Individuals infected with hepatitis C virus (HCV) are at high risk of developing progressive liver disease, including cirrhosis and hepatocellular carcinoma (HCC). How HCV infection causes liver destruction has been of significant interest for many years, and apoptosis has been proposed as one operative mechanism. In this study, we employed a tissue culture-adapted strain of HCV (JFH1_T) to test effects of HCV infection on induction of programmed cell death (PCD) in Huh-7.5 cells. We found that HCV infection reduced the proliferation rate and induced caspase-3-mediated apoptosis in the infected cell population. However, in addition to apoptosis, we also observed infected cells undergoing caspase-1-mediated pyroptosis, which was induced by NLRP3 inflammasome activation. By co-culturing HCV-infected Huh-7.5 cells with an HCV-non-permissive cell line, we also demonstrated induction of both apoptosis and pyroptosis in uninfected cells. Bystander apoptosis, but not bystander pyroptosis, required cell-cell contact between infected and bystander cells. In summary, these findings provide new information on mechanisms of cell death in response to HCV infection. The observation that both apoptosis and pyroptosis can be induced in bystander cells extends our understanding of HCV-induced pathogenesis in the liver.

Due to constitutive expression in cells targeted by human immunodeficiency virus (HIV), and immediate mode of viral restriction upon HIV entry into the host cell, APOBEC3G (A3G) and APOBEC3F (A3F) have been considered primarily as agents of innate immunity. Recent bioinformatic and mouse model studies hint at the possibility that mutation of the HIV genome by these enzymes may also affect adaptive immunity but whether this occurs in HIV-infected individuals has not been examined. We evaluated whether APOBEC-mediated mutations within common HIV CD8⁺ T cell epitopes can potentially enhance or diminish activation of HIV-specific CD8⁺ T cells from infected individuals. We compared ex vivo activation of CD8⁺ T lymphocytes from HIV-infected individuals by wild type HIV peptide epitopes and synthetic variants bearing simulated A3G/F-induced mutations by measuring interferon-γ (IFN-γ) production. We found that A3G/F-induced mutations consistently diminished HIV-specific CD8⁺ T cell responses against the common epitopes we tested. If this reflects a significant trend in vivo, then adaptation by HIV to enrich sequences that are favored for mutation by A3G/F (A3G/F hotspots) in portions of its genome that encode immunogenic CD8⁺ T cell epitopes would favor CTL escape. Indeed, we found the most frequently mutated A3G motif (CCC) is enriched up to 6-fold within viral genomic sequences encoding immunodominant CD8⁺ T cell epitopes in Gag, Pol and Nef. Within each gene, A3G/F hotspots are more abundant in sequences encoding epitopes that are commonly recognized due to their HLA restriction. Thus, in our system, mutations of the HIV genome, mimicking A3G/F activity, appeared to abrogate or severely reduce CTL recognition. We suggest that the physiological significance of this potential effect in facilitating CTL escape is echoed in the adaptation of the HIV genome to enrich A3G/F hotspots in sequences encoding CTL epitopes that are more immunogenic at the population level.

Hepatitis C virus (HCV) successfully evades the immune system and establishes chronic infection in ∼80% of cases. Immune evasion may involve modulating NK cell functions. Therefore, we developed a short-term assay to assess immediate effects of HCV-infected cells on ex vivo NK cytotoxicity and cytokine production. Natural cytotoxicity, Ab-dependent cell–mediated cytotoxicity, IFN-γ production, and TNF-α production were all significantly inhibited by short-term direct exposure to HCV-infected hepatoma–derived Huh-7.5 cells. Inhibition required cell-to-cell contact and increased together with multiplicity of infection and HCV protein levels. Blocking potential interaction between HCV E2 and NK CD81 did not abrogate NK cell inhibition mediated by HCV-infected cells. We observed no change in expression levels of NKG2D, NKG2A, NKp46, or CD16 on NK cells exposed to HCV-infected Huh-7.5 cells for 5 h or of human histocompatibility-linked leukocyte Ag E on HCV-infected compared with uninfected Huh-7.5 cells. Inhibition of ex vivo NK functions did correspond with reduced surface expression of the natural cytotoxicity receptor NKp30, and downregulation of NKp30 was functionally reflected in reduced anti-NKp30 redirected lysis of P815 cells. Infection of Huh-7.5 cells with HCV JFH1_T increased surface binding of an NKp30-IgG1 Fcγ fusion protein, suggesting upregulation of an antagonistic NKp30 ligand on HCV-infected cells. Our assay demonstrates rapid inhibition of critical NK cell functions by HCV-infected cells. Similar localized effects in vivo may contribute to establishment of chronic HCV infection and associated phenotypic and functional changes in the NK population.

Aging reflects long-term decline in physiological function and integrity. Changes arise at a variable pace governed by time-dependent and -independent mechanisms that are themselves complex, interdependent and variable. Molecular decay produces inferior cells that eventually dominate over healthy counterparts in tissues they comprise. In a form of biological entropy, progression from molecular through cellular to tissue level degeneration culminates in organ disease or dysfunction, affecting systemic health. To better understand time-independent contributors and their potential modulation, common biophysical bases for key molecular and cellular changes underlying age-related physiological deterioration must be delineated. This review addresses the potential contribution of cytomegalovirus (CMV)-driven T cell proliferation to cellular senescence and immunosenescence. We first describe molecular processes imposing cell cycle arrest, the foundation of cellular senescence, then focus on the unique distribution, phenotype and function of CMV-specific CD8⁺ T cells in the context of cellular senescence and “inflammaging”. Their features position CMV infection as a pathogenic accelerant of immune cell proliferation underlying immune senescence. In human immunodeficiency virus (HIV) infection, where increased inflammation and exaggerated anti-CMV immune responses accelerate immune senescence, CMV infection has emerged as a major factor in unhealthy aging. Thus, we speculate on mechanistic links between CMV-specific CD8⁺ T-cell expansion, immune senescence and prevalence of age-related disorders in HIV infection.

Activating receptor-mediated recognition of stress-induced ligands or IgG antibody bridging of tumor or pathogen-associated antigens to the FcγRIII CD16 triggers NK cells to kill transformed and infected cells with reduced HLA-I expression. According to the licensing hypothesis, NK cells become competent for activating receptor-mediated triggering after a formative encounter between a NK inhibitory receptor and its ligand. This general hypothesis is supported by murine and human studies, but to date, evidence of a role for such licensing in human ADCC is ambiguous. Inhibitory receptor interactions with HLA-C promote NK cell ADCC licensing, but interactions between KIR3DL1 and its HLA-Bw4 ligand may be insufficient. We investigated the impact of KIR3DL1 and HLA-Bw4 coexpression on NK cell ADCC using a robust, genuine target system of antibody-bearing EBV-transformed B lymphocytes. Although numbers of KIR3DL1⁺ NK cells were similar in HLA-Bw4⁺ and HLA-Bw4^– individuals, general levels of ADCC mediated against target cells were significantly higher in a group of HLA-Bw4⁺KIR3DL1⁺ individuals than in a comparable HLA-Bw4^– group. Flow cytometry demonstrated directly that a significantly higher fraction of KIR3DL1⁺ NK cells derived from HLA-Bw4⁺ compared with HLA-Bw4^– individuals produced IFN-γ following stimulation with ADCC targets. Murine FcR-bearing P815 target cells also triggered higher levels of CD16-mediated cytotoxicity by NK cells from HLA-Bw4⁺KIR3DL1⁺ individuals. These results indicate a prominent role for KIR3DL1/HLA-Bw4 interactions in licensing NK cells for CD16-mediated effector function.

Objective. Measuring NKG2C⁺CD57⁺natural killer (NK) cell expansion to investigate NK responses against human cytomegalovirus (HCMV) and assessing relationships with adaptive immunity against HCMV.Methods. Expansion of NKG2C⁺CD57⁺NK was measured in peripheral blood mononuclear cells (PBMC) from groups distinguished by HCMV and human immunodeficiency virus (HIV) infection status. Anti-HCMV antibody levels against HCMV-infected MRC-5 cell lysate were assessed by ELISA and HCMV-specific CD8⁺T cell responses characterized by intracellular flow cytometry following PBMC stimulation with immunodominant HCMV peptides.Results. Median NK, antibody, and CD8⁺T cell responses against HCMV were significantly greater in the HCMV/HIV coinfected group than the group infected with CMV alone. The fraction of CMV-specific CD8⁺T cells expressing CD28 correlated inversely with NKG2C⁺CD57⁺NK expansion in HIV infection.Conclusion. Our data reveal no significant direct relationships between NK and adaptive immunity against HCMV. However, stronger NK and adaptive immune responses against HCMV and an inverse correlation between NKG2C⁺CD57⁺NK expansion and proliferative reserve of HCMV-specific CD8⁺T cells, as signified by CD28 expression, indicate parallel evolution of NK and T cell responses against HCMV in HIV infection. Similar aspects of chronic HCMV infection may drive both NK and CD8⁺T cell memory inflation.