HCV-specific CD8 Research Articles

Hepatitis C virus chronicity is associated with a tox-1(high), Eomes(high), T-bet(dim) and PD1(high) phenotype of HCV-specific CD8+ T cells

Hepatitis C virus chronicity is associated with a tox-1(high), Eomes(high), T-bet(dim) and PD1(high) phenotype of HCV-specific CD8+ T cells

On the impact of hepatitis C virus and heterologous immunity on alloimmune responses following liver transplantation.

Virus-induced heterologous immunity is considered a barrier to transplantation tolerance. Yet, hepatitis C (HCV)-infected liver transplant (LT) patients occasionally achieve operational tolerance. We investigated the mechanisms through which HCV infection modulates donor-specific T cell responses following LT and the influence of HCV eradication. We generated T cell lines from HCV-infected LT and non-LT patients before and after HCV eradication and quantified alloreactive responses using cell lines expressing single-HLA class-I antigens in the presence/absence of PD-1/CTLA-4 blockade. HCV-specific CD8+ T cells cross-reacted with allogeneic class-I HLA molecules. HCV-positive LT recipients exhibited a higher proportion of CD8+ T cells coexpressing inhibitory receptors (PD-1/CTLA4) than HCV-negative LT, and their expression correlated with CXCL10 plasma levels. This resulted in decreased antidonor and third-party proliferative responses, which were significantly reversed by HCV eradication. PD-1/CTLA-4 blockade increased the proportion of HCV-specific CD8+ T cells reacting against donor only before viral clearance. In conclusion, HCV infection results in the generation of HCV-specific CD8+ T cells capable of reacting against allogeneic HLA molecules. Following LT, this results in a PD-1/CTLA4-dependent decrease in alloimmune responses. Our findings challenge the notion that heterologous immunity is necessarily detrimental in LT and provide an explanation for the association between HCV eradication and immune-mediated allograft damage.

MHC class II invariant chain-adjuvanted viral vectored vaccines enhances T cell responses in humans.

Strategies to enhance the induction of high magnitude T cell responses through vaccination are urgently needed. Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) plays a critical role in antigen presentation, forming MHC class II peptide complexes for the generation of CD4+ T cell responses. Preclinical studies evaluating the fusion of Ii to antigens encoded in vector delivery systems have shown that this strategy may enhance T cell immune responses to the encoded antigen. We now assess this strategy in humans, using chimpanzee adenovirus 3 and modified vaccinia Ankara vectors encoding human Ii fused to the nonstructural (NS) antigens of hepatitis C virus (HCV) in a heterologous prime/boost regimen. Vaccination was well tolerated and enhanced the peak magnitude, breadth, and proliferative capacity of anti-HCV T cell responses compared to non-Ii vaccines in humans. Very high frequencies of HCV-specific T cells were elicited in humans. Polyfunctional HCV-specific CD8+ and CD4+ responses were induced with up to 30% of CD3+CD8+ cells targeting single HCV epitopes; these were mostly effector memory cells with a high proportion expressing T cell activation and cytolytic markers. No volunteers developed anti-Ii T cell or antibody responses. Using a mouse model and in vitro experiments, we show that Ii fused to NS increases HCV immune responses through enhanced ubiquitination and proteasomal degradation. This strategy could be used to develop more potent HCV vaccines that may contribute to the HCV elimination targets and paves the way for developing class II Ii vaccines against cancer and other infections.

CD4+ T cell responses in human viral infection: lessons from hepatitis C.

Liver disease as a result of chronic hepatitis C virus (HCV) infection is a global problem. While some HCV infections resolve spontaneously, viral persistence associates with compromised T cell immunity. In this issue of the JCI, Chen et al. and Coss et al. explored virus-specific CD4+ T cell response during HCV infection. Both studies evaluated the HCV-specific T cells of patients with different courses of infection. Chen et al. revealed that initial CD4+ T cell responses are similar during early infection and that T cell failure resulted from loss of the virus-specific T cells themselves. Coss et al. showed that HCV-specific CD4+ T cells temporarily recovered in some women following childbirth. These studies contribute to our understanding of CD4+ T cell functionality during different natural courses of infection, with the notable implication that restoring CD4+ T cell immunity might contribute to controlling HCV infection.

306. CD8+ T-Cell Responses to Chronic Hepatitis C in Pregnancy

BackgroundChronic hepatitis C virus (HCV) infection is marked by stable, high -level viremia and a failed T-cell response. HCV-specific CD4+ helper T cells are rare, and CD8+ cytotoxic T cells are functionally exhausted or ineffective due to viral escape mutations. Postpartum, a subset of infected women experience a substantial drop in viremia. Preliminary data indicate that this unusual viral decline may be linked to a resurgence of HCV-specific CD4+ T cells producing Th1 cytokines. How improved CD4+ helper T-cell function might affect viral replication in this scenario is not established. Here we tested the hypothesis that improved CD4+ T cell help mediates control of chronic HCV replication through enhanced CD8+ T-cell function.MethodsWe examined plasma HCV RNA viral load (VL) and HCV-specific T-cell responses in 33 women with chronic HCV during the third trimester (T3) and at 3 months postpartum (3P). HCV-specific CD4+ and CD8+ T-cell IL2 and IFNγ responses were measured by intracellular cytokine staining following stimulation of peripheral blood mononuclear cells with peptide pools corresponding to the HCV proteins NS3, NS4a, and NS4B.ResultsMedian VL dropped from 5.87 log10 at T3 to 5.25 log10 at 3P (P < 0.0001, Wilcoxson signed rank), with a wide range from +0.4 log10 to -4.2 log10. The degree of decline correlated significantly with improved frequencies of HCV-specific CD4+ T cells producing IFNγ (P = 0.015 Spearman) but did not correlate with CD8+ T-cell changes. Nevertheless, improved T helper function correlated with increased HCV-specific CD8+ T-cell function (ΔCD4+IL2+ vs. ΔCD8+IFNγ+, P = 0.015 Spearman, graph 1; ΔCD4+IFNγ+ vs. CD8+IFNγ+ at 3P, P = 0.004 Spearman, graph 2).ConclusionDespite no significant association between virus-specific CD8+ T-cell Tc1 cytokine production and postpartum viral control, our data suggest that recovery of CD4+ T-cell help may augment CD8+ T-cell function. Further study incorporating viral genomic sequences to focus on intact class I epitopes is needed to clarify the relationship of improved CD8+ function and viral control in this unique model of immune restoration. Disclosures All authors: No reported disclosures.

303. A Surrogate Rodent Model for Studying Hepatitis C Virus-specific CD8 T-cell Impairment and Vaccine Prevention

BackgroundVirus-specific CD8 T cells are essential for control of acute hepatitis C virus (HCV) infections, yet spontaneously fail in most patients leading to lifelong chronicity and increased risk for severe liver diseases. Efforts to study HCV-specific CD8 T-cell impairment have been hampered by a lack of small animal models. Recently, we established a rat model of chronic HCV-like infection using a hepacivirus homolog identified in Rattus norvegicus. The nature of virus-specific CD8 T-cell immunity in this model has yet to be determined.MethodsUsing two MHC class I tetramers against epitopes located in the E1 and NS5B proteins, we tracked the induction and phenotype of virus-specific CD8 T cells during chronic infection. Responses to infection were similarly analyzed in immune rats that had been vaccinated against the NS3-5B proteins, a strategy that is effective in this experimental setting.ResultsVirus-specific CD8 T cells expanded vigorously in liver shortly after infection but did not develop into functional effectors based upon failure to produce cytokines (IFNγ, TNFα, IL-2, IL-4, IL-10, IL-17A) following peptide stimulation. Notably, subversion of responses was not due to viral escape from T-cell recognition, but rather an intrinsic defect in the antiviral response. Indeed, these populations expressed the inhibitory receptor programed cell death-1 and other markers consistent with an arrested effector-like state precluded from long-term memory formation (CD127-CD27+CD28+CD62L-GranzymeB+). In contrast, adenoviral immunization of naïve rats protected virus-specific T cells from functional impairment after infection and supported memory response development, including against the E1 epitope not encoded by vaccine.ConclusionTogether, our findings reveal a spontaneous failure of virus-specific CD8 T cells following rat hepacivirus challenge that is highly reminiscent of human HCV infections. Furthermore, these results highlight the utility and significance of this model for understanding mechanisms of HCV persistence and protective immunity necessary for the development of effective vaccines and immune interventions.Disclosures All authors: No reported disclosures.

Single-Dose Vaccination with a Hepatotropic Adeno-associated Virus Efficiently Localizes T Cell Immunity in the Liver with the Potential To Confer Rapid Protection against Hepatitis C Virus.

Hepatitis C virus (HCV) is a significant contributor to the global disease burden, and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4+ and CD8+ T cell immunity correlates with viral clearance in primary HCV infection, and intrahepatic CD8+ tissue-resident memory T (TRM) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4+ and CD8+ T cells, including CD8+ TRM cells, in the liver, given that HCV primarily infects hepatocytes. To achieve this, we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) nonstructural protein (NS5B) and a mutant perforin (pVAX-NS5B-PRF), as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4+ T helper (TH) cell and cytotoxic CD8+ T cell epitopes of NS5B in vivo, which were subsequently used to design a KdNS5B451-459 tetramer and analyze NS5B-specific T cell responses in vaccinated mice in vivo The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting TH and cytotoxic CD8+ T cell responses and intrahepatic CD8+ TRM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell-based vaccine against HCV should ideally result in localized T cell responses in the liver, this study describes primary observations in the context of HCV vaccination that can be used to achieve this goal.IMPORTANCE There are currently at least 71 million individuals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.

Hepatitis C Virus as a Unique Human Model Disease to Define Differences in the Transcriptional Landscape of T Cells in Acute versus Chronic Infection.

The hepatitis C virus is unique among chronic viral infections in that an acute outcome with complete viral elimination is observed in a minority of infected patients. This unique feature allows direct comparison of successful immune responses with those that fail in the setting of the same human infection. Here we review how this scenario can be used to achieve better understanding of transcriptional regulation of T-cell differentiation. Specifically, we discuss results from a study comparing transcriptional profiles of hepatitis C virus (HCV)-specific CD8 T-cells during early HCV infection between patients that do and do not control and eliminate HCV. Identification of early gene expression differences in key T-cell differentiation molecules as well as clearly distinct transcriptional networks related to cell metabolism and nucleosomal regulation reveal novel insights into the development of exhausted and memory T-cells. With additional transcriptional studies of HCV-specific CD4 and CD8 T-cells in different stages of infection currently underway, we expect HCV infection to become a valuable model disease to study human immunity to viruses.

HCV-specific CD4+ T cells of patients with acute and chronic HCV infection display high expression of TIGIT and other co-inhibitory molecules

The combined regulation of a network of inhibitory and activating T cell receptors may be a critical step in the development of chronic HCV infection. Ex vivo HCV MHC class I + II tetramer staining and bead-enrichment was performed with baseline and longitudinal PBMC samples of a cohort of patients with acute, chronic and spontaneously resolved HCV infection to assess the expression pattern of the co-inhibitory molecule TIGIT together with PD-1, BTLA, Tim-3, as well as OX40 and CD226 (DNAM-1) of HCV-specific CD4+ T cells, and in a subset of patients of HCV-specific CD8+ T cells. As the main result, we found a higher expression level of TIGIT+ PD-1+ on HCV-specific CD4+ T cells during acute and chronic HCV infection compared to patients with spontaneously resolved HCV infection (p < 0,0001). Conversely, expression of the complementary co-stimulatory receptor of TIGIT, CD226 (DNAM-1) was significantly decreased on HCV-specific CD4+ T cells during chronic infection. The predominant phenotype of HCV-specific CD4+ T cells during acute and chronic infection was TIGIT+, PD-1+, BTLA+, Tim-3−. This comprehensive phenotypic study confirms TIGIT together with PD-1 as a discriminatory marker of dysfunctional HCV-specific CD4+ T cells.

Elimination of hepatitis C virus has limited impact on the functional and mitochondrial impairment of HCV-specific CD8+ T cell responses

Hepatitis C virus (HCV)-specific CD8+ T cells are functionally impaired in chronic hepatitis C. Even though HCV can now be rapidly and sustainably cleared from chronically infected patients, the repercussions of HCV clearance on virus-specific CD8+ T cells remain elusive. Here, we aimed to investigate if HCV clearance by direct-acting antivirals (DAAs) could restore the functionality of exhausted HCV-specific CD8+ T cell responses. HCV-specific CD8+ T cells in peripheral blood were obtained from 40 patients with chronic HCV infection, during and 6 months following IFN-free DAA therapy. These cells were analyzed for comprehensive phenotypes, proliferation, cytokine production, mitochondrial fitness and response to immune-checkpoint blockade. We show that, unlike activation markers that decreased, surface expression of multiple co-regulatory receptors on exhausted HCV-specific CD8+ T cells remained unaltered after clearance of HCV. Likewise, cytokine production by HCV-specific CD8+ T cells remained impaired following HCV clearance. The proliferative capacity of HCV multimer-specific CD8+ T cells was not restored in the majority of patients. Enhanced in vitro proliferative expansion of HCV-specific CD8+ T cells during HCV clearance was more likely in women, patients with low liver stiffness and low alanine aminotransferase levels in our cohort. Interestingly, HCV-specific CD8+ T cells that did not proliferate following HCV clearance could preferentially re-invigorate their proliferative capacity upon in vitro immune-checkpoint inhibition. Moreover, altered mitochondrial dysfunction exhibited by exhausted HCV-specific CD8+ T cells could not be normalized after HCV clearance. Taken together, our data implies that exhausted HCV-specific CD8+ T cells remain functionally and metabolically impaired at multiple levels following HCV clearance in most patients with chronic hepatitis C. Our results might have implications in cases of re-infection with HCV and for HCV vaccine development. Direct-acting antiviral therapy results in cure of hepatitis C virus (HCV) in almost all treated patients. However, the impacts of HCV cure on immune responses remain controversial. Whether immune responses to HCV recover is important in cases of re-exposure, or for the resolution of extrahepatic manifestations. The main finding of our study was that HCV-specific T cells remain functionally impaired despite HCV clearance. This finding could explain the fact that HCV cure does not lead to protective immunity and that re-infections have frequently been observed.

Dynamic Changes in Ex Vivo T-Cell Function After Viral Clearance in Chronic HCV Infection

Direct-acting antiviral (DAA) agents can successfully treat chronic hepatitis C virus (HCV) infection. However, the ex vivo HCV-specific T-cell function following viral clearance remains unknown. We investigated functional alterations and phenotypic changes in ex vivo HCV-specific CD8+ T cells with a longitudinal analysis of 41 patients with chronic HCV infection who were undergoing DAA treatment. A patient subset exhibited a significantly increased T-cell response (mainly CD8+ T cells) at week 4 of treatment. However, this increased T-cell response diminished in later weeks. Relative to pretreatment levels, the ex vivo HCV-specific CD8+ T-cell frequency decreased at 12 weeks after the end of treatment, along with a decreased antigen-experienced CD8+ T-cell population. DAA treatment increased the proliferative capacity of HCV-specific CD8+ T cells, but this change was not correlated with ex vivo function. Patients experiencing viral breakthrough or relapse exhibited defective restoration of T-cell function. Our present results indicated that DAA-mediated viral clearance only transiently restored ex vivo T-cell function, suggesting a need to enhance T-cell function in DAA-treated patients.

Observed Changes in Natural Killer and T cell Phenotypes with Evaluation of Immune Outcome in a Longitudinal Cohort Following Sofosbuvir-Based Therapy for Chronic Hepatitis C Infection.

BackgroundChronic hepatitis C virus (HCV) infection diminishes immune function through cell exhaustion and repertoire alteration. Direct acting antiviral (DAA)-based therapy can restore immune cell subset function and reduce exhaustion states. However, the extent of immune modulation following DAA-based therapy and the role that clinical and demographic factors play remain unknown.MethodsWe examined natural killer (NK) cell, CD4+, and CD8+ T cell subsets along with activation and exhaustion phenotypes across an observational study of sofosbuvir-based treatment for chronic HCV infection. Additionally, we examined the ability of clinical variables and duration of infection to predict 12 weeks of sustained virologic response (SVR12) immune marker outcomes.ResultsWe show that sofosbuvir-based therapy restores NK cell subset distributions and reduces chronic activation by SVR12. Likewise, T cell subsets, including HCV-specific CD8+ T cells, show reductions in chronic exhaustion markers by SVR12. Immunosuppressive CD4+ regulatory T cells decrease at 4-weeks treatment and SVR12. We observe the magnitude and direction of change in immune marker values from pretreatment to SVR12 varies greatly among participants. Although we observed associations between the estimated date of infection, HCV diagnosis date, and extent of immune marker outcome at SVR12, our regression analyses did not indicate any factors as strong SVR12 outcome predictors.ConclusionOur study lends further evidence of immune changes following sofosbuvir-based therapy. Further investigation beyond SVR12 and into factors that may predict posttreatment outcome is warranted.

Chronic Hepatitis C Virus Infection Impairs M1 Macrophage Differentiation and Contributes to CD8+ T-Cell Dysfunction.

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment, not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver, including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a, M2b, and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions, respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics, such as increased CD86 expression and IL-10 secretion, compared to uninfected controls. In contrast, M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-γ. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls, M1 macrophages alone significantly increased CD8+ T cell IFN-γ expression in a cytokine-independent and cell-contact-dependent manner. However, M1 macrophages from HCV-infected individuals significantly decreased IFN-γ expression in CD8+ T-cells. Therefore, altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals, and aid in the mitigation of associated negative clinical outcomes.

PS-030-Cure of hepatitis C virus has limited impact on the functional and mitochondrial impairment of HCV-specific CD8+ T-cell responses

PS-030-Cure of hepatitis C virus has limited impact on the functional and mitochondrial impairment of HCV-specific CD8+ T-cell responses

FRI-147-Gradual restoration of HCV-specific CD8+ T cell frequency but persistence of altered phenotype in cirrhotic HCV-infected patients after successful direct-acting antiviral therapy

FRI-147-Gradual restoration of HCV-specific CD8+ T cell frequency but persistence of altered phenotype in cirrhotic HCV-infected patients after successful direct-acting antiviral therapy

ERAP1 allotypes shape the epitope repertoire of virus-specific CD8+ T cell responses in acute hepatitis C virus infection

Endoplasmic reticulum aminopeptidase 1 (ERAP1) polymorphisms are linked with human leukocyte antigen (HLA) class I-associated autoinflammatory disorders, including ankylosing spondylitis and Behçet's disease. Disease-associated ERAP1 allotypes exhibit distinct functional properties, but it remains unclear how differential peptide trimming in vivo affects the repertoire of epitopes presented to CD8+ T cells. The aim of this study was to determine the impact of ERAP1 allotypes on the virus-specific CD8+ T cell epitope repertoire in an HLA-B*27:05+ individual with acute hepatitis C virus (HCV) infection. We performed genetic and functional analyses of ERAP1 allotypes and characterized the HCV-specific CD8+ T cell repertoire at the level of fine epitope specificity and HLA class I restriction, in a patient who had acquired an HCV genotype 1a infection through a needle-stick injury. Two hypoactive allotypic variants of ERAP1 were identified in an individual with acute HCV infection. The associated repertoire of virus-derived epitopes recognized by CD8+ T cells was uncommon in a couple of respects. Firstly, reactivity was directed away from classically immunodominant epitopes, preferentially targeting either novel or subdominant epitopes. Secondly, reactivity was biased towards longer epitopes (10-11-mers). Despite the patient exhibiting favorable prognostic indicators, these atypical immune responses failed to clear the virus and the patient developed persistent low-level infection with HCV. ERAP1 allotypes modify the virus-specific CD8+ T cell epitope repertoire in vivo, leading to altered immunodominance patterns that may contribute to the failure of antiviral immunity after infection with HCV. Endoplasmic reticulum aminopeptidase 1 (ERAP1) plays a key role in antigen presentation. Genetic variants of ERAP1 (leading to distinct allotypes) are linked with specific autoinflammatory disorders, such as ankylosing spondylitis and Behçet's disease. We found that ERAP1 allotypes modified the repertoire of virus-specific CD8+ T cell epitopes in a patient with hepatitis C virus, leading to an altered pattern of immunodominance that may have contributed to the failure of antiviral immunity in this patient.

Alphavirus-based hepatitis C virus therapeutic vaccines: can universal helper epitopes enhance HCV-specific cytotoxic T lymphocyte responses?

Background:Antigen-specific T cell immune responses play a pivotal role in resolving acute and chronic hepatitis C virus (HCV) infections. Currently, no prophylactic or therapeutic vaccines against HCV are available. We previously demonstrated the preclinical potency of therapeutic HCV vaccines based on recombinant Semliki Forest virus (SFV) replicon particles. However, clinical trials do not always meet the high expectations of preclinical studies, thus, optimization of vaccine strategies is crucial. In efforts to further increase the frequency of HCV-specific immune responses in the candidate SFV-based vaccines, the authors assessed whether inclusion of three strong, so-called universal helper T cell epitopes, and an endoplasmic reticulum localization, and retention signal (collectively termed sigHELP-KDEL cassette) could enhance HCV-specific immune responses.Methods:We included the sigHELP-KDEL cassette in two of the candidate SFV-based HCV vaccines, targeting NS3/4A and NS5A/B proteins. We characterized the new constructs in vitro for the expression and stability of the transgene-encoded proteins. Their immune efficacy with respect to HCV-specific immune responses in vivo was compared with the parental SFV vaccine expressing the corresponding HCV antigen. Further characterization of the functionality of the HCV-specific CD8+ T cells was assessed by surface and intracellular cytokine staining and flow cytometry analysis.Results:Moderate, but significantly, enhanced frequencies of antigen-specific immune responses were achieved upon lower/suboptimal dosage immunization. In optimal dosage immunization, the inclusion of the cassette did not further increase the frequencies of HCV-specific CD8+ T cells when compared with the parental vaccines and the frequencies of effector and memory populations were identical.Conclusion:We hypothesize that the additional effect of the sigHELP-KDEL cassette in SFV-based vaccines depends on the immunogenicity, nature, and stability of the target antigen expressed by the vaccine.

Simultaneous Analysis of HCV-Specific CD4+ and CD8+ T Cells by Multicolor Flow Cytometry.

CD4 T cell responses are key to effective control of HCV infection. However, their precise mechanisms of action and the molecular programs leading to effective T help versus CD4 T cell failure remain elusive. Direct visualization of HCV-specific CD4 T cells using HLA class II tetramers holds the promise to better define the function and phenotype of these cells and to isolate them for further molecular analysis. Here we describe how to utilize peptide-MHC (pMHC) class II tetramers in multicolor flow cytometry to define the expression of molecules on the surface and within HCV-specific CD4 T cells, how to analyze HCV-specific CD4 and CD8 T cells in the same tube, and how to sort live HCV-specific CD4 T cells as single cells or T cell populations for further analysis by RNAseq or other methods.

Immune Correlates of Natural HIV Elite Control and Simultaneous HCV Clearance-Supercontrollers.

HIV-elite controllers are a minority group of HIV-infected patients with the ability to maintain undetectable HIV viremia for long time periods without antiretroviral treatment. A small group of HIV-controllers are also able to spontaneously clear the hepatitis C virus (HCV) whom we can refer to as “supercontrollers.” There are no studies that explore immune correlates looking for the mechanisms implicated in this extraordinary phenomenon. Herein, we have analyzed HCV- and HIV-specific T-cell responses, as well as T, dendritic and NK cell phenotypes. The higher HCV-specific CD4 T-cell polyfunctionality, together with a low activation and exhaustion T-cell phenotype was found in supercontrollers. In addition, the frequency of CD8 CD161high T-cells was related with HIV- and HCV-specific T-cells polyfunctionality. Interesting features regarding NK and plasmacytoid dendritic cells (pDCs) were found. The study of the supercontroller's immune response, subjects that spontaneously controls both chronic viral infections, could provide further insights into virus-specific responses needed to develop immunotherapeutic strategies in the setting of HIV cure or HCV vaccination.

Chemokine-Regulated Recruitment of Antigen-Specific T-Cell Subpopulations to the Liver in Acute and Chronic Hepatitis C Infection.

In hepatitis C virus (HCV) infection, virus-specific CD8+ T cells are recruited to the liver for antiviral activity. Multiple chemokine ligands are induced by the infection, notably interferon-inducible chemokine, CXCL10. In HCV, intrahepatic T cells express chemokine receptors (CCRs), including CXCR3, CXCR6, CCR1, and CCR5, but CCR expression on antigen-specific effector and memory T cells has not been investigated. Paired blood and liver samples were collected from subjects with chronic HCV for flow cytometric analysis of CCR expression on CD8+ T cells. Expression of these CCRs was then examined on HCV-specific CD8+ T-cell subpopulations in the blood from subjects with acute or chronic HCV. Relative to peripheral blood, the liver was enriched with CD8+ T cells expressing CCR2, CCR5, CXCR3, and CXCR6 either singly or in combinations. CXCR3 was preferentially expressed on HCV-specific CD8+ T cells in both acute and chronic phases of infection in blood. Both CXCR3 and CCR2 were overexpressed on HCV-specific CD8+CCR7+CD45RO+ (central memory) cells, whereas effector memory (CD8+CCR7-CD45RO+) cells expressed more CXCR6. CXCR3-mediated signals support the accumulation of HCV-specific CD8+ memory T cells in the infected liver, and emphasize the importance of the CXCL10/CXCR3 trafficking pathway during acute and chronic HCV infection.