Heterogeneity of the exhausted hbv-specific cd8 t cell population as defined by functional/phenotypic profiling

Chronic hepatitis B virus (HBV) infection represents a public health problem worldwide with approximately 250 million people chronically infected at increased risk of developing liver cirrhosis and cancer. Currently available antiviral treatments for chronic HBV infection are based on nucleos(t)ide analogues (NUC), which efficiently suppress viral replication but frequently require a lifelong administration and have limited effect on HBsAg concentrations, and on interferon alpha (IFN), which induces a sustained off-treatment viral suppression in only a minority of patients and is associated with severe side effects. Therefore, the development of new antiviral strategies to provide durable HBsAg loss with limited treatment duration is highly needed. During chronic HBV infection, virus-specific T lymphocytes are poorly responsive to antigenic stimulation and their responses are deeply dysfunctional. This functional impairment is the consequence of different inhibitory mechanisms, including the persistent exposure to high antigen loads, and the resulting exhaustion state is widely believed to contribute to viral persistence. Therefore, functional T cell reconstitution strategies represent possible candidates for the development of novel cure strategies with finite duration of treatment to improve the management of chronic patients and the prevention of HBV-associated hepatic complications. Available data from mouse and other human models of chronic virus infection indicate that exhausted virus-specific CD8 T cells are a heterogeneous dysfunctional population composed of variably differentiated subsets with different response to checkpoint blockade. In order to further characterize this phenomenon in HBV infection, we assessed how heterogeneous are exhausted HBV-specific T cells in chronic HBV patients and whether the analysis of exhausted CD8 T cell subsets co-existing in individual chronic patients can be helpful to predict response to immune modulators. For this purpose, a molecular characterization of T cell exhaustion in the HBV-specific T cell population in CHB patients was conducted by assessing the co-expression of transcription factors, co-inhibitory receptors and differentiation molecules (e.g. PD1, CD39, TOX, CD127, Bcl-2 and TCF1) that allowed to distinguish T cell subsets with different antiviral activity in different cohorts of CHB patients. In particular, co-expression levels of the inhibitory receptor PD-1 and the T cell memory-related marker CD127 distinguished three different T cell subsets: a PD1high CD127low/- cell subset selectively prevalent in naïve viremic patients; a PD1high and CD127high subset present in both CHB and in spontaneous resolver patient cohorts, irrespective of the clinical condition and the level of virus control; a PD1low/- CD127high subset associated with a more protective role in consideration of the preferential detection in patients who spontaneously controlled infection. To confirm that the prevalence of the PD1low/- and CD127high over CD127high CD127low/- subset in individual patients is an indication of better protection, T cell cytokine production was assessed in the different groups of patients. Total ex vivo cytokine levels (IFN- and TNF-) were significantly lower in chronic patients as compared to resolvers and also to flu-specific CD8 cells. The PD1highCD127low/- T cell subset was less efficient than the PD1low/-CD127high subset in terms of total cytokine production. Moreover, cytokine production by PD1low/-CD127high cells was similar in chronic patients and in NUC resolvers and subjects with spontaneous seroconversion. This phenotypic and functional T cell characterization allowed the identification of a distinctive CHB patient cohort with features more similar to resolved patients. To improve the predictive capacity of these profiles, other markers were studied. The PD-1highCD127low/- cells showed a significant correlation with the levels of the ecto-nuclease CD39, which leads to the generation of immune suppressive adenosine. On the other hand, the anti-apoptotic marker Bcl-2 and the memory-related transcription factor TCF1 were highly expressed within the CD127high subsets in both chronic and spontaneously resolved patients. Accordingly, the HMG-box exhaustion-associated transcription factor TOX displayed significantly higher expression in the more impaired PD1high CD127low/- cell subset in comparison with the less dysfunctional PD1low/- CD127high T cell subpopulation in chronically infected patients. The relative predominance of more or less exhausted T cell subsets seems to dictate the level of functional efficiency of the overall anti-viral CD8 T cell population and those patients who harbor a less inhibited virus-specific immune system with predominance of less exhausted CD8 T cell subsets may represent a CHB patient cohort with a better chance of response to immune modulatory interventions. This novel pathogenetic information could be applied for the development of future immune reconstitution strategies to cure HBV infection and to better monitor the results obtained with new anti-viral compounds.