RESUMEN

The newborn immune system is characterized by an impaired Th1-associated immune response. Hepatitis B virus (HBV) transmitted from infected mothers to newborns is thought to exploit the newborns' immune system immaturity by inducing a state of immune tolerance that facilitates HBV persistence. Contrary to this hypothesis, we demonstrate here that HBV exposure in utero triggers a state of trained immunity, characterized by innate immune cell maturation and Th1 development, which in turn enhances the ability of cord blood immune cells to respond to bacterial infection in vitro. These training effects are associated with an alteration of the cytokine environment characterized by low IL-10 and, in most cases, high IL-12p40 and IFN-α2. Our data uncover a potentially symbiotic relationship between HBV and its natural host, and highlight the plasticity of the fetal immune system following viral exposure in utero.

Asunto(s)

Citocinas/inmunología , Hepatitis B Crónica/inmunología , Inmunidad Innata/inmunología , Complicaciones Infecciosas del Embarazo/inmunología , Células TH1/inmunología , Adolescente , Adulto , Niño , Femenino , Sangre Fetal/citología , Humanos , Tolerancia Inmunológica/inmunología , Técnicas In Vitro , Recién Nacido , Interferón-alfa/inmunología , Interleucina-10/inmunología , Subunidad p40 de la Interleucina-12/inmunología , Interleucina-17/inmunología , Interleucina-1alfa/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Interleucina-8/inmunología , Embarazo , Factor de Necrosis Tumoral alfa/inmunología , Adulto Joven

RESUMEN

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.

Asunto(s)

Hepatitis C/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Enfermedad Aguda , Adolescente , Adulto , Antígeno CD56/análisis , Citotoxicidad Inmunológica , Femenino , Humanos , Interferón gamma/biosíntesis , Masculino , Persona de Mediana Edad , Subfamilia K de Receptores Similares a Lectina de Células NK/análisis , Receptores KIR/análisis

RESUMEN

BACKGROUND/AIMS: HCV-specific T cells in acute hepatitis C with subsequent chronic evolution are dysfunctional and most of them express PD-1. The aim of the study was to investigate to what extent the antiviral T cell function can be restored by reversing T cell exhaustion by PD-1/PD-L1 blockade and to assess whether this restoration is favored by IFN-alpha treatment. METHODS: PD-1 and PD-L1 expression was studied on T cells and dendritic cells, respectively, of 14 patients with acute hepatitis C and different evolutions of infection. The effect of anti-PD-L1 was analyzed on proliferation, cytokine production and cytolytic activity of CD4 and CD8 T cells. RESULTS: While PD-1 expression dropped concurrently with spontaneous or IFN-alpha induced HCV-RNA decline, PD-L1 levels on dendritic cells increased during IFN-alpha treatment. Anti-PD-L1 antibodies improved expansion and cytokine production but not the cytolytic activity of HCV-specific T cells. This restoration tended to be greater at lower levels of viremia and PD-1 expression and during PEG-IFNalpha treatment. CONCLUSIONS: PD-1/PD-L1 blockade has an immunoregulatory activity which may synergize with the antiviral effect of IFN-alpha therapy and should be thus explored further in long-lasting chronic HCV infections in the perspective of improving the efficacy of available antiviral treatments.

Asunto(s)

Antígenos CD/efectos de los fármacos , Antivirales/uso terapéutico , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Hepacivirus/inmunología , Hepatitis C Crónica/virología , Linfocitos T/inmunología , Viremia/virología , Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Antígeno B7-H1 , Proliferación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/patología , Citometría de Flujo , Estudios de Seguimiento , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/análisis , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/inmunología , Humanos , Interferón gamma/uso terapéutico , Receptor de Muerte Celular Programada 1 , ARN Viral/efectos de los fármacos , ARN Viral/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , Factores de Tiempo , Resultado del Tratamiento , Viremia/tratamiento farmacológico , Viremia/inmunología

RESUMEN

Dysfunctional CD8+ T cells present in chronic virus infections can express programmed death 1 (PD-1) molecules, and the inhibition of the engagement of PD-1 with its ligand (PD-L1) has been reported to enhance the antiviral function of these T cells. We took advantage of the wide fluctuations in levels of viremia which are typical of chronic hepatitis B virus (HBV) infection to comprehensively analyze the impact of prolonged exposure to different virus quantities on virus-specific T-cell dysfunction and on its reversibility through the blocking of the PD-1/PD-L1 pathway. We confirm that chronic HBV infection has a profound effect on the HBV-specific T-cell repertoire. Despite the use of a comprehensive panel of peptides covering all HBV proteins, HBV-specific T cells were rarely observed directly ex vivo in samples from patients with chronic infection, in contrast to those from patients with acute HBV infection. In chronic HBV infection, virus-specific T cells were detected mainly in patients with lower levels of viremia. These HBV-specific CD8+ T cells expressed PD-1, and their function was improved by the blocking of PD-1/PD-L1 engagement. Thus, a broad spectrum of anti-HBV immunity is expressed by patients with chronic HBV infection and this spectrum is proportional to HBV replication levels and can be improved by blocking the PD-1/PD-L1 pathway. This information may be useful for the design of immunotherapeutic strategies to complement and optimize available antiviral therapies.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Antígenos CD/análisis , Antígenos CD/inmunología , Proteínas Reguladoras de la Apoptosis/análisis , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Antígeno B7-H1 , Células Cultivadas , Hepatitis B/inmunología , Hepatitis B/virología , Hepatitis B Crónica/virología , Humanos , Tolerancia Inmunológica , Leucocitos Mononucleares , Estudios Longitudinales , Receptor de Muerte Celular Programada 1 , Subgrupos de Linfocitos T/inmunología , Viremia

RESUMEN

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.

Asunto(s)

Antígenos CD/biosíntesis , Proteínas Reguladoras de la Apoptosis/biosíntesis , Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Citometría de Flujo , Humanos , Memoria Inmunológica , Subgrupos Linfocitarios , Receptor de Muerte Celular Programada 1

RESUMEN

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.

Asunto(s)

Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Hepacivirus/patogenicidad , Hepatitis C/inmunología , Inmunidad Celular , Memoria Inmunológica , Enfermedad Aguda , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/virología , Antígenos de la Hepatitis C/inmunología , Humanos , Técnicas In Vitro , ARN Viral/análisis

RESUMEN

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.

Asunto(s)

Hepacivirus/inmunología , Hepatitis C/inmunología , Linfocitos T/inmunología , Proteínas Virales/inmunología , Adulto , Reacciones Cruzadas , Femenino , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad

RESUMEN

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.

Asunto(s)

Linfocitos T CD8-positivos/virología , Epítopos de Linfocito T/genética , Hepacivirus/inmunología , Hepatitis C/inmunología , Mutación , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Cartilla de ADN , Epítopos de Linfocito T/inmunología , Hepacivirus/genética , Humanos , Interferón gamma/biosíntesis , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/inmunología , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/inmunología , Proteínas Virales/inmunología , Viremia

RESUMEN

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.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C/virología , Enfermedad Aguda , Linfocitos T CD8-positivos/metabolismo , Reacciones Cruzadas/inmunología , Femenino , Hepatitis C/metabolismo , Hepatitis C/patología , Humanos , Interferón gamma/metabolismo , Cinética , Masculino

RESUMEN

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.

Asunto(s)

Reacción de Fase Aguda/inmunología , Linfocitos T CD4-Positivos/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/complicaciones , Hepatitis B/inmunología , Hepatitis C/complicaciones , Formación de Anticuerpos , Linfocitos T CD8-positivos/inmunología , Estudios de Casos y Controles , Progresión de la Enfermedad , Epítopos/inmunología , Antígeno HLA-A2/sangre , Antígeno HLA-A2/inmunología , Hepatitis B/fisiopatología , Hepatitis B/virología , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/sangre , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/etiología , Hepatitis B Crónica/inmunología , Humanos , Inmunidad Celular , Linfocitos T/inmunología , Carga Viral

RESUMEN

Hepatitis B virus (HBV) superinfection in chronic hepatitis C represents a natural model to investigate whether or not hepatitis C virus (HCV) can influence priming and maturation of antiviral T cells; whether or not HBV superinfection, which is known to determine control of HCV replication, can restore HCV-specific T cell responsiveness; and whether or not cytokines stimulated by HBV infection can contribute to HCV control. To address these issues, the function of CD8 cells specific for HBV and HCV was studied longitudinally in two chronic HCV patients superinfected with HBV. Patients with acute hepatitis B were also examined. Frequency and function of HBV tetramer+ CD8 cells were comparable in patients acutely infected with HBV with or without chronic HCV infection. HBV-specific CD8 cell function was efficiently expressed irrespective of serum HCV-RNA levels. Moreover, fluctuations of HCV viremia at the time of HBV superinfection were not associated with evident changes of CD8 responsiveness to HCV. Finally, no correlation was found between serum levels of interferon alpha, interleukin (IL)-12, IL-10, or IL-18 and control of HCV replication. In conclusion, HCV did not affect the induction of primary and memory HBV-specific CD8 responses. HCV-specific CD8 responses were undetectable when HCV-RNA was negative, showing that inhibition of HCV replication in the setting of a HBV superinfection was not sufficient to induce a restoration of CD8 reactivity against HCV.

Asunto(s)

Antivirales/metabolismo , Antígenos CD8/metabolismo , Portador Sano , Hepacivirus , Hepatitis B/inmunología , Hepatitis C Crónica/virología , Sobreinfección/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/sangre , Epítopos , Antígeno HLA-A2/inmunología , Hepacivirus/genética , Hepacivirus/inmunología , Hepacivirus/fisiología , Hepatitis B/virología , Virus de la Hepatitis B/inmunología , Humanos , Inmunidad , Estudios Longitudinales , Péptidos/genética , Péptidos/inmunología , Replicación Viral

RESUMEN

The aim of the present study was to clarify whether factor H mutations were involved in genetic predisposition to hemolytic uremic syndrome, by performing linkage and mutation studies in a large number of patients from those referred to the Italian Registry for Recurrent and Familial HUS/TTP. PCR and Western blot analyses were conducted to characterize the biochemical consequences of the mutations. Five mutations in the factor H gene were identified. Three, identified in two families and in a sporadic case, are heterozygous point mutations within the most C-terminal short consensus repeat 20 (SCR20) of factor H, resulting in single amino acid substitutions. The other two mutations introduce premature stop codons that interrupt the translation of factor H. A heterozygous nonsense mutation was identified in SCR8 in one family, and a homozygous 24-bp deletion within SCR20 was identified in a Bedouin family with a recessive mode of inheritance. Reverse transcription-PCR analysis of cDNA from peripheral blood leukocytes from the Bedouin family showed that the deletion lowered factor H mRNA levels. Although heterozygous mutations were associated with normal factor H levels and incomplete penetrance of the disease, the homozygous mutation in the Bedouin family resulted in severe reduction of factor H levels accompanied by very early disease onset. These data provide compelling molecular evidence that genetically determined deficiencies in factor H are involved in both autosomal-dominant and autosomal-recessive hemolytic uremic syndrome and identify SCR20 as a hot spot for mutations in the disease. The mutations identified here give an important hint to the relevance of the C-terminus of factor H in the control of the alternative complement activation pathway.

Asunto(s)

Factor H de Complemento/genética , Síndrome Hemolítico-Urémico/genética , Mutación , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Secuencia de Bases , Factor H de Complemento/química , Predisposición Genética a la Enfermedad , Humanos , Datos de Secuencia Molecular , Linaje , Polimorfismo Conformacional Retorcido-Simple