RESUMO
Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and ß) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8(+) T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8(+) cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8(+) CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8(+) OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.
Assuntos
Aminofenóis/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Quinase 3 da Glicogênio Sintase/metabolismo , Infecções por Herpesviridae/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Maleimidas/administração & dosagem , Receptor de Morte Celular Programada 1/metabolismo , Rhadinovirus/fisiologia , Proteínas com Domínio T/metabolismo , Linfócitos T Citotóxicos/imunologia , Aminofenóis/efeitos adversos , Animais , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Maleimidas/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/genética , RNA Interferente Pequeno/genética , Proteínas com Domínio T/genética , Linfócitos T Citotóxicos/virologia , Carga Viral/efeitos dos fármacos , Carga Viral/genéticaRESUMO
Immune checkpoint blockade using antibodies against negative co-receptors such as cytolytic T cell antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) has seen much success treating cancer. However, most patients are still not cured, underscoring the need for improved treatments and the possible development of small molecule inhibitors (SMIs) for improved immunotherapy. We previously showed that glycogen synthase kinase (GSK)-3α/ß is a central regulator of PD-1 expression, where GSK-3 inhibition down-regulates PD-1 and enhances CD8+ cytolytic T cell (CTL) function, reducing viral infections and tumor growth. Here, we demonstrate that GSK-3 also negatively regulates Lymphocyte Activation Gene-3 (LAG-3) expression on CD4+ and CD8+ T cells. GSK-3 SMIs are more effective than LAG-3 blockade alone in suppressing B16 melanoma growth, while their combination resulted in enhanced tumor clearance. This was linked to increased expression of the transcription factor, Tbet, which bound the LAG-3 promoter, inhibiting its transcription, and to increased granzyme B and interferon-γ1 expression. Overall, we describe a small molecule approach to inhibit LAG-3, resulting in enhanced anti-tumor immunity.