Figure 2.

CD28 activation is comparable to glycogen synthase kinase 3 (GSK-3) inactivation enhancing cytolytic function. (A) OT-1 CD8⁺ cytolytic T-cells (CTLs) were transfected with scrambled (left panel) or GSK-3 (right panel) small-interfering RNA (siRNA) prior to activation with OVA peptide and incubated with or without anti-CD28 or blocking CD28 (CTLA-4 IgG fusion protein). After 5 days CTLs were washed and counted before incubation with target (OVA-EL4) cells at the ratios shown for 4 h. Lactate dehydrogenase release was measured as an indication of target cell killing. Histogram depicts measurements normalized for background non-specific killing. OVA alone: light gray bars; anti-CD28: light blue bars; CTLA-4 IgG: dark blue bars (error bars based on triplicate values in individual experiments, data shown representative of four independent experiments). (B) Histogram showing MFI values of programmed cell death 1 (PD-1) expression as measured by flow cytometry. (C) Flow cytometry profiles of GZMB and Lamp-1 in either scrambled or GSK-3 siRNA transfected cells stimulated with Ova alone, or combined with anti-CD28 or CTLA-4 IgG. Error bars based on triplicate values in individual experiments; data shown representative of three independent experiments.