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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC Mar 1, 2011.
Published in final edited form as:
PMCID: PMC2924663
NIHMSID: NIHMS227847
Transient CD86 expression on HCV specific CD8+ T cells in acute infection is linked to sufficient IL-2 signaling1
Henry Radziewicz,* Chris C. Ibegbu,* Huiming Hon, Nathalie Bédard, Julie Bruneau,§ Kimberly A. Workowski, Stuart J. Knechtle, Allan D. Kirk, Christian P. Larsen, Naglaa H. Shoukry,§|| and Arash Grakoui*2
*Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322
Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital St-Luc, Montréal, Québec, Canada
Department of Surgery, Emory University School of Medicine, Atlanta, GA 30322
§Département de médecine familiale, Université de Montréal, Montréal, Québec Canada
||Département de médecine, Université de Montréal, Montréal, Québec Canada
2Corresponding author: Arash Grakoui, PhD Emory University School of Medicine 954 Gatewood Road, NE Atlanta, GA 30329 404-727-5850 phone 404-727-7768 fax ; arash.grakoui/at/emory.edu
Costimulatory signals via B7/CD28 family molecules (signal 2) are critical for effective adaptive CD8+ T cell immune responses. In addition to costimulatory signals, B7/CD28 family coinhibitory receptor/ligands have been identified that modulate immune responses. In acute HCV infection, PD-1, an inhibitory receptor in the CD28 family is highly expressed on virus specific CD8+ T cells, yet vigorous immune responses often still develop. We hypothesized that other costimulatory signals present during the acute phase of HCV infection would be important to counter this negative signaling. In this study, we report that (i) CD86 was highly expressed on HCV specific CD8+ T cells early in acute HCV infection and lost on transition to chronic HCV infection, (ii) Expression of CD86 was different from other “activation” markers since expression was delayed after in vitro TCR stimulation and required sufficient IL-2 signaling, and (iii) HCV specific CD8+ T cells in the liver of patients with chronic HCV infection were highly activated (CD69, CD38 and HLA-DR expression), but only a minority expressed CD86 or showed evidence of recent IL-2 signaling (low basal pSTAT5), despite persistent viremia. Our study identifies B7 ligand expression on HCV specific CD8+ T cells as a distinct marker of effective T cell stimulation with IL-2 signaling in acute HCV infection. Expression of costimulatory molecules such as CD86 early in HCV infection may be essential in overcoming inhibitory signals from the high-level of PD-1 expression also seen at this phase of infection.
Keywords: Human, T cells, hepatitis C virus, Costimulation, CD86, PD-1
A majority of patients infected with hepatitis C virus (HCV) do not spontaneously clear the virus and become chronically infected. It is hypothesized that in those individuals developing persistent infection, an effective adaptive T cell response either fails to develop during the acute phase of HCV infection or is lost on progression to the chronic phase of infection (reviewed by (1)). In the acute phase of HCV infection a clinical hepatitis is often observed and marked by a significant elevation in liver transaminase levels. In contrast, during chronic infection, a clinical hepatitis is often absent and only a mild elevation in liver transaminases is noted. This mild inflammation over many years can lead to liver fibrosis and eventual development of cirrhosis. Evaluation of HCV specific CD8+ T cells from the peripheral blood of patients with acute infection have shown that a majority are highly activated, expressing the markers CD69, CD38, and HLA-DR (2-7). Likewise, HCV specific CD8+ T cells during the chronic phase of infection, in the liver, are highly activated and prevalent (7-9) yet induce only mild liver injury as measured by serum liver transaminase levels. Currently, the loss of functionality of HCV specific CD8+ T cells despite the continued high level of activation that is seen in chronic infection (at the site of infection) is incompletely understood.
Recently, it has been reported that expression of PD-1, an inhibitory receptor in the B7/CD28 family, during chronic viral infection, was associated with a loss of T cell function and a state of “exhaustion” that could be reversed by PD-1 blockade (10). In chronic HCV infection, a high level of PD-1 was expressed on HCV specific CD8+ T cells infiltrating the liver of patients with chronic HCV infection (11-13), and expression of PD-1 on these cells contributed to their decreased function despite the high level of activation (12, 13). Somewhat surprisingly, high levels of PD-1 expression have also been noted on HCV specific CD8+ T cells in the acute phase of infection, yet these cells still induce significant liver injury (7, 13-15). We hypothesized that other stimulatory signals are important to counteract this high PD-1 expression to enable vigorous T cell responses in acute infection.
B7 ligands, such as CD80 and CD86, are typically described as being expressed on antigen presenting cells. However, these molecules can also be expressed on in vitro activated T cells (16-23) and expression of these ligands on T cells is hypothesized to be important in T:T cell interactions (17). For example, after 10 day stimulation with anti-CD3 and IL-2 >80% of human CD4+ and CD8+ T cells expressed CD80 (16), and stimulation of human PBMC with anti-CD3 and IL-2 led to maximal CD86 expression (60% of T cells) 3 weeks after stimulation (18). Expression of the B7 ligands CD80, CD86 and PD-L1 has also been identified directly ex vivo on CD3+ T cells from patients with HIV infection (24, 25) and autoimmune diseases (26, 27) and expression of these ligands has been hypothesized to be a marker of disease progression (24-27).
Despite these studies, the expression of B7 family ligands on HCV specific CD8+ T cells isolated from the peripheral blood or liver of patients with HCV infection has not been investigated. Since CD80 and CD86 expression on T cells can provide a costimulatory signal to other T cells (16, 18), study of the expression of these ligands on HCV specific CD8+ T cells in acute and chronic infection is important for a more complete understanding of the signals driving a functional adaptive T cell response to this virus. In the present study, we evaluated B7 ligand expression in four patients with acute HCV infection and detectable HCV specific tetramer responses. We report that HCV specific CD8+ T cells expressed high levels of CD86 in all of the patients in the acute phase of HCV infection at a time when significant liver injury was clinically apparent. We found that CD86 expression on CD8+ T cells was linked to effective TCR stimulation with sufficient IL-2 signaling. This differed from the expression of the activation markers CD69, CD38, HLA-DR and CD25 that occurred rapidly after TCR stimulation alone without addition of IL-2 to in vitro cultures of PBMC. Unlike in acute HCV infection, in chronic infection, despite the high level expression of activation markers, the majority of HCV specific CD8+ T cells did not express CD80 or CD86, even at the site of infection, and there was no evidence of recent common gamma-chain cytokine signaling. This study improves our understanding of the deficits in T cell stimulation at the site of HCV infection: HCV specific CD8+ T cells are activated partially (expression of CD69, CD38 and HLA-DR) but not effectively (low IL-2 signaling, low CD86 expression, low proliferation (7)). Furthermore, this study highlights the early loss of supportive cytokine stimulation of HCV specific CD8+ T cells during the waning response to HCV infection, and identifies B7 ligand expression (CD80 and CD86) on T cells as an indicator of effective TCR stimulation with supportive IL-2 signaling.
Subjects
Four patients with acute HCV infection as evidenced by HCV antibody seroconversion in the presence of a clinical syndrome of acute hepatitis, and 28 patients with chronic HCV infection (HCV antibody and HCV PCR positive) were enrolled in the study from the clinic or hospital of Emory University, Atlanta VA, Grady or the Montreal Acute Hep C Cohort (HEPCO) at Centre de Recherche du Centre Hospitalier de l'Universite de Montreal (CRCHUM), Hôpital St-Luc, Montreal as previously described (28, 29). The patient characteristics are summarized in Table I. Acutely infected patients are denoted by an “a” and chronic patients by a “c” preceding the patient number. Nine of the chronically infected patients were enrolled in the Emory Liver Transplant Program and liver specimens were procured at the time of hepatectomy for liver transplantation (explant liver). An “E” following the patient number denotes these patients. Patients a802, a240, a808, a4915, c113E, and c671 were HLA-A2 positive by FACS analysis, and this enabled analysis with HLA-A2 restricted tetramers. None of the patients with acute HCV infection spontaneously cleared viremia. a802 began therapy with pegylated interferon and ribavirin at day 80 of the study. a802 was also HIV positive and had an undetectable HIV viral load on highly active antiretroviral therapy (HAART) for approximately 9 months prior to our analysis. There were no changes in HIV viral load (remained undetectable throughout the study). a240, a808 and a4915 were not treated for HCV infection during the course of this study. The patients each provided informed consent, and the protocol (IRB #1358-2004) was approved by the local ethics committees of Emory University, the Atlanta VA Medical Center, and Grady Hospital and protocol (SL05.014) by the CRCHUM.
Table I
Table I
Patient Characteristics. Patients with acute HCV infection are denoted by an “a” preceding the HCV number, patients with chronic infection are denoted by a “c”. An “E” after the patient number denotes an (more ...)
HCV antibody testing, viral load determination, and genotyping
HCV antibody testing by ELISA was performed at the Emory Immunology Lab using a kit per the manufacturer's instructions (Abbott Diagnostics, Abbott Park, Ill), at the Atlanta VA Immunology lab (Bio-rad Laboratories, Hercules, CA) and at CRCHUM by standard Anti-HCV EIA1 and EIA2 tests (Abbott Diagnostics, Abbott Park, Ill). HCV viral load quantification was performed at the Emory Molecular Lab and Atlanta VA using a real-time RT-PCR assay (Roche Molecular Systems, Alameda CA) and at CRCHUM by automated COBAS AmpliPrep/COBAS Amplicor HCV test, version 2.0 (sensitivity 50 IU/ml) (Roche Molecular Systems, Inc., Branchburg, NJ). HCV genotyping was performed at the Emory Molecular Lab using a commercially available assay (Siemens Medical Solutions Diagnostics), at the Atlanta VA using a line probe assay (LIPA) (Bayer Diagnostics, Research Triangle Park, NC) and at CRCHUM using standard sequencing for the NS5B region by the Laboratoire de santé publique du Québec (LSPQ) (Ste-Anne-de-Bellevue, QC).
Peripheral blood mononuclear cell isolation
EDTA and heparin anticoagulated blood was collected from each patient and either used directly for FACS staining or for PBMC isolation. PBMCs were isolated using Ficoll-Paque PLUS density gradient (Amersham, Oslo, Norway), washed twice in PBS, and either analyzed immediately or cryopreserved in media containing 90% fetal calf serum (Hyclone) and 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO).
Liver biopsy
Liver tissue obtained by either ultrasound-guided needle biopsy, via transjugular fluoroscopic technique, or from explant liver was immediately put into RPMI-1640 medium (Gibco) containing 10% fetal calf serum (Hyclone, Logan, UT) for immunological assays. Another fragment was fixed in formalin for histological examination. Classification of the histological changes was performed using the Scheurer scoring system at the Emory University and Atlanta VA pathology labs.
Intrahepatic T cell isolation
The liver biopsy sample obtained by either ultrasound-guided needle biopsy or via transjugular fluoroscopic technique was washed three times with RPMI-1640 medium (Gibco, Carlsbad, CA) containing 10% fetal calf serum (Hyclone, Logan, UT) to remove cell debris and RBCs. Isolation of liver infiltrating lymphocytes was performed using an automated, mechanical disaggregation system (Medimachine, Becton Dickinson, San Jose, CA). The sample was inserted into a 50 mm Medicon and inserted into the Medimachine and run for 15 seconds. Disaggregated cells were removed using a syringe in the syringe port. The Medicon was rinsed twice with RPMI medium (Gibco, Carlsbad, CA) containing 10% fetal calf serum (Hyclone, Logan, UT) to ensure maximum cell recovery. Cells were used immediately for FACS staining. For explant liver samples, the liver section was first perfused with HBSS, sectioned using sterile scissors, resuspended in 0.5% collagenase in F12 media and disaggregated in a Stomacher 400 Circulator (Seward) set at 200 rpm for 20 minutes. The suspension was then washed in R10 and filtered using sterile autoclaved cheesecloth. To isolate liver infiltrating mononuclear cells (LIMC) the supernatant was resuspended in 40% percoll, layered over 70% percoll, and centrifuged at 2000 rpm for 10 minutes on low break. LIMC were harvested from the interface layer and washed in R10. Cells were used immediately for FACS, phosflow or culture.
Antibodies, HLA-A2 tetramers and flow cytometry
Cells were stained with FITC, PE, PerCP, APC and PB labeled monoclonal antibodies or tetramers according to the manufacturers’ instructions and flow cytometry performed using FACS Calibur (Becton Dickinson, San Jose, CA) or LSRII (Becton Dickinson, San Jose, CA). FACS data were analyzed with FlowJo software (Treestar). The following monoclonal antibodies from BD Pharmingen (BD Biosciences, San Jose, CA) were used: Anti-CD25 FITC, CD28 FITC, CD38 FITC, Ki67 FITC, IgG1k FITC, HLA-DR PE, CD86 PE, PD-L1 PE, PD-L2 PE, IgG1k PE, IgG2Ak PE, Granzyme B PE, Perforin FITC, Bcl-2 PE, CD8 PerCP and CD3 Pac Blue. CD80 PE, CD69 PE, CD127 PE and CD8 APC were all obtained from Beckman Coulter (Fullerton, CA). Anti-PD-1 PE conjugated antibody (clone EH12) was obtained from BioLegend (San Diego, CA). Dead cells were excluded by staining with 7AAD (BD Biosciences) or Alexa Fluor 430 (Invitrogen). HLA-A2 tetramers were specific for the following CD8+ T cell epitopes: HCV/1073: CINGVCWTV; HCV/ 1406: KLVALGINAV; CMV/NLV: NLVPMVATV. The tetramers were generated at the National Tetramer Core Facility at Emory University School of Medicine.
Analysis of STAT phosphorylation in T cells
The following monoclonal antibodies from BD Pharmingen (BD Biosciences, San Jose, CA) were used: anti-pSTAT1 A488 (clone 4a), pSTAT5 A488 (clone 47), pSTAT6 A488 (clone J71-773.58.11), IgG1k A488 (MOPC-21), CD86 PE (clone FUN-1), IgG1k PE (MOPC-21), and CD3 Pac Blue (SP34-2). CD8 PECy5 (B9.11) was obtained from Beckman Coulter (Fullerton, CA). For pSTAT analysis in HCV specific CD8+ T cells, whole blood or LIMC were first stained with tetramer for 10 minutes at room temperature, incubated with no stimulation, IL-2 (Roche), IL-7 (R/D Systems) or IL-15 (R/D Systems) for 10 minutes (with tetramer) at 37 degrees, then immediately fixed in Lyse/Fix buffer I (BD Biosciences), washed with PBS, permeabilized with Perm III buffer (BD Biosciences) at 4 degrees, washed in FACS buffer (PBS containing 0.5% BSA and 0.1% NaN3) and stained for 30 minutes with anti-pSTAT and other antibodies. For pSTAT analysis on cultured cells, cells were first washed in FACS buffer, then fixed with Fix Buffer I (BD Biosciences) prior to permeabilization. Flow cytometry was performed using FACS Calibur (Becton Dickinson, San Jose, CA) or LSRII (Becton Dickinson, San Jose, CA) cytometers. FACS data were analyzed with FlowJo software (Treestar).
Cell culture
1 × 106 PBMC or LIMC in 1 ml RPMI 1640/10% FCS (Hyclone) were cultured for 5 to 7 days as noted in the text. For specific antigen stimulation, HCV NS3 1073-1081 peptide (CINGVCWTV) (1 ug/ml), HCV NS3 1406-15 (KLVALGINAV) (1ug/ml), or CMV NLV peptide NLVPMVATV (1 ug/ml) was used (Genemed Synthesis, Inc). For nonspecific TCR stimulation, cells were incubated with anti-CD3 (Immunotech) at a concentration of 0.1 ug/ml. IL-2 (Roche), IL-7 (R/D Systems) or IL-15 (R/D Systems) was added to the culture media at the time of TCR stimulation at concentrations stated in the text. For some experiments, CD8+ T cells were negatively selected using a CD8 negative selection kit (Invitrogen) and then used for in vitro stimulation. Purity of CD8+ T cell selection was >95%. In some conditions, blocking antibody to CD86 (anti-CD86, BD Biosciences) at 10 ug/ml was added at the time of TCR stimulation. After stimulation, PBMC or isolated CD8+ T cells were washed in FACS buffer (PBS containing 0.5% BSA and 0.1% NaN3), surface stained for 20 min at room temperature, washed in FACS buffer, and fixed with 1% paraformaldehyde if not analyzed immediately on a flow cytometer. In some experiments, after stimulation, isolated CD8+ T cells were washed twice in FACS buffer and lysed with RLT Lysing Buffer (Qiagen) for use in rRT-PCR experiments. For the FACS analyses, dead cells were excluded with either with 7AAD (BD Biosciences) (unfixed cells) at the manufacturers instructions or using Alexa 430 staining in PBS at the time of surface staining (fixed cells). For intracellular antibody staining, the cells were washed twice with FACS buffer after surface staining, permeablized for 10 min at room temperature with 500 ul of Perm II Buffer (BD Biosciences), washed with FACS buffer, stained with the indicated intracellular antibodies (BD Biosciences) for 20 min at room temperature, then washed and fixed with 1% paraformaldehyde. Flow cytometry was performed using FACS Calibur (Becton Dickinson, San Jose, CA) or LSRII (Becton Dickinson, San Jose, CA) cytometers. FACS data were analyzed with FlowJo software (Treestar).
RNA preparation and cDNA synthesis
To determine mRNA expression profiling of selected genes the relative quantitative real-time PCR was performed. Cells were harvested and lysed immediately with 350 μl of RLT lysis buffer from RNeasy kit, then frozen at -80°C. Total RNA was extracted from collected samples by using the RNeasy kit (Qiagen), according to the protocol. Total RNA was eluted in 50 μl of water and optical density measurements were taken immediately. All total RNA was reverse-transcribed using a High-Capacity cDNA Archive Kit Protocol (Applied Biosystems Inc.).
Quantitative Real-Time PCR Analysis
The reaction was carried out on a 384-well optical plate (Applied Biosystems) in a 10-μl reaction volume with TaqMan Gene Expression Master Mix (Applied Biosystems). All sequences were amplified using the Applied Biosystems 7900HT Sequence Detection System with the PCR profile: 50° for 2 min, 95°C for 10 min, followed by 45 cycles at 95°C for 15 s, and 60°C for 1 min. Samples were tested in duplicate, in parallel with the housekeeping gene 18S. Real-Time StatMiner™ software (Integromics, Inc.) was used to perform a quality control for all runs and relative quantification delta-delta Ct analysis to calculate the fold differences between samples. For CD86 expression, primer and probe were designed and obtained from Applied Biosystems (part number 4329514T).
Statistical analysis
Results were graphed and analyzed using GraphPad Prism (v4). Comparisons between the expression of CD86 by FACS after anti-CD3 stimulation alone versus anti-CD3 and IL-2 were performed using the Mann-Whitney test. Comparison of fold changes in mRNA of CD86 was performed using paired T tests.
Early and transient expression of CD86 on HCV specific CD8+ T cells during acute HCV infection
The characteristics of four patients with acute HCV infection (a802, a240, a808 and a4915) are shown in Table I. Acute HCV infection was identified by HCV antibody seroconversion in the presence of a clinical syndrome of acute hepatitis. At the time of blood sampling, all the patients had significantly elevated liver transaminases. None of the patients spontaneously cleared HCV. Patient a802 was also HIV positive and had undetectable HIV viremia with highly active antiviral therapy (HAART) for approximately 9 months prior to acquiring HCV. The other three patients were HIV negative. These patients each had detectable HCV specific CD8+ T cell responses by tetramer analysis. Blood sampling from each of these patients was obtained at the earliest time points obtained after identification of acute HCV infection. Day 0 corresponds to the first time point sampled for this study not to Day 0 of infection since the exact time point of HCV acquisition was not known.
B7 ligand expression on HCV specific CD8+ T cells from the peripheral blood of each patient was measured at the earliest time points available for evaluation (day 0 of sampling for patients a802 and a240, day 25 for a808 and day 33 for a4915 (figure 1a). Approximately 30%-60% of HCV specific CD8+ T cells expressed CD86 at these early time points in HCV infection for all four patients (figure 1a). For a240, two HCV specific class I tetramer responses could be identified for two epitopes (HCV/1073 and HCV/1406), and HCV specific CD8+ T cells directed at both epitopes highly expressed CD86 (figure 1a). CD80 expression was not detected on HCV specific CD8+ T cells from a802, a240 or a4915 at the earliest time points (figure 1a) nor at any time point after infection (data not shown). In contrast, HCV specific CD8+ T cells from a808 expressed both CD80 and CD86 at the earliest time point studied (figure 1a). Approximately 30% of HCV specific CD8+ T cells expressed CD86 and 40% expressed CD80 at the earliest time point sampled for a808 (figure 1a). Neither CMV specific nor Flu specific CD8+ T cells expressed CD80 or CD86 during acute HCV infection (figure 1a). In addition, HCV specific CD8+ T cells did not express the other B7 ligands, PD-L1 or PD-L2 at the earliest time points sampled or at any time point of infection (data not shown).
Figure 1
Figure 1
Early and transient expression of CD80 and CD86 on HCV specific CD8+ T cells during acute HCV infection
Frequent blood sampling for a802 and a240 enabled a precise longitudinal assessment of CD86 expression on HCV specific CD8+ T cells. For both patients, high-level expression of CD86 on HCV specific CD8+ T cells was transient (figure 1b). After the first month of evaluation, less than 10% of HCV specific CD8+ T cells continued to express CD86 (figure 1b). For a802 and a240, an early and transient increase in CD86 expression on bulk CD8+ T cells was also noted (figure 1c). For a240 who was HIV negative, a peak of approximately 5% of bulk CD8+ T cells expressed CD86 at the earliest time points of HCV infection, which decreased to less than 1% of CD8+ T cells within the first month of follow-up (figure 1c). For a802, who was HIV positive, approximately 14% of CD8+ T cells expressed CD86 at the earliest time point, which decreased to approximately 11% after the first month (figure 1c). Since elevation of CD86 on CD3+ T cells has been reported in patients with HIV infection (24, 25), we hypothesize that continued elevation of CD86 on bulk CD8+ T cells from a802 was related to HIV infection. In contrast to these findings in acute HCV infection, for healthy donors, less than 1% of bulk CD8+ T cells typically expressed CD86 (figure 1d).
Lack of frequent blood sampling for a808 precluded a complete prospective evaluation of CD80 and CD86 expression for this patient, however, a peak of CD80 expression was noted on day 25 (40% of HCV specific CD8+ T cells) and by day 49 less than 5% of HCV specific CD8+ T cells expressed CD80 (figure 1e). CD86 expression was noted on approximately 30% of HCV specific CD8+ T cells on day 25, 25% of HCV specific CD8+ T cells at day 49 (figure 1e), and at the next available blood sampling at day 167 was less than 5% (data not shown). For a4915, 17 days after the initial time point, CD86 continued to be expressed on HCV specific CD8+ T cells (at day 50) (data not shown), but at the next available time point (day 130) no CD86 expression was found (data not shown).
The loss of detectable HCV specific CD8+ T cells expressing CD86 coincided with the loss of other activation markers, such as CD38 and HLA-DR, during the acute phase of infection (figures 1f and 1g); however, the decrease of CD86 expression to <10% of HCV specific CD8+ T cells preceded the loss of detectable HCV specific CD8+ T cells expressing HLA-DR and CD38 to <10%. In this regard, CD86 most resembled the early activation marker CD69 where a significant detectible population of HCV specific CD8+ T cells expressing CD69 was also lost very early during infection (figure 1f and 1g).
More specifically, for a802 (figure 1f), HLA-DR was expressed on >75% of HCV specific CD8+ T cells until anti-viral treatment initiation on day 80 of follow-up. Frequency of HCV specific CD8+ T cells expressing CD38 remained >50% until treatment initiation, although a substantial early fall in CD38 expression from nearly 100% of HCV specific CD8+ T cells to approximately 60% coincided with the decrease in CD86 expression (figure 1f). The early activation marker, CD69 was expressed on only approximately 25% of HCV specific CD8+ T cells at the earliest time point sampled for a802 and remained less than 10% after the first month (figure 1f).
For a240 (figure 1g), by day 25 only approximately 5% of HCV specific CD8+ T cells expressed CD86 while approximately 30% continued to express CD38 and 70% HLA-DR. As with a802, for a240, the level of CD69 expression most closely correlated with the level of CD86 expression. The costimulatory molecule, CD28, remained highly expressed at >75% on HCV specific CD8+ T cells throughout infection for both a802 and a240 (figures 1f and 1g).
Addition of IL-2 to culture media induces high-level expression of CD80 and CD86 on CD8+ T cells after TCR stimulation but is not required for CD69, CD38, HLA-DR or CD25 expression after brief in vitro culture of PBMC
We next investigated the signals driving CD80 and CD86 expression on CD8+ T cells and compared this to the expression of other activation markers in response to the same stimulation (figure 2a). Though T cell receptor (TCR) stimulation via brief in vitro culture of PBMC with anti-CD3 monoclonal antibody led to a significant level (>25%) expression of CD69, CD38, HLA-DR and CD25 within 1-2 days this led to only a low level expression of CD80 and CD86 (<25% of bulk CD8+ T cells) (figure 2a). In particular, anti-CD3 stimulation alone led to very high levels of CD69 expression (>50% of CD8+ T cells) within 1-2 days (figure 2a). Significant expression of CD86 on bulk CD8+ T cells after brief in vitro culture (7 days) with anti-CD3 could be achieved by addition of IL-2 to the culture media (figure 2b). Furthermore, increasing amounts of IL-2 led to increasing CD86 expression (figure 2b). We further assessed the importance of IL-2 for CD86 expression after brief in vitro culture on sorted CD8+ T cells from 11 patients with chronic HCV infection (c128, c144, c148, c152, c157, c161, c163, c167, c176, c177, c181) utilizing a negative bead selection protocol (Invitrogen). After TCR stimulation without IL-2 added to the culture media, minimal CD86 expression was detected on the sorted CD8+ T cells (figure 2c). Addition of IL-2 led to consistent and significant CD86 expression on the CD8+ T cells after TCR stimulation with anti-CD3 (figure 2c). Though expression of CD69 could be enhanced by co-culture with IL-2 among the sorted CD8+ T cells, TCR stimulation alone with anti-CD3 led to significant expression of CD69 in distinct contrast with B7 ligand upregulation (data not shown). Hence, though CD86 expression during acute HCV infection (figure 1) most resembled CD69 expression in terms of the timing of expression (both seen transiently at the earliest phase of acute infection only), the signal driving expression of CD86 was unique in its dependence on additional cytokine signaling and in its delayed expression in vitro.
Figure 2
Figure 2
Addition of IL-2 in culture induces high-level expression of CD80 and CD86 after TCR stimulation but is not required for CD69, CD38, HLA-DR or CD25 expression
We also investigated the signals driving CD80 and CD86 expression on virus specific CD8+ T cells from the peripheral blood (figure 2d). Fresh PBMC from a808 (day 0) was used in 5-day culture to assess the ability of TCR stimulation plus IL-2 to increase CD86 expression on HCV specific CD8+ T cells (figure 2d). Scant frequency of detectible HCV specific CD8+ T cells could be obtained after 5-day culture for the no stimulation condition (figure 2d). This was in concordance with our previous work indicating that a large fraction of these HCV specific CD8+ T cells at this early time point were highly susceptible to cytokine withdrawal apoptosis (7). 5-day stimulation with HCV peptide plus IL-2 led to high expression of CD86 (figure 2d) and CD80 (data not shown) to nearly 100% of HCV specific CD8+ T cells.
Expression of CD86 on virus specific CD8+ T cells after in vitro culture with IL-2 was not unique to HCV infection
After 5-day culture of fresh PBMC from a healthy donor (HD 209), culture with CMV peptide alone led to only low level of CD86 expression on CMV specific CD8+ T cells (figure 2e). Culture with CMV peptide plus IL-2 (50 U/ml) led to high expression of CD86 (figure 2e). Culture with CMV peptide plus the common gamma-chain cytokines, IL7 (5ng/ml) and IL-15 (5ng/ml), also led to high level expression of CD86 on CMV specific CD8+ T cells (figure 2e).
De novo production of CD86 on CD8+ T cells
Two mechanisms exist by which CD86 (and other B7 ligands) could be expressed on CD8+ T cells in general. The first is via de novo generation in highly stimulated cells (16, 30) and the second is by a relatively recently described mechanism termed “trogocytosis” whereby T cells capture surface molecules through the immunological synapse from antigen-presenting cells (31-33). Both mechanisms have been reported to occur in T cells, trogocytosis occurring after stimulation for 24h or less and endogenous production occurring after 3 days of stimulation (30). In support of de novo production of CD86 on CD8+ T cells, we demonstrated that sorted CD8+ T cells without the presence of CD86 expressing, antigen presenting cells from which to “steal” CD86, expressed CD86 after TCR stimulation with IL-2 supplementation in 7-day culture (figure 2c). In addition, sorted CD8+ T cells from these patients with chronic HCV infection (c128, c134, c144, c148, c152, c157, c161, c163, c167, c176, c177, c181) increased the mRNA expression of CD86 after in vitro culture with anti-CD3 and IL-2 in comparison to no stimulation (figure 3a) and in comparison to stimulation with anti-CD3 alone (figure 3b). For the sorting experiments for c134, quantity of sorted CD8+ T cells only allowed mRNA analysis and not flow cytometry analysis. In further support of the ability for HCV specific CD8+ T cells to synthesize B7 ligands de novo after stimulation, we have generated an HCV specific CD8+ T cell line that has been propagated via anti-CD3+ IL-2 stimulation without antigen presenting cells. These cells highly expressed CD86 (figure 3c).
Figure 3
Figure 3
De novo production of CD86 on CD8+ T cells
Early loss of STAT5 phosphorylation of HCV specific CD8+ T cells in a patient with acute HCV infection
STAT5 phosphorylation (pSTAT5) in T cells is critical for signal transduction after common gamma-chain cytokine binding (34). No previous study has evaluated pSTAT5 directly ex vivo or in vitro on antigen specific CD8+ T cells using tetramer analysis. Since we noted a dependence on common gamma-chain cytokine signaling for maximal CD80 and CD86 expression of HCV specific CD8+ T cells in vitro we assessed the level of pSTAT5 in T cells directly ex vivo from fresh blood of a808 during acute infection (figure 4). For the other three acutely infected patients only frozen PBMC was available, precluding an ex vivo, basal pSTAT analysis for these patients. We found an increase in the basal level of pSTAT5 in HCV specific CD8+ T cells in comparison to pSTAT1 or pSTAT6 for a808 (Figure 4a). This increase in pSTAT5 was also detected in CD3+ T cells in comparison to CD3+ T cells from a patient with chronic HCV infection (figure 4b). Prospective evaluation of a808 showed that the increased level of detectable pSTAT5 in HCV specific CD8+ T cells as well as in CD3+ T cells was lost early in the acute phase of infection, since by day 49 of blood sampling we were unable to detect increased pSTAT5 (figure 4c). These findings in HCV infection contrast the recent report in HIV infection, where increased basal pSTAT5 was noted in bulk CD4+ and CD8+ T cells even in chronic HIV infection (35), which likely indicated the persistent high-level of immune activation that often characterizes HIV infection.
Figure 4
Figure 4
Early loss of STAT5 phosphorylation in HCV specific CD8+ T cells in a patient with acute HCV infection
We also assessed the ability of HCV specific CD8+ T cells during early HCV infection to further signal through common gamma cytokines and found that brief culture with IL-15 in particular increased pSTAT5 (figure 4d, red box). Though the concentrations of IL-2 (100 U/ml), IL-7 (5 ng/ml) and IL-15 (5 ng/ml) were adequate to cause phosphorylation of STAT5 on bulk CD3+ T cells after very brief culture (10 minutes) (note the shift in the non-tetramer CD3+ T cells in figure 4d), there was a lack of increase of pSTAT5 via IL-2 and IL-7 stimulation of HCV specific CD8+ T cells. This deficit in pSTAT5 to IL-7 and IL-2 stimulation in HCV specific CD8+ T cells may be related to the low level of IL-7 receptor (CD127) and the IL-2 receptor (CD25) characterizing the HCV specific CD8+ T cells from a808 at this time point (day 25) (figure 4e). CD127 expression, in particular, has also been previously reported to be of low level on HCV specific CD8+ T cells during acute infection (7, 28, 36-38). Our finding of relatively low pSTAT5 in HCV specific CD8+ T cells after IL-2 stimulation in vitro in acute HCV infection corresponds with deficits in signaling of CD4+ and CD8+ T cells seen in chronic HIV infection where increased basal level of pSTAT5 correlated with poor pSTAT5 signaling in response to further IL-2 stimulation (35).
Low level of CD80, CD86 and basal pSTAT5 expression in HCV specific CD8+ T cells in the liver of patients with chronic HCV infection despite high-level expression of activation markers CD69, CD38 and HLA-DR
In order to fully understand the deficits in the adaptive T cell response to HCV infection it is important to study immune cells at the site of infection. Studying bulk CD8+ T cells from the liver of five patients with chronic HCV infection (c159, c280, c147E, c671, and c684), we found that the majority of CD8+ T cells expressed the activation markers CD69, CD38 and HLA-DR (figure 5a). Despite high-level expression of these activation markers in the liver of patients with chronic HCV infection only a minority of CD8+ T cells expressed the B7 ligands, CD80 and CD86. We identified two patients with chronic HCV infection and a detectable HCV specific CD8+ T cell response by tetramer analysis in the liver (c671, c113e). In previous work, we (7) and others (8, 9) found high-level expression of the activation markers CD69, CD38 and HLA-DR on HCV specific CD8+ T cells from the liver of patients with chronic HCV. Nearly 100% of HCV specific CD8+ cells in the liver of patients with chronic HCV infection expressed these activation markers (7). Despite expressing high-levels of these activation markers (7), the frequency of liver infiltrating HCV specific CD8+ T cells from c671 and c113e that expressed CD86 (approx. 14%) or CD80 (<1%) was low despite persistent HCV viremia (figure 5b). Similarly, in blood, no detectible CD80 or CD86 expression was found on HCV specific CD8+ T cells from c671 (figure 5b). C113e did not have a detectible HCV specific CD8+ T cell response in the peripheral blood.
Figure 5
Figure 5
Low CD80, CD86 and phosphorylated STAT5 in the liver of patients with chronic HCV infection
Evaluating the basal level of pSTAT5 in bulk CD3+ T cells and in HCV specific CD8+ T cells for c113E, no basal elevation in recent common gamma-chain cytokine signaling could be detected in CD3+ T cells or HCV specific CD8+ T cells (figure 5c, upper plots), nor a difference in pSTAT5 in the liver versus blood (figure 5c, lower plot). We analyzed ex vivo, basal pSTAT5 in the blood and liver of 5 additional patients with chronic HCV infection (c128E, c135E, c140E, c142E, c270) and did not detect an increase in the basal level of pSTAT5 in CD8+ T cells in blood or liver of these patients with chronic HCV infection (data not shown).
We further evaluated for the presence of any underlying impairment in IL-2, IL-7 or IL-15 signaling of liver infiltrating CD3+ T cells that could explain the low expression of CD80 and CD86 despite persistent activation (figure 5d). We found no deficit in pSTAT5 in response to IL-2 or IL-15 (figure 5d). In comparison, we found a relatively lower level of pSTAT5 for the IL-7 condition for the liver CD3+ T cells (figure 5d). We also investigated the ability of HCV specific CD8+ T cells in the liver to signal after exposure to IL-2, IL-7 and IL-15 (figure 5e). Similar to c166 (figure 5d), for patient c113E, liver derived CD3+CD8+ T cells increased pSTAT5 after exposure to IL-2 and IL-15 efficiently (figure 5e, upper row). However, again, there was lower pSTAT5 for the IL-7 condition (figure 5e upper row). Mirroring these liver CD3+CD8+ T cells, liver HCV specific CD8+ T cells also efficiently increased pSTAT5 after brief exposure to IL-2 and IL-15; however, we noted lower pSTAT5 after IL-7 exposure (figure 5e, lower row). We hypothesize that this deficit in IL-7 signaling in the liver that is seen in these patients may be related to the decreased frequency of CD127 expression seen on bulk T cells and HCV specific CD8+ T cells in the liver of patients with chronic HCV infection in general (11). This contrasts the deficit in pSTAT5 expression in acute HCV where HCV specific CD8+ T cells showed deficiency in pSTAT5 to both IL-2 and IL-7 exposure (figure 4d). Future studies with larger cohorts of acute and chronically infected patients will be important to characterize the noted deficits in pSTAT5 signaling. The defect in pSTAT5 phosphorylation of HCV specific CD8+ T cells in the liver in response to IL-7 that we detected could contribute to the lack of CD80 and CD86 expression seen in the majority of CD8+ T cell in the liver of patients with chronic HCV infection. However, overall, we conclude that there is not an overriding, underlying deficit in the ability of HCV specific CD8+ T cells from the liver to respond to cytokine (particularly IL-2) that might explain the lack of B7 ligand expression on HCV specific CD8+ T cells in the liver. Rather, based on these findings, we hypothesize that a dearth of common gamma chain cytokine at the site of chronic HCV infection explains the low frequency of CD80 or CD86 expression on HCV specific CD8+ T cells in the liver.
In further support of a lack of an underlying deficit in the ability of liver infiltrating T cells to signal after exposure to cytokines, 5-day in vitro culture of liver CD8+ T cells with anti-CD3 and IL-2 led to high expression of CD86 (figure 6a). High-level CD80 expression was also seen on CD8+ T cells after culture with anti-CD3 and IL-2 (figure 6b). Comparing blood and liver for c101E, 5-day culture with IL-2 alone led to a higher expression of CD80 and CD86 for liver CD8+ T cells (approx. 30%) compared with blood (9.1% for CD80, 2.8% for CD86) (figure 6b). In contrast, anti-CD3 culture alone led to greater expression of CD86 on CD8+ T cells in blood compared with liver. We hypothesize that these findings indicate that liver infiltrating T cells of patients with chronic HCV infection recently received stimulation via their TCR without supporting cytokine. As such, they respond poorly to further TCR stimulation alone (via anti-CD3) but do respond rapidly to IL-2 alone (or the combination of anti-CD3+IL-2) by increased expression of CD80 and CD86.
Figure 6
Figure 6
Liver infiltrating CD8+ T cells express CD80 and CD86 after in vitro stimulation with IL-2
Expression of CD86 is linked to STAT5 signaling
Our studies identified the importance of IL-2 signaling for the expression of CD86 on T cells. In fact, without IL-2 in the culture media, minimal CD86 expression was observed on sorted CD8+ T cells (figure 2c). However, we had noted low-level CD86 expression on CD8+ T cells after in vitro stimulation of whole PBMC from some patients with anti-CD3 alone (figure 2a and figure 6b) in contrast to a lack of CD86 expression after anti-CD3 stimulation alone of sorted CD8+ T cells. Thus, we investigated the level of pSTAT5 expression in relation to CD86 expression in cell culture after TCR stimulation with anti-CD3 alone (figure 7a) and the level of pSTAT5 expression after anti-CD3 stimulation alone in PBMC versus sorted CD8+ T cells (figure 7b and figure 7c). The anti-CD3 and IL-2 conditions are shown as positive controls. Importantly, we found that anti-CD3 stimulation alone of PBMC also led to transient levels of STAT5 phosphorylation in CD8+ T cells (25% noted at day 1) and to transient, low-level expression of CD86 (figure 7a, upper row). Addition of IL-2 to anti-CD3 stimulation led to a greater frequency of CD8+ cells expressing pSTAT5 and to prolonged pSTAT5 expression as expected (figure 7, lower row). Evaluating CD8+ T cells in culture over time showed that all CD8+ T cells eventually expressing CD86 also expressed pSTAT5, whether stimulated with anti-CD3 alone or with anti-CD3 and IL-2 (figure 7a). Since we did not see expression of CD86 on sorted CD8+ T cells in culture after anti-CD3 stimulation alone (figure 2c), we repeated the pSTAT5 experiment on sorted CD8+ T cells (figure 7b and figure 7c). In concordance with the importance of IL-2 signaling for CD86 expression, there was no pSTAT5 expression on sorted CD8+ T cells after anti-CD3 stimulation alone in contrast with the findings in PBMC (figure 7b). With the anti-CD3 stimulation alone of PBMC, approximately 20-30 % of CD8+ T cells expressed pSTAT5 at day 1 (figure 7a and figure 7c). Consistently, we found a lack of pSTAT5 expression after anti-CD3 stimulation alone of sorted CD8+ T cells in contrast to anti-CD3 stimulation of PBMC (data not shown). We hypothesize that IL-2 released by CD4+ T cells in cultured PBMC after anti-CD3 stimulation alone contributed to the expression of CD86 on CD8+ T cells in this condition and that lack of CD4+ T cells in sorted CD8+ T cells led to a lack of pSTAT5 expression (figure 7) and lack of CD86 expression (figure 2a) after anti-CD3 stimulation alone. Based on these studies, we conclude that pSTAT5 expression in CD8+ T cells is critical for CD86 expression after TCR stimulation of bulk PBMC and sorted CD8+ T cells.
Figure 7
Figure 7
Expression of CD86 is tightly linked to STAT5 phosphorylation after TCR stimulation
B7/CD28 family molecules play a central role in the generation and modulation of the adaptive T cell immune response. A balance of costimulatory and coinhibitory signaling governs the activation and function of the responding T cells (reviewed in (21)). Classically, it is the B7 ligand expressed on the antigen presenting cell signaling to the CD28 family receptor on the T cell that directs the T cell response. However, a number of studies have also identified B7 ligand expression on T cells (16-23), and linked its expression to an enhancement of the T cell response (16). Expression of CD80 on a human CD4+ T cell clone enhanced a mixed lymphocyte reaction (MLR) to resting peripheral blood responder T cells (16) and expression of CD86 on anti-CD3 stimulated and paraformaldehyde fixed human T cells enhanced interferon-gamma production and proliferation of naïve CD4+ T cells responding to suboptimal concentrations of anti-CD3 (18). Furthermore, fixed CD86+ but not CD86- T cells induced an MLR response that was partly decreased by neutralizing anti-CD86 monoclonal antibodies (18). Despite these studies, the importance of B7 ligand expression on virus specific CD8+ T cells is not known.
In the acute phase of infection, the inhibitory receptor, PD-1, is often highly expressed on HCV specific CD8+ T cells (7, 15), so we hypothesized that other costimulatory signals are important at this early phase of infection to enable an effective immune response. In the current study, we demonstrate that the B7 ligand, CD86, is highly expressed on HCV specific CD8+ T cells during the early acute phase of infection and not during the later phase of acute infection or during chronic infection even at the site of infection in the liver. Significant CD86 expression on HCV specific CD8+ T cells was not detected at any later time points for all of the acutely infected patients developing chronic infection nor for any other chronically infected patients that we evaluated. For some patients in the acute phase of HCV infection, HCV specific CD8+ T cells also expressed CD80, though expression was not seen in other patients with acute infection. Currently, why CD80 is expressed on HCV specific CD8+ T cells from some patients with acute infection but not others is not well understood. We hypothesize that this may be related to differing kinetics of CD86 and CD80 expression or to different levels of signaling required for expression of CD86 and CD80.
In this study, we investigated the significance of B7 ligand expression on T cells and found that high-level expression was delayed after brief in vitro culture (5-7 days) and was linked with recent common gamma-chain cytokine signaling. This was in contrast with other “activation markers” such as CD69, CD38, HLA-DR or CD25 whose expression could rapidly (1-3 days) be induced by TCR stimulation alone after brief in vitro culture of PBMC. Hence, our findings support the hypothesis that B7 ligand expression on T cells is a unique marker that identifies recent stimulation via TCR in the presence of sufficient supportive cytokine. The lack of B7 ligand expression on liver infiltrating HCV specific CD8+ T cells in chronic infection, despite high-level expression of activation markers, highlights a critical deficit in supportive cytokine signaling that contributes to the waning immune response to HCV. Recent studies in mice demonstrate IL-7 can be produced by hepatocytes themselves and that IL-7 is important in regulating the expansion of T cells in response to LPS (39). Though hepatocyte IL-7 was not found to be important in pathogen-specific CD8+ T cell proliferation in this study (39), improved understanding of the cytokine milieu in the liver of patients with hepatotropic viral infection is clearly important.
IL-2, in particular, has been shown to be important in the generation of effective immune responses to HCV infection, and secretion of IL-2 by CD4+ T cells during the acute phase infection is critical for sustained and effective adaptive CD8+ T cell responses (38, 40, 41). CD4+ T cells from patients with self-limited evolution of infection produced considerably more IL-2 in response to HCV recombinant proteins compared with patients with chronically evolving disease (38, 40, 41). In the chimpanzee model of HCV infection, depletion of CD4+ T cells prior to infection led to an inability to clear viremia (42). On progression to chronic HCV infection, a preferential loss of IL-2 secreting CD4+ T cells has been noted (43), and HCV specific CD8+ T cells from the peripheral blood of patients with chronic infection have an impaired ability to proliferate that can be rescued in vitro by exposure to IL-2 (44). Our study further supports the critical loss of IL-2 during progression to chronic infection and identifies ex vivo pSTAT5 signaling and expression of B7 ligands (CD86 and CD80) as important markers of recent effective signaling. Though our study highlights the role of common gamma-chain cytokines such as IL-2 in the expression of CD86 on T cells, future studies will need to investigate the role of other inflammatory cytokines in the expression of CD86 or CD80 on T cells during HCV infection. Furthermore, determining whether the level of CD86 expression or the timing of CD86 expression is a determinant of viral clearance versus persistence will require longitudinal studies with larger numbers of acute patients.
Studies of liver infiltrating HCV specific CD8+ T cells are critical to understand the failure of the immune response seen in most patients with HCV infection. Previous studies have demonstrated high activation state of these cells in the liver but poor functionality in the chronic phase of infection (7-9). A number of factors likely contribute to the waning immune response and include high-level expression of PD-1 (11-13), infiltration by Tregs ((45) and reviewed by (46)), and a loss of CD4+ T cell help (43). We hypothesize that a central feature of each of these mechanisms is the loss of IL-2 signaling on HCV specific CD8+ T cells. Recent studies on the mechanism of action of PD-1 signaling indicate the possibility that PD-1 signaling might directly prevent STAT5 phosphorylation via activation of the SHP-2 phosphatase (45, 47). In addition, one of the proposed mechanisms of action of Tregs is to act as an “IL-2-sink” and depleting the immunological milieu of supportive cytokine (48). Our study is the first characterize pSTAT5 on virus specific CD8+ T cells using tetramers. Our findings indicate an early loss of pSTAT5 that occurs in the acute phase of infection and a lack of high-level pSTAT5 in liver infiltrating HCV specific CD8+ T cells despite persistent infection and persistent activation. Future studies will need to determine the relative contribution of PD-1 signaling, Treg infiltration and CD4+ T helper cell loss in the reduction of pSTAT5 in HCV specific CD8+ T cells.
Clearly, differences in the acute versus chronic immune response are evident in HCV infection and this can be seen in the differing clinical responses and degree of liver injury as measured by ALT in the patients infected with HCV. Based on our study, we hypothesize that other B7 molecules, and in particular, CD86, that are expressed at this early phase of infection provide costimulatory signals via T:T interactions, that enhance the immune response at this early stage of infection. In this study, we assessed for the ability of HCV specific CD8+ T cells expressing CD86 to function as antigen presenting cells in a T:T dependent manner by sorting on CD3+CD8+ T cells from fresh PBMC from a808 (day 0) and culturing in the presence of HCV peptide, HCV peptide plus IL-2, and HCV peptide plus anti-CD86 with/without IL-2 (data not shown). We were unable to demonstrate an ability of HCV specific CD8+ T cells to present antigen in this manner, indicating that these T cells functioned poorly in presenting antigen despite expression of B7 ligands (data not shown). Though we did not see evidence of direct antigen presentation by HCV specific CD8+ T cells to other T cells, we hypothesize that expression of CD86 on HCV specific CD8+ T cells is important for other T:T interactions by providing costimulation to neighboring T cells interacting with an antigen presenting cell or via an cell autonomous costimulatory signal. Unfortunately, given the difficulty in separating the effect of CD86 expression on antigen presenting cells from CD86 expression on T cells during in vitro assays, we were unable to directly demonstrate this effect.
In this study, we found that CD86 expression was lost early during HCV infection (figure 1) despite persistent high-level PD-1 expression on HCV specific CD8+ T cells in acute infection (7, 15). This loss of CD86 expression also coincided with decreased liver inflammation as measured by ALT levels. Thus, we hypothesize that, as HCV progresses to chronic infection, the persistent negative signals via receptors such as PD-1 and the loss of positive signals via CD86 on T cells, tip the balance in favor of a waning response. Net negative costimulatory/coinhibitory signals to T cells at this phase of infection may be adaptive for a host that is unable to clear a virus, and waning CD86 expression may be a mechanism to prevent further high-level liver damage. If, indeed, CD86 expression on HCV specific CD8+ T cells is shown to provide direct costimulation to other HCV specific CD8+ T cells in acute infection, prolonging or modulating CD86 expression on these T cells may also be a mechanism that can be utilized to enhance future therapies for patients with chronic HCV infection.
Acknowledgements
The authors would like to thank Francie Lasseter, Beverly Weaver, Melissa K. Osborn, Ana Howells-Ferrerira and the Emory Transplant clinical group for patient cohort coordination. We would like to also thank Benton Lawson and Natalia Kozyr for assistance with rRT-PCR experiments, Enrique Martinez and Naasha Talati for patient referrals, and the patients who agreed to participate.
Non-standard abbreviations
LIMCliver infiltrating mononuclear cells
pSTATphosphorylated STAT

Footnotes
1We would like to acknowledge the support from the Bill and Melinda Gates Foundation Grand Challenges in Global Health (AG, GC#12 to Rafi Ahmed), EVC/CFAR Immunology Core P30 AI050409 (HR, CI, AG), Cancer Research Institute Investigator Award (AG), the Yerkes Research Center Base Grant RR-00165, the Canadian Institutes for Health Research (CIHR) {MOP-74524 (NHS, JB)} and the Fonds de la Recherche en Santé du Quebec (FRSQ) AIDS and Infectious Disease Network (SIDA-MI) (NHS, JB), FRSQ Senior clinical research award (JB), CIHR New Investigator Award (NHS), and the Public Health Service {K08 AI072191 (HR), and AI070101 (AG)}.
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