Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA
Correspondence
Philip Stevenson (at University of Cambridge)
pgs27{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Present address: Division of Immunology, Walter and Eliza Hall Institute of Medical Research, PO Box Royal Melbourne Hospital, Victoria 3050, Australia.
Present address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
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MAIN TEXT |
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Murine gammaherpesvirus-68 (MHV-68) persists in immunocompetent mice after intranasal infection without causing overt disease (Virgin & Speck, 1999; Nash et al., 2001
), but is lethal to CD4+ T cell-deficient I-Ab-/- mice after 34 months due to chronic lytic virus replication (Cardin et al., 1996
). CTL control the acute infection in I-Ab-/- mice. However, even though the CTL specific for two immunodominant MHV-68 lytic cycle epitopes (Stevenson et al., 1999
) are maintained numerically during the chronic illness (Stevenson et al., 1998
), they cannot suppress lytic replication completely, even when boosted to high levels by post-exposure, epitope-specific vaccination (Belz et al., 2000
). These boosted MHV-68-specific CTL were recently shown to be functionally abnormal (Liu et al., 2002
). There was both decreased expression of CD44 and reduced cytotoxicity in comparison with I-Ab+/+ controls. This has raised the possibility that our earlier analysis of MHV-68 infection in otherwise unmanipulated I-Ab-/- mice missed some subtle defect in the virus-specific CD8+ T cell response. We have thus looked in more detail at this question of CD8+ T cell functional integrity for MHV-68-specific CTL generated in conventional and CD4+ T cell-deficient mice.
Previously, we documented the persistence of MHV-68-specific CD8+ T cells in I-Ab-/- mice by MHC class I/peptide tetramer staining and by peptide-induced IFN- production (Stevenson et al., 1998
). However, recent data (Appay et al., 2000
; Liu et al., 2002
) have raised the possibility that such criteria are not sufficient to rule out functional deficits. Surface phenotypes may change and the capacity for cytotoxicity may be lost, even when total numbers and IFN-
production are maintained. In order to assess the MHV-68-specific CD8+ T cell population further, we first analysed CD8+ tetramer+ T cells by flow cytometry for cell surface adhesion molecules and other activation markers (Fig. 1
).
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We then determined the cytotoxic activity of MHV-68-specific CTL from I-Ab+/+ and I-Ab-/- mice (Fig. 2). Spleen cells were recovered and used directly to kill peptide-pulsed syngeneic targets. The splenocytes of MHV-68-infected I-Ab-/- mice showed at least as good cytotoxicity against epitope-pulsed targets as equivalent I-Ab+/+ populations (Fig. 2A
). The cytotoxic activity of spleen cell populations late in infection thus reflected the frequencies of antigen-specific CTL measured by tetramer and intracellular IFN-
staining (Stevenson et al., 1998
): recognition of the H-2Kb-p79 epitope was maintained at a similar level in I-Ab+/+ and I-Ab-/- mice, while the H-2Db-p56 population was preferentially expanded in the I-Ab-/- group. There was no evidence for a loss of cytotoxic activity against either immunodominant epitope in the absence of CD4+ T cells. The ex vivo killing of virus-infected targets by all spleen cell populations was low, variable and usually undetectable above the background lysis of uninfected targets (data not shown). This probably reflected the action of viral evasion genes such as K3 (Boname & Stevenson, 2001
). Because the peptide concentrations used in cytotoxicity assays were saturating (Stevenson et al., 1999
), it was possible that these assays detected relatively low affinity CTL that would contribute little to in vivo immunity. Thus we titrated the peptide requirement for cytotoxicity, comparing early and late populations and I-Ab+/+ and I-Ab-/- mice (Fig. 2B
). A similar level of sensitivity to antigen was observed in all populations, with cytotoxicity titrating out at peptide concentrations of 10-1010-13 M. Thus there was no evidence for a loss of CTL responsiveness to low levels of antigen late in infection of the I-Ab-/- mice.
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Our conclusion is that the virus-specific CD8+ T cell populations in MHV-68-infected but otherwise unmanipulated I-Ab-/- mice show no signs of functional exhaustion, even though these cells probably encounter viral antigens fairly frequently (Belz & Doherty, 2001). The difference between the normal CTL function observed here and the compromised function in populations further expanded by antigenic challenge (Liu et al., 2002
) suggests that the level of CD8+ T cell stimulation determines the necessity of CD4+ T cell help. In situations with high antigen loads there may be intense competition for limited resources, for example cytokines, such that functional impairment results. The peak cytotoxicity against MHV-68 was not impaired in the I-Ab-/- mice, but the numbers involved are small compared with lymphocytic choriomeningitis virus infection (Butz & Bevan, 1998
; Zajac et al., 1998
). Immune evasion by MHV-68 (Boname & Stevenson, 2001
) may contribute to the relative lack of antigen presented, and is a more likely explanation than a CTL deficiency for the incapacity of I-Ab-/- mice to control chronic infection. In contrast to MHV-68, vaccinia virus provides a large stimulus to murine CTL and presumably stretches the response sufficiently to reveal an effect of CD4+ T cell deficiency. A lack of CD8+ T cell exhaustion can perhaps be seen as one more aspect of the adaptation of a herpesvirus to its natural host.
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ACKNOWLEDGEMENTS |
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Received 10 September 2002;
accepted 2 October 2002.