Revisiting the PD-1 pathway

Abstract

Programmed Death-1 (PD-1; CD279) is an inhibitory receptor induced in activated T cells. PD-1 engagement by its ligands, PD-L1 and PD-L2, maintains peripheral tolerance but also compromises anti-tumor immunity. Blocking antibodies against PD-1 or its ligands have revolutionized cancer immunotherapy. However, only a fraction of patients develop durable antitumor responses. Clinical outcomes have reached a plateau without substantial advances by combinatorial approaches. Thus, great interest has recently emerged to investigate, in depth, the mechanisms by which the PD-1 pathway transmits inhibitory signals with the goal to identify molecular targets for improvement of the therapeutic success. These efforts have revealed unpredictable dimensions of the pathway and uncovered novel mechanisms involved in PD-1 and PD-L1 regulation and function. Here, we provide an overview of the recent advances on the mechanistic aspects of the PD-1 pathway and discuss the implications of these new discoveries and the gaps that remain to be filled.

Fig. 1. Posttranslational modifications regulate PD-1 expression.

(A) In the endoplasmic reticulum (ER), PD-1 is glycosylated at residues N49 and N74, which are subsequently fucosylated (red triangle) in the Golgi apparatus, resulting in sustained expression of PD-1 at the cell membrane and transmission of inhibitory signals. Genetic depletion or pharmacologic inhibition of Fut8 fucosyltransferase decreases PD-1 fucosylation, expression, and inhibitory signaling, resulting in increased T cell activation. (B) FBXO38 ubiquitin ligase mediates a K48-linked ubiquitination of PD-1 at K233, resulting in PD-1 internalization and proteasomal degradation. Genetic ablation or down-regulation of FBXO38 results in increased PD-1 expression, leading to enhanced inhibitory signaling and T cell suppression. (C) Expression of PD-L1 in tumor cells is stabilized by glycosylation. This is antagonized by GSK3β, which binds to the nonglycosylated form of PD-L1, leading to phosphorylation at T180 and S184, and β-TrCP–mediated PD-L1 ubiquitination and proteasomal degradation. EGFR-mediated signals inhibit GSK3β-mediated PD-L1 phosphorylation and degradation and promote PD-L1 stabilization and immunosuppressive function. Antibodies targeting glycosylated PD-L1 (anti–gPD-L1) block interaction with PD-1 and induce PD-L1 internalization and degradation. (D) In cancer cells, TNFR (TNF receptor)–mediated signaling results in IKKβ (inhibitor of nuclear factor κB kinase β)–mediated p65 activation and nuclear translocation, leading to transcription of CSN5, which stabilizes PD-L1 by direct deubiquitination or by inhibiting PD-L1 ubiquitination, resulting in enhanced immunosuppressive activity. CMTM4/6 associates with PD-L1 at the cell surface, reducing its ubiquitination and increasing the half-life of PD-L1 protein.

Fig. 2. Interaction modes of PD-1 and PD-L1.

(A) According to the canonical PD-1/PD-L1 interaction, PD-L1 expressed on APCs or tumor cells interacts with PD-1 expressed on T cells in trans to attenuate activation mediated by TCR/MHC (major histocompatibility complex) and CD28/B7-1 interactions. Blocking antibodies against PD-1 or PD-L1 alleviate T cell inhibition by preventing trans PD-1/PD-L1 interaction. (B) When PD-1 and PD-L1 are coexpressed on APCs or tumor cells, PD-1 binds to PD-L1 in cis, diminishing the ability of PD-L1 to bind PD-1 expressed on T cells in trans. (C) PD-L1/B7-1 interaction in cis on APCs or tumor cells disrupts PD-1/PD-L1 binding in trans, resulting in diminished PD-1–mediated T cell inhibition. Binding of PD-L1/B7-1 in cis does not disrupt the binding of B7-1 to CD28, and costimulatory effects of B7-1/CD28 interaction are preserved. In contrast, binding of PD-L1/B7-1 in cis disrupts the B7-1/CTLA-4 axis. (D) Adding a blocking anti–PD-L1 antibody to disrupt PD-1/PD-L1 interaction can also disrupt PD-L1/B7-1 interaction and allow released B7-1 to bind to CTLA-4 and deliver inhibitory signals. In this case, a blocking anti–CTLA-4 antibody might be beneficial by preventing CTLA-4–mediated T cell inhibition. (E) PD-L1/B7-1 interaction in cis on APCs prevents regulatory T cell (T_reg) CTLA-4–mediated trans-endocytosis of B7-1 that leads to B7-1 depletion from APC surface. (F) Anti–PD-L1 antibody can have a negative impact on immunotherapy by disrupting PD-L1/B7-1 interaction and allowing B7-1 binding to CTLA-4 on T_regs, CTLA-4–mediated trans-endocytosis of B7-1, and diminished B7-1–mediated costimulation. Such negative effect of anti–PD-L1 antibody might be alleviated by an anti–CTLA-4 antibody.

Fig. 3. PD-1/SHP-2 interaction modes.

(A) Two-step binding model, according to which SHP-2 C-SH2 binds to PD-1 pY-ITSM with strong affinity, recruiting PD-1 to SHP-2, while PD-1 pY-ITIM binds to N-SH2, displacing it from the PTP site to activate the phosphatase. (B) Dimerization model, according to which SHP-2 bridges two pY-ITSM residues on two PD-1 molecules via its N-SH2 and C-SH2 domains forming a PD-1:PD-1 dimer and inducing SHP-2 activation.

Fig. 4. Signaling through PD-L1 protects tumor cells from IFN-mediated cytotoxicity.

PD-L1 expressed on tumor cells engages PD-1 to deliver inhibitory signals to T cells. PD-L1 may also deliver inhibitory signals to tumor cells to attenuate IFN-mediated cytotoxicity through a STAT3/caspase-7–dependent pathway. The conserved RMLDVEKC motif of PD-L1 is required to counteract IFN toxicity, while the DTSSK motif prevents this function. Thus, PD-L1 provides tumor cells with a dual escape mechanism from T cell–dependent cytotoxicity. IFNAR1, interferon alpha/beta receptor alpha chain.

Fig. 5. PD-1 regulates the differentiation and lineage fate commitment of myeloid progenitors during cancer-mediated emergency myelopoiesis and determines the efficiency of T cell antitumor responses.

(A) During cancer-driven emergency myelopoiesis, PD-1 is up-regulated on CMPs but mostly in GMPs and inhibits signaling and metabolic reprogramming mediated by growth factors driving emergency myelopoiesis, resulting in accumulation of immature myeloid cells and immunosuppressor MDSCs, and decreased systemic output of effector myeloid cells. (B) PD-1 ablation in myeloid cells promotes signaling and metabolic reprogramming mediated by growth factors of emergency myelopoiesis and leads to the output of effector myeloid cells with improved antigen-presenting function that drive T effector memory cell responses and antitumor protection. HSC, hematopoietic stem cell; CMP, common myeloid progenitor; GMP, granulocyte/monocyte progenitor; MDP, monocyte/dendritic cell progenitor; CDP, common dendritic cell progenitor; DC, dendritic cell; CSF, cancer-produced soluble factor.