Department of Immunology, St Jude Childrens Research Hospital, 332 North Lauderdale, Memphis, TN 38101, USA1
Author for correspondence: David Topham. Present address: David H. Smith Center for Vaccine Biology & Immunology, Aab Institute for Biomedical Sciences, University of Rochester Medical Center, 601 Elmwood Avenue, Box 609, Rochester, NY 14642-8609, USA. Fax +1 716 273 2452. e-mail david_topham{at}urmc.rochester.edu
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Abstract |
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Introduction |
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What then are the relative roles of perforin and Fas in the CD8+ T cell-mediated control of respiratory infection with a large DNA virus? Intranasal challenge with murine gammaherpesvirus 68 (HV-68) (Cardin et al., 1996
; Nash & Sunil-Chandra, 1994
; Virgin & Speck, 1999
) is characterized by a lytic phase in epithelial cells (particularly of the respiratory tract) and a persistent, latent infection of B lymphocytes and macrophages (Cardin et al., 1996
; Sunil-Chandra et al., 1992
; Virgin & Speck, 1999
; Weck et al., 1999
). Depletion experiments in B cell-deficient µMT and B6 mice have demonstrated that the infection can be controlled by either CD4+ or CD8+ T cells, but not when T cells are absent (Christensen et al., 1999
; Stevenson et al., 1999c
). Control of the infection in P-/- mice was shown to differ little from P+/+ mice, leading to the conclusion that the perforin/granzyme pathway is not required to deal with this virus (Usherwood et al., 1997
). However, these experiments did not consider the role of the CD4+ T cells. More recent experiments showed that CD4+ T cells are important antiviral effector cells for this infection, and operate through IFN-
(Christensen et al., 1999
). Whether CD8+ T cells can operate via a similar mechanism, or are dependent on another cytokine or cytotoxic process, is not known. The current analysis focuses on the control of lytic
HV-68 infection in the lung by CD8+ T cells operating in the absence of the CD4+ subset. The contribution of IFN-
was investigated in B6 (P+/+), P-/- and Fas-/- congenic mice, while chimeric animals with P+/+ or P-/- T cells and Fas+/+ or Fas-/- infected target cells were used to dissect the requirements for perforin- and Fas-mediated cytotoxicity.
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Methods |
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Virus, infection and sampling.
HV-68 (clone G2.4), obtained from A. A. Nash (Edinburgh, UK), was grown in owl monkey kidney cells [OMK; CRL 1566 (ATCC)] (Cardin et al., 1996
). For infection, animals were anaesthetized with Avertin (2,2,2-tribromoethanol) prior to intranasal (i.n.) inoculation with 104 p.f.u.
HV-68 in 30 µl PBS. After infection, at the times indicated, the animals were given a lethal dose of Avertin and bled from the axilla. The inflammatory cells from the lung were obtained by broncheoalveolar lavage (BAL) (Allan et al., 1990
), and the draining mediastinal lymph node (MLN) and the spleen were made into a single cell suspensions for analysis. Lungs were removed and frozen at -70 °C for later titration of infectious virus.
Titration of infectious and latent virus in tissue samples.
The presence of infectious virus, or cells releasing infectious virus, was determined by plaque assay on NIH 3T3 cells (ATCC CRL 1658) (Cardin et al., 1996 ). Briefly, lung tissue was first disrupted in a Dounce homogenizer, and then frozen and thawed three time to lyse the cells and release virus. Dilutions of the homogenate were then adsorbed onto 3T3 monolayers for 1 h at 37 °C, overlaid with carboxymethylcellulose (CMC), and incubated at 37 °C for 5 days, At the end of the incubation, the overlay was removed, and the cells were fixed with methanol and stained with Giemsa prior to enumeration of the plaques. To determine the number of latently infected cells in the spleen, an infectious centre (i.c.) assay was performed by co-culturing various numbers of the spleen cells with 5x105 NIH 3T3 cells at 37 °C under a CMC overlay for 6 days, followed by removal of the overlay, and then fixation and staining with methanol and Giemsa. Aliquots of spleen cells were also sonicated to disrupt the cells and assayed for infectious virus.
Monoclonal antibody treatment and flow cytometry.
CD4 T cells were depleted by treatment of the animals with the GK1.5 MAb to CD4, and IFN- was neutralized by treatment with the XMG1.2 MAb (Christensen et al., 1999
; Stevenson et al., 1998
). Treatment was begun 5 days prior to infection and continued on alternating days throughout the course of the experiment. The prevalence of various lymphocyte subsets and the efficacy of the depletion regimen were examined by flow cytometry. Briefly, single cell suspensions were stained with phycoerythrin (PE)-conjugated 53-6.72 MAb to CD8 and FITC-conjugated RM-4-4 MAb to CD4, or PE-conjugated 30-H12 MAb to Thy1.2 and FITC-conjugated RA3-6B2 MAb to B220 prior to analysis on a FACScan (Becton Dickinson) using CellQuest software. In vitro T cell depletions and B cell depletions were similarly analysed by staining the cells with B220FITC and PE-conjugated 2C11 to CD3 to confirm removal of the appropriate subset. For identification of Ig heavy chain allotype expression, the cells were stained with FITC or PE conjugated DS-1 MAb to mouse IgMa and AF6-78 MAb to mouse IgMb. All conjugated antibodies were obtained from Pharmingen.
Radiation chimeras.
To study control of infection in the lung, Fas+/+ B6 mice and Fas-/- lpr mice were lethally irradiated with 950 rad and reconstituted 24 h later with 2x107 BM cells from either P+/+ or P-/- naïve mice. The animals were rested for 12 weeks to allow reconstitution of the immune cells before infection with -HV68. As above, beginning 1 day prior to infection and continuing every 2 to 3 days, some of the animals were treated with GK1.5 to remove the CD4 T cells, and XMG1.2 MAb to neutralize IFN-
(Christensen et al., 1999
; Topham et al., 1997
). To study the control of persistently infected B cells, two additional sets of chimeric animals were constructed by reconstituting irradiated (950 rad) B6 mice with 1x107 T cell-depleted Fas+/+ B6 BM or Fas-/- lpr BM. Two weeks later, the animals received 5x107 B cell-depleted spleen cells from perforin intact B6 or P-/- naïve mice. The T-depleted bone marrow was prepared by staining the BM with the anti-Thy1.2 IgM MAb AT83 prior to lysis with a 1:5 mixture of rabbit and guinea pig complement (Cedar Lane Laboratories, Ontario, Canada) (Topham & Doherty, 1998
). The efficacy of the T cell depletion was assessed by FACS subsequent to staining with MAb to CD3
(2C11) and was >99%. The depletion of the B cells from the donor spleens was accomplished by staining the cells with anti-class II MHC MAb to IAb (ATCC TIB120) followed by incubation of the cells with a mixture of anti-rat Ig and anti-mouse Ig magnetic beads (Dynal), and later harvesting the non-adherent cells after placing against a magnet. The remaining cells were <1% B220+ at the end of the protocol. In a second experiment, the same techniques of enrichment were employed except that the recipients were thymectomized, congenic C57BL6/Thy1.1/IgHa mice which differ in their Ig heavy chain allotype expression (IgHa vs IgHb).
Intracellular IFN-
staining.
The non-adherent BAL cell population was cultured for 6 h in 96-well round-bottom plates at 58x105 cells per well in complete medium containing 10 µg/ml Brefeldin A (Epicenter Technologies, Madison, WI, USA) with or without 110 µM HV-68-specific peptides, p56 or p79 (Stevenson et al., 1998
, 1999a
). After culture, the cells were placed on ice, washed in PBSBrefeldin A (10 µg/ml), stained with anti-CD8FITC as above, washed again, and fixed with 1% formaldehyde. The cells were permeabilized in 0·5% saponin (Sigma) before staining with anti-IFN-
PE (Pharmingen) for 30 min on ice. The cells were then washed and analysed on the FACScan as above.
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Results |
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Uncontrolled infection in the absence of perforin and Fas
What remains unclear from the analysis of P-/- and lpr mice are the individual contributions of Fas and perforin. IFN- does not appear to be an essential mediator of the antiviral activity of the cells remaining after CD4 T cell depletion, though there was some effect on latency in the lpr mice (Table 1
, Fig. 1
). The observation that
HV-68 is controlled in the lung regardless of whether the animals were missing either Fas or perforin alone suggested that keeping at least one of these modes of cytotoxic action intact is sufficient. Alternatively, there could be another mechanism of antiviral control (cytotoxic or otherwise) that had not yet been identified in these experiments. If there is a further means of control, this should emerge in a situation where both perforin and Fas are absent. We thus made radiation bone marrow chimeras using P-/- BM to provide the CD8+ CTL, and lpr mice as recipients or B cell donors to limit Fas expression either in the lung or B cells respectively. With such mice, the Fas pathway is retained among the lymphocytes to regulate T cell homeostasis.
To examine the issue of controlling lytic virus in the lung, lethally irradiated (950 rad) Fas+/+ (B6) or Fas-/- (lpr) recipients were reconstituted with P+/+ or P-/- bone marrow. The chimeras were held for 12 weeks to allow for T cell maturation, and then treated with anti-CD4 MAb (to deplete the CD4+ T cells) or a control rat Ig. At 10 and 15 days after infection (1314 weeks after reconstitution), the recipient animals had similar splenic cellularity and proportions of CD8+ T cells (Table 2). Analysis of the inflammatory exudate by BAL revealed a strong inflammatory cellular infiltrate that consisted of primarily CD8+ T cells (Table 2
). Assaying the BAL cells for reactivity to the two most prominent class I MHC restricted epitopes of
HV-68 (p56 and p79) showed clearly that virus-specific CD8+ T cells were indeed present and responsive in all groups of the chimeric mice (Fig. 2
).
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Discussion |
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Another interpretation might consider whether CD4+ and CD8+ T cells act indirectly through the recruitment of natural killer (NK) cells. Although the potential of NK cells was not directly explored here, experiments done with doubly (CD4 and CD8) T-depleted mice (data not shown), or irradiated mice which received no T cells (Table 3), gave no evidence that NK cells are a major player in controlling
HV-68 respiratory infection. If NK cells do have a role, they are more likely to be important early (57 days) in the infection as they are for other viruses (Hussell & Openshaw, 1998
; Sarawar et al., 1993
; Tay et al., 1999
), rather than the day 1040 time-points examined here. In fact, at 10 days after infection, the proportion of CD8+ T cells in the BAL of the chimeric animals was 8495%, leaving little room for NK cells. Even so, NK effector cells have been shown to use the perforin or, in tumour models, Fas-mediated cytotoxicity (Kodama et al., 1999
; Rosen et al., 2000
; Tay & Welsh, 1997
; van den Broek et al., 1995
, 1996
). Consequently, the NK effectors would also be compromised in the experimental models described here. Cytotoxicity alone, whether achieved through Fas, perforin or both has a limited capacity to eliminate the cells producing infectious virus from the lung (Doherty et al., 1997
; Stevenson et al., 1999b
). This may be relevant to the situation where an individual continues to shed infectious virus while maintaining a degree of control that obscures disease. When both Fas and perforin are compromised as they are in the P-/-
Fas-/- chimeras, control of replicating virus is ineffectual, even in the presence of CD4+ T cells and Ig-secreting B cells. This suggests that some contact-dependent cytotoxic effector activity is always necessary, and that soluble cytotoxic mediators like TNF-
at best play an ancillary role for the control of this infection in the lung (Fleck et al., 1998
; Liu et al., 1999
). It still seems reasonable to assume that control of this virus (and certainly other viruses) by CD8+ T cells is enhanced by IFN-
, in spite of the observation that it is not essential.
The high incidence of lethal disease seen consistently in P-/-Fas+/+ chimeric mice raises questions about the specificity of Fas-directed CTL activity. We did not observe this consequence in unirradiated P-/- mice, which theoretically have similar cytotoxic machinery. That this lethal effect is an immunopathogenic consequence related to the expression of Fas is suggested by the fact that animals with the same P-/- cells in a Fas-/- host show no evidence of disease (Fig. 4
). This is in spite of the fact that these mice do not control virus replication (Fig. 3
). Although the P-/-
Fas+/+ chimeric animals appear to be well reconstituted (Table 2
) and have a reasonable number of antigen-specific CD8+ T cells at the site of infection (Fig. 2
), the availability of effector cells may be somewhat more limiting than in their non-chimeric P-/- counterparts. The net consequence is the slowed clearance of lytic virus in the chimeras, which in turn leads to greater damage resulting from Fas ligation by the P-/- CD8+ effectors. Thus the race between the virus and the immune effectors in this scenario favours the virus for too long, with the immune system trying to catch up, and many lung cells being damaged in the process. It is possible that the irradiation used to produce these chimeric animals may also increase Fas expression or otherwise alter lung physiology to enhance their susceptibility to Fas-mediated lysis. Exposure of the lung to chemotherapeutic agents like bleomycin results in an increase of Fas expression by lung epithelial cells (Kuwano et al., 1999
, 2000
). In either case the suggestion is that Fas-mediated cytotoxicity is more promiscuous, causing proximal uninfected Fas+ cells to also be destroyed (Smyth et al., 1998
).
Perforin-mediated cytotoxicity is generally more rapid and exquisitely targeted to the antigen-positive cells (Kagi et al., 1994 ; Lowin et al., 1994
b). For example, P-/- CTL do not read out in a conventional 6 h 51Cr-release assay while P+/+ CTL do (Kagi et al., 1994
). The cell death mechanisms involving Fas are very old in the phylogenetic sense, though both the perforin/granzyme B (Kagi et al., 1994
; Lowin et al., 1994
a
, 1995
; Mullbacher et al., 1999b
) and Fas-ligation pathways operate to initiate apoptosis via activation of the same caspases (Bird et al., 1998
; Trapani et al., 1998
). It seems clear that the rapid and efficient perforin-mediated cytotoxic pathway is the more desirable and efficient means for CTL to control this and other virus infections of the lung (Kagi et al., 1994
; Topham et al., 1997
). Perhaps the evolution of the perforin/granzyme cytotoxic pathway reflects the fact that Fas-directed CTLs cause too much collateral damage and are too dangerous to be used for normal virus control.
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Acknowledgments |
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Footnotes |
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c Present address: Institute of Medical Microbiology and Immunology, The Panum Institute, Blegdamsvej 3c, Building 22.5.16, DK-2200 Copenhagen N, Denmark.
d Present address: Becton-Dickinson/Transduction Laboratories, Inc., Lexington, KY 40511, USA.
e Present address: The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Victoria, Australia.
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Received 9 January 2001;
accepted 28 March 2001.