Programmed death receptor 1 (PD‐1) ligand Fc fusion proteins reduce T‐cell proliferation in vitro independently of PD‐1

Abstract

AbstractProgrammed death receptor 1 (PD‐1) is an inhibitory receptor on T cells shown to restrain T‐cell proliferation. PD‐1 immune checkpoint blockade has emerged as a highly promising approach in cancer treatment. Much of our understanding of the function of PD‐1 is derived from in vitro T‐cell activation assays. Here we set out to further investigate how T cells integrate inhibitory signals such as PD‐1 in vitro using the PD‐1 agonist, PD‐1 ligand 1 (PD‐L1) fusion protein (PD‐L1.Fc), coimmobilized alongside anti‐CD3 agonist monoclonal antibody (mAb) on plates to deliver PD‐1 signals to wild‐type and PD‐1−/− CD8+ T cells. Surprisingly, we found that the PD‐L1.Fc fusion protein inhibited T‐cell proliferation independently of PD‐1. This PD‐L1.Fc inhibition was observed in the presence and absence of CD28 and interleukin‐2 signaling. Binding of PD‐L1.Fc was restricted to PD‐1–expressing T cells and thus inhibition was not mediated by the interaction of PD‐L1.Fc with CD80 or other yet unknown binding partners. Furthermore, a similar PD‐1–independent reduction of T‐cell proliferation was observed with plate‐bound PD‐L2.Fc. Hence, our results suggest that the coimmobilization of PD‐1 ligand fusion proteins with anti‐CD3 mAb leads to a reduction of T‐cell engagement with plate‐bound anti‐CD3 mAb. This study demonstrates a nonspecific mechanism of T‐cell inhibition when PD‐L1.Fc or PD‐L2.Fc fusion proteins are delivered in a plate‐bound coimmobilization assay and highlights the importance of careful optimization of assay systems and reagents when interpreting their influence on T‐cell proliferation.