The structural features that distinguish PD-L2 from PD-L1 emerged in placental mammals

Abstract

Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells. PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110_PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance to monitor real-time binding of recombinantly-expressed and -purified proteins, we found that W110_PD-L2 acts as an "elbow" that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Furthermore, we identified a "latch" between the C and D β-strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110_PD-L2 "elbow" and a C-D region "latch." Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2-affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.

Keywords: PD-L2; T-cell biology; glycoprotein structure; immune checkpoint; immune receptors/ligands; immunotherapy; programmed cell death protein 1 (PD-1); programmed death ligand 1 (PD-L1); protein evolution; protein structure.

Figure 1.

W110 of PD-L2 acts as an elbow to hinder PD-1 binding. A and B, ribbon diagrams of the murine PD-1 IgV domain (gray) in complex with the IgV and IgC domains of human PD-L1 (blue) (PDB code 3BIK) (A) and hPD-L2 (green) (hPD-L2 sequence threaded onto PDB code 3BP5) (B). C and D, upper panels show surface electrostatic representation of mPD-1 with the G-strands of PD-L1 (blue) (C) or PD-L2 (green) (D) in ribbon and stick representation. Lower panels show front faces of IgV domains of PD-L1 (blue) (C) and PD-L2 (green) (D) with β-strand lettering and A121_PD-L1 and W110_PD-L2 highlighted in red. E–G, SPR sensorgrams of the indicated PD-ligand analytes injected over immobilized PD-1. H, representative, normalized binding curves. I, affinity measurements from independent experiments. J, dissociation rates from independent experiments. Dissociation rates exceeding the range of accurate measurement are shown as >0.4 s⁻¹. Unpaired t tests: *, p < 0.05; **, p < 0.01; ****, p < 0.0001; RU, response units.

Figure 2.

Atypical C–D region of PD-L2 forms a latch that enhances PD-1 binding. A, ribbon representation of the IgV domain of PD-L2 with the C–D latch residues represented as sticks. B and C, SPR sensorgrams of the indicated PD-ligand analytes injected over immobilized PD-1. D, representative, normalized binding curves. E, affinity measurements from independent experiments. F, dissociation rates from independent experiments. Dissociation rates exceeding the range of accurate measurement are shown as >0.4 s⁻¹. Unpaired t tests: **, p < 0.01; ****, p < 0.0001; ns, not significant; RU, response units.

Figure 3.

Trp-110 elbow and C–D latch of PD-L2 evolved contemporaneously with placental mammal radiation. Phylogenetic analysis of the PD-L2 IgV domain. PD-L2 emerged from a gene duplication from a primordial PD-L1 between lobe-finned fish and amphibian divergence (blue hexagon). The Trp-110 elbow (red) and C–D latch (green) of PD-L2 emerged exclusively in placental mammals (red hexagon).

Figure 4.

Effect of PD-ligand affinity for PD-1 on T-cell inhibition. A–G, proliferation of primary human CD4⁺ T-cell blasts in response to anti-CD3 and anti CD-28 antibodies was measured by CFSE dilution without (A) or with the indicated ligand absorbed at the indicated concentration. H, cumulative data from multiple independent experiments with the indicated absorbed concentration. I, correlation between T-cell inhibition and affinity for PD-ligand variants. A–I, data are from a single healthy donor. J, IL-2 release from primary human T cells in response to SEE bound to Raji B cells with or without ectopic and equal expression of PD-ligands (24 h co-culture). Data are from three healthy donors assessed independently. K, effect of 20 μg/ml Nivolumab or the indicated PD-ligand Fc fusion protein on IL-2 secretion from human PBMCs in response to increasing concentrations of SEB superantigen. Data shown represent independent experiments from four healthy donors. Bar graphs: paired t tests: *, p < 0.05; **, p < 0.01. Dose-response curves: two-way analysis of variance; ****, p < 0.0001; ns, not significant.