T cell receptor signaling and phenotypic landscape of exhausted virus-specific CD8 T cells in chronic LCMV infection

Abstract

A hallmark of chronic infections is the presence of exhausted CD8 T cells characterized by a perturbed transcriptional program, sustained co-expression of multiple inhibitory receptors, progressive loss of effector function, and alteration of normal memory development, driven by repeated T cell receptor (TCR) engagement. Significant effort has been put into characterizing exhausted cells and understanding their differentiation. Most of the studies focused on a murine model system of virus-specific CD8 T cells isolated from the spleen of animals chronically infected with Lymphocytic Choriomeningitis virus (LCMV), a prototypical chronic infection with active viral replication. This thesis focuses on characterizing in vivo TCR signaling and the heterogeneity of exhausted CD8 T cells isolated from different tissues in established chronic LCMV infection. First, we validated the use of the TCR signaling reporter Nr4a1-GFP expressing GFP under the control of the Nr4a1 promoter as a proxy for TCR signaling in vivo. We evaluated basal TCR signaling in CD8 T cells in steady state and after heterologous viral challenge. In homeostatic conditions, basal Nr4a1-GFP levels decreased in MHC class I-deficient hosts. Additionally, the higher expression of Nr4a1-GFP in infected hosts with a heterologous virus was MHC class I-dependent. These results suggest that the basal Nr4a1-GFP signal is faithfully reporting tonic signaling triggered by interactions between MHC class I loaded with self-antigens and the TCR. Next, we evaluated TCR signaling in virus-specific CD8 T cells as reported by Nr4a1-GFP during chronic infection. We found that the in vivo signaling in exhausted CD8 T cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both α-PD-L1 blockade and adoptive transfer of naïve target cells increased TCR signaling in vivo, demonstrating that engagement of co-inhibitory receptors such as PD-1 profoundly curtails TCR signaling in vivo. Last, we assessed the heterogeneity of exhausted LCMV-specific cells in various tissues by single-cell RNAseq. Our data revealed that exhausted CD8 T cells are heterogeneous and adopt organ-specific signatures, suggesting that the tissue microenvironment has a major impact in shaping the phenotype and function of virus-specific CD8 T cells during chronic viral infection. Furthermore, exhausted cells were grouped into five main functional subpopulations based on hallmark markers: proliferating, memory-like TCF1hi, effector-like CXCR6loCX3CR1hi, intermediate CXCR6+CX3CR1int, and in an advanced exhaustion state CXCR6hiCX3CR1-. Additionally, adoptive transfer experiments of sorted homogeneous functional phenotypes suggested that, while the functional phenotype is mostly preserved at the population level, exhausted CD8 T cells display considerable plasticity and change their phenotype depending on the tissue of residence.