System Dynamics of PD-1 Signaling in T Cells

PROJECT SUMMARY/ABSTRACT Adaptive immune responses are governed by T cell receptor (TCR) signaling, which determines the fates and activities of T cells (helper, effector, etc.). The TCR and its signaling partners integrate antigen-recognition signals and second signals, which carry information about the context in which antigen presentation is occurring. Second signals can be either stimulatory or inhibitory: stimulatory signals are essential for T cell activation, whereas inhibitory signals (also called checkpoints) are responsible for T cell exhaustion and antigen tolerance. Stimulatory second signals are generated by the innate arm of the immune system when, for example, signaling by Toll-like receptors induces expression of the B7-family ligands B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells. Expression of B7-1/B7-2 indicates that antigen presentation is occurring within the context of an ongoing innate immune response. B7-1 and B7-2 are recognized by CD28, a TCR coreceptor that potently enhances TCR-generated T-cell activation signals. Inhibitory second signals arise during the course of chronic stimulation of TCR signaling. They are important for limiting the collateral damage caused by an immune response and avoidance of autoimmunity, but they can also be deleterious. For example, tumor cells commonly express the B7-family ligands B7-H1 (PD-L1/CD274) and B7-DC (PD- L2/CD273), which are recognized by PD-1, a TCR coreceptor that inhibits TCR-generated T-cell activation signals. B7-H1/B7-DC expression conveys immune privilege to tumor cells. For these and other reasons, it is imperative that we improve our basic understanding of checkpoint signaling. Here, we propose to characterize the dynamics of PD-1-regulated tyrosine phosphorylation in Jurkat E6-1, HuT 78, and TALL-104 cells, CRISPR-engineered cells derived from these parental cell lines, and primary human CD8+ cells. We will apply quantitative mass spectrometry (MS) to obtain an unbiased, nearly comprehensive picture of phosphotyrosine (pTyr) site abundances with and without PD-1/CD28 coreceptor signaling in populations of T cells over time and across conditions. Concurrently, using fluorescence microscopy and engineered SH2 domain affinity reagents, we will characterize single-molecule patterns of multisite phosphorylation for TCR, CD28, and PD-1. We will also measure membrane-recruitment lifetimes for individual cytosolic signaling partners of these receptors. The resulting data will be used to drive the formulation and parameterization of a detailed mechanistic model for TCR signaling accounting for the effects of CD28 and PD-1 coactivation. Although PD-1 is viewed as a platform for recruitment of phosphatases that counteract activation signals from kinases, we will evaluate specific hypotheses about how PD-1 could potentially generate positive signals for T-cell activation. These hypotheses are motivated by the fact that the best characterized signaling partners of PD-1 are protein tyrosine phosphatases, SHP1 and SHP2, which are known to promote cell activation in other contexts by, for example, mediating the dephosphorylation of inhibitory pTyr sites. Model predictions will be tested.