Project Summary T cell responses are continually regulated in quality and magnitude in order to meet the changing requirements during immune challenge with commensals and infectious organisms. On specific form of adaptation is called T cell exhaustion and occurs when the source of a non-self antigen cannot be eliminated, such as during chronic viral infections or in cancer. An exhausted T cell response exerts residual control over pathogen replication or malignant growth, while at the same time minimizing the immunopathological consequences of ongoing immune activation. At the level of the individual T cell, exhaustion is characterized by gene expression programs that facilitate attenuation of T cell receptor signal strength at multiple levels, including the expression of co-inhibitory receptors, such as PD-1. In settings where reinvigoration of exhausted T cell responses is desirable, this can in many cases be achieved by interrupting the PD-1 inhibitory pathway through use of blocking antibodies against PD-1 or its ligand PD- L1. However, it is not known how reinvigoration of the response occurs at the single cell level and what cellular interactions and signals govern this process as compared to the priming of nave or the re-activation of memory T cells. Can PD-1 blockade instantaneously release the effector activities of exhausted T cells, or does it initiate a gradual re-differentiation into fully competent effector cells? Are all or only subpopulations of exhausted cells capable of recovery? Does PD-1 blockade merely prevent exhaustion in cells that are de novo recruited to the response? What are the requirements for co-stimulation and interaction with specialized dendritic cells? To address such questions we have developed a mouse tumor model that allows us to track the response of individual exhausted T cells to PD-1 pathway blockade at high temporal and spatial resolution using multiphoton intravital microscopy in tumor tissue. The use of functional fluorescent reporter systems allow us to monitor TCR-dependent signaling activity in T cells in addition to their effects on local antigen-presenting cells as well as tumor cells, in order to map the kinetics and heterogeneity of recovery of T cell function upon binding of blocking anti-PD-1 antibodies. Using this imaging approach in combination with genetic models, we will also examine the role of local co-stimulation and tumor-associated dendritic cells in restoring the function of exhausted tumor-infiltrating T cells.