Restauración de la respuesta celular citotóxica específica en la infección crónica por virus de la hepatitis C mediante la modulación de vías inductoras de anergia y apoptosis

Abstract

Background: During chronic hepatitis C virus (HCV) infection, HCV-specific cytotoxic T cells (CTLs) lack adequate effector functions and fail to control HCV. This impairment could be related with programmed death-1 (PD-1) and interleukin (IL)-7 receptor (CD127) regulation. Engagement of PD-1 and its ligand, PD-L1, delivers a negative signal to the T cell receptor (TCR) activation pathways, avoiding proliferation and IL-2 production. On the other hand, CD127 plays an essential role in mature lymphocyte survival by counteracting apoptosis induction after antigen encounter through Bcl2-interacting mediator (Bim) down-regulation, enhancing IL-2 secretion and life span. Therefore, it could be possible that HCV infection could modulate PD-1 and CD127 expression in order to impair HCV-specific CTL reactivity through an induction of an anergic and pro-apoptotic state to favour viral persistence. Objectives: 1.- To describe HCV specific CTL response according to viral control, paying especial attention to PD-1 and CD127 expression. 2.- To analyse the effect of PD-1 and CD127 pathways modulation on HCV-specific CTL reactivity. Materials and Methods: Non-experimental cross-sectional analytical study. HCVspecific CTL frequency, proliferation, γ-IFN production, and PD-1/CD127 phenotypes (dependent variables) according to viral control (independent variable) were analysed. HLA-A2+ HCV infected patients were recruited and divided into two groups according to their serum HCV RNA and alanine transaminase (ALT) levels; defined as persistent infection (PI), and resolved infection (RI). Peripheral blood (PBMC) and intrahepatic (IHMC) mononuclear cells were obtained. Directly ex-vivo and after specific stimulation, HCV-specific CTL frequency against three immunodominant epitopes (NS31406-1415, NS31073-1081 y Core132-140) was tested using labelled pentameric HLAA2/peptide complexes (Pent). Afterwards, PD-1/CD127 and γ-IFN production on HCVspecific CTLs were analysed. HCV-specific CTL proliferation after specific in-vitro challenge in presence or absence of blocking anti-PD-L1 antibodies, to block PD-1/PDL1 pathway, and z-VAD-fmk to block apoptosis cascade, induced by IL-7 deprivation, were also tested. Bim expression was analysed after specific in-vitro challenge in presence of z-VAD-fmk. In order to test whether HCV sequence variation could affect to HCV-specific CTL response, NS31406-1415 and NS31073-1081 genes were sequenced in selected cases. Results: RI and PI patients displayed a low frequency of HCV-specific CTLs in peripheral blood (PB), but these cells were sequestered into the liver in PI patients. Directly ex-vivo, HCV-specific CTLs from PB displayed a higher PD-1 and lower CD127 expression in PI with regard to RI cases. Interestingly, PD-1/CD127 phenotype on HCV-specific CTLs showed a positive and negative correlation respectively with viraemia in PI cases. Moreover, this PD-1/CD127 gradient was also observed between PB and liver in PI cases. Proliferation and γ-IFN production on HCV-specific CTLs from RI cases were higher than in PI cases. Specifically, proliferation and γ-IFN secretion 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 also observed in some PI patients, and these maintained expansion ability but they did not target the infecting virus. Finally, HCV-specific CTL proliferation in PI increased after PD-1/PD-L1 interaction blocking, and after anti-apoptotic treatment. Bim expression on HCV-specific CTLs from PI patients was also enhanced. Conclusions: HCV-specific CTL response is still present in persistent infection but its reactivity is impaired. This impairment correlates with PD-1 up-regulation and CD127 down regulation. Particularly, during chronic HCV infection non-reactive PD-1+ HCVspecific CD8+ cells targeting the virus are CD127-/Bim+ and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in-vitro reactivity. Modulation of these two pathways could be considered as a future therapeutic tool for HCV chronic infection treatment.