FUNCTIONAL AND TRANSCRIPTIONAL RESTORATION OF EXHAUSTED VIRUS-SPECIFIC T LYMPHOCYTES FROM PATIENTS WITH CHRONIC HEPATITIS B

Background and Aims: The current therapy for chronic hepatitis B (CHB) is mainly based on direct acting antiviral drugs that efficiently suppress virus replication, but don’t eradicate HBV and frequently require lifelong administration. Therefore, novel anti-HBV therapies should induce complete HBV cure in a short definite time of treatment. During CHB, HBV-specific T cells gradually lose their anti-viral functions in a process known as T cell exhaustion, which is associated with a deregulated CD8 transcriptional profile that underlies a number of altered biological processes. Among them, an impaired mitochondrial function with ROS overproduction as well as NAD depletion are believed to be crucial. These observations suggest that mitochondrial antioxidants, such as Mitoquinone, and NAD precursors, such as nicotinamide mononucleotide (NMN), perhaps in combination with inhibition of CD38, one of the principal NAD consumers, may represent possible therapeutic strategies aimed at restoring HBV-specific CD8 T cell functions. Methods: HBV Core18-27- specific CD8 T cells from chronic HBV patients were expanded in vitro by HBV peptide stimulation in the presence or absence of Mitoquinone or NMN plus CD38 inhibitors (CD38i). Influenza(Flu)- specific CD8 cells from the same patients were expanded at the same experimental conditions without any treatment and used as controls for the definition of reference transcriptional features of memory CD8 T cells associated with control of infection. Virus-specific CD8 T cells were then sorted and subjected to low input RNASeq analysis by SMART-Seq. Differentially expressed genes were identified by DESeq2 v1.34.0, with the lfcShrink analysis (s value<0.05). The softwares Shinygo and GSEA were also used. Results: HBV-specific CD8 cells from cultures treated with either therapy showed a significant reduction of PD1 expression and a trend towards a decreased CD38 expression. In addition, a number of differentially expressed genes were identified comparing cultures treated with immune-modulating strategies and untreated control cells. Interestingly, in parallel with antiviral function enhancement, both treatments induced the development of transcriptional memory T cell features. Among these, the down-regulation of several genes controlling intracellular Ca2+ uptake, which may be potentially responsible for reduced T cell activation, and regulating the glycolytic metabolism could be observed, with an enhancement of the fatty-acid β-oxidation. Interestingly, GSEA analysis showed a significant association of gene expression profiles in HBV-specific CD8 T cells from treated cultures with control fully functional FLU-specific CD8 T cells. Conclusions: Administration of mitochondria-targeted antioxidant compounds and NAD supplementation represent strategies able to correct the deregulated transcriptional profile in exhausted HBV-specific CD8 T cells from CHB patients, by acting at several levels, including T cell metabolism.