Valeria Barili

Lack of full CD8 functional restoration after antiviral treatment for acute and chronic hepatitis C virus infection

Background Hepatitis C virus (HCV) persistence is associated with impaired CD8 functions. Whether functional restoration of CD8 T cells chronically exposed to antigen can be obtained once the antigen is removed remains to be clarified. Objective To determine whether clearance of HCV by antiviral treatment can fully restore the antiviral function of HCV-specific CD8 cells. Design Peripheral blood HCV-, Flu- and cytomegalovirus (CMV)-specific CD8 cells were quantified by tetramer staining in 28 patients whose HCV infection resolved after peginterferon or peginterferon/ribavirin treatment for either acute or chronic hepatitis and in eight subjects with acute HCV infection which resolved spontaneously for comparison. HCV-specific CD8 cells were evaluated for their phenotypic and functional characteristics by comparing different patient groups and CD8 cells with different viral specificities in the same patients. Results Sustained viral response (SVR) did not lead to full maturation of a functional memory CD8 cell response. In particular, SVR in chronic infection was associated with a greater level of T cell dysfunction than responders after acute infection, who showed HCV-specific CD8 responses comparable to those of spontaneous resolvers but weaker than those of Flu-specific CD8 cells. Higher programmed death (PD)-1 expression was detected on HCV than on Flu- and CMV-specific CD8 cells and the effect of PD-1/PD-L1 blockade was better in SVRs after chronic than after acute HCV infection. Conclusion A better restoration of HCV-specific CD8 function was detectable after SVR in patients with acute hepatitis than in those with chronic disease. Thus, the difficulty in achieving a complete restoration of the antiviral T cell function should be considered in the design of immunomodulatory therapies.

Natural killer cell phenotype modulation and natural killer/T‐cell interplay in nucleos(t)ide analogue‐treated hepatitis e antigen‐negative patients with chronic hepatitis B

Natural killer (NK) and hepatitis B virus (HBV)‐specific T cells are functionally impaired in chronic hepatitis B (CHB). Understanding to what extent nucleos(t)ide analogue (NUC) therapy can improve T‐ and NK‐cell responses is important in the perspective of immunomonitoring strategies for a safe and earlier NUC withdrawal and of novel combination therapies based on modulation of antiviral immunity. To gain further insights into T/NK‐cell interplay, we studied NK‐cell phenotype and function in hepatitis B e antigen–negative chronic HBV patients either untreated (25) or NUC treated (36 hepatitis B surface antigen [HBsAg]+ and 10 HBsAg–/hepatitis B surface antibody [anti‐HBs]+). Interferon‐gamma, interleukin‐2, and tumor necrosis factor alpha (TNF‐α) production by HBV‐specific T cells was also analyzed in NUC‐treated patients. NK cells from chronic naïve patients showed an “inflammatory” phenotype defined by increased expression of TNF‐related apoptosis‐inducing ligand (TRAIL), CD38, and Ki67 that significantly declined upon viremia suppression and alanine aminotransferase normalization induced by NUC therapy. Reversion to a quiescent NK‐cell phenotype was associated with restoration of the HBV‐specific T‐cell function. T‐ and NK‐cell responses showed an inverse correlation, with an opposite behavior in individual NUC‐treated patients. NK‐cell depletion as well as TRAIL and NKG2D pathway blockade induced a significant improvement of the HBV‐specific T‐cell function. Conclusions: NK cells can express regulatory activity on T cells in NUC‐treated patients with prevalent inhibition of CD4 T cells, likely needed to limit persistent T‐cell activation. NK‐cell phenotype is modulated by NUC therapy and its reversion to quiescence mirrors efficient HBV‐specific T‐cell responses. Thus, changes of NK‐cell phenotype may predict acquisition of antiviral control before anti‐HBs seroconversion and represent the groundwork for future studies aimed at assessing whether NK phenotyping can be translated into the clinical practice to guide NUC suspension.(Hepatology 2015;62:1697–1709)

Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B

Hepatitis B virus (HBV)-specific CD8 T cells are functionally exhausted in chronic hepatitis B infection, and this condition can be corrected only partially through the modulation of inhibitory pathways, which suggests that a more complex molecular interplay underlies T cell exhaustion. To gain broader insight into this process and identify additional targets for the restoration of T cell function, we compared the transcriptome profiles of HBV-specific CD8 T cells from patients with acute and chronic disease with those of HBV-specific CD8 T cells from patients able to resolve HBV infection spontaneously and influenza (FLU)-specific CD8 T cells from healthy participants. The results indicate that exhausted HBV-specific CD8 T cells are markedly impaired at multiple levels and show substantial downregulation of various cellular processes centered on extensive mitochondrial alterations. A notable improvement of mitochondrial and antiviral CD8 functions was elicited by mitochondrion-targeted antioxidants, which suggests a central role for reactive oxygen species (ROS) in T cell exhaustion. Thus, mitochondria represent promising targets for novel reconstitution therapies to treat chronic hepatitis B infection.

MicroRNA cloning and sequencing in osteosarcoma cell lines: differential role of miR-93

BackgroundStudies show that abnormalities in non-coding genes can contribute to carcinogenesis; microRNA levels may modulate cancer growth and metastatic diffusion.MethodMicroRNA libraries were built and sequenced from two osteosarcoma cell lines (MG-63 and 143B), which differ in proliferation and transmigration. By cloning and transfection, miR-93, expressed in both cell lines, was then investigated for its involvement in osteosarcoma progression.ResultsSix of the 19 miRNA identified were expressed in both cell lines with higher expression levels of miR-93 in 143B and in primary osteosarcoma cultures compared to normal osteoblasts. Interestingly, levels of miR-93 were significantly higher in metastases from osteosarcoma than in paired primary tumours. When 143B and MG-63 were transfected with miR-93, clones appeared to respond differently to microRNA overexpression. Ectopic expression of miR-93 more significantly increased cell proliferation and invasivity in 143B than in MG-63 clones. Furthermore, increased mRNA and protein levels of E2F1, one of the potential miR-93 targets, were seen in osteosarcoma cellular clones and its involvement in 143B cell proliferation was confirmed by E2F1 silencing.ConclusionAlthough further studies are needed to evaluate miRNA involvement in osteosarcoma progression, miR-93 overexpression seems to play an important role in osteosarcoma cell growth and invasion.

NK cell phenotype modulation and NK/T cell interplay in nucleos(t)ide analogue treated HBeAg‐ patients with chronic hepatitis B

Natural killer (NK) and hepatitis B virus (HBV)‐specific T cells are functionally impaired in chronic hepatitis B (CHB). Understanding to what extent nucleos(t)ide analogue (NUC) therapy can improve T‐ and NK‐cell responses is important in the perspective of immunomonitoring strategies for a safe and earlier NUC withdrawal and of novel combination therapies based on modulation of antiviral immunity. To gain further insights into T/NK‐cell interplay, we studied NK‐cell phenotype and function in hepatitis B e antigen–negative chronic HBV patients either untreated (25) or NUC treated (36 hepatitis B surface antigen [HBsAg]+ and 10 HBsAg–/hepatitis B surface antibody [anti‐HBs]+). Interferon‐gamma, interleukin‐2, and tumor necrosis factor alpha (TNF‐α) production by HBV‐specific T cells was also analyzed in NUC‐treated patients. NK cells from chronic naïve patients showed an “inflammatory” phenotype defined by increased expression of TNF‐related apoptosis‐inducing ligand (TRAIL), CD38, and Ki67 that significantly declined upon viremia suppression and alanine aminotransferase normalization induced by NUC therapy. Reversion to a quiescent NK‐cell phenotype was associated with restoration of the HBV‐specific T‐cell function. T‐ and NK‐cell responses showed an inverse correlation, with an opposite behavior in individual NUC‐treated patients. NK‐cell depletion as well as TRAIL and NKG2D pathway blockade induced a significant improvement of the HBV‐specific T‐cell function. Conclusions: NK cells can express regulatory activity on T cells in NUC‐treated patients with prevalent inhibition of CD4 T cells, likely needed to limit persistent T‐cell activation. NK‐cell phenotype is modulated by NUC therapy and its reversion to quiescence mirrors efficient HBV‐specific T‐cell responses. Thus, changes of NK‐cell phenotype may predict acquisition of antiviral control before anti‐HBs seroconversion and represent the groundwork for future studies aimed at assessing whether NK phenotyping can be translated into the clinical practice to guide NUC suspension.(Hepatology 2015;62:1697–1709)

Natural killer cells phenotypic characterization as an outcome predictor of HCV-linked HCC after curative treatments.

ABSTRACT NK-cell number and function have been associated with cancer progression. A detailed analysis of phenotypic and functional characteristics of NK-cells in HCC is still lacking. NK-cell function is regulated by activating and inhibitory receptors determined by genetic factors and engagement with cognate ligands on transformed or infected cells. We evaluated phenotypic and functional characteristic of NK-cells in HCC patients undergoing curative treatment in relation to clinical outcome. NK-cells from 70 HCC patients undergoing resection or ablative treatment, 18 healthy volunteers and 12 cirrhotic patients with HCV-infection (controls) were phenotypically characterized. Unsupervised clustering based on the frequency of cells expressing different phenotypic NK-cell markers segregated HCC patients into different cohorts that were compared for outcome. NK-cell cytokine production and cytotoxicity were compared between cohorts with different overall survival (OS) and time to disease recurrence (TTR). By multivariate analysis, age, Child-Pugh class and NK-cell phenotypic clustering could independently identify patients with significantly different OS. NK-cells from patients with better outcome expressed higher levels of cytotoxic granules and CD3ζ and lower levels of natural cytotoxic receptors (NCRs) that were co-expressed with the inhibitory receptor NKG2A known to negatively regulate NCR function. Cytotoxic function and IFNγ production were significantly lower in the cohort of patients with worse outcome compared to controls (p < 0.05). Our results show a role for NK-cells in the control of HCC progression and survival providing the basis for the development of immunotherapeutic strategies to potentiate NK-cell response.

Strategies to overcome HBV-specific T cell exhaustion: checkpoint inhibitors and metabolic re-programming

HBV-specific T cells play a key role in antiviral protection and failure to control HBV is associated with severely dysfunctional T cell responses. Therefore, functional T cell reconstitution represents a potential way to treat chronically infected patients. The growing understanding of the dysregulated transcriptional/epigenetic and metabolic programs underlying T cell exhaustion allows to envisage functional T cell reconstitution strategies based on the combined/sequential use of compounds able to induce decline of antigen load, checkpoint modulation, metabolic and epigenetic reprogramming with possible boosting of functionally restored responses by specific vaccines.