The differential phenotype and functionality of CMV- and EBV-specific CD8 T cells in patients with chronic hepatitis C is associated with an altered plasma cytokine milieu

Abstract

Persistent infections such as chronic hepatitis C (CHC) are often characterized by an ongoing inflammation and thus altered cytokine milieu due to the constant antigen stimulation. It is still under debate whether such persistent infections may also affect immune responses towards other pathogens. We therefore investigated the phenotype and function of CD8 T cells specific for common pathogens like CMV and EBV using PBMC from HLA-A2+ CHC patients (n= 37) and healthy blood donors (n= 31). Total and CMV/EBV-specific CD8 T cells were assessed for expression of PD-1, Tim-3 and 2B4 ex vivo and after in vitro stimulation with cognate peptides as well as their functionality investigated. We further comprehensively analysed serum profiles of 50 cytokines/chemokines in these CHC patients and in further healthy individuals (n= 35). We demonstrate here that ex vivo expression of PD-1, Tim-3 and 2B4 on total CD8 T cells were 1.6-, 1.5- and 1.5-fold higher in patients with CHC compared to healthy individuals, respectively. Similarly, expression of PD-1, Tim-3 and 2B4 were likewise increased on the CMV/EBV-specific CD8 T cells in CHC patients (CMV: 1.2 fold, 3.2 fold 1.1 fold, EBV: 1.2 fold, 2.0 fold, 1.1 fold, respectively) compared to healthy humans despite their ex vivo frequencies being similar. Importantly, this increased expression culminated in a yet even stronger proliferation and cytokine production by CMV/EBV-specific CD8 T cells in patients with CHC upon peptide stimulation. In order to investigate which role cytokines/chemokines might play in this altered phenotype and activation status during CHC, we compared the plasma cytokine/chemokine levels between healthy individuals and CHC patients using ONE-WAY ANOVA and Principal Component Analysis. Here, IFN-α2, SDF-1α, HGF, MIG, SCF, SCGF-β, CTACK, ICAM-1, VCAM-1, and IP-10 were present at significantly higher levels, while PDGF-bb, G-CSF and GRO-α were lower in CHC patients. We then correlated the levels of these cytokines to the ex vivo expression of PD-1, 2B4 and Tim-3 on both bulk and CMV/EBV-specific CD8 T cells in these CHC patients. Here, levels of IP-10, ICAM-1, VCAM-1, LIF and GRO-α showed to be positively correlating with PD-1 and 2B4 expression on both cell populations. Importantly, the impact on Tim-3 expression differed with plasma levels of SDF-1, INF-α2, PDGF-bb and G-CSF positively correlating while VCAM-1, ICAM-1 and CTACK were a negatively associated on both cell populations. In conclusion, our results suggest an important influence of the cytokine microenvironment in shaping the phenotype and function of antiviral CD8 T cells possibly by affecting the expression of co-regulatory molecules.