Oroya, A. | bioRxiv Science

bioRxiv2025

GSK-3 regulates CD4-CD8 cooperation needed to generate super-armed CD8+ cytolytic T cells against tumors

While immune checkpoint blockade (ICB) has revolutionized cancer treatment, the key T-cell signaling pathways responsible for its potency remain unclear. GSK-3 is an inhibitory kinase that is most active in resting T-cells. In this study, we demonstrate that GSK-3 facilitates PD-1 blockade, an effect seen by modulating CD4 T-cell help for CD8+ CTL responses against ICB resistant tumors. We show that GSK-3 controls metabolic reprogramming towards glycolysis and synergizes with PD-1 to induce a transcriptional program that reduces suppressive CD4+ Treg numbers while generating super-armed effector-memory CD8+ CTLs that express an unprecedented 7/9 granzymes from the genome. Crucially, we found that GSK-3 cooperates with PD-1 blockade to determine the dependency of CD8+ CTLs on help from CD4+ T-cells. Our study unravels a novel cooperative PD-1 blockade-dependent signaling pathway that potentiates CTL responses against tumors, offering a new strategy to overcome immunotherapy resistance by modulating CD4+ helper and CD8+ cytotoxic functions. SignificanceThis study demonstrates for the first time that GSK-3 controls the crosstalk between CD4+ and CD8+ T cells, synergizing with anti-PD-1 therapy to overcome resistance to checkpoint blockade and to generate super-armed CD8+ effector cells in cancer immunotherapy. This newly uncovered GSK-3-dependent CD4-CD8 T-cell crosstalk mechanism presents a new approach to enhance anti-PD-1 immunotherapy.

immunology↗

bioRxiv2024

PD-1 endocytosis unleashes the cytolytic potential of check-point blockade in tumor immunity

PD-1 immune checkpoint blockade (ICB) is now a promising first-line treatment for many cancers. While the steric blockade of PD-1 binding to its ligand plays a role, the role of internalisation in promoting the efficacy of ICB has not been explored. In this study, we show that PD-1 internalisation also contributes by unlocking the full cytolytic potential of ICB in cancer immunotherapy. We found that anti-mouse and human PD-1 downregulate a subset of PD-1 surface receptors on T-cells with high-density surface PD-1 leaving T-cells with intermediate expression resistant to further internalisation. Down regulation was seen on both CD4 and CD8 cells but was maximally effective on CD8 effector cells. In human T-cells, nivolumab outperformed pembrolizumab in terms of rate and efficacy. We also found that PD-1 internalisation depended on bivalent antibody (Ab)-induced crosslinking, while monovalent Ab sterically blocked PD-1 without inducing endocytosis. Immunologically, while both monovalent and bivalent Ab limited B16-PD-L1 tumor growth, bivalent Ab was significantly more effective. In molecular terms, while both antibodies increased granzyme B (GZMB) expression in CD8+ cytolytic T-cells, the induction of the second key cytolytic pore-forming mediator, perforin, was dependent on the blockade and internalisation mediated by bilavent anti-PD-1. Our findings unveil a novel mechanism in checkpoint blockade where steric blockade combined with the removal of PD-1 from the cell surface by endocytosis can complement and optimize therapy. The targeting of PD-1 internalisation holds promise for enhancing anti-tumor immunity and improving PD-1 checkpoint blockade therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/591549v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1cbb458org.highwire.dtl.DTLVardef@ad15eborg.highwire.dtl.DTLVardef@17a2bf6org.highwire.dtl.DTLVardef@1dc5ba0_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefBen Saad et al define the mechanism of PD-1 inhibitory endocytosis and show that the removal of surface PD-1 by endocytosis plays a role in complementing and optimizing checkpoint blockade. Targeting PD-1 internalisation holds promise for enhancing anti-tumor immunity and improving the efficacy of PD-1 checkpoint blockade therapy.

immunology↗