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Background & Aims: Natural killer (NK) cells are essential early after infection, not only for viral containment but also for timely and efficient induction of adaptive responses. An inhibitory effect of hepatitis C virus (HCV)-E2 proteins on NK cells has been reported, but the features of NK cell responses in the acute phase of hepatitis C are still largely undefined. Therefore, the aim of this study was to characterize the function and phenotype of NK cells in the acute phase of infection and compare individuals with chronic and self-limited outcomes., Methods: Twenty-two individuals with acute HCV infection, 14 with chronic evolution, and 8 with self-limited infection, were studied using NK phenotypic and functional assays., Results: An increased expression of NKG2D on both CD56(bright) and CD56(dim) NK cells was detected in patients with acute HCV, irrespective of the outcome, as compared with healthy controls. Also, interferon gamma production and cytotoxicity by NK cells were higher in individuals with acute HCV infection than in healthy controls. Subset analysis showed increased interferon gamma production in both NK cell subsets carrying group 1 and group 2 HLA-C-specific killer cell immunoglobulin-like receptors. However, increased CD107a was noted only on NK cells expressing the group 1 HLA-C-specific killer cell immunoglobulin-like receptor and was maximal in self-limited infection., Conclusions: Our data show that in the acute phase of HCV infection, NK cells are activated regardless of outcome, with no evidence of a suppressive effect of HCV on NK cell function., (2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Hepatitis C virus (HCV)-specific CD8 cell exhaustion may represent a mechanism of HCV persistence. The inhibitory receptor PD-1 has been reported to be up-regulated in exhausted CD8 cells. Therefore, we studied PD-1 expression longitudinally during acute HCV infection. Most HCV-specific CD8 cells expressed PD-1 at the time of acute illness, irrespective of the final outcome. PD-1 expression declined with the acquisition of a memory phenotype and recovery of an efficient CD8 cell function in resolving HCV infections, whereas high levels were maintained when HCV persisted and HCV-specific CD8 cells remained dysfunctional. Blocking PD-1/PDL-1 interaction with an anti-PDL-1 antibody improved the capacity of expansion of virus-specific CD8 cells.

A timely, efficient, and coordinated activation of both CD4 and CD8 T cell subsets following HCV infection is believed to be essential for HCV control. However, to what extent a failure of the individual T cell subsets can contribute to the high propensity of HCV to persist is still largely undefined. To address this issue, we analyzed the breadth, vigor, and quality of CD4 and CD8 responses simultaneously with panels of peptides covering the entire HCV sequence or containing the HLA-A2-binding motif, and with recombinant HCV proteins in 16 patients with acute HCV infection by tetramer staining, ELISPOT, and intracellular cytokine staining for interferon gamma, interleukin (IL)-2, IL-4, and IL-10. Our results indicate that at clinical onset, CD8 responses are similarly weak and narrowly focused in both self-limited and chronically evolving infections. At this stage, CD4 responses are deeply impaired in patients with a chronic outcome as they are weak and of narrow specificity, unlike the strong, broad and T helper 1-oriented CD4 responses associated with resolving infections. Only patients able to finally control infection show maturation of CD8 memory sustained by progressive expansion of CD127+ CD8 cells. Thus, a poor CD8 response in the acute stage of infection may enhance the overall probability of chronic viral persistence. In conclusion, the presence of functional CD4 responses represents one of the factors dictating the fate of infection by directly contributing to control of the virus and by promoting maturation of protective memory CD8 responses.

Detection of hepatitis C virus (HCV)-specific T cell response after exposure to hepatitis C in anti-HCV-positive or anti-HCV-negative patients has been associated with an ability to successfully control the infection. However, cross-reactivity between common human pathogens and HCV sequences has been demonstrated. The aim of this study was to investigate the impact of T cell cross-reactivity on HCV-specific T cell responses and their detection in HCV infected and non-infected subjects. The magnitude, function, and cross-reactivity of HCV peptide reactive T cells were studied in non-HCV-infected newborns and adults using a broad array of HCV peptides (601 peptides) spanning the entire HCV sequence. Comparisons were made with responses present in recovered and in chronically HCV-infected patients. HCV peptide reactive T cells are detectable in adults irrespective of previous HCV exposure and cross-reactivity between HCV peptides, and sequences of common pathogens, such as human herpes virus 1, can be demonstrated. Furthermore, the comprehensive magnitude of HCV-peptide reactive T cells present in chronically HCV-infected patients is similar and in some cases even lower than that of HCV-peptide reactive T cell response found in HCV-negative adults. In conclusion, the presence of oligo-specific HCV-peptide reactive T cells in humans does not always reflect a demonstration of previous HCV contact, whereas cross-reactivity with other common pathogens can potentially influence the HCV-specific T cell profile. The conspicuous deficit of HCV-peptide-specific T cells found in chronically HCV-infected patients confirms the profound collapse of virus-specific T cell response caused by HCV persistence.

Evasion from protective CD8 responses by mutations within immunodominant epitopes represents a potential strategy of HCV persistence. To investigate the pathogenetic relevance of this mechanism, a careful search for immunodominant CD8 epitopes was conducted in six patients with chronic evolution of HCV infection by analyzing their global CD8 response with a panel of overlapping synthetic peptides covering the overall HCV sequence and by studying the CD8 frequency by tetramer staining. Immunodominant responses were followed longitudinally from the time of acute onset in relation to the evolution of the epitopic sequences. Although intensity of CD8 responses and frequency of HCV-specific CD8 cells declined over time in all patients, mutations emerged in only three of the six acute patients studied. Variant sequences were less efficiently recognized by CD8 cells than parental epitopes and were poorly efficient in inducing a CD8 response in vitro. CD8 epitopes undergoing mutations were targeted by high avidity CD8 cells more efficient in effector function. Our data support the view that immunodominant CD8 responses are affected by inhibitory mechanisms operating early after infection and that the emergence of escape mutations represents an additional mechanism of virus evasion from those CD8 responses that are functionally preserved.

To characterize acute-phase hepatitis B virus (HBV)-specific T cell responses associated with self-limited and persistent HBV infections, we compared a patient with acute HBV/HCV coinfection, who was able to control HCV but developed chronic hepatitis B, with patients who resolved acute HBV infection spontaneously. Acute-phase CD4 responses were efficient in self-limited infections but undetectable in the coinfected patient with HBV persistence. CD8 responses were multispecific irrespective of the outcome of infection, but the CD8 repertoire associated with HBV persistence lacked the most dominant specificities detectable in self-limited infections. In conclusion, insufficient CD4 help and defective CD8 repertoire may play a role at the early stages of infection in influencing HBV persistence.

Hepatitis C virus (HCV) can cause liver disease of variable severity. Expansion of preexisting memory CD8 T cells by cross-reactivity with a new heterologous virus infection has been shown in mice to shape the repertoire of the primary response and to influence virus-related immunopathology. To determine whether this mechanism can influence the course of HCV infection, we analyzed the features of the HCV-specific CD8 T cell response in eight patients with acute HCV infection, two of whom had a particularly severe illness. Patients with severe hepatitis, but not those with mild disease, showed an extremely vigorous CD8 T cell response narrowly focused on a single epitope (NS3 1073-1081), which cross-reacted with an influenza neuraminidase sequence. Our results suggest that CD8 T cell cross-reactivity influences the severity of the HCV-associated liver pathology and depicts a model of disease induction that may apply to different viral infections.

Hepatitis B and hepatitis C viruses (HBV and HCV) are both noncytopathic and can cause acute and chronic infections of the liver. Although they share tropism for the same organ, development of chronic hepatitis is much more frequent following HCV infection, suggesting different mechanisms of viral persistence. In this study, we show that circulating HBV- and HCV-specific tetramer-positive CD8 cells during the acute phase of hepatitis B and C belong almost entirely to an effector-memory subset (CCR7(-) CD45RA(-)). Despite this phenotypic similarity, HBV- and HCV-specific CD8 cells show striking functional differences. HBV-specific tetramer-positive CD8 cells express high perforin content ex vivo, expand vigorously, and display efficient cytotoxic activity and gamma interferon (IFN-gamma) production upon peptide stimulation. A comparable degree of functional efficiency is maintained after the resolution of hepatitis B. In contrast, HCV-specific CD8 cells in the acute phase of hepatitis C express significantly lower levels of perforin molecules ex vivo and show depressed CD8 function in terms of proliferation, lytic activity, and IFN-gamma production, irrespective of the final outcome of the disease. This defect is transient, because HCV-specific CD8 cells can progressively improve their function in patients with self-limited hepatitis C, while the CD8 function remains persistently depressed in subjects with a chronic evolution.

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.

Cell-mediated immune responses to hepatitis B (HBV) and hepatitis C virus (HCV) antigens are vigorous and multispecific in acute, self-limited infections. Moreover, the prevalent cytokine pattern of circulating virus-specific T cells from patients who recover spontaneously from acute hepatitis is Th1-like. Longitudinal analysis of the T cell response to HCV antigens from the early stages of HCV infection in patients who recover from hepatitis and those who do not indicates that weaker responses and a prevalent Th2 pattern of cytokine production is associated with viral persistence and chronic evolution of disease. Although similar sequential studies are missing in hepatitis B, the observation that HBV-specific T cell responses are very weak or totally undetectable in the peripheral blood of patients with long-lasting chronic hepatitis B suggests that strength and quality of virus-specific T cell responses at the early stages of infection may influence the final outcome of both hepatitis B and C. While T cell hyporesponsiveness seems to be an important determinant for HBV persistence once chronic hepatitis has developed, this mechanism appears to be less critical in chronic HCV infection, because the vigor and quality of HCV-specific T cell responses seem to improve as a function of the duration of infection. This is shown by the finding that HCV-specific CD4- and CD8-mediated responses are easily detectable in the peripheral blood of patients with long-lasting chronic hepatitis C and that production of Th1 cytokines predominates within their livers. HCV therefore seems to be able to persist even in the face of an active T cell response and to acquire the capacity to survive within a host environment apparently unfavorable to its persistence. The high variability of HCV may explain its efficiency in escaping immune surveillance.