February 2003
Volume 44, Issue 2
Free
Immunology and Microbiology  |   February 2003
Impaired Killing of HCMV-Infected Retinal Pigment Epithelial Cells by Anti-pp65 CD8+ Cytotoxic T Cells
Author Affiliations
  • Sophie Allart
    From the National Institute of Health and Medical Research (INSERM), Toulouse, France;
  • Jacqueline Lulé
    From the National Institute of Health and Medical Research (INSERM), Toulouse, France;
  • Brigitte Serres
    Ophthalmology Service, University Hospital Center Purpan, Toulouse, France; and
  • Thomas Jones
    Infectious Disease Section, Wyeth-Ayerst Research, Pearl River, New York.
  • Jean-Luc Davignon
    From the National Institute of Health and Medical Research (INSERM), Toulouse, France;
  • François Malecaze
    Ophthalmology Service, University Hospital Center Purpan, Toulouse, France; and
  • Christian Davrinche
    From the National Institute of Health and Medical Research (INSERM), Toulouse, France;
Investigative Ophthalmology & Visual Science February 2003, Vol.44, 665-671. doi:10.1167/iovs.02-0547
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Sophie Allart, Jacqueline Lulé, Brigitte Serres, Thomas Jones, Jean-Luc Davignon, François Malecaze, Christian Davrinche; Impaired Killing of HCMV-Infected Retinal Pigment Epithelial Cells by Anti-pp65 CD8+ Cytotoxic T Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(2):665-671. doi: 10.1167/iovs.02-0547.

      Download citation file:


      © 2017 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

purpose. Host defense against infection by human cytomegalovirus (HCMV) is ensured in great part by cytotoxic CD8+ T lymphocytes (CTLs) directed against the tegument protein pp65. The hyperimmediate release of incoming pp65 into the major histocompatibility complex (MHC) class I pathway after fusion of the virus with the cell membrane provides a very early mechanism of defense. In retinal pigment epithelial (RPE) cells HCMV is known to enter through endocytosis. This study was conducted to determine whether this means of penetration into the cells would allow the virus to elude immune surveillance.

methods. Infection of RPE cells with HCMV AD169 was performed for 6 hours, 48 hours, and 8 days. Expression of intracellular pp65 in RPE cells and in the astrocytoma reference cell line U373MG was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. Killing of both HCMV-infected cell lines by HLA-A2–restricted CD8+ CTLs directed against pp65 was monitored by 51Cr-release assays.

results. RPE cells were not lysed by CTLs directed against incoming pp65, contrary to U373MG. Moreover, both cell lines were not killed by anti-pp65 CTLs later after infection, because of the MHC class-I–downregulating effect of HCMV unique short (US2-11) proteins.

conclusions. In RPE cells, both HCMV entry through endocytosis and the immunosuppressive effect of US proteins could allow the virus to evade immune surveillance at any stage of infection, which could promote viral spreading within the retina.

Human cytomegalovirus (HCMV), a member of the β-herpesviruses, is latent and harmless in most immunocompetent people. However, immunocompromised hosts such as transplant recipients can be severely affected by HCMV infection. It is the cause of a wide range of disabilities (mental retardation, hearing loss, developmental delay) in some children whose mothers are newly infected during pregnancy. HCMV is also among the most harmful infections faced by patients with acquired immunodeficiency syndrome (AIDS). HCMV infection can act as a direct cause of retinitis and blindness in patients with end-stage AIDS. 1 2 3 One major component of the very early immune control of HCMV replication is a CD8+ cytotoxic T lymphocyte (CTL) response directed against the tegument protein pp65 (pUL83). 4 5 Indeed, pp65 has been shown to be internalized immediately after the fusion of both virions and dense bodies envelope with the cell membrane 6 and thus is available for presentation to specific CD8+ CTLs without de novo synthesis. 7 Moreover, we have shown recently that incoming pp65 contained in apoptotic infected cells can serve as an antigen source for cross-presentation by dendritic cells to CTLs. 8 The observation that CTLs responsive to pp65 are present at a high frequency in blood donors suggests that this antigen is immunodominant. 5 Recognition of infected cells by anti-pp65 CTLs very early after adsorption of a virus could avoid replication and spreading of the virus. The relevance of this very early presentation of pp65 to CTLs is emphasized, because HCMV develops strategies to escape from CTL responses throughout infection. Among these processes, those inducing a downregulation of major histocompatibility complex (MHC) class I molecule transport to the cell surface involve viral proteins encoded by multiple independent loci within the unique short (US) region of the HCMV genome. 9 10 It has been shown that in human retinal pigment epithelial (RPE) cell lines and endothelial cells, which are two relevant targets of HCMV infection, 11 the virus enters through endocytosis in vitro and is thus sequestered in vesicles. 12 In light of this observation we hypothesized that RPE cells may possess characteristics that allow the virus to escape from very early control by anti-pp65 CTLs and thus favor the establishment of viral persistence and pathogenicity. 
In this study, we report that HCMV-infected RPE cells were not lysed by HLA-A2-restricted cytotoxic CD8+ T-cell lines directed against incoming pp65, contrary to U373MG astrocytoma cells, although they expressed the tegument protein. We showed that a threshold amount of incoming pp65 in RPE cells was not responsible for resistance to CTL recognition. Because RPE cells that had been infected with pp65 recombinant adenovirus were sensitive to lysis by CTLs, we suggest that after infection with HCMV, incoming pp65 was not available for processing and recognition by T cells. Finally, we produce evidence that later after infection, even though amounts of pp65 increased due to de novo synthesis, both cell types were resistant to killing by anti-pp65 CTLs due to downregulation of MHC class I molecules mediated by the expression of proteins encoded in the US2-11 region of the viral genome. Together, these data suggest that control of HCMV-infected RPE is not ensured by anti-pp65 CTLs at any stage of infection, which may facilitate spread of the virus within the retina. 
Material and Methods
Cell Cultures
Primary human RPE cell lines were established from donor eyes (Purpan Hospital, Toulouse, France), according to the tenets of the Declaration of Helsinki. RPE cells were isolated with trypsin, resuspended in Ham-F10 medium supplemented with 20% fetal calf serum (FCS; Invitrogen, Groningen, The Netherlands) and 10 μg/mL anti-mycoplasma (Oflocet; Roussel, Paris, France) and transferred to a 75-cm2 culture flask. Cells formed monolayers and were typically hexagonal at confluence. Homogeneity was confirmed by positive immunostaining with monoclonal antibodies (mAbs) to cytokeratins (MNF 116 clone; Dako, Trappes, France). Cell surface expression of HLA-A2 on RPE cells was confirmed by flow cytometry analysis with a mouse mAb (clone BB7-2, HB82; American Type Culture Collection [ATCC] Manassas, VA)). Cultures from two different primary cell lines were used at passages 4 to 11 for further experiments. HLA-A2–positive astrocytoma cells U373MG were from ATCC. Cells were maintained in RPMI medium (Invitrogen) containing 10% FCS supplemented with sodium pyruvate and antibiotics. U373MG cells transduced with IE1 cDNA were a gift from Susan Michelson (Pasteur Institute, Paris, France). 
Viruses
HCMV AD169 was from ATCC, and construction of the mutant AD169, with the US2-11 genome region (HCMVΔ2-11) deleted, has been described. 13 These viruses were propagated in human foreskin fibroblasts (HFF) in Dulbecco’s modified Eagle’s medium supplemented with 5% FCS. Viruses were collected when cytopathic effects were more than 90%. Supernatants were clarified of cell debris by centrifugation at 1500g for 10 minutes at 4°C and stored at −70°C until use. Virus titers were determined by plaque-forming unit titration in HFFs, according to standard procedures. A multiplicity of infection (MOI) of 3 was used for all the experiments. Type 2 adenoviruses recombinant for pp65 (pUL83) sense (Ad-pp65s) or antisense (Ad-pp65as; a gift of Marina Cavazana, National Institute of Health and Medical Research, Unit 429, Paris, France) were used at 50 MOI during 24 hours before chromium-release assays. 
Generation of Anti-pp65 CD8+ T-Cell Lines
HLA typing was performed by the Central Laboratory of Immunology (Elie Ohayon, Toulouse, France). CTLs directed against pp65 were generated from peripheral blood mononuclear cells (PBMCs) of HLA-A2 healthy HCMV-seropositive donor V as described by Arrode et al. 8 Briefly, PBMC (2 × 106 cells/mL) were cultured in 24-well plates in RPMI medium containing 10% human serum AB, 1% minimal essential medium with no essential amino acids, and 10 mM HEPES (all from Invitrogen). Culture was stimulated with a mixture containing 5 μg/mL of a pp65-derived peptide corresponding to a known CTL epitope (NLVPMVATV, N9V), 5 synthesized by Neosysteme (Strasbourg, France), and 5 μg/mL of a recombinant chimeric protein IE1-pp65 associated with a nanoparticulate carrier (SMBV; a gift from Biovector Therapeutics, Labege, France) to provide CD4+ helper T cells. 14 At days 3 and 7, recombinant human IL-7 (rhIL-7 supplied as a kind gift by Sanofi-Synthelabo, Labege, France) was added at a concentration of 25 ng/mL. CTL activity was tested in a 51Cr-release assay at days 9 to 12, as will be described further. 
Chromium Release Assay
Targets cells (U373MG or RPE cells) were seeded in six-well plates and infected with the following viruses: HCMV AD169, Ad-pp65s, Ad-pp65as, or HCMVΔ2-11. At various times after infection, cells were labeled with [51CrNa] (ICN Biomedicals, Orsay, France) at 100 μCi per well for 2 hours and washed three times in RPMI-10% FCS. Effector T cells were incubated in RPMI-FCS for 4 hours at 37°C with 5 × 103 target cells per well in a U-bottomed 96-well plate, at different effector-to-target (E-T) ratios. Alternatively, target cells were preincubated overnight at 37°C with N9V peptide at a 500-nM final concentration. An irrelevant HLA-B35-binding peptide I9Y (IPSINVHHY, Neosysteme), 15 was used as the negative control. The percentage of specific 51Cr release was calculated as follows (cpm, counts per minute): [(cpm experimental release minus cpm spontaneous release)/(cpm maximum release minus cpm spontaneous release)] × 100. Spontaneous release was always less than 15% of the maximum release. 
Indirect Immunofluorescence Assay
One day before the assay, U373MG and RPE cells were seeded on chamber slides (Labtek; Nunc, Napierville, IL) at 6 × 104 cells per well. Cells were infected with HCMV (MOI = 3) for either 6 hours or 48 hours and then processed as follows: after fixation for 10 minutes in PBS containing 2% saccharose and 5% formaldehyde, cells were permeabilized with a solution containing 10% saccharose, 0.5% NP40 and 1% FCS 1% in PBS for 5 minutes, washed and incubated (30 minutes, 37°C) with mouse mAb directed against pp65 (Cinapool 1:20; Argene Biosoft, Varilhes, France). Cells were then washed three times in PBS and incubated with a 1:200 dilution of a rhodamine-conjugated anti-mouse IgG (Coulter-Immunotech SA, Marseille, France) for 30 minutes. Slides were washed again three times, mounted, and examined, either on a fluorescence microscope (Leitz, Wetzlar, Germany) or on a confocal laser scanning microscope (model 410; Carl Zeiss, Oberkochen, Germany). Images were edited on computer (Photoshop; Adobe, San Jose, CA). 
Metabolic Labeling and Immunoprecipitation
Cells were infected with HCMV in six-well culture plates. At various times after infection, they were washed in PBS, incubated in methionine+cysteine–free medium (Invitrogen) for 1 hour, and labeled with [35S] methionine + [35S] cysteine (200 μCi/mL; NEN, Cologne, Germany) for 2 hours. Monolayers were treated with trypsin (Invitrogen), washed, and either stocked at −20°C or immediately lysed by incubation for 45 minutes on ice in lysis buffer (5 mM EDTA, 150 mM NaCl, 1 mM MgCl2, 0.05 mM phenylmethylsulfonyl fluoride [PMSF], 50 mM Tris [pH 7.6], and 1% NP40). After a 2-hour preclearing with protein G–conjugated Sepharose beads (Amersham Pharmacia Biotech, Saclay, France), antibody was added to the lysate, and the mixture was incubated for 2 hours at 4°C. Mouse anti-pp65 mAb (a gift of Giuseppe Gerna, Policlinica San Matteo, Pavia, Italy) was used. Protein G–conjugated beads were then added, and incubation continued overnight at 4°C. After washing, the beads were pelleted and boiled for 5 minutes in 5% β-mercaptoethanol reducing Laemmli sample buffer. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gels. Gels were fixed, incubated in Amplify (Amersham Pharmacia Biotech), vacuum dried, and exposed to autoradiograph film (Hyperfilm-MP; Amersham Pharmacia Biotech). 
Flow Cytometry
Cells were cultured in six-well plates and infected with HCMV as indicated. For pp65 labeling, cells were detached with trypsin and washed with PBS-20% human serum (PBS-HS). After fixation for 15 minutes in 1% paraformaldehyde (PFA), cells were permeabilized overnight with 100% methanol and washed again with PBS-HS. Anti-pp65 mAb (Cinapool; Argene Biosoft, Varilhes, France) was applied directly onto pelleted cells. Labeling with a mouse IgG1 (Dakopatts, A/S, Glostrup, Denmark) served as an isotypic control. After incubation for 45 minutes at 37°C and a wash with PBS-HS, cells were resuspended in goat anti-mouse F(ab′)2 conjugated to FITC (Sigma, Lyon, France), diluted to 1:50, and incubated for a further 30 minutes at 4°C. Cells were then washed and analyzed with a cell sorter (EPICS Elite; Beckman Coulter, Hialeah, FL). HLA-A2 labeling was performed as follows: cells were harvested, washed with PBS and 3% FCS, and then incubated with BB7-2 mAb for 45 minutes at 4°C. Treatment with secondary antibody and cytofluorometric analysis were performed as described earlier. 
Western Blot Analysis
At different times after infection, monolayers of U373MG or RPE cells were harvested, washed, and either stocked at −20°C or immediately lysed by incubation for 5 minutes on ice in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% desoxycholate, and 0.1% SDS [pH 8]). Lysates were homogenized and centrifuged for 15 minutes at 10,000 rpm. Supernatants were collected, and total proteins were quantified by protein assay (Bio-Rad, Marnes-la-Coquette, France). Proteins (30 μg) were boiled for 5 minutes in 5% β-mercaptoethanol reducing Laemmli sample buffer, and samples were separated by SDS-PAGE in 12.5% polyacrylamide gel, and transferred to nitrocellulose (Hybond C; Amersham International). Immunoblots were stained with ponceau red to visualize total proteins contained in each slot and probed alternatively with the following antibodies: a mouse anti-pp65 mAb (a gift of Giuseppe Gerna, Policlinica San Matteo, Pavia, Italy) at 1:3000 dilution; a mouse mAb specific for HCMV immediate early proteins (IE1 [UL122] and IE2 [UL123], E13 hybridoma supernatant (provided by Marie Christine Mazeron, Paris, France) at 1:10 dilution. Antigen recognition was determined with a peroxidase-conjugated polyclonal anti-mouse antibody (1:10,000 dilution; Sigma) and an enhanced chemiluminescence detection kit (ECL; Amersham Pharmacia Biotech). 
Results
Analysis of pp65 Expression in U373MG and RPE Cells
Before measuring the CTL activity of anti-pp65 CD8+ T cells, we evaluated the expression of pp65 in infected RPE and U373MG cells. Qualitative and quantitative analyses of intracellular pp65 expression were assessed by flow cytometry, fluorescence microscopy, and Western blot analysis. First, we examined the number of infected RPE cells by flow cytometric analysis of pp65 expression. This approach allowed us to detect intracellular protein only at day 8 postinfection (pi) but not at 6 hours and 48 hours pi (data not shown). Based on a previous report showing that fluorescence microscopy is more sensitive in detecting pp65 than flow cytometry, 16 immunostaining of RPE cells and analysis by confocal microscopy were performed. At 6 hours pi (Fig. 1A) , a granular cytoplasmic fluorescence was observed in 100% of RPE cells from which 15% exhibited a nuclear localization, as previously demonstrated by Bodaghi et al. 12 Microscopic examination and counting of fluorescent pp65-positive RPE cells revealed that the number of positive nuclei increased to 71% at 48 hours pi, suggesting that viral infection had progressed. Regarding U373MG, 100% cells were pp65 positive at 6 and 48 hours after infection (data not shown). 
Kinetics of pp65 expression was analyzed by Western blot analysis and immunoprecipitation of metabolically labeled cells in both cell lines. pp65 was detected by Western blot analysis in both cell lines at 6 hours pi and increased over time, even though biosynthesis of pp65 started later in RPE cells (72 hours pi) than in U373MG cells (24 hours pi). Because no metabolic labeling was obtained after immunoprecipitation of cell lysates, at 6 hours pi we suggest that in both cell lines, pp65 originated from the virus inoculum. 
Impaired Killing of RPE Cells in Response to Incoming pp65
HLA-A2 restricted CD8+ CTL lines directed against pp65 were raised from PBMCs of HCMV-seropositive donor V, as described in the Materials and Methods section. The cell lines used expressed CD3, were more than 80% CD8 positive, and produced IFN-γ, as described in previous publications. 8 17 We hypothesized that CD8+ T cell-mediated lysis of RPE cells would be impaired compared with that of U373MG cells. Figure 2A shows specific lysis of both U373MG and RPE cells pulsed with N9V peptide in the presence of CTLs from donor V. When an irrelevant peptide I9Y known to be presented by HLA-B35 was used, no lysis of targets was observed. In Figure 2B , U373MG cells were infected with HCMV AD169 for 6 hours and 48 hours and used as targets for CTLs. U373MG cells were killed at 6 hours pi, presumably due to incoming pp65, because no protein had been synthesized at this time point. However, as will be presented in detail in the next paragraph, they became partially or totally resistant to lysis at 48 hours pi, depending on the set of experiments. Because PBMCs were expanded in the presence of the chimeric IE1-pp65 protein, we ensured that killing of infected cells was not mediated by anti-IE1 CTLs. To this end, CTLs were assayed in the presence of IE1-transfected U373MG cells as targets. In these conditions, no lysis was observed (data not shown), demonstrating that killing of HCMV-infected cells occurred through recognition by anti-pp65 CTLs. Regarding RPE cells, we took into account data from Detrik et al. 18 showing that replication of HCMV progressed very slowly, compared with that in fibroblasts. Accordingly, infection of RPE cells with HCMV was performed at 6 hours and 48 hours and extended to 8 days before incubation with CTLs. Contrary to U373MG cells, HCMV-infected RPE cells were not killed by CTLs after 6 hours of infection, despite the presence of incoming pp65 (Fig. 2B) . In addition, at later time points of infection (48 hours and 8 days) no more significant cytotoxicity against RPE cells was observed (Fig. 2B) . Furthermore, cells were still resistant to lysis, even when infection was performed at the higher MOI of 60 (data not shown), suggesting that impaired lysis was not due to a threshold amount of pp65. It is noteworthy that the lysis observed on U373MG cells has been consistently observed on MRC5 fibroblast cells in similar conditions. 8 17 We used U373MG cells as controls in our experiments because, contrary to fibroblasts, they constitutively expressed a sufficient amount of HLA-A2 at their surface, without treatment with IFN-γ. 
To investigate whether pp65 could be processed into relevant HLA-A2–binding peptides in RPE cells, infections with recombinant adenoviruses containing sense or antisense pp65 gene (pp65s or pp65as) were performed and the infected cells used as targets for CTLs. Figure 3 shows that lysis occurred only when cells were infected with the sense pp65 adenovirus, demonstrating that RPE cells could present naturally processed endogenous pp65 peptides to HLA-A2–restricted CTLs. 
To examine whether HCMV infection could interfere with pathways of T-cell cytotoxicity, cells were infected with HCMV, as described earlier and then pulsed with N9V peptide. Figure 4 shows that RPE and U373MG cells were lysed in these conditions by anti-pp65 CTLs, excluding that inhibition of CD8+ T-cell cytolysis pathways by HCMV infection could account for impaired killing. 
Altogether, these results demonstrate that impaired killing of HCMV-infected RPE by anti-pp65 CTLs was due neither to viral interference with pathway of T-cell cytotoxicity, nor to inability of cells to process endogenous pp65 and to present peptides to CTLs. 
Proteins encoded in the US2-11 genomic region are responsible for HCMV escape from anti-pp65 CTLs both in U373MG and RPE cells at late time points of infection. 
As suggested earlier, even though U373MG and RPE cells had sustained amounts of pp65 in a late stage of infection, they were resistant to lysis by CTLs. It has been widely documented that HCMV inhibits MHC class I antigen presentation by a sequential multistep process involving a family of US region-encoded glycoproteins. 9 10 Ectopic expression of US proteins, namely US2, US3, US6, and US11 resulted in a defective expression of MHC class I-peptide complexes at the cell surface. To address this question in U373MG and RPE cells, infection with either HCMV AD169 or HCMVΔ2-11 (Δ2-11) was performed and the cells used as targets for CTLs. 
Figure 5A shows that in U373MG cells infected with Δ2-11, sensitivity to lysis by anti-pp65 CTLs was restored at 48 hours pi, showing that the expression of US2-11–encoded proteins was responsible for immune escape from CTL lysis. It is notable that after infection with the Δ2-11, viral lysis was more efficient at 48 hours pi than at 6 hours pi. This could be due to the presence of a higher amount of pp65, as shown in Figure 1B . Accordingly, flow cytometric analyses showed that in U373MG cells, HCMV-mediated downregulation of membranous expression of HLA-A2 was not observed in the presence of Δ2-11 (Fig. 5B) . In contrast, RPE cells that had been infected with Δ2-11 for 6 and 48 hours were still resistant to CTLs, probably due to a defect in the recognition of incoming pp65 at 6 hours pi (Fig. 2B) , and to a still-insufficient level of pp65 accessible to the cytosolic class I machinery at 48 hours pi, in accordance with Figure 1B . Sensitivity to CTLs was restored at day 8 pi (Fig. 5A) in accordance with the absence of MHC class I downregulation (Fig. 5B) . This could be ascribed to the absence of the immunosuppressive US2-11 proteins and, in addition, to an increased amount of cytosolic pp65 caused by de novo synthesis. Flow cytometry, used as described earlier, showed that levels of pp65 expression in infected cells were identical, regardless of the presence (AD169) or absence (Δ2-11) of the US2-11 fragment in HCMV strains (data not shown). These data suggest that late after infection, anti-pp65 CTLs failed to lyse HCMV-infected U373MG and RPE cells due to the effect of the immunosuppressive proteins encoded in the US2-11 region. 
Discussion
In this study we showed impaired killing of HCMV-infected RPE cells by anti-pp65 CD8+ CTLs. We demonstrated that this was due neither to the absence of incoming pp65 into cells nor to their inability to process and present pp65-derived peptide to CTLs. Furthermore, we showed that later, after infection, both cell lines were resistant to killing by CTLs due to the expression of the immunosuppressive HCMV US proteins. It is well known that in fibroblasts, entry of virus through fusion leads to an efficient uptake of viral particles and to a cytosolic delivery of nucleocapsid and tegument proteins. 6 Accordingly, our results showed that in the astrocytoma reference cell line U373MG, where HCMV entry by fusion exists, cytosolic delivery of incoming pp65 was responsible for activation of anti-pp65 CD8+ CTLs as we previously demonstrated in these cells and in MRC5 fibroblasts. 8 17 We can assume that fusion entry of HCMV into cells may be a prerequisite for efficient control of infection by anti-pp65 CTLs. 
According to previous studies showing that, in RPE cells, entry of HCMV occurs through an unusual endocytic pathway, followed by sequestration of viral particles, 12 we made the assumption that this would be detrimental to pp65 delivery into the cytosol for processing into the MHC class I machinery. Even though we have no direct evidence that no pp65 protein at all was expressed into the cytosol of infected cells, either in native form or degraded into peptides, no CTL activation was recovered, even when very high MOIs were used, suggesting that provided cytosolic pp65 was not properly processed into HLA-A2–binding peptides. 
We demonstrated that later, after infection, as expression of UL83 gene permitted an increase of intracellular pp65 level in both RPE and U373MG cells, the downregulating effect of proteins encoded in the US2-11 region on MHC class I expression was responsible for the absence of lysis of both cell lines by anti-pp65 CTLs. It has been shown that US2-, US6-, or US11-transfected cells are unable to present endogenous antigens to CTLs and were therefore resistant to CTL lysis. 19 20 21 However, our study is the first to show that proteins encoded by the whole US2-11 region interfered with CTL recognition of an endogenous HCMV antigen in a context of HCMV infection. Even though we have no data demonstrating the specific effect of US2, US3, US6, and US11, as extensively reported, 9 13 we demonstrated that after 6 hours of infection none of these proteins was able to efficiently inhibit presentation of incoming pp65. In contrast, after 48 hours of infection the cumulative effect of the different US proteins was evidenced by using the HCMVΔ2-11 virus, especially in U373MG cells, where CTL lysis was highly restored. Regarding RPE cells, despite a partial MHC class I downregulation at 48 hours pi as shown by flow cytometric analysis (Fig. 5) , cell sensitivity to CTLs was not significantly recovered by using the Δ2-11 virus, probably because of a poor level of processable pp65 (Fig. 1B) , because later on (8 day pi), as expression of UL83 gene increased, HCMVΔ2-11–infected cells were clearly sensitive to lysis. 
Taken together, our results suggest that anti-pp65 CTL response within the first hours after infection could be an efficient way to control the spread of virus before expression of the immunosuppressive activity of the US proteins. In conclusion, we can assume that impaired killing by CTLs directed against incoming pp65 or any other incoming tegument protein may contribute to spreading and to constitution of HCMV reservoirs within the retina. 
RPE cells are in vivo targets of HCMV infection and replication, 11 even though the in vitro replication of the virus is very delayed, compared with fibroblasts. 12 18 The best known incidence of HCMV infection in immunodeficient HIV+ patients is retinitis and blindness. 3 The pathophysiological mechanisms involved in HCMV retinitis have not been entirely defined. The presence of HCMV in areas of retinal cotton-wool spots as well as breaches in retinal microvasculature allowing viral permeation of the blood–retina barrier have been suggested in pathogenesis. 22 23 Infection of the RPE as an important part of the blood–retina barrier was also suspected to be crucial in the development of retinitis. 1 24 Although the eye is an immune-privileged site, 25 RPE cells may acquire, under inflammatory conditions, the capacity to function as antigen-presenting cells so that recognition by activated CTLs directed against viral antigens could induce cell death. We suggest that impaired killing of HCMV-infected RPE cells by CTLs directed against incoming tegument proteins could provide the virus with an additional trick for eluding host surveillance. It has also been shown recently that HCMV infection upregulates constitutive Fas ligand (FasL), which impairs neutrophil binding to infected RPE cells. 26 The incidence of both mechanisms may contribute to viral spreading in the retina and to pathogenesis after reactivation in immunocompromised hosts. In two studies, it has been shown that growth kinetics of HCMV was altered in phagocytic macrophages and that in these cells the virus accumulated in cytoplasmic vacuoles and survived through disruption of the microtubule network to evade lysosomal fusion. 27 28 The authors suggested that disruption of the microtubule network could alter the trafficking of proteins involved in antigen-presentation pathways, providing a novel mechanism for latency and evasion of immune recognition. Nevertheless, there are no data available showing that macrophages are resistant to CTLs directed against incoming pp65. 
Generally, T lymphocytes are dangerous to the host’s central nervous system, and these cells can be thus considered a double-edged sword for the host. Whether impaired killing by CD8+T cells is beneficial either for the host, according to the vital functions of RPE cells, or for the persistence and spread of the virus is an open question. 
Overall, it is worth hypothesizing that in cells such as epithelial cells, endothelial cells, and monocytes, both HCMV entry through endocytosis and the immunosuppressive effect of US proteins could allow the virus to elude immune surveillance at any stage of infection and to constitute reservoirs. Studies of the physiological relevance and mechanisms of viral entry in various cell types could provide new clues to fight against HCMV. 
 
Figure 1.
 
RPE cells are positive for incoming pp65. RPE cells were either not infected (ni) or were infected with HCMV AD169 during 6 hours (6 hours pi). Anti-pp65 labeling and localization were examined by immunostaining followed by analysis under a confocal laser scanning microscope (A). Expression of pp65 in U373MG and RPE cells was examined throughout infection by immunoblotting of cell lysates (IB) and immunoprecipitation of [35S] methionine–[35S] cysteine–labeled cells (IP) with an anti-pp65 mAb (B).
Figure 1.
 
RPE cells are positive for incoming pp65. RPE cells were either not infected (ni) or were infected with HCMV AD169 during 6 hours (6 hours pi). Anti-pp65 labeling and localization were examined by immunostaining followed by analysis under a confocal laser scanning microscope (A). Expression of pp65 in U373MG and RPE cells was examined throughout infection by immunoblotting of cell lysates (IB) and immunoprecipitation of [35S] methionine–[35S] cysteine–labeled cells (IP) with an anti-pp65 mAb (B).
Figure 2.
 
Analysis of HCMV-infected U373MG and RPE cell sensitivity to killing by anti-pp65 CD8+T cells. Anti-pp65 CTLs from donor V were cultured and cytotoxicity determined in a 51Cr-release assay using as targets HLA-A2+ U373MG or RPE cells that had been either pulsed overnight with HLA-A2–binding peptide (N9V) or irrelevant peptide (I9Y) at 500 nM (A) or infected with HCMV AD169 strain at an MOI of 3 for 6 hours (6 hours pi), 48 hours (48 hours pi), or 8 days (8d pi) (B). E-T ratios were as indicated.
Figure 2.
 
Analysis of HCMV-infected U373MG and RPE cell sensitivity to killing by anti-pp65 CD8+T cells. Anti-pp65 CTLs from donor V were cultured and cytotoxicity determined in a 51Cr-release assay using as targets HLA-A2+ U373MG or RPE cells that had been either pulsed overnight with HLA-A2–binding peptide (N9V) or irrelevant peptide (I9Y) at 500 nM (A) or infected with HCMV AD169 strain at an MOI of 3 for 6 hours (6 hours pi), 48 hours (48 hours pi), or 8 days (8d pi) (B). E-T ratios were as indicated.
Figure 3.
 
RPE cells were able to present endogenously derived pp65 peptides to CTLs. HLA-A2 positive U373MG and RPE cells were either pulsed overnight with the HLA-A2–binding peptide N9V or irrelevant peptide I9Y at 500 nM or infected with pp65 recombinant adenoviruses Ad-pp65s and Ad-pp65as, sense and antisense, respectively. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 3.
 
RPE cells were able to present endogenously derived pp65 peptides to CTLs. HLA-A2 positive U373MG and RPE cells were either pulsed overnight with the HLA-A2–binding peptide N9V or irrelevant peptide I9Y at 500 nM or infected with pp65 recombinant adenoviruses Ad-pp65s and Ad-pp65as, sense and antisense, respectively. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 4.
 
Infection of U373MG and RPE cells with HCMV did not inhibit T-cell cytotoxicity. U373MG and RPE cells were either mock infected or infected with HCMV AD169 (MOI = 3), as indicated, for 6 hours (N9V-6 hours pi) or 48 hours (N9V-48 hours pi) and then pulsed overnight with N9V peptide at 500 nM. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 4.
 
Infection of U373MG and RPE cells with HCMV did not inhibit T-cell cytotoxicity. U373MG and RPE cells were either mock infected or infected with HCMV AD169 (MOI = 3), as indicated, for 6 hours (N9V-6 hours pi) or 48 hours (N9V-48 hours pi) and then pulsed overnight with N9V peptide at 500 nM. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 5.
 
Proteins encoded in the US2-11 genomic region were responsible for escape of HCMV from anti-pp65 CTLs, both in U373MG and RPE cells. U373MG and RPE cells were infected for the indicated times with either HCMV AD169 (AD) or HCMVΔ2-11 (Δ2-11) and used as targets for HLA-A2-restricted anti-pp65 CTLs in a 51Cr-release assay (A). Cells pulsed with the irrelevant peptide I9Y were used as a negative control. E-T ratios were as indicated. Expression of HLA-A2 at the surface of U373MG and RPE cells was detected by staining with a mouse mAb and analyzed by flow cytometry (B).
Figure 5.
 
Proteins encoded in the US2-11 genomic region were responsible for escape of HCMV from anti-pp65 CTLs, both in U373MG and RPE cells. U373MG and RPE cells were infected for the indicated times with either HCMV AD169 (AD) or HCMVΔ2-11 (Δ2-11) and used as targets for HLA-A2-restricted anti-pp65 CTLs in a 51Cr-release assay (A). Cells pulsed with the irrelevant peptide I9Y were used as a negative control. E-T ratios were as indicated. Expression of HLA-A2 at the surface of U373MG and RPE cells was detected by staining with a mouse mAb and analyzed by flow cytometry (B).
The authors thank G. Cassar for technical assistance with flow cytometry analyses. 
Bodaghi, B, Michelson, S. (1999) Cytomegalovirus: virological facts for clinicians Ocular Immunol Inflamm 7,133-137 [CrossRef]
Britt, WJ, Alford, C. (1996) Cytomegalovirus Fields, BN Knipe, DM Howley, PMet al eds. Fields’ Virology ,2493-2523 Lippincott-Raven Philadelphia.
Jabs, DA, Barlett, JG. (1997) AIDS and ophthalmology: a period of transition Am J Ophthalmol 124,227-233 [CrossRef] [PubMed]
Gyulai, ZV, Endresz, K, Burian, S, et al (2000) Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs J Infect Dis 181,1537-1546 [CrossRef] [PubMed]
Wills, MA, Carmichael, J, Mynard, K, et al (1996) The human cytotoxic T-lymphocytes (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL J Virol 70,7569-7579 [PubMed]
Topilko, A, Michelson, S. (1994) Hyperimmediate entry of human cytomegalovirus virions and dense bodies into human fibroblasts Res Virol 145,75-82 [CrossRef] [PubMed]
McLaughlin-Taylor, EH, Pande, SJ, Forman, B, et al (1994) Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes J Med Virol 43,103-110 [CrossRef] [PubMed]
Arrode, G, Boccaccio, C, Lulé, J, et al (2000) Incoming human cytomegalovirus pp65 (UL83) contained in apoptotic infected fibroblasts is cross presented to CD8+T cells by dendritic cells J Virol 74,10018-10024 [CrossRef] [PubMed]
Loenen Wil, AM, Bruggeman, CA, Wiertz, EJHJ. (2001) Immune evasion by human cytomegalovirus: lessons in immunology and cell biology Semin Immunol 13,41-49 [CrossRef] [PubMed]
Ploegh, HL. (1998) Viral strategies of immune evasion Science 280,248-253 [CrossRef] [PubMed]
Rao, NA, Zhang, J, Ishimoto, S. (1998) Role of retinal vascular endothelial cells in development of CMV retinitis Trans Am Ophthalmol Soc 96,111-126 [PubMed]
Bodaghi, B, Slobbe-Van Drunen, ME, Topilko, A, et al (1999) Entry of human cytomegalovirus into retinal pigment epithelial and endothelial cells by endocytosis Invest Ophthalmol Vis Sci 40,2598-2607 [PubMed]
Jones, TR, Hanson, L, Lei Sun,, Slater, JS, Stenberg, RM, Campbell, AE. (1995) Multiple independent loci within the human cytomegalovirus unique short region down regulate expression of major histocompatibility complex class I heavy chain J Virol 69,4830-4841 [PubMed]
Vaz-Santiago,, Lulé, J, Rohrlich, P, et al (2001) Ex vivo stimulation and expression of both CD4+ and CD8+ T cells from PBMC of human cytomegalovirus (HCMV) seropositive blood donors using a soluble recombinant chimeric protein IE1-pp65 J Virol 75,7840-7847 [CrossRef] [PubMed]
Gavin, MA, Gilbert, MJ, Riddell, SR, Greenberg, PD, Bevan, MJ. (1993) Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes J Immunol 151,3971-3980 [PubMed]
Imbert-Marcille, BM, Robillard, N, Poirier, AS, et al (1997) Development of a direct quantification of cytomegalovirus antigenemia by flow cytometry J Clin Microbiol 35,2665-2626 [PubMed]
Gallot, G, Vivien, R, Ibish, C, et al (2001) Purification of Ag-specific T lymphocytes after direct peripheral blood mononuclear cell stimulation followed by CD25 selection: application to CD4+ or CD8+ cytomegalovirus phosphoprotein pp65 epitope determination J Immunol 167,4196-4206 [CrossRef] [PubMed]
Detrick, B, Rhame, J, Wang, Y, Nagineni, CN, Hooks, JJ. (1996) Cytomegalovirus replication in human retinal pigment epithelial cells. Altered expression of viral early proteins Invest Ophthalmol Vis Sci 37,814-825 [PubMed]
Berger, C, Xuereb, C, Johnson, S, et al (2000) Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8+ cytotoxic T lymphocytes J Virol 74,4465-4473 [CrossRef] [PubMed]
Hengel, H, Koopmann, JO, Flohr, T, et al (1997) A viral ER-resident glycoprotein inactivates the MHC-encoded peptide transporter Immunity 6,623-632 [CrossRef] [PubMed]
Lehner, P, Karttunen, J, Wilkinson, G, Cresswell, P. (1997) The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation Proc Natl Acad Sci USA 94,6904-6909 [CrossRef] [PubMed]
Gonzalez, CR, Wiley, CA, Arevalo, JF, et al (1996) Polymerase chain reaction detection of cytomegalovirus and human immunodeficiency virus-1 in the retina of patients with acquired immune deficiency syndrome with and without cotton-wool spots Retina 16,305-311 [PubMed]
Glasgow, BJ. (1997) Evidence for breaches of the retinal vasculature in acquired immune deficiency syndrome angiopathy: a fluorescent microsphere study Ophthalmology 104,753-760 [CrossRef] [PubMed]
Pereira, L, Maidji, E, Tugizov, S, Jones, T. (1995) Deletion mutants in human cytomegalovirus glycoprotein US9 are impaired in cell-cell transmission and in altering tight junctions of polarized human retinal pigment epithelial cells Scand J Dis Suppl 99,82-87
Ferguson, TA, Griffith, TS. (1997) A vision of cell death: insight immune privilege Immunol Rev 156,167-184 [CrossRef] [PubMed]
Cinatl, J, Jr, Blaheta, R, Bittoova, M, et al (2000) Decreased neutrophil adhesion to human cytomegalovirus infected retinal pigment epithelial cells is mediated by virus-induced up-regulation of Fas-ligand independent of neutrophil apoptosis J Immunol 165,4405-4413 [CrossRef] [PubMed]
Fish, KN, Brit, W, Nelson, JA. (1996) A novel mechanism for persistence of human cytomegalovirus in macrophages J Virol 70,1855-1862 [PubMed]
Fish, KN, Depto, AS, Moses, AV, Britt, W, Nelson, JA. (1995) Growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages J Virol 69,3037-3043
Figure 1.
 
RPE cells are positive for incoming pp65. RPE cells were either not infected (ni) or were infected with HCMV AD169 during 6 hours (6 hours pi). Anti-pp65 labeling and localization were examined by immunostaining followed by analysis under a confocal laser scanning microscope (A). Expression of pp65 in U373MG and RPE cells was examined throughout infection by immunoblotting of cell lysates (IB) and immunoprecipitation of [35S] methionine–[35S] cysteine–labeled cells (IP) with an anti-pp65 mAb (B).
Figure 1.
 
RPE cells are positive for incoming pp65. RPE cells were either not infected (ni) or were infected with HCMV AD169 during 6 hours (6 hours pi). Anti-pp65 labeling and localization were examined by immunostaining followed by analysis under a confocal laser scanning microscope (A). Expression of pp65 in U373MG and RPE cells was examined throughout infection by immunoblotting of cell lysates (IB) and immunoprecipitation of [35S] methionine–[35S] cysteine–labeled cells (IP) with an anti-pp65 mAb (B).
Figure 2.
 
Analysis of HCMV-infected U373MG and RPE cell sensitivity to killing by anti-pp65 CD8+T cells. Anti-pp65 CTLs from donor V were cultured and cytotoxicity determined in a 51Cr-release assay using as targets HLA-A2+ U373MG or RPE cells that had been either pulsed overnight with HLA-A2–binding peptide (N9V) or irrelevant peptide (I9Y) at 500 nM (A) or infected with HCMV AD169 strain at an MOI of 3 for 6 hours (6 hours pi), 48 hours (48 hours pi), or 8 days (8d pi) (B). E-T ratios were as indicated.
Figure 2.
 
Analysis of HCMV-infected U373MG and RPE cell sensitivity to killing by anti-pp65 CD8+T cells. Anti-pp65 CTLs from donor V were cultured and cytotoxicity determined in a 51Cr-release assay using as targets HLA-A2+ U373MG or RPE cells that had been either pulsed overnight with HLA-A2–binding peptide (N9V) or irrelevant peptide (I9Y) at 500 nM (A) or infected with HCMV AD169 strain at an MOI of 3 for 6 hours (6 hours pi), 48 hours (48 hours pi), or 8 days (8d pi) (B). E-T ratios were as indicated.
Figure 3.
 
RPE cells were able to present endogenously derived pp65 peptides to CTLs. HLA-A2 positive U373MG and RPE cells were either pulsed overnight with the HLA-A2–binding peptide N9V or irrelevant peptide I9Y at 500 nM or infected with pp65 recombinant adenoviruses Ad-pp65s and Ad-pp65as, sense and antisense, respectively. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 3.
 
RPE cells were able to present endogenously derived pp65 peptides to CTLs. HLA-A2 positive U373MG and RPE cells were either pulsed overnight with the HLA-A2–binding peptide N9V or irrelevant peptide I9Y at 500 nM or infected with pp65 recombinant adenoviruses Ad-pp65s and Ad-pp65as, sense and antisense, respectively. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 4.
 
Infection of U373MG and RPE cells with HCMV did not inhibit T-cell cytotoxicity. U373MG and RPE cells were either mock infected or infected with HCMV AD169 (MOI = 3), as indicated, for 6 hours (N9V-6 hours pi) or 48 hours (N9V-48 hours pi) and then pulsed overnight with N9V peptide at 500 nM. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 4.
 
Infection of U373MG and RPE cells with HCMV did not inhibit T-cell cytotoxicity. U373MG and RPE cells were either mock infected or infected with HCMV AD169 (MOI = 3), as indicated, for 6 hours (N9V-6 hours pi) or 48 hours (N9V-48 hours pi) and then pulsed overnight with N9V peptide at 500 nM. Cells were used as targets in a 51Cr-release assay at different E-T ratios.
Figure 5.
 
Proteins encoded in the US2-11 genomic region were responsible for escape of HCMV from anti-pp65 CTLs, both in U373MG and RPE cells. U373MG and RPE cells were infected for the indicated times with either HCMV AD169 (AD) or HCMVΔ2-11 (Δ2-11) and used as targets for HLA-A2-restricted anti-pp65 CTLs in a 51Cr-release assay (A). Cells pulsed with the irrelevant peptide I9Y were used as a negative control. E-T ratios were as indicated. Expression of HLA-A2 at the surface of U373MG and RPE cells was detected by staining with a mouse mAb and analyzed by flow cytometry (B).
Figure 5.
 
Proteins encoded in the US2-11 genomic region were responsible for escape of HCMV from anti-pp65 CTLs, both in U373MG and RPE cells. U373MG and RPE cells were infected for the indicated times with either HCMV AD169 (AD) or HCMVΔ2-11 (Δ2-11) and used as targets for HLA-A2-restricted anti-pp65 CTLs in a 51Cr-release assay (A). Cells pulsed with the irrelevant peptide I9Y were used as a negative control. E-T ratios were as indicated. Expression of HLA-A2 at the surface of U373MG and RPE cells was detected by staining with a mouse mAb and analyzed by flow cytometry (B).
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×