M. Calvino

Cyclosporine A-induced apoptosis in renal tubular cells is related to oxidative damage and mitochondrial fission

Cyclosporine A (CsA) nephrotoxicity has been linked to reactive oxygen species (ROS) production in renal cells. We have demonstrated that the antioxidant Vitamin E (Vit E) abolished renal toxicity in vivo and in vitro models. As one of the main sources of intracellular ROS are mitochondria, we studied the effects of CsA on several mitochondrial functions in LLC-PK1 cells. CsA induced ROS synthesis and decreased reduced glutathione (GSH). The drug decreased mitochondrial membrane potential (ΔΨm) and induced physiological modifications in both the inner (IMM) and the outer mitochondrial membranes (OMM). In the IMM, CsA provoked mitochondrial permeability transition pores (MPTP) and cytochrome c was liberated into the intermembrane space. CsA also induced pore formation in the OMM, allowing that intermembrane space contents can reach cytosol. Furthermore, CsA altered the mitochondrial dynamics, inducing an increase in mitochondrial fission; CsA increased the expression of dynamin related protein 1 (Drp1) that contributes to mitochondrial fission, and decreased the expression of mitofusin 2 (Mfn2) and optic atrophy protein 1 (Opa1), proteins involved in the fusion process. All these phenomena were related to apoptosis. These effects were inhibited when cells were treated with the antioxidant Vit E suggesting that they were mediated by the synthesis of ROS.

Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1+/CD127− HCV-specific CD8+ cells targeting the virus in chronic hepatitis C virus infection

PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8(+) cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8(+) cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1(-)/CD127(+) cells from RI cases was preserved, while it was impaired on PD-1(+)/CD127(-) cells from PI patients. PD1(+)/CD127(+) population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8(+) cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8(+) cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8(+) cells targeting the virus are PD-1(+)/CD127(-)/Bim(+) and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity.

298 SUSTAINED VIROLOGIC RESPONSE IN HIGH VIRAEMIC GENOTYPE-1 HCV INFECTION CORRELATES WITH PERIPHERAL HCV-SPECIFIC CYTOTOXIC RESPONSE RESTORATION

Background and Aims: Programmed cell death-1 receptor (PD-1) plays pivotal roles in regulating host immune responses. This study aimed to characterize PD-1 expression in patients with HBVrelated acute-on-chronic liver failure (ACLF) and further evaluate its prognostic significance and functions associated with the disease progression. Methods: Sixty-four patients with HBV-related ACLF (Early, n = 23; Intermediate, n = 21; Late, n = 20) and twenty healthy controls (HC) were enrolled in this study. Flow cytometric, immunohistochemical and functional analyses were employed in the study. Results: Our study found that the expression of PD-1 on circulating CD8T cells was significantly upregulated in ACLF patients from early, intermediate to late stage. Both immunohistochemical staining and flow cytometric analyses revealed a further enhanced PD-1-expression in the liver compared with its counterparts in the peripheral blood in ALCF patients. Moreover, we found the expression of PD-L1, the ligand for PD-1, was also greatly enhanced on the hepatocytes. Correlation analysis with the clinical parameters showed that the expression of PD-1 on CD8T cells positively correlated with MELD Score (model for endstage liver disease) and aspartate aminotransferase (AST), and negatively correlated with prothrombin activity (PTA). Notably, longitudinal study showed that there were gradually decreased PD1-expressions on CD8T cells in patients with improved prognosis, compared with sustained or even increased expressions in those with poor prognosis. Furthermore, the expression of several common g-chain cytokines, including IL-2, IL-7 and IL-15, were greatly upregulated in the liver of ACLF patients, which would contribute to the upregulation of PD-1 on CD8T cell. To evaluate the function of PD-1 on CD8T cells, the in-vitro assays showed that blocking PD-L1 could revitalize the abilities of CD8T cells in cytokine-secretion and proliferation. Conclusions: PD-1 upregulation could mitigate pathogenic CD8Tcell responses, whereas sustained overexpression of PD-1 on CD8T cells is associated with poor prognosis, indicating an uncontrollable immune injury in the liver of ACLF patients. Therefore, PD-1 expression on CD8-T cells could serve as a potential prognostic marker for patients with HBV-related ACLF, which would be valuable for better clinical management and new therapy development.

Pd1(+) Regulatory T Cells Are Increased in Helicobacter pylori-Associated Gastritis

INTRODUCTION: Regulatory T cells (Treg) (CD4+CD25+FoxP3+) play a fundamental role in modulating the balance between inflammation and immune tolerance and are identified as a factor that contributes to bacterial persistence and to infection chronicity. It is emerging that Treg include different functional and phenotypic subpopulations according to the expression intensity of surface CD25 molecule (IL-2 receptor alpha-chains). Moreover, more recently it has been described that they can be subclassified based on the surface expression of programmed death receptor 1 (PD1). PD-1 is a negative co-stimulatory molecule that plays an important role in the balance and regulation of adaptive immune responses. PD1/PDL1 engagement can down-regulate dramatically T cell activation. AIM: To evaluate Treg cell subtypes in relation with phenotype CD25 and PD1 in H. pylori(+) and H. pylori () biopsies specimens from patients suffering gastritis. METHODS: We analyzed CD4(+) lymphocytes from 16 gastric biopsy specimens of patients with gastritis. H. pylori infection was diagnosed with histology and rapid urease test. Afterwards, it was confirmed by RT PCR assay with SYBR Green I fluorochrome, performed on gastric biopsies using a set of primers for the ureC gene specific for the bacteria. 12 cases of H. pylori(+)-gastritis and 4 of gastritis where H. pylori was not detected were studied. After disintegrating biopsies with a needle, lymphocytes were isolated stirring the remaining tissue in a collagenase-DNase solution (100 U collagenase/mL and 0.1 mg DNase/mL) at 37°C for 2h. The cell suspension was then filtered through mesh and the staining for flow cytometry was performed. The following antibodies were used: anti-PD1-FITC, anti-CD4-PECy5 and anti-CD25-APC. For intracellular labeling with anti-Foxp3-PE, cells were previously permeabilized using Cytofix/ Cytoperm solution. Cells were classified in CD25+ and CD25++ according to the fluorescence of this antibody (cut-off was determined with CD8(+) fluorescence). RESULTS: The percentage of CD4+CD25++Foxp3+ was elevated 5-fold in H. pylori(+) compared to H. pylori(-) samples. The number of CD4+CD25+Foxp3+ was similar in both cases. The subpopulation of CD4+CD25++Foxp3+PD1+ cells in biopsies H. pylori(+) were increased 1.7-fold compared to those found in biopsies H. pylori(-). CONCLUSIONS: H. pylori infection is associated with a marked increase of Treg subpopulation expressing PD1 in gastric mucosa. The use of antibodies anti-PD1 inhibiting only such subset of cells should be investigated as a potential new therapy to reduce gastric inflammation associated with H. pylori infection

679 CD127/PD-1 PHENOTYPE DEFINES THE HCV-SPECIFIC CYTOTOXIC T CELL REACTIVITY AFTER ANTIGEN ENCOUNTER DURING CHRONIC HEPATITIS C VIRUS INFECTION

Methods: 20 HCV-seronegative HLA-A2+ individuals without risk for HCV-acquisition were studied. HCV-NS3(1073)-specific cell lines were generated by short culturing of CD8 T-cells with APCs pulsed with HCV-NS3(1073) peptide. After one-week in-vitro stimulation proliferation by CFSE, after three weeks cytokine responses and tetramer binding were analyzed. Individuals were categorized into non-responder and responder based on proliferation and tetramer frequency. TCR repertoire-analysis of tetramer-sorted cells was performed by RT-PCR with Vb-specific primer and subcloning and sequencing of the CDR3-region. TCR repertoire of HCV-seronegative responder was compared with the TCR repertoire of patients with acute HCV-infection. Results: In 8/20 individuals, HCV-NS3(1073)-specific cells proliferated and variable frequencies (0.4–76%) of tetramer positive cells were documented. TCR reper-analysis of the highly responding individual showed usage of only TCR repertore-Vb6.2. Subcloning of Vb6.2 revealed an oligoclonal TCR repertoire. Interestingly, the CDR3-region of the dominant clone (Vb6.2-QEAGAP-Jb2.2) revealed the presence of similar amino acids found in patients with acute HCV-infection (Acute HCV motifs: Vb6.2-LxGxP-Jb 2.7 and Vb6.2ExAxG-Jb 2.3). Conclusions: Our study confirmed that HCV-NS3(1073)-specific CD8 T-cells exist in HCV-seronegative individuals. The variability between individuals is consistent with the concept of private specificity. The identified TCR repertoire was oligoclonal reminiscent to previously identified cross-reactive responses in mouse models and similar to clones in patients with acute HCVinfection. We suggest that this clone may cross-react during acute HCV-infection leading either to protection, immune pathology or immune-escape due to its oligoclonality. The polyspecificity of memory T-cells needs to be considered when developing peptide based vaccines.