Ineffective CD8+ T-Cell Immunity to Adeno-Associated Virus Can Result in Prolonged Liver Injury and Fibrogenesis
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.08.004
ISSN1525-2191
AutoresJessica Spahn, Robert H. Pierce, Ian Nicholas Crispe,
Tópico(s)Hepatitis B Virus Studies
ResumoChronic viral hepatitis depends on the inability of the T-cell immune response to eradicate antigen. This results in a sustained immune response accompanied by tissue injury and fibrogenesis. We have created a mouse model that reproduces these effects, based on the response of CD8+ T cells to hepatocellular antigen delivered by an adeno-associated virus (AAV) vector. Ten thousand antigen-specific CD8+ T cells undergo slow expansion in the liver and can precipitate a subacute inflammatory hepatitis with stellate cell activation and fibrosis. Over time, antigen-specific CD8+ T cells show signs of exhaustion, including high expression of PD-1, and eventually both inflammation and fibrosis resolve. This model allows the investigation of both chronic liver immunopathology and its resolution. Chronic viral hepatitis depends on the inability of the T-cell immune response to eradicate antigen. This results in a sustained immune response accompanied by tissue injury and fibrogenesis. We have created a mouse model that reproduces these effects, based on the response of CD8+ T cells to hepatocellular antigen delivered by an adeno-associated virus (AAV) vector. Ten thousand antigen-specific CD8+ T cells undergo slow expansion in the liver and can precipitate a subacute inflammatory hepatitis with stellate cell activation and fibrosis. Over time, antigen-specific CD8+ T cells show signs of exhaustion, including high expression of PD-1, and eventually both inflammation and fibrosis resolve. This model allows the investigation of both chronic liver immunopathology and its resolution. The liver has unique immunological properties that often complicate the ability of the immune system to effectively eliminate hepatotropic pathogens such as hepatitis B and hepatitis C viruses (HBV and HCV). A significant percentage of people infected with these viruses generate ineffective CD8+ T-cell responses that fail to eliminate the pathogen but are capable of causing prolonged liver damage, often resulting in fibrosis, cirrhosis, and hepatocellular carcinoma. For the development of more effective therapies, it is necessary to understand the mechanism by which CD8+ T cells are activated and the reasons for their failure. The CD8+ T-cell response to hepatocellular-derived antigen is an area of active research. Several studies have shown that hepatocytes are capable of directly activating CD8+ T cells.1Bertolino P. Bowen D.G. McCaughan G.W. Fazekas de St Groth B. Antigen-specific primary activation of CD8+ T cells within the liver.J Immunol. 2001; 166: 5430-5438PubMed Google Scholar, 2Lee Y.C. Lu L. Fu F. Li W. Thomson A.W. Fung J.J. Qian S. Hepatocytes and liver nonparenchymal cells induce apoptosis in activated T cells.Transplant Proc. 1999; 31: 784Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 3Bertolino P. Trescol-Biemont M.C. Rabourdin-Combe C. Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival.Eur J Immunol. 1998; 28: 221-236Crossref PubMed Scopus (205) Google Scholar, 4Wuensch S.A. Spahn J. Crispe I.N. Direct, help-independent priming of CD8+ T cells by adeno-associated virus-transduced hepatocytes.Hepatology. 2010; 52: 1068-1077Crossref PubMed Scopus (29) Google Scholar This is unusual, in that primary activation of T cells generally occurs in the lymph node by professional antigen-presenting cells. CD8+ T-cell activation on hepatocytes, however, does not always generate effective immune responses and can even cause the T cells to undergo apoptosis.3Bertolino P. Trescol-Biemont M.C. Rabourdin-Combe C. Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival.Eur J Immunol. 1998; 28: 221-236Crossref PubMed Scopus (205) Google Scholar Hepatocyte-driven activation of CD8+ T cells also leads to the generation of a unique population of PD-1hi cells.4Wuensch S.A. Spahn J. Crispe I.N. Direct, help-independent priming of CD8+ T cells by adeno-associated virus-transduced hepatocytes.Hepatology. 2010; 52: 1068-1077Crossref PubMed Scopus (29) Google Scholar These two outcomes, together with poor activation of CD4+ T cells,4Wuensch S.A. Spahn J. Crispe I.N. Direct, help-independent priming of CD8+ T cells by adeno-associated virus-transduced hepatocytes.Hepatology. 2010; 52: 1068-1077Crossref PubMed Scopus (29) Google Scholar likely contribute to the tolerogenic environment in the liver and the propensity of hepatotropic pathogens to cause chronic infections and long-term hepatitis. Many mouse models devised to study hepatitis cause only acute liver damage. The bile duct ligation model does result in chronic injury, but it is less relevant to the immunopathology of viral hepatitis because it is not initiated by a T-cell response. Nonetheless, valuable information has been obtained from these models. The concanavalin A and lipopolysaccharide models have demonstrated the importance of cytokines such as tumor necrosis factor α (TNFα) and interferon γ (IFNγ) in causing liver damage.5Sass G. Heinlein S. Agli A. Bang R. Schumann J. Tiegs G. Cytokine expression in three mouse models of experimental hepatitis.Cytokine. 2002; 19: 115-120Crossref PubMed Scopus (185) Google Scholar, 6Tiegs G. Hentschel J. Wendel A. A T cell-dependent experimental liver injury in mice inducible by concanavalin A.J Clin Invest. 1992; 90: 196-203Crossref PubMed Scopus (958) Google Scholar The HBV transgenic model has shown the ability of these cytokines to also control infection in a noncytopathic manner,7G Guidotti L.G. Ishikawa T. Hobbs M.V. Matzke B. Schreiber R. Chisari F.V. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes.Immunity. 1996; 4: 25-36Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar and the lymphocytic choriomeningitis virus model has described a hepatotropic virus that is eliminated by an effective CD8+ T-cell response.8Zinkernagel R.M. Haenseler E. Leist T. Cerny A. Hengartner H. Althage A. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay?.J Exp Med. 1986; 164: 1075-1092Crossref PubMed Scopus (167) Google Scholar We have previously used an adeno-associated virus (AAV) model of hepatocellular antigen delivery to study the resulting CD8+ T-cell response. The delivery of a transgene to the liver using AAV as a vector leads to hepatocyte-restricted expression.9Wuensch S.A. Pierce R.H. Crispe I.N. Local intrahepatic CD8+ T cell activation by a non-self-antigen results in full functional differentiation.J Immunol. 2006; 177: 1689-1697PubMed Google Scholar Approximately 2% to 5% of hepatocytes are transduced with these AAV vectors.10Snyder R.O. Miao C.H. Patijn G.A. Spratt S.K. Danos O. Nagy D. Gown A.M. Winther B. Meuse L. Cohen L.K. Thompson A.R. Kay M.A. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.Nat Genet. 1997; 16: 270-276Crossref PubMed Scopus (497) Google Scholar Because patients with HCV typically have 5% (but sometimes up to 60%) infected hepatocytes,11Gosálvez J. Rodríguez-Iñigo E. Ramiro-Díaz J.L. Bartolomé J. Tomás J.F. Oliva H. Carreño V. Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis.Hepatology. 1998; 27: 1428-1434Crossref PubMed Scopus (36) Google Scholar the number of antigen-expressing hepatocytes obtained using this vector is analogous to the number infected by HCV. In an AAV study of acute hepatitis, the administration of transgene-specific CD8+ T cells resulted in liver damage that is mediated by both IFNγ and TNFα.12Giannandrea M. Pierce R.H. Crispe I.N. Indirect action of tumor necrosis factor-alpha in liver injury during the CD8+ T cell response to an adeno-associated virus vector in mice.Hepatology. 2009; 49: 2010-2020Crossref PubMed Scopus (15) Google Scholar CD8+ T cells activated in this manner express a high level of the inhibitory receptor PD-1, but they are also capable of cytotoxicity against antigen-expressing splenocytes.4Wuensch S.A. Spahn J. Crispe I.N. Direct, help-independent priming of CD8+ T cells by adeno-associated virus-transduced hepatocytes.Hepatology. 2010; 52: 1068-1077Crossref PubMed Scopus (29) Google Scholar, 9Wuensch S.A. Pierce R.H. Crispe I.N. Local intrahepatic CD8+ T cell activation by a non-self-antigen results in full functional differentiation.J Immunol. 2006; 177: 1689-1697PubMed Google Scholar When an AAV-OVA vector is used (expressing the whole ovalbumin protein), activation of the CD8+ T cells occurs in a CD4+ T-cell help-independent manner. CD4+ T cells specific for ovalbumin peptides are not activated in this model, based on the absence of carboxyfluorescein succinimidyl ester (CFSE) dilution, down-regulation of CD62L, and up-regulation of CD44. Furthermore, in mice lacking CD4+ T cells, OT-1 CD8+ T cells are activated and proliferate to the same extent as when CD4+ T cells are present.4Wuensch S.A. Spahn J. Crispe I.N. Direct, help-independent priming of CD8+ T cells by adeno-associated virus-transduced hepatocytes.Hepatology. 2010; 52: 1068-1077Crossref PubMed Scopus (29) Google Scholar These experimental models share some features with chronic viral hepatitis, but the injury is acute and self-limiting. To create chronic immunopathology against the same persistent antigen, we used an adoptive transfer of antigen-specific CD8+ T cells at a cell dose much closer to the natural precursor frequency. With the present study, we show that the activation of 1 × 104 OT-1 CD8+ T cells against hepatocyte-derived antigen results in slower clonal expansion, which can be accompanied by long-term inflammation, liver damage, and fibrosis. This immunopathology mimics HCV in many respects, including chronicity, hepatocellular injury, and fibrogenesis. In addition, the strength of the T-cell response dictates the ability of those cells to eliminate vector expression, directly affecting hepatitis. C57BL/6, B6.SJL-PtprcaPepcb/BoyJ (CD45.1 transgenic), B6.PL-Thy1a/CyJ (Thy1.1 transgenic), and C57BL/6J-Tyrc-2J/(B6 albino) male mice were purchased from the Jackson Laboratory (Bar Harbor, ME). OT-1 transgenic mice,13Hogquist K.A. Jameson S.C. Heath W.R. Howard J.L. Bevan M.J. Carbone F.R. T cell receptor antagonist peptides induce positive selection.Cell. 1994; 76: 17-27Abstract Full Text PDF PubMed Scopus (2266) Google Scholar whose T cells recognize the SIINFEKL peptide, on either the CD45.1 or Thy1.1 background were maintained in house. All animals receiving the AAV-eGFP or AAV-eGFP-SIINFEKL vector were C57BL/6 males between 6 and 7 weeks old. All experiments were approved by the Institutional Animal Care and Use Committee. AAV2 vectors containing either enhanced green fluorescent protein (eGFP) or eGFP-SIINFEKL fusion protein under the control of the human elongation factor α (EF1α) promoter were purchased from the Viral Vector Core Facility of the Columbus Children's Research Institute (Columbus, OH). Mice aged 7 to 8 weeks were anesthetized with tribromoethanol (Avertin; Sigma-Aldrich, St. Louis, MO) at a dose of 0.017 mL/g body weight. A small incision was made just below the sternum, and the right lobe of the liver was exposed. Using a 29G insulin syringe, 60 μL of vector (5.88 × 1010 DNase-resistant particles) was injected directly into the liver. The peritoneal cavity was sutured using absorbable sutures (Vicryl; Ethicon, Somerville, NJ), and the skin was closed with wound clips. CD8+ T cells were isolated from the spleens and lymph nodes of OT-1 transgenic mice. Organs were homogenized between the frosty sides of two slides. After a washing, the cell suspension was layered on top of Lympholyte M density gradient separation medium (Cedarlane Laboratories, Burlington, ON, Canada) and spun at 650 × g for 20 minutes at room temperature. The interface was then collected and counted. The CD8a+ T-cell isolation kit for isolation of untouched CD8+ T Cells was used to purify OT-1 cells resulting in more than 90% purity (Miltenyi Biotec, Auburn, CA; Bergisch Gladbach, Germany). Cells were then labeled with CFSE dye (Invitrogen, Carlsbad, CA) and adoptively transferred into mice through the tail vein in a volume of 200 μL at 3 weeks after AAV injection. These cells were detected by flow cytometry using the allotypic marker CD45.1 after gating on CD8+ T cells. Mice were anesthetized using ketamine and xylazine (100 mg/kg ketamine; 10 mg/kg xylazine) and then were given an intraperitoneal injection of the substrate, d-luciferin (214 μg/g body weight; Xenogen Biosciences-Caliper Life Sciences, Alameda, CA). After 5 minutes, mice were placed in the imaging chamber of the Xenogen in vivo imaging system (IVIS-100). A grayscale image of the mouse was captured with a 10-cm field of view, a 0.2-second exposure time, an f/16 aperture, and an open filter. Bioluminescence data were then acquired with mice positioned supine, to image the ventral surface. Acquisition time was 5 minutes. Blood was collected via cardiac puncture and allowed to coagulate at room temperature for 2 to 3 hours. Coagulated blood was then centrifuged and the serum was collected. Samples were sent to the Strong Health Clinical Laboratories (Rochester, NY) or Phoenix Central Laboratory (Everett, WA) for measurement of alanine transaminase (ALT, U/L). Experimental animals were sacrificed using CO2, and PBS was injected into the portal vein to flush out peripheral lymphocytes from the liver. Livers were then manually homogenized through a tea strainer using a 5-mL syringe plunger. The cell suspension was then digested in RPMI 1640 medium containing 0.05% collagenase IV (Sigma-Aldrich) and 0.002% DNase I (Sigma-Aldrich) at 37°C for 40 minutes with gentle agitation. After digestion and washing, the cell pellet was resuspended in 2.75 mL 40% OptiPrep cell separation medium (Accurate Chemical & Scientific Corporation, Westbury, NY) and overlaid with 2.25 mL of serum-free RPMI 1640 medium. The gradient was spun at 1015 × g for 25 minutes at 4°C. The interface was collected and stained for flow cytometry. Cell surface staining was performed using PBS and 1% bovine serum albumin (Sigma-Aldrich). The following antibodies were used: CD8 (53–6.7), CD45.1 (A20), Thy1.1 (OX-7), CD44 (IM7), PD-1 (RMP1-30), CD127 (A7R34), DR5 (MD5-1), and CD62L (MEL-14). Antibodies were purchased from eBioscience (San Diego, CA), BD Biosciences (San Jose, CA), or BioLegend (San Diego, CA). Streptavidin in Pacific Orange was purchased from Invitrogen. Flow cytometric analysis was performed on either a FACSCalibur or LSR II system (BD Biosciences). Data were analyzed using FlowJo software version 6.4.7 (Tree Star, Ashland, OR). Whole livers were homogenized using a mortar and pestle and liquid nitrogen. RNA was isolated using TRIzol reagent (Invitrogen) according to manufacturer's instructions. Genomic DNA was isolated using phenol-chloroform (Sigma-Aldrich). Custom primers and TaqMan MGB probes for eGFP were designed by Applied Biosystems (Carlsbad, CA). The sequences for the primers and probes were as follows: 5′-GCTACCCCGACCACATGAAG-3′ (forward primer), 5′CGGGCATGGCGGACTT-3′ (reverse primer) and 5′CAGCACGACTTCTTC-3′ (probe). Primer and probe assays were also purchased from Applied Biosystems for CXCL9, CXCL10, ICAM-1, and VCAM-1. For cDNA synthesis, an Applied Biosystems high-capacity cDNA reverse transcription kit was used. PCR reactions were performed using Applied Biosystems TaqMan universal PCR master mix, no AmpErase UNG. All results for real-time quantitative PCR (qPCR) were normalized to β-actin, using an intronic actin sequence for genomic DNA. For quantitative reverse transcription PCR (qRT-PCR), results were normalized to β-actin using an exonic actin sequence. Reverse transcription reactions were performed at the Functional Genomic Center, University of Rochester, as was qPCR using an Applied Biosystems 7900HT sequence detection system; alternatively, these reactions were performed at Seattle Biomedical Research Institute on an Applied Biosystems 7500Fast. Data were analyzed by the ΔCt method, as 2−(GFP Mean Ct − Actin Mean Ct). The University of Rochester Medical Center Department of Pathology and Laboratory Medicine or Merck Research Laboratories (Palo Alto, CA) was given formalin-fixed slices of liver tissue after harvest. Tissue was then embedded into paraffin blocks. To detect the presence of hepatitis, H&E staining was performed. Sirius Red was used to demonstrate fibrosis. Formalin-fixed, paraffin-embedded tissue blocks were also stained for CD3, F4/80, and α-smooth muscle actin (SMA). The CD3 antibody (DakoCytomation, Glostrup, Denmark; Carpinteria, CA) was polyclonal and used at a dilution of 1:200 or 0.6 μg/mL. The F4/80 antibody (eBioscience) was used at 1:400 or 0.5 μg/mL. The anti-SMA antibody (DakoCytomation) was used at a 1:150 dilution. Data were analyzed using Prism software version 5 (GraphPad Software, La Jolla, CA). Most experiments were analyzed using a Mann-Whitney test, and data are reported as means ± SEM. When indicated, experiments were pooled and analyzed using two-way analysis of variance. Significance was determined by a P value of <0.05. To study CD8+ T-cell activation and the associated immunopathology in the liver, an adeno-associated virus-based replication-defective vector was used to deliver a fusion protein of enhanced green fluorescent protein and the ovalbumin peptide SIINFEKL (AAV-GFP-SIINFEKL). By directly injecting this vector into the liver, hepatocyte-restricted expression is achieved.9Wuensch S.A. Pierce R.H. Crispe I.N. Local intrahepatic CD8+ T cell activation by a non-self-antigen results in full functional differentiation.J Immunol. 2006; 177: 1689-1697PubMed Google Scholar At 3 weeks after vector transduction, OT-1 T cells from a transgenic mouse line whose CD8+ T cells specifically recognize the SIINFEKL peptide in the context of the H-2kb molecule were adoptively transferred into the host. AAV-GFP-SIINFEKL-treated mice without OT-1 CD8+ T cells were used as controls. In previous studies, 5 × 106 OT-1 T cells were administered, cell numbers peaked at day 3 and contracted thereafter, and this resulted in acute liver damage; however, this injury rapidly resolved, rendering this a limited model for the analysis of chronic liver immunopathology.12Giannandrea M. Pierce R.H. Crispe I.N. Indirect action of tumor necrosis factor-alpha in liver injury during the CD8+ T cell response to an adeno-associated virus vector in mice.Hepatology. 2009; 49: 2010-2020Crossref PubMed Scopus (15) Google Scholar Badovinac et al14Badovinac V.P. Haring J.S. Harty J.T. Initial T cell receptor transgenic cell precursor frequency dictates critical aspects of the CD8(+) T cell response to infection.Immunity. 2007; 26: 827-841Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar have shown that the number of adoptively transferred transgenic cells has a significant influence on the outcome of the CD8+ T-cell response, affecting the proliferative potential, the kinetics of expansion, and the phenotype of the T cells. To gain a better understanding of chronic liver inflammation and to more closely mimic the number of endogenous CD8+ T-cell precursors, we induced liver immunopathology using 1 × 104 OT-1 CD8+ T cells. To determine the kinetics of OT-1 CD8+ T-cell expansion using this number of cells, we used OT-1 T cells that express firefly luciferase under control of the human CD2 promoter. These T cells, after administration of the substrate luciferin, produce bioluminescence in the presence of ATP. Using live whole-mouse imaging with a Xenogen IVIS instrument, we were able to obtain longitudinal data showing OT-1 CD8+ T-cell accumulation in various anatomical sites. The OT-1 CD8+ T-cell localization data are collected in the form of photons emitted in a given area, and this measure is directly correlated with the number of OT-1 CD8+ T cells in a tissue.15Azadniv M. Dugger K. Bowers W.J. Weaver C. Crispe I.N. Imaging CD8+ T cell dynamics in vivo using a transgenic luciferase reporter.Int Immunol. 2007; 19: 1165-1173Crossref PubMed Scopus (23) Google Scholar Representative images for this type of data are shown in Figure 1A. Detection of the OT-1 CD8+ T cells was first seen on day 13 in the area corresponding to the liver, and 10 days later a large expansion of these cells had occurred. This was seen most drastically in one mouse (Figure 1A, bottom left); although in this mouse there is some bioluminescent signal in the lower abdomen, the vast majority of light is being emitted from the area corresponding to the liver. OT-1 CD8+ T cells reached a peak number at approximately day 15, but remained elevated for at least 1 week thereafter (Figure 1B). The bioluminescent signal was significantly stronger than the control level at all points after day 13 (P < 0.05). To confirm these findings, mice were harvested every 5 days after adoptive transfer from day 10 to day 35. Flow cytometry was used to detect the OT-1 CD8+ T cells by gating on CD8+ T cells and the allotypic marker CD45.1. This alternative method corroborated the previous findings obtained using live imaging (Figure 1, C and D). A peak in OT-1 CD8+ T-cell frequency was reached between days 10 and 15, after which the frequency decreased; thus, the OT-1 T cells were approximately 35% of all CD8+ T cells (Figure 1D). Even out to day 35, however, the CD8+ T-cell compartment consisted of approximately 10% OT-1 cells. In two such experiments, we estimated the absolute number of OT-1 T cells by multiplying the total viable leukocyte count by the percentage of viable cells that were OT-1 T cells. In one experiment, the peak cell number was 1.8 × 106 OT-1 cells at day 20; in the other, it was 2.0 × 106 OT-1 cells at day 15. If we were to assume that all of the input 1 × 104 OT-1 T cells were participating in the local clonal expansion, and that our recovery of liver leukocytes was 100% effective, we would calculate the magnitude of expansion to be approximately 200-fold. However, it is certain that only a subset of the input OT-1 T cells are participating, and that the liver leukocyte recovery is very inefficient. Therefore, all we can say is that the magnitude of clonal expansion in the liver is very much greater than 200-fold. Throughout this time course, OT-1 CD8+ T cells in the spleen and lymph node never exceeded 2.7% and 0.34% of total CD8+ T cells, respectively (data not shown), indicating that the OT-1 CD8+ T cells were activated in the liver, as we have reported previously.9Wuensch S.A. Pierce R.H. Crispe I.N. Local intrahepatic CD8+ T cell activation by a non-self-antigen results in full functional differentiation.J Immunol. 2006; 177: 1689-1697PubMed Google Scholar Staining of liver sections with anti-CD3 at various time points after adoptive transfer also showed an increase in CD3+ cells, starting at day 5 and peaking at day 15 (Figure 2). In principle, these cells could be a mixture variously of OT-1 T cells, polyclonal host CD8+ T cells, CD4+ T cells, NK-T cells, or TCRγδ cells. From flow cytometric analyses (Figure 1, C and D), we can conclude that only a subset (10% to 35%) of the CD8+ T cells were in fact OT-1, and although their numbers argue that the OT-1 cells must have undergone many rounds of cell division, they were still only a minority of the CD3+ cells in the infiltrate. CD8+ T-cell activation is characterized by the production of the multifunctional cytokine IFNγ (reviewed by Harty et al16Harty J.T. Tvinnereim A.R. White D.W. CD8+ T cell effector mechanisms in resistance to infection.Annu Rev Immunol. 2000; 18: 275-308Crossref PubMed Scopus (550) Google Scholar). Signaling through the IFNγ receptor is necessary for OT-1 CD8+ T-cell-mediated acute liver damage.12Giannandrea M. Pierce R.H. Crispe I.N. Indirect action of tumor necrosis factor-alpha in liver injury during the CD8+ T cell response to an adeno-associated virus vector in mice.Hepatology. 2009; 49: 2010-2020Crossref PubMed Scopus (15) Google Scholar We therefore looked at expression of IFNγ during the time that OT-1 CD8+ T cells were present in the liver. RNA was harvested from the livers of mice treated with AAV-GFP-SIINFEKL with or without OT-1 T cells every 5 days. Quantitative RT-PCR shows that there was a significant increase in the expression of IFNγ when OT-1 CD8+ T cells were activated in the liver at days 10 and 25 (P = 0.008 and P = 0.014, respectively) (see Supplemental Figure S1 at http://ajp.amjpathol.org). These results show that the activation of OT-1 CD8+ T cells in response to hepatocyte-derived antigen leads to an increase in the expression of IFNγ. Infections of the liver frequently develop into a chronic state in which activation of the CD8+ T cells is suboptimal and not capable of eliminating the pathogen.17Spangenberg H.C. Viazov S. Kersting N. Neumann-Haefelin C. McKinney D. Roggendorf M. von Weizsäcker F. Blum H.E. Thimme R. Intrahepatic CD8+ T-cell failure during chronic hepatitis C virus infection.Hepatology. 2005; 42: 828-837Crossref PubMed Scopus (134) Google Scholar, 18Gruener N.H. Lechner F. Jung M.C. Diepolder H. Gerlach T. Lauer G. Walker B. Sullivan J. Phillips R. Pape G.R. Klenerman P. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus.J Virol. 2001; 75: 5550-5558Crossref PubMed Scopus (458) Google Scholar This may be due to the unique environment of the liver and the ability of CD8+ T cells to be activated directly on parenchymal cells in the liver without CD4+ T-cell help.4Wuensch S.A. Spahn J. Crispe I.N. Direct, help-independent priming of CD8+ T cells by adeno-associated virus-transduced hepatocytes.Hepatology. 2010; 52: 1068-1077Crossref PubMed Scopus (29) Google Scholar Because of the substantial increase in OT-1 CD8+ T-cell numbers after activation by hepatocellular antigen and in view of the fact that this resulted in increased IFNγ, we next wanted to obtain a more direct representation of the activation state of the OT-1 CD8+ T cells. Using flow cytometry, we analyzed OT-1 CD8+ T cells for proliferation and activation markers at various time points after the adoptive transfer of 1 × 104 OT-1 T cells. We documented OT-1 CD8+ T-cell division by labeling the T cells with the dye CFSE, but even at early time points the OT-1 CD8+ T cells had diluted the CFSE to background levels, indicating that the cells had undergone at least eight rounds of proliferation.19Hodgkin P.D. Lee J.H. Lyons A.B. B cell differentiation and isotype switching is related to division cycle number.J Exp Med. 1996; 184: 277-281Crossref PubMed Scopus (323) Google Scholar Because of the small number of cells adoptively transferred, it was not possible to use CFSE to visualize earlier proliferation and activation events. The small number of cells also made it impossible to detect the OT-1 CD8+ T cells in a negative control given AAV-GFP without the SIINFEKL peptide. We therefore used host CD8+ T cells from each animal as an internal control for the activated OT-1 CD8+ T cells. The OT-1 cells had high expression of the inhibitory receptor PD-1 up to day 35 after adoptive transfer (Figure 3). These cells were also low in CD62L (the lymph node homing receptor), indicating that they had been activated. With less consistency, we sometimes saw that OT-1 CD8+ T cells had low expression of IL7R-α (CD127). The combination of high PD-1 expression and low CD127 expression is a phenotype characteristic of exhausted CD8+ T cells.20Urbani S. Amadei B. Tola D. Massari M. Schivazappa S. Missale G. Ferrari C. PD-1 expression in acute hepatitis C (HCV) invection is associated with HCV-specific CD8+ exhaustion.J Virol. 2006; 80: 11398-11403Crossref PubMed Scopus (464) Google Scholar, 21Bengsch B. Spangenberg H.C. Kersting N. Neumann-Haefelin C. Panther E. von Weizsäcker F. Blum H.E. Pircher H. Thimme R. Analysis of CD127 and KLRG1 expression on hepatitis C virus-specific CD8+ T cells reveals the existence of different memory T-cell subsets in the peripheral blood and liver.J Virol. 2007; 81: 945-953Crossref PubMed Scopus (94) Google Scholar, 22Radziewicz H. Ibegbu C.C. Fernandez M.L. Workowski K.A. Obideen K. Wehbi M. Hanson H.L. Steinberg J.P. Masopust D. Wherry E.J. Altman J.D. Rouse B.T. Freeman G.J. Ahmed R. Grakoui A. Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.J Virol. 2007; 81: 2545-2553Crossref PubMed Scopus (385) Google Scholar Given that the OT-1 CD8+ T cells with this phenotype also expressed activation markers, their exact functional status was difficult to interpret. Kupffer cell prominence is a common phenomenon in chronic hepatitis.23Roberts J.M. Searle J.W. Cooksley W.G. Histological patterns of prolonged hepatitis C infection.Gastroenterol Jpn. 1993; 28: 37-41Crossref PubMed Scopus (54) Google Scholar, 24Bach N. Thung S.N. Schaffner F. The histological features of chronic hepatitis C and autoimmune chronic hepatitis: a comparative analysis.Hepatology. 1992; 15: 572-577Crossref PubMed Scopus (481) Google Scholar In light of this feature of hepatitis, we next looked at the presence of Kupffer cells in the liver. F4/80 staining of liver sections from mice treated with AAV-GFP-SIINFEKL with or without OT-1 CD8+ T cells showed an increase in Kupffer cells in the presence of antigen-activated OT-1 T cells (Figure 4). Quantitation of this increase shows F4/80 message expression as measured by qRT-PCR (see Supplemental Figure S2 at http://ajp.amjpathol.org). Activation of monocytes by IFNγ leads to increased expression of the chemokines CXCL9 (known also as monokine induced by gamma-IFN, or MIG)
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