T-cells.