Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

Several genome-wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular JAK-STAT pathway. Effects unique to IL28B that would distinguish it from IFN-α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN-α or IL28B, we sought to identify functional differences between IFN-α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN-α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 h of stimulation with IFN-α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV infected cells, the rapid response after stimulation with IFN-α was blunted, and the induction pattern resembled that caused by IL28B. In conclusion, we describe the kinetics of gene induction as being fundamentally different for stimulations with either IFN-α or IL28B in hepatocytes suggesting distinct roles of these cytokines within the immune response. Furthermore, we demonstrate that the observed differences are substantially altered by infection with the hepatitis C virus.

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Background & Aims There is an unclear relationship between T-cell expression of inhibitory receptors and their ability to control viral infections. Studies of human immune cells have been mostly limited to T cells from blood, which is not always the site of infection. We investigated the relationship between T-cell location, expression of inhibitory receptors, maturation, and viral control using blood and liver cells from patients with hepatitis C virus (HCV) and other viral infections. Methods We analyzed 36 liver samples from HCV antibody-positive patients (30 from patients with chronic HCV infection, 5 from patients with sustained virologic responses [SVRs] to treatment, and 1 from a patient with spontaneous clearance), with 19 paired blood samples, and 51 liver samples from HCV-negative patients, with 17 paired blood samples. Intrahepatic and circulating lymphocytes were extracted; T-cell markers and inhibitory receptors were quantified, for total and virus-specific T cells, by flow cytometry. Results Levels of the markers PD-1 and 2B4 (but not CD160, TIM3, or LAG3) were increased on intrahepatic T cells from healthy and diseased liver tissues, compared to T cells from blood. HCV-specific intrahepatic CD8+ T-cells from patients with chronic HCV infection were distinct in that they expressed TIM3 along with PD1 and 2B4. In comparison, HCV-specific CD8+ T cells from patients with SVRs and T cells that recognized cytomegalovirus (CMV) lacked TIM3, but expressed higher levels of LAG3; these cells also had different memory phenotypes and proliferative capacity. Conclusions T cells from liver express different inhibitory receptors than T cells from blood, independent of liver disease. HCV-specific and CMV-specific CD8+ T cells can be differentiated based on their expression of inhibitory receptors; these correlate with their memory phenotype and levels of proliferation and viral control.

Summary Epidemiological evidence suggests that chronic infections impair immune responses to unrelated pathogens and vaccines. The underlying mechanisms, however, are unclear and distinguishing effects on priming versus development of immunological memory has been challenging. We investigated whether bystander chronic infections impact differentiation of memory CD8+ T cells, the hallmark of protective immunity against intracellular pathogens. Chronic bystander infections impaired development of memory CD8+ T cells in several mouse models and humans. These effects were independent of initial priming and were associated with chronic inflammatory signatures. Chronic inflammation negatively impacted the number of bystander CD8+ T cells and their memory development. Distinct underlying mechanisms of altered survival and differentiation were revealed with the latter regulated by the transcription factors T-bet and Blimp-1. Thus, exposure to prolonged bystander inflammation impairs the effector to memory transition. These data have relevance for immunity and vaccination during persisting infections and chronic inflammation.

Summary Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection but these are not well-studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that were linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.

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T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional, and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation towards a memory-like profile. However, functionally, the cells showed little improvement and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, T cell stimulation duration impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for development of functional T cell memory.