Pathogenic Role for Virus-Specific CD4 T Cells in Mice with Coronavirus-Induced Acute Encephalitis
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.051308
ISSN1525-2191
AutoresDaniela Anghelina, Lecia L. Pewe, Stanley Perlman,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoAcute viral encephalitis is believed to result from direct virus destruction of infected cells and from virus-induced host immune response, but the relative contribution of each remains largely unknown. For example, C57BL/6 (B6) mice infected with mouse hepatitis virus (JHM strain, JHMV) develop severe encephalitis, with death occurring within 7 days. Here, we show that the host response to a single JHMV-specific immunodominant CD4 T-cell epitope is critical for severe disease. We engineered a recombinant JHMV with mutations in the immunodominant CD4 T-cell epitope (rJ.MY135Q). Infection of naïve B6 mice with this virus resulted in mild disease with no mortality. However, introduction of a CD4 T-cell epitope from Listeria monocytogenes into rJ.MY135Q generated a highly virulent virus. The decrease in disease severity was not due to a switch from Th1 to Th2 predominance in rJ.MY135Q-infected mice, an effect on CD8 T-cell function, or differential expression of tumor necrosis factor-α by JHMV-specific CD4 T cells. These results show that the response to a single virus-specific CD4 T-cell epitope may contribute to a pathogenic host response in the setting of acute viral disease and that abrogation of this response ameliorates clinical disease without diminishing virus clearance. Acute viral encephalitis is believed to result from direct virus destruction of infected cells and from virus-induced host immune response, but the relative contribution of each remains largely unknown. For example, C57BL/6 (B6) mice infected with mouse hepatitis virus (JHM strain, JHMV) develop severe encephalitis, with death occurring within 7 days. Here, we show that the host response to a single JHMV-specific immunodominant CD4 T-cell epitope is critical for severe disease. We engineered a recombinant JHMV with mutations in the immunodominant CD4 T-cell epitope (rJ.MY135Q). Infection of naïve B6 mice with this virus resulted in mild disease with no mortality. However, introduction of a CD4 T-cell epitope from Listeria monocytogenes into rJ.MY135Q generated a highly virulent virus. The decrease in disease severity was not due to a switch from Th1 to Th2 predominance in rJ.MY135Q-infected mice, an effect on CD8 T-cell function, or differential expression of tumor necrosis factor-α by JHMV-specific CD4 T cells. These results show that the response to a single virus-specific CD4 T-cell epitope may contribute to a pathogenic host response in the setting of acute viral disease and that abrogation of this response ameliorates clinical disease without diminishing virus clearance. Mice infected with the JHM strain of mouse hepatitis virus (JHMV), a neurotropic coronavirus, develop acute and chronic diseases of the central nervous system. The JHMV-infected mouse is most often studied as a model of chronic demyelination because it has similarities to the disease observed in humans with multiple sclerosis.1Stohlman SA Hinton DR Viral induced demyelination.Brain Pathol. 2001; 11: 92-106Crossref PubMed Scopus (143) Google Scholar, 2Stohlman SA Bergmann CC Perlman S Mouse Hepatitis Virus.in: Ahmed R Chen I John Wiley & Sons, Ltd., New York1998: 537-557Google Scholar Myelin destruction in these animals occurs as a direct consequence of virus clearance and is largely immune-mediated because it does not occur to a significant extent in mice lacking T or B cells (lethally irradiated mice, mice with severe combined immunodeficiency, or mice lacking recombination activation enzyme gene 1 [RAG1−/−]). Less is known about the role of the adaptive immune response in JHMV-mediated acute encephalitis. After intranasal inoculation, virus spreads from the olfactory bulb transneuronally to its primary, secondary, and tertiary connections. By day 7 post inoculation (p.i.), virus is partly cleared from the front of the brain but is present at high levels in more distal regions such as the medial parabrachial nucleus and brainstem reticular formation. Most notably, JHMV only rarely infects the CA1 and CA3 regions of the hippocampus, unlike other viruses that cause acute encephalitis, such as HSV-1.3Barnett E Cassell M Perlman S Two neurotropic viruses, herpes simplex virus type I and mouse hepatitis virus, spread along different neural pathways from the main olfactory bulb.Neuroscience. 1993; I57: 1007-1025Abstract Full Text PDF Scopus (117) Google Scholar, 4Barnett E Perlman S The olfactory nerve and not the trigeminal nerve is the major site of CNS entry for mouse hepatitis virus, strain JHM.Virology. 1993; 194: 185-191Crossref PubMed Scopus (85) Google Scholar Clinical signs of disease first become apparent at approximately 5 days p.i., and mice are moribund by 7 to 8 days p.i.3Barnett E Cassell M Perlman S Two neurotropic viruses, herpes simplex virus type I and mouse hepatitis virus, spread along different neural pathways from the main olfactory bulb.Neuroscience. 1993; I57: 1007-1025Abstract Full Text PDF Scopus (117) Google Scholar, 5Perlman S Evans G Afifi A Effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain.J Exp Med. 1990; 172: 1127-1132Crossref PubMed Scopus (96) Google Scholar Neutrophils, macrophages, and NK cells are detected as early as day 3 p.i. with T-cell infiltration first noted at day 5 p.i.6Williamson JS Sykes KC Stohlman SA Characterization of brain-infiltrating mononuclear cells during infection with mouse hepatitis virus strain JHM.J Neuroimmunol. 1991; 32: 199-207Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 7Haring JS Pewe LL Perlman S High magnitude, virus-specific CD4 T-cell response in the central nervous system of coronavirus-infected mice.J Virol. 2001; 75: 3043-3047Crossref PubMed Scopus (40) Google Scholar The innate immune response may be important in the development of disease; consistent with this, evidence of cytokine and chemokine dysregulation in mice with JHMV-induced fatal acute encephalitis has been reported.8Rempel JD Murray SJ Meisner J Buchmeier MJ Differential regulation of innate and adaptive immune responses in viral encephalitis.Virology. 2004; 318: 381-392Crossref PubMed Scopus (69) Google Scholar However, other observations are consistent with a role for the T-cell response in the development of clinical disease. Thus, although high titers of infectious virus are detected in mice dying from acute encephalitis, titers decline slightly from days 5 to 7 p.i. as mice deteriorate clinically and virus is cleared from ventral portions of the brain.5Perlman S Evans G Afifi A Effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain.J Exp Med. 1990; 172: 1127-1132Crossref PubMed Scopus (96) Google Scholar, 8Rempel JD Murray SJ Meisner J Buchmeier MJ Differential regulation of innate and adaptive immune responses in viral encephalitis.Virology. 2004; 318: 381-392Crossref PubMed Scopus (69) Google Scholar, 9Weiner LP Pathogenesis of demyelination induced by a mouse hepatitis virus (JHM virus).Arch Neurol. 1973; 28: 298-303Crossref PubMed Scopus (246) Google Scholar JHMV-specific CD4 and CD8 T cells are not detected in appreciable numbers until day 6 p.i., 1 to 2 days before the death of the animals and concomitant with the onset of virus clearance.7Haring JS Pewe LL Perlman S High magnitude, virus-specific CD4 T-cell response in the central nervous system of coronavirus-infected mice.J Virol. 2001; 75: 3043-3047Crossref PubMed Scopus (40) Google Scholar These studies raised the possibility that virus-specific T cells, although required for virus clearance,2Stohlman SA Bergmann CC Perlman S Mouse Hepatitis Virus.in: Ahmed R Chen I John Wiley & Sons, Ltd., New York1998: 537-557Google Scholar, 10Williamson JS Stohlman SA Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells.J Virol. 1990; 64: 4589-4592Crossref PubMed Google Scholar are critical for the development of severe clinical disease in mice with acute encephalitis. Observations from other models of JHMV-mediated disease also suggest a role for T cells in enhanced clinical disease, independent of any role that these cells have in demyelination. For example, RAG1−/− mice infected with an attenuated strain of JHMV generally remain asymptomatic until approximately 14 to 18 days p.i., at which point they develop signs of fatal encephalitis. However, adoptive transfer of total splenocytes from JHMV-immune mice at 4 days p.i., which results in partial virus clearance and the development of clinical disease (hindlimb paresis and mild encephalitis), shortens the asymptomatic period to 11 days p.i. The development of clinical disease is accelerated further if CD4 T-cell-enriched splenocytes are transferred, with clinical signs of severe encephalitis detected as early as 9 to 10 days p.i. and death occurring over the subsequent 24 to 48 hours.11Pewe L Haring J Perlman S CD4 T-cell-mediated demyelination is increased in the absence of gamma interferon in mice infected with mouse hepatitis virus.J Virol. 2002; 76: 7329-7333Crossref PubMed Scopus (49) Google Scholar, 12Wu GF Dandekar AA Pewe L Perlman S CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination.J Immunol. 2000; 165: 2278-2286Crossref PubMed Scopus (173) Google Scholar, 13Wu GF Perlman S Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus.J Virol. 1999; 73: 8771-8780Crossref PubMed Google Scholar At least three CD4 T-cell epitopes are recognized in C57BL/6 (B6) mice infected with JHMV, with a large fraction of the response (up to 25%) directed against an epitope encompassing residues 133 to 147 of the transmembrane (M) protein (epitope M133).7Haring JS Pewe LL Perlman S High magnitude, virus-specific CD4 T-cell response in the central nervous system of coronavirus-infected mice.J Virol. 2001; 75: 3043-3047Crossref PubMed Scopus (40) Google Scholar, 14Xue S Perlman S Antigen specificity of CD4 T cell response in the central nervous system of mice infected with mouse hepatitis virus.Virology. 1997; 238: 68-78Crossref PubMed Scopus (30) Google Scholar It is well established that virus clearance is delayed in many viral central nervous system (CNS) infections if the CD4 T-cell response is completely abrogated, with a consequence of more severe disease.15Njenga MK Pavelko K Baisch J Lin X David C Leibowitz JL Rodriguez M Theiler's virus persistence and demyelination in major histocompatibility complex class II-deficient mice.J Virol. 1996; 70: 1729-1737Crossref PubMed Scopus (66) Google Scholar, 16Houtman JJ Fleming JO Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM.J Neurovirol. 1996; 2: 101-110Crossref PubMed Scopus (102) Google Scholar, 17Liu T Chambers TJ Yellow fever virus encephalitis: properties of the brain-associated T-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for CD4+ lymphocytes.J Virol. 2001; 75: 2107-2118Crossref PubMed Scopus (75) Google Scholar, 18Murali-Krishna K Ravi V Manjunath R Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: requirement for L3T4+ T cells.J Gen Virol. 1996; 77: 705-714Crossref PubMed Scopus (88) Google Scholar, 19Weidinger G Henning G ter Meulen V Niewiesk S Inhibition of major histocompatibility complex class II-dependent antigen presentation by neutralization of gamma interferon leads to breakdown of resistance against measles virus-induced encephalitis.J Virol. 2001; 75: 3059-3065Crossref PubMed Scopus (21) Google Scholar Therefore, to probe the potentially pathogenic role of CD4 T cells in JHMV-mediated acute encephalitis, we used reverse genetics to engineer a virus with mutations in the immunodominant epitope M133. CD4 T cells in mice infected with this virus still recognize subdominant JHMV-specific CD4 T-cell epitopes. Our results show that elimination of the CD4 T-cell response to this single epitope resulted in a reduction in mortality from 100 to 0%. Specific pathogen-free 5- to 6-week-old B6 and BALB/c mice (National Cancer Institute, Bethesda, MD) and RAG1−/− mice (Jackson Laboratories, Bar Harbor, ME) were inoculated intranasally (i.n.) with 4 to 8 × 104 plaque forming units of virus in 12 μl of Dulbecco's modified Eagle's medium. Mice were examined and weighed daily. In all experiments, surviving mice were euthanized at 16 days p.i. Virus was harvested from the infected CNS and titered by plaque assay on HeLa cells expressing the cellular receptor for mouse hepatitis virus, CEACAM1 (HeLa-MHVR). In some experiments, B6 mice were inoculated with 3 to 5 × 106 colony forming units of attenuated recombinant actA-deficient strain of Listeria monocytogenes 1 month before infection with virus. These L. monocytogenes (LM)-immune mice were kindly provided by Dr. J. Haring, University of Iowa. All animal studies were approved by the University of Iowa Animal Care and Use Committee. JHMV was grown and titered as described previously.20Perlman S Schelper R Bolger E Ries D Late onset, symptomatic, demyelinating encephalomyelitis in mice infected with MHV-JHM in the presence of maternal antibody.Microb Pathog. 1987; 2: 185-194Crossref PubMed Scopus (87) Google Scholar A chimeric recombinant virus encoding the feline S protein (designated fMHV-JHM clone B4c) was used as a recipient for targeted recombination.21Ontiveros E Kuo L Masters PS Perlman S Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.Virology. 2001; 290: 230-238Crossref Scopus (74) Google Scholar, 22Kuo L Godeke GJ Raamsman MJ Masters PS Rottier PJ Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier.J Virol. 2000; 74: 1393-1406Crossref PubMed Scopus (296) Google Scholar All recombinant viruses encoding the MHV S protein were propagated in mouse 17Cl-1 cells and titered on HeLa-MHVR cells. Recombinant viruses were generated by targeted recombination as described previously.21Ontiveros E Kuo L Masters PS Perlman S Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.Virology. 2001; 290: 230-238Crossref Scopus (74) Google Scholar, 22Kuo L Godeke GJ Raamsman MJ Masters PS Rottier PJ Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier.J Virol. 2000; 74: 1393-1406Crossref PubMed Scopus (296) Google Scholar In brief, a plasmid containing genes 2 to 7 of JHM.IA21Ontiveros E Kuo L Masters PS Perlman S Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.Virology. 2001; 290: 230-238Crossref Scopus (74) Google Scholar was used as the substrate for RNA synthesis. We used overlapping extension polymerase chain reaction (PCR) to mutate epitope M133 (Tyr to Gln at position 135; pJ.MY135Q). The two inner primers used were 5′-GTACCGTGCAAGTTAGACC-3′ (forward) and 5′-GGGTCTAACTTGCACGGTAC-3′ (reverse) (mutations responsible for Tyr to Gln change at amino acid M135 are underlined). The outer primers were 5′-CTACCAATGGACGGCCGACGAGG-3′ (forward; nucleotides 29,174 to 29,196) and 5′-CCAGATCGGCTAGCAGGTGCAGACC-3′ (reverse; nucleotides 30,429 to 30,453). The overlapping PCR product was subcloned into pCR2.1-TOPO (pCR2.1.MY135Q). A DNA fragment was excised from pCR2.1.MY135Q with MfeI and NheI and inserted into pJHM.IA. Donor RNAs were transcribed using T7 polymerase and transfected into feline cells (AK-D) previously infected with fMHV-JHM (a recombinant MHV strain encoding the feline surface (S) glycoprotein). fMHV-JHM does not infect murine cells, but recombinant virus expressing the MHV S protein does, allowing for efficient selection of recombinant virus on 17Cl-1 murine cells. Recombinant virus rJ.MY135Q was propagated as previously described (Figure 1).21Ontiveros E Kuo L Masters PS Perlman S Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.Virology. 2001; 290: 230-238Crossref Scopus (74) Google Scholar A second set of recombinants was also engineered in which a CD4 T-cell epitope from LM-encompassing residues 190 to 201 of listeriolysin O (epitope LLO190, NEKYAQAYPNVS23Geginat G Schenk S Skoberne M Goebel W Hof H A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes.J Immunol. 2001; 166: 1877-1884Crossref PubMed Scopus (128) Google Scholar) was inserted into gene 4 of rJ.MY135Q by overlapping extension PCR, resulting in rJ.MY135Q.LLO190. We showed previously that insertions into gene 4 did not affect virus growth in tissue culture or the ability to cause acute encephalitis.21Ontiveros E Kuo L Masters PS Perlman S Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.Virology. 2001; 290: 230-238Crossref Scopus (74) Google Scholar Outer primers were 5′-CCAAGCAATTCAGTGATAGTAGTACGC-3′ (forward) and 5′-CCTCTTGAACTACCAAG-3′ (reverse). Inner primers were 5′-GCTCAAGCTTATCCAAATGTAAGTATTGGTCCATTTCTAGTAGCA-3′ (forward) and 5′-GCTTGAGCATATTTTTCATTGGCCATAACTACTTGCTGCC-3′ (reverse) (LLO190 epitope is underlined). PCR products were prepared, subcloned, and eventually inserted into pJ.MY135Q (pJ.MY135Q.LLO190) (Figure 1). Recombinant virus was isolated as described above. In all cases, viruses were sequenced to confirm the presence of the introduced mutations before inoculation into mice. To control for any unwanted mutations that might have occurred during the process of targeted recombination, at least two independent isolates of each recombinant virus were analyzed in these studies. Mice were infected with virus intranasally. At day 7, mice were sacrificed, and RNA was harvested from one-half of each brain as described previously.24Pewe L Wu G Barnett EM Castro R Perlman S Cytotoxic T cell-resistant variants are selected in a virus-induced demyelinating disease.Immunity. 1996; 5: 253-262Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar To confirm that no changes occurred in epitopes M133Y135Q or LLO190 during passage in mice, cDNA was prepared, and PCR products encompassing the two epitopes were sequenced. Confluent 17Cl-1 monolayers in 12-well plates were infected with viruses at a multiplicity of infection of 1, as described previously.21Ontiveros E Kuo L Masters PS Perlman S Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.Virology. 2001; 290: 230-238Crossref Scopus (74) Google Scholar Samples were harvested at the times indicated, and viral titers were determined on HeLa-MHVR cells. In all experiments in which virus titers were measured, cells and supernatants were combined before determining titer. Equal amounts of rJ and rJ.MY135Q were diluted in Dulbecco's modified Eagle's medium buffered to pH 6.0, 7.0, or 8.0, prepared as previously described.25Krueger DK Kelly SM Lewicki DN Ruffolo R Gallagher TM Variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion.J Virol. 2001; 75: 2792-2802Crossref PubMed Scopus (75) Google Scholar Viruses were incubated at 37°C for the indicated times and subsequently titered on HeLa-MHVR cells. Cells were prepared from infected brains and analyzed for cytokine production after stimulation with JHMV-specific peptides as previously described.12Wu GF Dandekar AA Pewe L Perlman S CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination.J Immunol. 2000; 165: 2278-2286Crossref PubMed Scopus (173) Google Scholar Peptides corresponding to the immunodominant CD8 T-cell epitope recognized in B6 mice (epitope S510) or irrelevant peptide (Ova 257–264) were used at a final concentration of 1 μmol/L. Peptides corresponding to the CD4 T-cell epitopes M133, M133Y135Q, LLO190, S333, and S358 were used at a final concentration of 5 μmol/L, except for peptide LLO190, which was used at 8 μmol/L. Briefly, cells were washed, permeabilized, and incubated in blocking buffer containing 10% rat serum and anti-FcγRIII/II Ab (2.4G2). Cells were then stained with fluorescein isothiocyanate (FITC) anti-mouse CD8α monoclonal antibody (mAb) (Ly-2, clone 53–6.7) or FITC anti-mouse CD4 mAb (L3T4, clone GK1.5), respectively. Cells were stained for intracellular IFN-γ or tumor necrosis factor-α (TNF-α) using phycoerythrin-conjugated anti-IFN-γ or allophycocyanin-conjugated anti-TNF-α, respectively. All reagents were purchased from BD Pharmingen (San Diego, CA). After washing and fixation, cells were analyzed using a FACScan Flow Cytometer (BD Biosciences, Mountain View, CA). The number of lymphocytes harvested from each infected brain ranged from 1 × 106 to 3 × 106. Cells were prepared from infected brains and incubated in blocking buffer containing 10% rat serum and anti-FcγRIII/II Ab (2.4G2). Cells were then stained with PerCP-conjugated rat anti-CD45 (mAb 30-F11; BD Pharmingen), rat PE-conjugated rat anti-F4/80 (macrophages/microglia, cl BM-8; Caltag Laboratories, Burlingame, CA), and FITC-conjugated anti-Ly6G (neutrophils, mAb 1A8; BD Pharmingen) and then analyzed using a FACScan Flow Cytometer. Macrophages/microglia were identified as CD45hi/intF4/80+Ly6G−, whereas neutrophils were CD45hiF4/80−Ly6G+. For immunohistochemistry, brain and spinal cord sections were fixed in zinc formalin and processed as previously described.11Pewe L Haring J Perlman S CD4 T-cell-mediated demyelination is increased in the absence of gamma interferon in mice infected with mouse hepatitis virus.J Virol. 2002; 76: 7329-7333Crossref PubMed Scopus (49) Google Scholar Sections were probed with antibody directed against the JHMV nucleocapsid (N) protein (mAb 5B188.2, 1:10,000; kindly provided by Dr. M. Buchmeier [The Scripps Research Institute, La Jolla, CA]) followed by biotinylated goat anti-mouse (1:100; Jackson Immunoresearch, West Grove, PA). Sections were developed by sequential incubation with strepavidin-horseradish peroxidase conjugate and diaminobenzidine (Sigma, St. Louis, MO). Cells were harvested from the CNS of infected mice and stained with MHC class I/S510 (CSLWNGPHL) tetrameric complexes, obtained from the National Institutes of Health Tetramer Core Facility, Atlanta, GA, as described previously.26Pewe L Heard SB Bergmann CC Dailey MO Perlman S Selection of CTL escape mutants in mice infected with a neurotropic coronavirus: quantitative estimate of TCR diversity in the infected CNS.J. Immunol. 1999; 163: 6106-6113Crossref PubMed Google Scholar Mononuclear cells were harvested from the brains of B6 mice infected with rJ or rJ.M135Q and analyzed in direct ex vivo chromium release cytotoxicity assays as previously described.27Castro RF Perlman S CD8+ T cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity.J Virol. 1995; 69: 8127-8131Crossref PubMed Google Scholar Target cells were EL-4 cells coated with peptide at a final concentration of 1 μmol/L or left uncoated. The percent specific release was defined as 100 × (experimental release − spontaneous release)/(total release [detergent-treated] − spontaneous release). Maximum spontaneous release was <10% in all experiments. IL-5 ELISPOT assays were performed as described previously.28Varga SM Wissinger EL Braciale TJ The attachment (G) glycoprotein of respiratory syncytial virus contains a single immunodominant epitope that elicits both Th1 and Th2 CD4+ T cell responses.J Immunol. 2000; 165: 6487-6495Crossref PubMed Scopus (111) Google Scholar Briefly, nitrocellulose-based 96-well plates (Millititer HA, Millipore, Bedford, MA) were coated overnight at 4°C with 5 μg/ml anti-IL-5 (clone TRFK5; eBioscience. San Diego, CA), 2.5 μg/ml anti-CD3 (clone 145-2C11; eBioscience), and 2.5 μg/ml anti-CD28 mAb (clone 37-51; eBioscience) diluted in phosphate-buffered saline (PBS), washed the next day with PBS, and blocked with RPMI-10% fetal calf serum. After washing with PBS, CNS-derived lymphocytes from two rJ-infected mice and five rJ.MY135Q mice were added to the wells in triplicate (105 to 2 × 105 cells/well), in a total volume of 200 μl/well. Plates were incubated at 37°C for 48 hours and washed with PBS-0.05% Tween 20 (Sigma), followed by sequential incubation with biotinylated anti-IL-5 (clone TRFK4; eBioscience) and avidin-peroxidase (1/400 dilution; Sigma). Spots were visualized using 3-amino-9-ethylcarbazole (Sigma). Plates were analyzed using an immunospot analyzer (Cellular Technology Laboratory, Cleveland, OH) according to the manufacturer's instructions. The number of spots per 1 × 104 CD4 T cells was calculated from the frequency of CD4+ cells as determined by fluorescence activated cell sorting (FACS). Total RNA was isolated from brains using Tri Reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer's instructions. cDNA was prepared as previously described24Pewe L Wu G Barnett EM Castro R Perlman S Cytotoxic T cell-resistant variants are selected in a virus-induced demyelinating disease.Immunity. 1996; 5: 253-262Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar and was subjected to PCR as follows. Two microliters of cDNA was added to a 23-μl PCR cocktail containing 2× SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and 0.2 μmol/L each of sense and antisense primers (Integrated DNA Technologies, Coralville, IA). Amplification was then performed in an Applied Biosystems Prism 7700 thermocycler. Specificity of the amplification was confirmed using melting curve analysis. Data were collected and recorded by the Prism 7700 software and expressed as a function of threshold cycle. Specific primer sets used for TNF-α and a murine housekeeping gene were as follows: TNF-α forward, 5′-GCCTCTTCTCATTCCTGCTT-3′; TNF-α reverse, 5′-GGTGGTTTGCTAGCACGTG-3′; HPRT forward, 5′-CCTCATGGACTGATTATGGAC-3′; HPRT reverse, 5′-CAGATTCAACTTGCGCTCATC-3′. TNF-α RNA abundance was calculated using methods described previously.29Pewe L Zhou H Netland J Tangadu C Olivares H Shi L Look D Gallagher TM Perlman S A severe acute respiratory syndrome-associated coronavirus-specific protein enhances virulence of an attenuated murine coronavirus.J Virol. 2005; 79: 11335-11342Crossref PubMed Scopus (85) Google Scholar A two-tailed unpaired Student's t-test was used to analyze differences in mean values between groups. All results are expressed as means ± SEM. P values of <0.05 were considered statistically significant. Recent advances in coronavirus reverse genetics make it possible to introduce mutations into the JHMV genome.30Masters PS Reverse genetics of the largest RNA viruses.Adv Virus Res. 1999; 53: 245-264Crossref PubMed Google Scholar, 31Yount B Denison MR Weiss SR Baric RS Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59.J Virol. 2002; 76: 11065-11078Crossref PubMed Scopus (243) Google Scholar We used one of these methods, targeted recombination,30Masters PS Reverse genetics of the largest RNA viruses.Adv Virus Res. 1999; 53: 245-264Crossref PubMed Google Scholar to introduce a mutation into epitope M133, the immunodominant CD4 T-cell epitope recognized in B6 mice. Using a previously described motif for peptides binding to the I-Ab molecule,32Wall KA Hu J-Y Currier P Southwood S Sette A Infante A A disease-related epitope of Torpedo acetylcholine receptor: residues involved in I-Ab binding, self-nonself discrimination, and TCR antagonism.J Immunol. 1994; 152: 4526-4536Crossref PubMed Google Scholar we identified a tyrosine at position 135 of the M protein as a likely anchor residue for binding to the MHC class II molecule. In preliminary experiments, we showed that a peptide containing the Y135Q change was no longer recognized by CD4 T cells harvested from the CNS of mice acutely infected with JHMV. This mutation was introduced into JHMV to create a recombinant virus, rJ.MY135Q, as described in Materials and Methods and Figure 1. Two independent isolates were identified, and the presence of the mutation was confirmed by sequence analysis after amplification in tissue culture cells. To confirm the loss of recognition of the epitope in the context of infectious virus, lymphocytes were harvested from the CNS of mice inoculated with wild-type recombinant virus (rJ) or rJ.MY135Q and analyzed for interferon-γ (IFN-γ) production after stimulation with peptide M133 (Figures 2, A and B; Table 1). As expected, rJ but not rJ.MY135Q elicited an epitope M133-specific CD4 T-cell response. Furthermore, mice infected with rJ.MY135Q did not mount a de novo CD4 T-cell response to the variant M133 epitope (Figure 2B, right panel).Table 1Antigen Specificity of CD4 T Lymphocytes Harvested from CNS of Mice at Day 7 p.i.VirusNo. of mice% CD4M133*No. and percentage of virus-specific CD4 T cells after subtracting background (no peptide).LLO190*No. and percentage of virus-specific CD4 T cells after subtracting background (no peptide).S333*No. and percentage of virus-specific CD4 T cells after subtracting background (no peptide).S358*No. and percentage of virus-specific CD4 T cells after subtracting background (no peptide).%n (×104)%n (×103)%n (×103)%n (×103)rJ62.8 ± 0.521.2 ± 1.21.6 ± 0.3N.D.N.D.2.4 ± 0.12.0 ± 0.210.2 ± 0.97.5 ± 1.5rJ.MY135Q122.3 ± 0.200N.D.N.D.3.1 ± 0.51.6 ± 0.46.8 ± 1.23.3 ± 0.7rJ.MY135Q.LLO190113.6 ± 0.3004.3 ± 0.83.3 ± 0.72.1 ± 0.41.7 ± 0.48.0 ± 0.75.7 ± 0.8N.D., not determined.* No. and percentage of virus-specific CD4 T cells after subt
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