In Vitro Assays to Study PD‐1 Biology in Human T Cells

Our understanding of programmed cell death 1 (PD‐1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD‐1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD‐1 ligation on short‐term T cell signaling, long‐term T cell function, and the structural consequences of PD‐1 ligation with PD‐1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short‐term assay to examine the signaling cascade triggered by PD‐1. We describe a phospho flow cytometry method to determine how PD‐1 ligation alters the level of CD3ζ phosphorylation on Tyr142, which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate‐bound assay that is useful to examine the long‐term consequences of PD‐1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen‐based assay to evaluate T cell responses to therapeutic agents targeting the PD‐1/PD‐L axis, as well as immune synapse formation in the presence of PD‐1 engagement. Finally, in Basic Protocol 4 we outline a tetramer‐based method useful to interrogate the quality of PD‐1/PD‐L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD‐1 signaling in T cells and the functional consequences of various PD‐1‐based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD‐1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate‐based ligand binding assay to study PD‐1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD‐1 ligation Basic Protocol 3: In vitro APC/T cell co‐culture system to evaluate therapeutic interventions targeting the PD‐1/PD‐L1 axis Support Protocol 2: Microscopy‐based approach to evaluate the consequences of PD‐1 ligation on immune synapse formation Basic Protocol 4: Tetramer‐based approach to study PD‐1/PD‐L1 interactions