PD-1 and PD-L1 on the same cell bind in cis with high affinity

The PD-1 pathway, consisting of the co-inhibitory receptor PD-1 on T cells and its ligand (PD-L1) on antigen-presenting cells (APCs), is a major mechanism of tumor immune evasion. PD-1 and PD-L1 blockade antibodies have produced remarkable clinical activities against a subset of cancers. Binding between T cell-intrinsic PD-1 and APC-intrinsic PD-L1 triggers inhibitory signaling to attenuate the T cell response. Here, we report that PD-1 is co-expressed with PD-L1 on tumor cells and tumor-infiltrating APCs. Using reconstitution and cell culture assays, we demonstrate that the co-expressed PD-1 binds to PD-L1 in cis. Such interaction inhibits the ability of PD-L1 to bind T cell-intrinsic PD-1 in trans and, in turn, represses canonical PD-L1/PD-1 inhibitory signaling. Selective blockade of tumor-intrinsic PD-1 frees up tumor-intrinsic PD-L1 to inhibit T cell signaling and cytotoxicity. Our study uncovers another dimension of PD-1 regulation, with important therapeutic implications.

Introduction

Hui et al., 2017 Hui E.

Cheung J.

Zhu J.

Su X.

Taylor M.J.

Wallweber H.A.

Sasmal D.K.

Huang J.

Kim J.M.

Mellman I.

Vale R.D. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Parry et al., 2005 Parry R.V.

Chemnitz J.M.

Frauwirth K.A.

Lanfranco A.R.

Braunstein I.

Kobayashi S.V.

Linsley P.S.

Thompson C.B.

Riley J.L. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Sheppard et al., 2004 Sheppard K.A.

Fitz L.J.

Lee J.M.

Benander C.

George J.A.

Wooters J.

Qiu Y.

Jussif J.M.

Carter L.L.

Wood C.R.

Chaudhary D. PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta. Yokosuka et al., 2012 Yokosuka T.

Takamatsu M.

Kobayashi-Imanishi W.

Hashimoto-Tane A.

Azuma M.

Saito T. Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. Keir et al., 2008 Keir M.E.

Butte M.J.

Freeman G.J.

Sharpe A.H. PD-1 and its ligands in tolerance and immunity. To date, mechanistic studies of PD-1 have been largely focused on its role on T cells. Absent on naive T cells, PD-1 is inducibly expressed on T cells by T cell antigen receptor (TCR) signal and then acts as a molecular brake to prevent uncontrolled T cell activity. Upon binding to its ligand PD-L1 on the antigen-presenting cell (APC), a pair of tyrosines within the cytoplasmic tail of PD-1 becomes phosphorylated and recruits the protein tyrosine phosphatases SHP2 and SHP1, which dephosphorylate both the TCR and co-stimulatory signaling components (). These biochemical events ultimately lead to the attenuation of T cell proliferation, cytokine production, and cytolytic activities ().

In stark contrast to the intensively studied PD-L1/PD-1 trans interaction, the existence and functional consequence of the cis interaction are unknown. Challenges for this effort include the co-expression of PD-L1 and PD-1 on both APCs and T cells, signaling in both directions, and the potential crosstalk with other signaling axes.

In this work, we investigated whether PD-1 and PD-L1 interact in cis and how the potential cis interaction regulates classical PD-1 signaling outputs using well-defined in vitro reconstitution, cellular reconstitution, and cell culture assays. In both HEK293T cells and liposomes reconstituted with both PD-1 and PD-L1, we determined their molecular proximity using Förster resonance energy transfer (FRET). We next asked whether the presence of cis-PD-1 impacts the ability of PD-L1 to engage PD-1 in trans, using a liposome–bilayer conjugation assay, a cell-bilayer assay, and APC-T cells assays with multiple signaling readouts. Finally, we determined how antibody blockade of cis-PD-1 affects T cell signaling and cytotoxicity.