Negative Regulation of Hepatitis C Virus Specific Immunity Is Highly Heterogeneous and Modulated by Pegylated Interferon-Alpha/Ribavirin Therapy

<div><p>Specific inhibitory mechanisms suppress the T-cell response against the hepatitis C virus (HCV) in chronically infected patients. However, the relative importance of suppression by IL-10, TGF-β and regulatory T-cells and the impact of pegylated interferon-alpha and ribavirin (PegIFN-α/ribavirin) therapy on these inhibitory mechanisms are still unclear. We revealed that coregulation of the HCV-specific T-cell responses in blood of 43 chronic HCV patients showed a highly heterogeneous pattern before, during and after PegIFN-α/ribavirin. Prior to treatment, IL-10 mediated suppression of HCV-specific IFN-γ production in therapy-naive chronic HCV patients was associated with higher HCV-RNA loads, which suggests that protective antiviral immunity is controlled by IL-10. In addition, as a consequence of PegIFN-α/ribavirin therapy, negative regulation of especially HCV-specific IFN-γ production by TGF-β and IL-10 changed dramatically. Our findings emphasize the importance of negative regulation for the dysfunctional HCV-specific immunity, which should be considered in the design of future immunomodulatory therapies.</p> </div

Chronic HCV infected patients show dysfunctional HCV-specific T-cell responses.

<p>Forty-three chronic HCV-infected patients were investigated (21 therapy-naive and 22 PegIFN-α/ribavirin experienced). T-cell proliferation (cpm) to HCV (<b>A</b>) or CMV (<b>B</b>) were determined at day 5 upon stimulation of PBMC, and IFN-γ production (<b>C</b>) at day 3. The sensitivity of the IFN-γ ELISA was 20 pg/mL, and therefore data of 17 patients are shown as one single horizontal line in the graph. For all graphs, patients showing significant responses are depicted to the left (YES) and those without are shown to the right (NO).</p

Irrespective of viral outcome, regulation of HCV-specific IFN-γ production by TGF-β increases during and up to 24 weeks after PegIFN-α/ribavirin therapy.

<p>Individual data of previously therapy-naive chronic HCV patients before, during and after therapy are shown. Patients 1–13 showed an SVR, patients 14–21 did not achieve an SVR (non-SVR). (<b>A</b>) Green squares reflect patients with a significant increase in either HCV-specific proliferation or (<b>B</b>) IFN-γ production after neutralization of TGF-β. White squares reflect the absence of regulation by TGF-β. Grey squares reflect missing data. Histograms to the right side show percentages of patients with significant TGF-β driven regulation of HCV-specific responses at the indicated timepoints.</p

Regulation of HCV-specific immunity by IL-10 is associated with higher HCV-RNA levels in therapy-naive patients, however not linked to success of PegIFN-α/ribavirin therapy.

<p>(<b>A</b>) HCV-RNA levels of 21 therapy-naive chronic HCV patients with or without regulation by IL-10 of HCV-specific IFN-γ production. (<b>B</b>) Individual data of 21 previously therapy-naive chronic HCV patients before and 24 weeks after ending therapy are shown. Patients 1–13 showed an SVR, patients 14–21 did not achieve an SVR (non-SVR). (<b>A</b>) Green squares reflect patients with a significant increase in either HCV-specific proliferation or (<b>B</b>) IFN-γ production after neutralization of the IL-10R. White squares reflect the absence of regulation by IL-10. Grey squares reflect missing data. Histograms to the right side show percentages of patients with significant IL-10 driven regulation of HCV-specific responses at the indicated timepoints.</p

Multiple regulatory mechanisms control HCV-specific T-cell reactivity in PBMC from chronic HCV infected patients.

<p>(<b>A</b>) Individual data of 43 patients on regulation of HCV-specific T- cell proliferation and (<b>B</b>) IFN-γ production are shown (number 1–21 are therapy-naive; 22–43 PegIFN-α/ribavirin experienced patients). Patient numbers are identical to the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049389#pone-0049389-t001" target="_blank">Table 1</a>, and numbers of non-genotype 1 patients are depicted in red. Red squares depict HCV-specific T-cell proliferation or IFN-γ production without <i>in vitro</i> blockade of any regulatory pathway (baseline response). Green squares reflect patients with a significant increase of HCV-specific responses after IL-10R or TGF-β neutralization, or depletion of Treg. White squares reflect the absence of a baseline response or regulation. The experiments were performed similar as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049389#pone-0049389-g001" target="_blank">Figure 1A</a>. Histograms to the right hand side show percentages of patients with HCV-specific responses at baseline, or significant regulation of these responses by respectively IL-10, TGF-β or Treg. The quantitative data of T cell proliferation and IFN-γ production is presented as cpm and IFN-γ levels in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049389#pone.0049389.s003" target="_blank">Figure S3</a> to allow evaluation of the strength of the individual responses. (<b>C</b>) Serum IL-10 and TGF-β levels and blood CD4+FoxP3+ Treg as a proportion of leukocytes are presented in naïve and treatment experienced patients and healthy controls.</p

Patient characteristics.

<p>Abbreviations: n.d., not determined within 3 months before start of therapy; n.a., not applicable.</p

γGT, ALT and AST in humans and chimpanzees chronically infected with HCV.

<p>Interquartile range and the median (horizontal line) of γGT, ALT and AST in U/ml in chimpanzees and patients chronically infected with HCV.</p

Characteristics of chimpanzee population.

<p>Characteristics of the chimpanzee population studied, the HCV-genotype, the time since HCV exposure, HCV-RNA load, serum IP-10levels, and the liver enzymes levels ALT, AST and γGT. The average values and the range are shown.</p

Relation between IP-10 levels and aminotransferases in serum.

<p>Relation between IP-10 and liver enzymes γGT, ALT and AST from HCV-infected individual chimpanzees (A) and HCV-infected patients (B) A significant correlation between the two parameters is defined as r²>0.85 and p<0.05 where “r²” is a measure for correlation and “p” is a measure for the quality if this correlation.</p

Frequencies of Circulating MAIT Cells Are Diminished in Chronic HCV, HIV and HCV/HIV Co-Infection and Do Not Recover during Therapy

<div><p>Objective</p><p>Mucosal-associated invariant T (MAIT) cells comprise a subpopulation of T cells that can be activated by bacterial products and cytokines to produce IFN-γ. Since little is known on MAIT cells during HCV infection, we compared their phenotype and function in comparison to HIV and HCV/HIV co-infected patients, and determined the effect of IFN-α-based and direct-acting antiviral therapy on MAIT cells of HCV patients.</p><p>Methods</p><p>Blood samples from patients with chronic HCV (CHCV), virologically suppressed HIV, acute HCV/HIV co-infection (AHCV/HIV) and healthy individuals were examined by flowcytometry for phenotype and function of MAIT and NK cells.</p><p>Results and Conclusions</p><p>Compared to healthy individuals, the frequency of CD161⁺Vα7.2⁺ MAIT cells was significantly decreased in patients with CHCV, HIV and AHCV/HIV co-infection. CD38 expression on MAIT cells was increased in AHCV/HIV patients. MAIT cells were responsive to IFN-α <i>in vitro</i> as evidenced by enhanced frequencies of IFN-γ producing cells. IFN-α-based therapy for CHCV decreased the frequency of IFN-γ⁺ MAIT cells, which was still observed 24 weeks after successful therapy. Importantly, even after successful IFN-α-based as well as IFN-α-free therapy for CHCV, decreased frequencies of MAIT cells persisted. We show that the frequencies of MAIT cells are reduced in blood of patients with CHCV, HIV and in AHCV/HIV co-infection compared to healthy individuals. Successful therapy for CHCV did not normalize MAIT cell frequencies at 24 weeks follow up. The impact of HIV and HCV infection on the numbers and function of MAIT cells warrant further studies on the impact of viral infections and the antimicrobial function of MAIT cells.</p></div