ELISPOT Analysis of Hepatitis C Virus Protein-Specific IFN-γ-Producing Peripheral Blood Lymphocytes in Infected Humans with and without Cirrhosis

Anthony, Donald D.; Post, Anthony B.; Valdez, Hernán; Murphy, Madeleine; Heeger, Peter S.

doi:10.1006/clim.2001.5018

Cited by 36 publications

(27 citation statements)

References 46 publications

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“…Studies in subjects with chronic HCV infection indicate that secretion of IFN-γ might be linked to the degree of liver damage (39,40). In our study, HCV-specific responses were detectable in 30-40% of coinfected or HCV-infected women and IFN-γ responses only in 9% of coinfected women.…”

Section: Discussionsupporting

confidence: 45%

Reduced Type 1 and Type 2 Cytokines in Antiviral Memory T Helper Function Among Women Coinfected with HIV and HCV

et al. 2005

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Bias in cytokine responses has been proposed as a contributing mechanism to pathogenesis in persistent HIV or hepatitis C virus (HCV) infections. We investigated whether coinfection with HCV modifies the profile of antigen-specific cytokine secretion in women persistently infected with HIV compared to women with single HIV or HCV infection. The T helper response to HIV, HCV and cytomegalovirus (CMV) as a positive viral control was dominated by type 1 cytokines ( interleukin-[IL] 2, interferon-[IFN] γ and tumor necrosis factor-[TNF] α), with IFN-γ as the most abundantly secreted. IL-4, IL-5 and IL-10 were low in healthy controls and patients. Robust CMV-specific responses contrasted with curtailed HCV-specific responses in HCV-infected women. The overall anti-viral profile was dominated by Th1 cytokines even in coinfected women but both type 1 and type 2 responses were reduced in HIV-infected women and more extensively in women with HCV/HIV coinfection.

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Section: Discussionsupporting

confidence: 45%

Reduced Type 1 and Type 2 Cytokines in Antiviral Memory T Helper Function Among Women Coinfected with HIV and HCV

et al. 2005

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“…A response was defined as IFN-␥-producing frequencies that were threefold greater than the background frequency and the presence of Ն50 spot-forming units/10 6 cells. Using similar criteria, we previously observed no responses in healthy or disease control subjects (2,3,36,43). Proliferative responses were measured using the same cells, after transfer from ELISPOT plates to 96-well plates at 20 h, to complete a 5-day culture.…”

Section: Methodsmentioning

confidence: 97%

“…Cells were cultured for 20 h in RPMI 1640 medium (Gibco BRL, Grand Island, NY) with the addition of penicillin, streptomycin, L-glutamine, and 3% human serum (Gemini Bio-Products, Woodland, CA) at 37°C, and ELISPOT plates were developed as previously described (2). The resulting spots were counted using a computer-assisted ELISPOT image analyzer (Cellular Technologies Limited, Cleveland, OH) and predetermined criteria based on size, shape, and colorimetric density (21).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis C Virus-Specific T-Cell Gamma Interferon and Proliferative Responses Are More Common in Perihepatic Lymph Nodes than in Peripheral Blood or Liver

Moonka

Milkovich

Rodrı́guez

et al. 2008

J Virol

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The activation state, differentiation state, and functions of liver lymphocytes and perihepatic lymph nodes during chronic hepatitis C virus (HCV) infection are not well understood. Here, we performed phenotypic and functional analyses of freshly prepared lymphocytes isolated from the livers, perihepatic lymph nodes, and peripheral blood compartments of chronic HCV-infected and disease control subjects with end-stage liver disease undergoing liver transplantation. We measured lymphocyte subset frequency and memory T-cell gamma interferon (IFN-␥) and proliferative responses to HCV peptide and control viral antigens in direct ex vivo assays. We found higher frequencies of CD4 cells in the lymph node compartment than in the other compartments for both HCV-infected and disease control subjects. Lymph node CD4 and CD8 cells less commonly expressed the terminal differentiation marker CD57, a finding consistent with an earlier differentiation state. In HCV-infected subjects, HCV-specific IFN-␥-producing and proliferative responses were commonly observed in the lymph node fraction, while they were uncommonly observed in the peripheral blood or liver fractions. In contrast, control viral CD4 protein antigen and CD8 peptide antigen-specific IFN-␥ responses were commonly observed in the periphery and uncommonly observed in the lymph nodes of these same subjects. These findings are consistent with a selective defect in HCV-specific T-cell effector function or distribution in patients with advanced chronic HCV infection. The high frequency of HCV-reactive T cells in perihepatic lymph nodes indicates that a failure to generate or sustain T-lymphocyte HCV reactivity is not responsible for the paucity of functional cells even in end-stage liver disease.During hepatitis C virus (HCV) infection, both CD4 and CD8 virus-specific lymphocytes are thought to play important roles both in control/clearance of viral infection and in hepatocellular damage (7,9,13,23,26,32,33,37,38,40). Priming of these cells during acute infection and sustained activation of memory cells during chronic infection are thought to take place in regional lymph nodes that drain the tissue sites of infection. Chronic HCV infection is characterized by low frequencies of HCV-reactive T cells in circulation (16,34,(42)(43)(44). While there are a number of potential explanations for these meager responses, the relative distribution levels of HCVreactive T cells in lymph nodes, liver, and peripheral blood are not well recognized. We therefore examined the phenotype and function of HCV-specific T cells within the regional lymph nodes, livers, and peripheral blood of HCV-infected individuals, using freshly isolated tissue samples in order to obtain an improved understanding of the dominant T-cell dynamics during HCV infection and to establish a foundation for improved models of compartmentalized T-cell activation during chronic viral hepatitis. Results indicate similar paucities of functional HCV-specific T cells in the liver and periphery, while HCVreactive T cells ar...

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“…Cells were placed into 96-well round-bottom plates for 48 h at 37°C then transferred to ELISPOT plates (Whatman) precoated with IFN-␥ capture Ab (4 g/ml clone 2G1; Endogen) and incubated for 20 h at 37°C. The plates were then developed and analyzed as described previously (30,31). In control assays that included TLR ligand and DC in the absence of T cells, no IFN-␥ production was observed.…”

Section: Activation Of Naive Cd4 T Cellsmentioning

confidence: 99%

TLR Ligand-Dependent Activation of Naive CD4 T Cells by Plasmacytoid Dendritic Cells Is Impaired in Hepatitis C Virus Infection

Yonkers¹,

Rodrı́guez²,

Milkovich³

et al. 2007

The Journal of Immunology

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Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-α or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-γ. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-α did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-α) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.

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ELISPOT Analysis of Hepatitis C Virus Protein-Specific IFN-γ-Producing Peripheral Blood Lymphocytes in Infected Humans with and without Cirrhosis

Cited by 36 publications

References 46 publications

Reduced Type 1 and Type 2 Cytokines in Antiviral Memory T Helper Function Among Women Coinfected with HIV and HCV

Reduced Type 1 and Type 2 Cytokines in Antiviral Memory T Helper Function Among Women Coinfected with HIV and HCV

Hepatitis C Virus-Specific T-Cell Gamma Interferon and Proliferative Responses Are More Common in Perihepatic Lymph Nodes than in Peripheral Blood or Liver

TLR Ligand-Dependent Activation of Naive CD4 T Cells by Plasmacytoid Dendritic Cells Is Impaired in Hepatitis C Virus Infection

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