Glycogen Synthase Kinase 3 Inactivation Compensates for the Lack of CD28 in the Priming of CD8(+) Cytotoxic T-Cells: Implications for anti-PD-1 Immunotherapy

The rescue of exhausted CD8(+) cytolytic T-cells (CTLs) by anti-Programmed Cell Death-1 (anti-PD-1) blockade has been found to require CD28 expression. At the same time, we have shown that the inactivation of the serine/threonine kinase glycogen synthase kinase (GSK)-3α/β with small-interfering RNAs (siRNAs) and small molecule inhibitors (SMIs) specifically down-regulates PD-1 expression for enhanced CD8(+) CTL function and clearance of tumors and viral infections. Despite this, it has been unclear whether the GSK-3α/β pathway accounts for CD28 costimulation of CD8(+) CTL function. In this article, we show that inactivation of GSK-3α/β through siRNA or by SMIs during priming can substitute CD28 co-stimulation in the potentiation of cytotoxic CD8(+) CTL function against the EL-4 lymphoma cells expressing OVA peptide. The effect was seen using several structurally distinct GSK-3 SMIs and was accompanied by an increase in Lamp-1 and GZMB expression. Conversely, CD28 crosslinking obviated the need for GSK-3α/β inhibition in its enhancement of CTL function. Our findings support a model where GSK-3 is the central cosignal for CD28 priming of CD8(+) CTLs in anti-PD-1 immunotherapy.