1 Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8096, St Louis, MO 63110, USA
2 Department of Molecular Microbiology & Pathogenesis, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8096, St Louis, MO 63110, USA
3 Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St Louis, MO 63104, USA
Correspondence
Patrick M. Stuart
stuart{at}vision.wustl.edu
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ABSTRACT |
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INTRODUCTION |
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Corneas removed from patients requiring corneal transplants due to HSK contain both CD4+ and CD8+ T cells that are specific for HSV-encoded antigens (Koelle et al., 2000). In most models of HSK, T cells are critical to the development of corneal lesions, as athymic nude mice do not display signs of HSK (Metcalf et al., 1979
) unless adoptive transfer of T cells is performed (Russell et al., 1984
). While most investigators believe that the CD4+ subset of T cells mediates this disease, some reports implicate CD8+ T cells as having a major role in primary HSV keratitis (Akova et al., 1993
; Doymaz & Rouse, 1992
; Hendricks & Tumpey, 1990
; Niemialtowski & Rouse, 1992
). Furthermore, when most corneas are immunohistochemically stained for T cell subsets, the predominant subset found appears to be dependent on the model system employed (Doymaz & Rouse, 1992
; Hendricks & Tumpey, 1990
). Further complicating the issue are reports by Ghiasi et al. (1999
, 2000)
showing that both CD4 and CD8 knockout (KO) mice made on the C57BL/6 background demonstrate increased disease when compared with normal C57BL/6 mice. They interpreted their results as indicating that both of these T cell subsets play a role in herpetic corneal disease and that their function can be both destructive and protective. Additional studies assessing the function of T cell subsets in HSV latency found that CD8+ T cells in mice with HSV-infected trigeminal ganglia are responsible for maintaining latency (Khanna et al., 2003
; Liu et al., 2000
, 2001
).
An understanding of the cellular interactions between virus-specific immune cells and cells of the cornea and nervous system are crucial in determining the underlying mechanisms of HSK. To examine more fully the role of CD4+ and CD8+ cells during primary HSK, we utilized mice deficient in CD4+ or CD8+ T cells. To determine whether host genetic background influences the role of T cell subsets in recurrent corneal disease, we performed our experiments in HSV-susceptible (BALB/c) and HSV-resistant (C57BL/6) strains of mice. Our findings indicated that disease was associated with the presence of CD4+ T cells and that, when these cells were absent, little disease was evident. Furthermore, transfer of CD8+ T cells from infected mice provided significant protection against the development of primary HSK.
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METHODS |
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Mice and primary infection.
Investigations with mice conformed to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 (B6) and BALB/c mice were purchased from the National Cancer Institute (Fredrick, MD, USA). The B6.129-cd4tm1Mak and B6.129-cd8tm1Mak mice were generously provided by Dr Tak Mak (University of Toronto) and maintained in our colony. For the purposes of this report, these mice are referred to as B6-CD4 KO and B6-CD8 KO mice, respectively. We also bred the B6-CD4 KO and B6-CD8 KO mice with BALB/c mice for a minimum of 10 generations. The resultant strains were designated C.129S(B6)-cd4tm1 and C.129S(B6)-cd8tm1, referred to here as C-CD4 KO and C-CD8 KO, respectively. Due to the occasional leakiness' of the KO phenotype, all mice underwent flow cytometric analysis of peripheral blood lymphocytes. Only those mice in which the targeted cell type could not be detected were used in these studies (Haskova et al., 2000). It should be noted that we have never observed a mouse that tested negative for the targeted cell type prior to infection display those cells at any time following infection (data not shown). Normal and KO mice (812 weeks old) were infected as described previously (Rader et al., 1993
). Briefly, following corneal scarification, 2x107 p.f.u. HSV-1 KOS strain or 5x106 p.f.u. HSV-1 McKrae strain in 5 µl MEM-EBS was placed onto the surface of both corneas of BALB/c (HSV-sensitive) or C57BL/6 (HSV-resistant) mice, respectively.
Clinical evaluation.
On the designated days after virus infection, a masked observer examined mouse eyes through a binocular-dissecting microscope and scored clinical disease. Stromal opacification was rated on a scale of 04, where 0 indicated clear stroma, 1 indicated mild stromal opacification, 2 indicated moderate opacity with discernible iris features, 3 indicated dense opacity with loss of defined iris detail except pupil margins, and 4 indicated total opacity with no posterior view. Corneal neovascularization was evaluated as described previously (Stuart et al., 2003) using a scale of 08, where each of four quadrants of the eye is evaluated for the number of vessels that have grown into them. Periocular disease, in the form of blepharitis, was measured in a masked fashion on a semi-quantitative scale as described previously (Smith et al., 2000).
Titration of virus from tissues.
Eye swab material was collected and assayed for virus by standard plaque assay as described previously (Rader et al., 1993). Trigeminal ganglia and 6 mm biopsy punches of periocular skin were removed and placed in pre-weighed tubes containing 1 mm glass beads and 1 ml medium. Trigeminal ganglia and periocular skin homogenates were prepared by freezing and thawing the samples, mechanically disrupting in a Mini-Beadbeater-8 (Biospec Products) and sonicating. Homogenates were assayed for virus by standard plaque assay and the amount of virus was expressed as p.f.u. (ml tissue homogenate)1.
Virus reactivation assay.
Trigeminal ganglia were removed from infected mice 3040 days post-infection. To assess reactivation, individual trigeminal ganglia were dissociated (Kennedy et al., 1980) and plated on collagen-coated 12-well plates. Supernatants were assayed every 12 h for progeny virus from 1 to 5 days post-plating.
Limiting dilution assay for latency.
Between 28 and 35 days post-infection, trigeminal ganglia were removed from infected mice. Trigeminal ganglia were pooled from three to four mice, washed once in Dulbecco's minimal essential medium (DMEM) and 3 ml dissociation buffer was added per ganglion pool. The mixture was incubated in a shaking incubator at 37 °C for 1 h, then pelleted at 2500 r.p.m. and the supernatant discarded. The cells in the pellets were resuspended in DMEM and added to collagen-coated plates; twofold serial dilutions, starting with 5x105 cells per well, were performed. Cells were incubated overnight at 37 °C. At this time, 5x104 Vero cells were added in 1 ml serum-free DMEM containing penicillin, fungizone and streptomycin to each collagen well. Wells were monitored for cytopathic effect (CPE) and 100 µl supernatant was removed from each well on days 3, 5, 9, 12 and tested for the presence of virus by adding to 48-well plates containing 2x104 Vero cells per well. These wells were then judged for CPE at day 6.
In vivo T cell depletion.
BALB/c mice were treated with either anti-CD4 (clone GK1.5) or anti-CD8 (clone H-35) to remove targeted T cell subsets, or with diluent as a control. Treatment consisted of three injections given on days 2, 3 and 5 post-infection. Mice were monitored for effectiveness of in vivo depletion between 2 and 3 weeks post-infection by flow cytometry of peripheral blood lymphocytes (Haskova et al., 2000). Treatment with anti-CD4 antibody resulted in 8590 % depletion of CD4+ T cells. Because this treatment did not remove more than 95 % of the CD4+ T cells, representative HSV-infected mice were tested for the functional presence of CD4+ T cells by testing them for specific delayed-type hypersensitivity (DTH) responses to HSV antigenic preparations (Keadle et al., 2002b
). For all mice tested, no mice displayed DTH responses above background levels (data not shown). Mice treated with anti-CD8 antibody resulted in more than 98 % depletion of CD8+ T cells.
T cell isolation and adoptive transfer.
C-CD4 KO mice were infected with 2x107 p.f.u. HSV-1 KOS strain and killed 23 weeks following infection. Single-cell suspensions of spleen and lymph node cells were prepared. These cells were then fractionated as follows: a T cell-enriched population was isolated by passing cells over a nylon wool column followed by treatment with anti-B220 (clone J11d) and anti-MHC class II (clone 11.3.1) plus complement (Cedarlane Laboratories) to deplete B cells and monocytes further. A T cell-depleted population was isolated by treatment with anti-Thy (clone HO 13-4.6) and anti-CD8 (clone 3.155) plus complement. A control population was treated with an irrelevant antibody (clone W6/32) plus complement. All of the antibodies used in these depletions were derived from culture supernatants of the indicated hybridoma clones. Following depletion, each of the resultant groups was evaluated for the presence of the targeted cell population and less than 1 % of the targeted cells were found. BALB/c and C-CD8 KO mice were then injected with these fractions using 2x107 cells for adoptive transfer. Mice were subsequently challenged with 2x107 p.f.u. HSV-1 KOS strain and the disease monitored for 5 weeks.
Assays of antibody titres.
Serum was collected from mice at weekly intervals following infection and examined for HSV-specific antibody content as described previously (Geiss et al., 2000). Briefly, for ELISA, serial fourfold dilutions of mouse serum were incubated for 2 h in duplicate wells of a 96-well plate coated with purified HSV-1 glycoprotein. Biotinylated goat anti-mouse IgG was subsequently used in a colorimetric assay to determine specific IgG levels based on comparison with a standard curve generated as described previously (Geiss et al., 2000
).
Statistical analyses.
All statistical analyses were performed with the aid of Sigma Stat for Windows, version 2.0 (Jandel, Corte Madera, CA). Student's unpaired t-test was used to compare corneal disease scores and virus and antibody titre data. Fisher's exact 2 test was used to compare the limiting dilution assay data.
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RESULTS |
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DISCUSSION |
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The role of CD8+ T cells remains a complicated and controversial question. Mice that have CD8+ but not CD4+ T cells exhibit similar or reduced corneal disease when compared with normal mice and they also display no differences when compared with wild-type mice in signs of neurological disease or increased mortality following HSV-1 infection. In contrast, mice lacking CD8+ T cells have a higher incidence of gross neurological pathology and have a slightly, although not significantly, greater incidence of mortality. Since the disease phenotype for mice lacking CD8+ T cells does not appear to involve compromised virus clearance or increased viral loads in the trigeminal ganglia, we propose that CD8+ T cells might be acting as regulators of disease by controlling the activity of those cells that mediate HSK. Data shown in Fig. 6 are consistent with this hypothesis. When we transferred T cells from infected C-CD4 KO mice, recipient C-CD8 KO mice were significantly protected from disease. Thus, the addition of CD8+ T cells to CD8 KO mice prevented the development of HSK following infection.
It is unlikely that protection from HSK in mice receiving T cells by adoptive transfer was due to contaminating B cells producing protective levels of anti-HSV-1 antibodies (Keadle et al., 2002b; Shimeld et al., 1990
). Firstly, there were no differences in protection when C-CD8 KO mice were given unfractionated cells or B cell-depleted cells from infected CD4 KO mice. Secondly, when C-CD4 KO mice were evaluated for anti-HSV antibodies (Fig. 5
), there were significantly less than seen in either C-CD8 KO or BALB/c mice, which is consistent with previous reports that mice lacking CD4+ T cells are significantly compromised in their ability to generate anti-HSV-1 protective antibody responses (Chan et al., 1985
; Ghiasi et al., 1997
; Irie et al., 2002
; Morrison & Knipe, 1997
).
Since several reports (Khanna et al., 2003; Liu et al., 2000
, 2001
) have implicated a direct role for CD8+ T cells in maintaining the latent phenotype of HSV-1 in trigeminal ganglia, we thought it an attractive hypothesis that the underlying mechanism for increased neurological pathology was an inability to maintain latency effectively in CD8 KO mice. However, when trigeminal ganglia from parental, C-CD4 KO and C-CD8 KO mice were compared for kinetics of virus production following infection, reactivation rates or viral loads as determined by limiting dilution analysis, no significant differences were noted. Because we used more than one virus/mouse strain combination in these studies, we feel more confident in our conclusion that the increased disease we observed in CD8 KO mice is not due to persistent virus production, increased rates of reactivation or increased viral load in trigeminal ganglia.
Regulatory T cells have experienced a renaissance in their importance in controlling immune responses (Shevach, 2000). While most of the recent literature has focused on CD4+CD25+ T cells with regulatory activity (Field et al., 2001
; Piccirillo & Shevach, 2001
; Skelsey et al., 2003
), there also exist CD8+ T cells that express regulatory functions (Cosmi et al., 2003
; Ferguson et al., 2002
, 2003
). These CD8+ T regulatory cells are found in both humans (Cortesini et al., 2002
; Filaci & Suciu-Foca, 2002
) and mice (Ferguson et al., 2002
; Jiang & Chess, 2000
; Jiang et al., 2001
) and operate in a variety of antigen-specific responses (Ferguson et al., 2002
; Nakamura et al., 2003
) and diseases (Jiang et al., 2001
; Taneja et al., 2002
; Zhang et al., 2002
). They have also been implicated in viral and parasitic infections (Hafalla et al., 2003
). Finally, there is evidence that CD8+ T regulatory cells mediate antigen-specific tolerance in models of anterior chamber-associated immune deviation (Nakamura et al., 2003
; Skelsey et al., 2003
), as well as controlling T cells that infiltrate the eyes of mice with autoimmune anterior uveitis (Zhang et al., 2002
).
Concomitant with resolution of HSK lesions associated with the activity of CD4+ Th1 cells is an increase in cytokines associated with immunosuppression (Niemialtowski & Rouse, 1992). We hypothesize that the cells that mediate resolution of corneal disease are most likely CD8+ T regulatory cells. While it is possible that CD4+ T cells may also be involved, as has been suggested recently (Suvas et al., 2003
), we have no evidence to support their involvement in regulating HSK. We do know from this work that, in the absence of CD4+ T cells, a population of CD8+ T cells is generated that protects mice from disease and that this is not by more efficient clearance of virus. Thus, we propose that these cells act by regulating the CD4+ Th1 cells.
In conclusion, our data best support the hypothesis that primary HSK is mediated by T cells bearing the CD4+ phenotype and that CD8+ cells protect the mice from HSK by a mechanism that does not involve more efficient virus clearance or an inability to maintain latency, but likely involves their ability to regulate the function of disease-mediating CD4+ T cells. Future studies will focus on further defining these CD8+ T cells and the specific means that they use to regulate corneal disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chan, W. L., Lukig, M. L. & Liew, F. Y. (1985). Helper T cells induced by an immunopurified herpes simplex virus type I (HSV-I) 115 kilodalton glycoprotein (gB) protect mice against HSV-I infection. J Exp Med 162, 13041318.[Abstract]
Cortesini, R., Renna-Molajoni, E., Cinti, P., Pretagostini, R., Ho, E., Rossi, P. & Suciu-Foca Cortesini, N. (2002). Tailoring of immunosuppression in renal and liver allograft recipients displaying donor specific T-suppressor cells. Hum Immunol 63, 10101018.[CrossRef][Medline]
Cosmi, L., Liotta, F., Lazzeri, E. & 10 other authors (2003). Human CD8+CD25+ thymocytes sharing phenotypic and functional features with CD4+CD25+ regulatory thymocytes. Blood 102, 41074114.
Doymaz, M. Z. & Rouse, B. T. (1992). Herpetic stromal keratitis: an immunopathologic disease mediated by CD4+ T lymphocytes. Invest Ophthalmol Vis Sci 33, 21652173.[Abstract]
Ferguson, T. A., Herndon, J., Elzey, B., Griffith, T. S., Schoenberger, S. & Green, D. R. (2002). Uptake of apoptotic antigen-coupled cells by lymphoid dendritic cells and cross-priming of CD8+ T cells produce active immune unresponsiveness. J Immunol 168, 55895595.
Ferguson, T. A., Stuart, P. M., Herndon, J. M. & Griffith, T. S. (2003). Apoptosis, tolerance and regulatory T cells. Old wine, new wineskins. Immunol Rev 193, 111123.[CrossRef][Medline]
Field, E. H., Matesic, D., Rigby, S., Fehr, T., Rouse, T. & Gao, Q. (2001). CD4+CD25+ regulatory cells in acquired MHC tolerance. Immunol Rev 182, 99112.[CrossRef][Medline]
Filaci, G. & Suciu-Foca, N. (2002). CD8+ T suppressor cells are back to the game: are they players in autoimmunity? Autoimmun Rev 1, 279283.[CrossRef][Medline]
Geiss, B. J., Smith, T. J., Leib, D. A. & Morrison, L. A. (2000). Disruption of virion host shutoff activity improves the immunogenicity and protective capacity of a replication-incompetent herpes simplex virus type 1 vaccine strain. J Virol 74, 1113711144.
Ghiasi, H., Cai, S., Nesburn, A. B. & Wechsler, S. L. (1997). MHC-II but not MHC-I responses are required for vaccine-induced protection against ocular challenge with HSV-1. Curr Eye Res 16, 11521158.[CrossRef][Medline]
Ghiasi, H., Perng, G.-Y., Nesburn, A. B. & Wechsler, S. L. (1999). Either CD4+ or CD8+ T cell function is sufficient for clearance of infectious virus from trigeminal ganglia and establishment of herpes simplex virus type 1 latency in mice. Microb Pathog 27, 387394.[CrossRef][Medline]
Ghiasi, H., Cai, S., Perng, G.-Y., Nesburn, A. B. & Wechsler, S. L. (2000). Both CD4+ and CD8+ T cells are involved in protection against HSV-1 induced corneal scarring. Br J Ophthalmol 84, 408412.
Gilliet, M. & Liu, Y. J. (2002a). Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells. J Exp Med 195, 695704.
Gilliet, M. & Liu, Y. J. (2002b). Human plasmacytoid-derived dendritic cells and the induction of T-regulatory cells. Hum Immunol 63, 11491155.[CrossRef][Medline]
Hafalla, J. C., Morrot, A., Sano, G., Milon, G., Lafaille, J. J. & Zavala, F. (2003). Early self-regulatory mechanisms control the magnitude of CD8+ T cell responses against liver stages of murine malaria. J Immunol 171, 964970.
Haskova, Z., Usui, N., Ferguson, T. A., Pepose, J. S. & Stuart, P. M. (2000). CD4+ T cells are critical in corneal but not skin allograft rejection. Transplantation 69, 483488.[CrossRef][Medline]
Hendricks, R. L. & Tumpey, T. M. (1990). Contribution of virus and immune factors to herpes simplex virus type 1-induced corneal pathology. Invest Ophthalmol Vis Sci 31, 19291939.[Abstract]
Irie, H., Aita, K., Koyama, A. H., Fukuda, A., Yoshida, T. & Shiga, J. (2002). The role of donor CD4+ T cells in the reconstitution of oral immunity by herpes simplex virus type 1 in severe combined immunodeficiency mice. J Infect Dis 185, 409416.[CrossRef][Medline]
Jiang, H. & Chess, L. (2000). The specific regulation of immune responses by CD8+ T cells restricted by the MHC class Ib molecule, Qa-1. Annu Rev Immunol 18, 185216.[CrossRef][Medline]
Jiang, H., Braunstein, N. S., Yu, B., Winchester, R. & Chess, L. (2001). CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice. Proc Natl Acad Sci U S A 98, 63016306.
Keadle, T. L., Morris, J. L., Pepose, J. S. & Stuart, P. M. (2002a). CD4+ and CD8+ cells are key participants in the development of recurrent herpetic stromal keratitis in mice. Microb Pathog 32, 255262.[CrossRef][Medline]
Keadle, T. L., Morrison, L. A., Morris, J., Pepose, J. S. & Stuart, P. M. (2002b). Therapeutic immunization with a virion host shutoff (vhs) defective, replication-incompetent HSV-1 strain limits recurrent herpetic ocular infection. J Virol 76, 36153625.
Kennedy, P. G., Lisak, R. P. & Raff, M. C. (1980). Cell type-specific markers for human glial and neuronal cells in culture. Lab Invest 43, 342351.[Medline]
Khanna, K. M., Bonneau, R. H., Kinchington, P. R. & Hendricks, R. L. (2003). Herpes simplex virus-specific memory CD8+ T cells are selectively activated and retained in latently infected sensory ganglia. Immunity 18, 593603.[CrossRef][Medline]
Koelle, D. M., Reymond, S. N., Chen, H. & 7 other authors (2000). Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans. J Virol 74, 1093010938.
Liu, T., Khanna, K. M., Chen, X., Fink, D. J. & Hendricks, R. L. (2000). CD8+ T cells can block herpes simplex virus type 1 (HSV-1) reactivation from latency in sensory neurons. J Exp Med 191, 14591466.
Liu, T., Khanna, K. M., Carriere, B. N. & Hendricks, R. L. (2001). Gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons. J Virol 75, 1117811184.
Maertzdorf, J., Verjans, G. M., Remeijer, L., van der Kooi, A. & Osterhaus, A. D. (2003). Restricted T cell receptor beta-chain variable region protein use by cornea-derived CD4+ and CD8+ herpes simplex virus-specific T cells in patients with herpetic stromal keratitis. J Infect Dis 187, 550558.[CrossRef][Medline]
Metcalf, J. F., Hamilton, D. S. & Reichert, R. W. (1979). Herpetic keratitis in athymic (nude) mice. Infect Immun 26, 11641171.[Medline]
Miller, J. K., Laycock, K. A., Umphress, J. A., Hook, K. K., Stuart, P. M. & Pepose, J. S. (1996). A comparison of recurrent versus primary HSK in inbred mice. Cornea 15, 497504.[Medline]
Morrison, L. A. & Knipe, D. M. (1997). Contributions of antibody and T cell subsets to protection elicited by immunization with a replication-defective mutant of herpes simplex virus type 1. Virology 239, 315326.[CrossRef][Medline]
Nakamura, T., Sonoda, K. H., Faunce, D. E., Gumperz, J., Yamamura, T., Miyake, S. & Stein-Streilein, J. (2003). CD4+ NKT cells, but not conventional CD4+ T cells, are required to generate efferent CD8+ T regulatory cells following antigen inoculation in an immune-privileged site. J Immunol 171, 12661271.
Newell, C. K., Martin, S., Sendele, D., Mercadal, C. M. & Rouse, B. T. (1989). Herpes simplex virus-induced stromal keratitis: role of T-lymphocyte subsets in immunopathology. J Virol 63, 769775.[Medline]
Nicholson, S. M., Dal Canto, M. C., Miller, S. D. & Melvold, R. W. (1996). Adoptively transferred CD8+ T lymphocytes against TMEV-induced demyelinating disease in BALB/c mice. J Immunol 156, 12761283.[Abstract]
Niemialtowski, M. G. & Rouse, B. T. (1992). Phenotype and functional studies on ocular T cells during herpetic infections of the eye. J Immunol 148, 18641870.
Pepose, J. S., Leib, D. A., Stuart, P. M. & Easty, E. L. (1996). Herpes simplex virus diseases: anterior segment of the eye. In Ocular Infection and Immunity, pp. 905932. Edited by J. S. Pepose, G. A. N. Holland & K. R. Wilhelmus. St Louis, MO: Mosby.
Piccirillo, C. A. & Shevach, E. M. (2001). Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells. J Immunol 167, 11371140.
Rader, K. A., Ackland-Berglund, C. E., Miller, J. K., Pepose, J. S. & Leib, D. A. (1993). In vivo characterization of site-directed mutations in the promoter of the herpes simplex virus type 1 latency-associated transcripts. J Gen Virol 74, 18591869.[Abstract]
Russell, R. G., Nasisse, M. P., Larsen, H. S. & Rouse, B. T. (1984). Role of T-lymphocytes in the pathogenesis of herpetic stromal keratitis. Invest Ophthalmol Vis Sci 25, 938944.[Abstract]
Shevach, E. M. (2000). Regulatory T cells in autoimmmunity. Annu Rev Immunol 18, 423449.[CrossRef][Medline]
Shimeld, C., Hill, T. J., Blyth, W. A. & Easty, D. L. (1990). Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system. J Gen Virol 71, 681687.[Abstract]
Skelsey, M. E., Mayhew, E. & Niederkorn, J. Y. (2003). CD25+, interleukin-10-producing CD4+ T cells are required for suppressor cell production and immune privilege in the anterior chamber of the eye. Immunology 110, 1829.[CrossRef][Medline]
Smith, T. J., Ackland-Berglund, C. E. & Leib, D. A. (2000). Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice. J Virol 74, 35983604.
Stuart, P. M., Pan, F., Plambeck, S. & Ferguson, T. A. (2003). FasLFas ligand interactions regulate neovascularization in the cornea. Invest Ophthalmol Vis Sci 44, 9398.
Suvas, S., Kumaraguru, U., Pack, C. D., Lee, S. & Rouse, B. T. (2003). CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses. J Exp Med 198, 889901.
Taneja, V., Taneja, N., Paisansinsup, T., Behrens, M., Griffiths, M., Luthra, H. & David, C. S. (2002). CD4 and CD8 T cells in susceptibility/protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: implications for rheumatoid arthritis. J Immunol 168, 58675875.
Thomas, J. & Rouse, B. T. (1997). Immunopathogenesis of herpetic ocular disease. Immunol Res 16, 375386.[Medline]
Verjans, G. M., Remeijer, L., van Binnendijk, R. S., Cornelissen, J. G., Volker-Dieben, J. H., Baarsma, S. G. & Osterhaus, A. D. (1998). Identification and characterization of herpes simplex virus-specific CD4+ T cells in corneas of herpetic stromal keratitis patients. J Infect Dis 177, 484488.[Medline]
Youinou, P., Colin, J. & Mottier, D. (1985). Immunological analysis of the cornea in herpetic stromal keratitis. J Clin Lab Immunol 17, 105106.[Medline]
Youinou, P., Colin, J. & Ferec, C. (1986). Monoclonal antibody analysis of blood and cornea T lymphocyte subpopulations in herpes simplex keratitis. Graefes Arch Clin Exp Ophthalmol 224, 131133.[Medline]
Zhang, X., Jiang, S., Manczak, M., Sugden, B. & Adamus, G. (2002). Phenotypes of T cells infiltrating the eyes in autoimmune anterior uveitis associated with EAE. Invest Ophthalmol Vis Sci 43, 14991508.
Received 19 February 2004;
accepted 18 March 2004.