الفهرس 
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Abstract
About 170 million people are infected with hepatitis C virus (HCV), which may cause severe liver disease and liver cancer. 71 million individuals are chronically infected with important variations according to the geographical area. There is markedly high prevalence in the Middle East about 14.7% in Egypt. After acute infection, approximately 20% of infections are spontaneously cleared before the establishment of chronicity while a majority of healthy adults will develop persistent viremia. HCV-specific cytotoxic T lymphocytes play an important role in HCV control during acute infection. After the resolution of infection, a population of the virus-specific memory CD8+ T cells protects the host against reinfection. These subsets of memory cells differ with respect to their half-lives, their anatomical localization, their proliferative potential, their immediate effector function, their requirements for maintenance, their phenotype and their protective potential.
The aim of the present study is to identify the role of CD127, CCR7, CD45RA and CD45RO expressed on the CD8+ T cells in the clearance of HCV infection through studying the chronic HCV patients versus seronegative high-risk individuals.
Our study included 60 individuals divided into 2 main groups: chronic HCV group (20 chronic HCV patients) and seronegative high risk group (40 healthcare workers “HCWs”). Both groups were subjected to flow cytometric analysis of PBMCs to determine the percentage of expression of CD3, CD8 and CD127 markers on CD8+ T cells. PBMCs from both groups were subjected to CFSE proliferation assay in response to HCV specific peptides(core, NS3\NS4, NS5) and the second group was divided into 2 subgroups according to proliferation index(cut-off<2) into: positive proliferation index high risk group (+PIHRG) (20 HCWs) and negative proliferation index high risk group (-PIHRG) (20 HCWs). Flow cytometric analysis of HCV specific CD8+ T cells was done for all subjects in the three groups (chronic HCV patients, “+PIHRG”,” –PIHRG”) to determine the expression of CD127, CCR7, CD45RO, and CD45RA on proliferating and non- proliferating fractions of HCV specific CD8+ T cells.
Primarily CD127 expression was studied on CD8+ T cells in peripheral blood without CFSE stimulation. Only total T cell population (CD3+) was significantly lower in the chronic HCV patients versus both the (-PIHRG) and the (+PIHRG) groups and this may be explained by the decrease in cell populations other than cytotoxic T cells such as CD4+ T cells and NK T cells. Then we studied the surface expression of CD127 on CD8+ T cells after stimulation with different HCV peptides using the CFSE proliferation assay. We found that CD3+CD8+CD127+ cell population was significantly higher on the proliferating fraction more than on the non-proliferating fraction of HCV CD8+ T cells in the all studied groups in response to different HCV peptides. However, the frequency of CD127 expression on peripheral HCV-specific CD8+ cells (despite viral persistence in chronic patients) suggests the absence of ongoing TCR triggering and /or antigen recognition in peripheral blood.
Upon comparing this population between different studied groups, only a significant difference was found between (+PIHRG) and (–PIHRG) as regards CD3+CD8+CD127+ cell in response to NS5, which was higher among the (+PIHRG).
On the other hand, a significant increase was found in the expression of CD45RA “marker of early-differentiated memory T cells” on the non-proliferating fraction HCV specific CD8+ T cells while CD45RO ”marker of terminally differentiated memory T cells” was significantly increased on the proliferating fraction of virus CD8+T cells in the all studied groups in response to different peptides. We found that expression of CD45RO was significantly increased on HCV CD8+ T cells in (–PIHRG) more than in the chronic HCV group while expression of CD45RA was significantly increased on HCV CD8+ T cells in the chronic HCV group more than in the
(–PIHRG) in response to different peptides. This defect of CD45 isoform switching on long-standing memory CD8+ T cells in chronic HCV patients suggests the absence of ongoing TCR triggering in peripheral blood despite viral persistence. We also found that CD45RO was significantly increased on HCV CD8+ T cells in (–PIHRG) more than in the (+PIHRG) group while CD45RO was significantly increased on of HCV CD8+ T cells in (–PIHRG) more than in the (+PIHRG) group in response to NS5.
As regards CCR7 “marker of central memory CD8+ T cells” ,a significant increase was found in its expression on the proliferating fraction of HCV specific CD8+ T cells in the high risk groups in response to different HCV peptides. This finding was concomitant with the high proliferative capacity of central memory CD8+ T cells in response to cognate antigen. In two group comparative study it was obvious that expression of CCR7 on HCV specific CD8+ T cells was significantly increased in the (+PIHRG) and the (-PIHRG) more than in the chronic HCV patients in response to core peptide and NS3/NS4 peptide. While expression of CCR7 was significantly increased on HCV CD8+ T cells in the (+PIHRG) more than in the (-PIHRG) in response to NS3/NS4 peptide. The overall expression of trafficking molecules like CCR7 on virus specific memory CD8+ T cells is significantly altered following multiple, subsequent antigenic stimulations. As antigen experience increases, memory CD8+ T cells drastically down regulate the expression of CCR7 related to homing to peripheral tissue where they have high propensity for reinfection.
In conclusion and according to our findings, chronic HCV patients showed predominance of less differentiated central memory phenotype of HCV CD8+ T cells (CD45RA+) in peripheral blood. Persistent viremia drastically down regulate the expression of CCR7 on virus specific CD8+ T cells that are ineffective in clearing the virus as in chronic patients the sub threshold signal levels established by HCV different proteins may provide anergy of memory cytotoxic T cells that are devoid of effector functions which was evident by high expression of CD45RA. More focused research studying this cell population with its different subsets will help in immune response restoration as an interesting therapeutic tool in viral clearance and exploite the design of more therapeutic approaches for chronic HCV infected patients.