Affinity purification mass spectrometry analysis of PD-1 uncovers new checkpoint modulators

Introduction: Antibodies targeting PD-1 have elicited clinical responses in cancer. Nevertheless, responses are limited and a better understanding of the signaling pathways downstream PD-1 should provide new therapeutic targets.

Materials and methods: We used affinity purification mass spectrometry to uncover novel proteins associated with PD-1. PD-1 ligation is known to induce the binding of the phosphatase SHP2 to the cytoplasmic tail of PD-1, which dephosphorylates proximal signaling downstream of the T cell receptor, hence preventing the activation of T cells.

Results and discussion: We identified SHP2 as a PD-1 interacting protein, and in addition we identified 17 novel PD-1-interacting proteins. Two of the more abundant proteins, EFHD2 and SAP, were functionally analyzed for their contribution to PD-1 inhibitory responses.

Silencing EFHD2 abrogated PD-1 inhibitory effects, including elimination of the ability of PD-1 to inhibit cytokine secretion. Mechanistically, EFHD2 co-localized with PD-1 in the immunological synapse where it contributed to PD-1 clustering. In contrast, SAP silencing augmented PD-1 functions while over-expression of SAP blocked PD-1 effects. SAP inhibits the activity of SHP2, thus eliminating the inhibitory signaling of PD-1.

Conclusion: We identified proteins that modulate PD-1 function and might serve as targets for cancer therapy. Additional proteomics-based analysis will be utilized in Sheba Medical Center to discover novel immunotherapy targets.