CTLA-4 is a co-receptor on T-cells that controls peripheral tolerance and the development of autoimmunity. Immune check-point blockade (ICB) uses monoclonal antibodies (MAbs) to block the binding of inhibitory receptors (IRs) to their natural ligands. A humanized antibody to CTLA-4 was first approved clinically followed by the use of antibody blockade against PD-1 and its ligand PD-L1. Effective anti-tumor immunity requires the activation of tumor-specific effector T-cells, the blockade of regulatory cells and the migration of T-cells into the tumor. Here, we review data implicating CTLA-4 and PD-1 in the motility of T-cells with a specific reference to the potential exploitation of these pathways for more effective tumor infiltration and eradication.

The impact of PD-1 immune checkpoint therapy prompts exploration of other strategies to downregulate PD-1 for cancer therapy. We previously showed that the serine/threonine kinase, glycogen synthase kinase, GSK-3α/β, is a central regulator of PD-1 transcription in CD8+ T cells. Here, we show that the use of small-molecule inhibitors of GSK-3α/β (GSK-3i) to reduce pcdc1 (PD-1) transcription and expression was as effective as anti–PD-1 and PD-L1–blocking antibodies in the control of B16 melanoma, or EL4 lymphoma, in primary tumor and metastatic settings. Furthermore, the conditional genetic deletion of GSK-3α/β reduced PD-1 expression on CD8+ T cells and limited B16 pulmonary metastasis to the same degree as PD-1 gene deficiency. In each model, GSK-3i inhibited PD-1 expression on tumor-infiltrating lymphocytes, while increasing Tbx21 (T-bet) transcription, and the expression of CD107a+ (LAMP1) and granzyme B (GZMB) on CD8+ T cells. Finally, the adoptive transfer of T cells treated ex vivo with a GSK-3 inhibitor delayed the onset of EL4 lymphoma growth to a similar extent as anti–PD-1 pretreatment. Overall, our findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8+ T-cell function in cancer therapy to a similar degree as PD-1–blocking antibodies. Significance: These findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8+ T-cell function in cancer therapy to a similar degree as PD-1 blocking antibodies, offering a next-generation approach in the design of immunotherapeutic approaches for cancer management. Cancer Res; 78(3); 706–17. ©2017 AACR.

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/β 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.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti–PD-1 immunotherapy. We showed that phospho-eIF4E–deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E–mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8⁺ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1⁺PD-1⁺CD8⁺ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti–PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to 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.

The past 25 years have seen enormous progress in uncovering the receptors and signaling mechanisms on T-cells that activate their various effecter functions. Until the late 1980s, most studies on T-cells had focused on the influx of calcium and the levels of cAMP/GMP in T-cells. My laboratory then uncovered the interaction of CD4 and CD8 co-receptors with the protein-tyrosine kinase p56^lckwhich are now widely accepted as the initiators of the tyrosine phosphorylation cascade leading to T-cell activation. The finding explained how immune recognition receptors expressed by many immune cells, which lack intrinsic catalytic activity, can transduce activation signals via non-covalent association with non-receptor tyrosine kinases. The discovery also established the concept that a protein tyrosine phosphorylation cascade operated in T-cells. In this vein, we and others then showed that the CD4- and CD8-p56^lckcomplexes phosphorylate the TCR complexes which led to the identification of other protein-tyrosine kinases such as ZAP-70 and an array of substrates that are now central to studies in T-cell immunity. Other receptors such as B-cell receptor, Fc receptors and others were also subsequently found to usesrckinases to control cell growth. In T-cells, p56^lckdriven phosphorylation targets include co-receptors such as CD28 and CTLA-4 and immune cell-specific adaptor proteins such as LAT and SLP-76 which act to integrate signals proximal to surface receptors. CD4/CD8-p56^lckregulated events in T-cells include intracellular calcium mobilization, integrin activation and the induction of transcription factors for gene expression. Lastly, the identification of the targets of p56^lckin the TCR and CD28 provided the framework for the development of chimeric antigen receptor (CAR) therapy in the treatment of cancer. In this review, I outline a history of the development of events that led to the development of the “TCR signaling paradigm” and its implications to immunology and immunotherapy.