Immunological control of a murine gammaherpesvirus ... - CiteSeerX

Journal of General Virology (1999), 80, 477–483. Printed in Great Britain ...................................................................................................................................................................................................................................................................................

Immunological control of a murine gammaherpesvirus independent of CD8M T cells P. G. Stevenson, R. D. Cardin, J. P. Christensen and P. C. Doherty St Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA

Adult thymectomized C57BL/6J mice were depleted of T cell subsets by MAb treatment either prior to, or after, respiratory challenge with murine gammaherpesvirus-68. Protection against acute infection was maintained when either the CD4M or the CD8M T cell population was greatly diminished, whereas the concurrent removal of both T cell subsets proved invariably fatal. The same depletions had little effect on mice with established infection. The results indicate firstly that both CD4M and CD8M T cells play a significant part in dealing with the acute infection, and secondly that virus-specific antibody contributes to controlling persistent infection with this gammaherpesvirus.

Introduction Gammaherpesviruses characteristically establish persistent, asymptomatic infections, but can re-emerge after immune suppression to cause disease (Rickinson & Kieff, 1996). A major goal is thus to define the cellular mechanisms of normal virusspecific immunity. The murine gammaherpesvirus MHV-68 (Efstathiou et al., 1990 ; Virgin et al., 1997) provides a tractable model of gammaherpesvirus pathogenesis in conventional mice. After intranasal (i.n.) infection, MHV-68 replicates acutely in the respiratory epithelium and then persists as latent virus both in the lung (Stewart et al., 1998) and in B cells (SunilChandra et al., 1992). Analysis to date suggests that CD8+ T cells control the infection ; CD8-depleted BALB\c mice were unable to eliminate virus from the lung after i.n. infection (Ehtisham et al., 1993) and CD8+ T cell-deficient β M−/− mice # failed to clear infectious virus from the spleen after high-dose intraperitoneal (i.p.) exposure (Weck et al., 1996). However, mice deficient in perforin, a major mediator of CD8+ T cell effector function (Kagi et al., 1994), have no obvious deficit in immune protection (Usherwood et al., 1997). Also, MHC class II-deficient mice that lack CD4+ T cells (Grusby et al., 1991) control virus infection initially, but later suffer a lethal recrudescence of the epithelial infection (Cardin et al., 1996). Chronic vascular disease is observed after high-dose i.p. challenge of mice deficient in IFN-γ or its receptor (Weck et al., 1997). The present experiments analyse the contributions of Author for correspondence : Peter C. Doherty. Fax j1 901 495 3107. e-mail peter.doherty!stjude.org

0001-5896 # 1999 SGM

CD4+ and CD8+ T cells to the control of the acute and persistent phases of MHV-68 infection in C57BL\6J (B6) mice.

Methods

Mice and T cell depletions. Female B6 mice were purchased from Jackson Laboratories and, apart from MHV-68 infection, kept under specific-pathogen-free conditions. The mice were thymectomized (ATx) at 6–8 weeks of age, followed by a 3 week recovery period. To deplete T cell subsets, mice were given 5i0n5 ml i.p. injections, at 2 day intervals, of ascitic fluid containing MAbs to CD8 (2.43) or CD4 (GK1.5).

Viruses. The mice were infected i.n. with 600 p.f.u. MHV-68 under Avertin anaesthesia. Virus titres were determined using NIH 3T3 fibroblast monolayers by plaque assay of freeze–thawed lung tissue homogenates or infective centre assay of single cell suspensions from lymphoid organs (Cardin et al., 1996).

Flow cytometry. Lymphocytes from the ATx mice were washed in ice-cold PBS, blocked by incubation with 10 % normal mouse serum, and stained with anti-CD8β–FITC and anti-CD4–PE (RM4-4) (Pharmingen). The cells were washed once after a 30 min incubation on ice, and analysed on a FACScan using Cellquest v3.1 software (Becton-Dickinson).

CTL restimulation and assay. The protocol previously described for BALB\c mice (Stevenson & Doherty, 1998) was adapted to the B6 strain. Briefly, naive B6 spleen cells were infected with MHV-68 (0n1 p.f.u. per cell) for 1 h in RPMI (Life Technologies), supplemented with penicillin (60 µg\ml), glutamine (2 mM), 10 % FCS (Hyclone) and 2mercaptoethanol (55 µM) (complete medium), washed once, irradiated (3000 rad), and incubated (10' per ml) with responder lymphocytes (1n5i10& per ml) for 5 days in complete medium at 37 mC with 5 % CO . # Viable lymphocytes were recovered by centrifugation on Ficoll (Fisher Scientific) and incubated for 5 h with MHV-68-infected (10 p.f.u. per cell) or uninfected, &"Cr-labelled H-2b embryonic fibroblasts (ATCC 2214EHH

P. G. Stevenson and others

Fig. 1. Survival of ATx mice after T cell subset depletion and MHV-68 infection. Depletion of both CD4+ and CD8+ T cells significantly increased mortality (P 0n0001 ; chi-squared test), while depletion of either CD4+ or CD8+ T cells alone had no significant effect. No mice died after day 40.

CRL) before harvesting supernatants for γ-counting. The &"Cr release from targets with medium alone was 20 % of the total &"Cr release with Triton X-100.

ELISA. Virus-specific serum IgG was measured by ELISA (Stevenson & Doherty, 1998). Briefly, serum dilutions were incubated on Nunc Maxisorp immunoplates (Life Technologies) coated with 0n05 % Triton X100-disrupted virus, and bound IgG was detected with an alkaline phosphatase-conjugated rabbit anti-mouse Fcγ antiserum (Sigma) and a nitrophenylphosphate substrate (Sigma). Antibody titres were determined by comparison with a standard pool of immune sera.

ELISpot assay of CD4M and CD8M T cells. Nitrocellulosebottomed 96-well plates (Millipore) were coated overnight at 4 mC with rat anti-mouse IFN-γ (10 µg\ml ; Pharmingen), washed 5i with PBS, and blocked for 1 h at 37 mC with complete medium. Feeder cells were incubated for 1 h at 37 mC in complete medium with 1 µM AGPHNDMEI, a prominent H-2Db-restricted viral peptide epitope (P. G. Stevenson, G. T. Belz & P. C. Doherty, unpublished results), infected with MHV-68 (1 p.f.u. per cell), or left untreated. All feeders were then irradiated (3000 rad), washed twice in complete medium, added (5i10& per well) to duplicate 3i dilutions of responder cells, and incubated for 40–44 h in complete medium with 10 U\ml IL-2 (Boehringer Mannheim) at 37 mC in 5 % CO . Secreted cytokine was detected with biotinylated rat # anti-mouse IFN-γ and streptavidin–alkaline phosphatase (Dako). The plates were washed 5i with PBS–0n05 % Tween 20 after each incubation, and the spots that were visualized with a BCIP\NBT substrate (Sigma) EHI

Fig. 2. Lung virus titres after T cell subset depletion and MHV-68 infection. Each bar shows the meanpSD titres from three to six mice. All the doubly depleted mice had died by 78 days after infection. The dashed lines represent the lower and upper limits of detection in this assay (log10 l 1n0 and log10 l 5n5, respectively). Either CD4 or CD8 depletion significantly impaired virus clearance before day 20 (P 0n002 ; chi-squared test), but not at later time-points.

were counted microscopically. The mean number of spots with untreated feeders ( 10 per well) was subtracted from the mean number of spots with MHV-68-infected or peptide-pulsed feeders (20–200 spots per well) to give the number of peptide-specific T cells. In separate control experiments (not shown), CD8 depletion decreased the number of peptide-specific spots by 95 %, while neither CD4 nor NK1.1 depletion had a significant effect. Similarly, CD4 depletion decreased the number of virus-specific spots by 95 %, while depletion of CD8+ or NK1.1+ cells had no effect.

Results T cell depletion before MHV-68 infection

The ATx B6 mice were depleted of none, one or both T cell subsets by MAb treatment on days k4, k2, 0, j2 and j4 relative to i.n. MHV-68 challenge. Mice lacking both CD4+ and CD8+ T cells became progressively debilitated and succumbed 2–5 weeks after infection (Fig. 1) when virus titres in the lung were invariably high (Fig. 2). Virus clearance from the lung was delayed in mice depleted of either CD4+ or CD8+ T cells alone (Fig. 2), but these mice remained well and suffered no significant mortality (Fig. 1). Fig. 3 shows functional assays of virus-specific immunity in the early (days 16–19) and late (days 78–103) stages after i.n. infection. When mice lacking both T cell subsets were moribund (16–19 days after infection),

Gammaherpesvirus cell-mediated immunity

Fig. 3. Analysis of splenic T cell subsets after MAb depletion prior to MHV-68 infection. (A) Efficacy of T cell depletion. The residual CD4+ T cells in CD4-depleted and doubly depleted mice, and the residual CD8+ T cells in CD8-depleted and doubly depleted mice, were determined by flow cytometry and are expressed as percentages of the undepleted values (100 %). Each bar gives the mean from three to six mice. All doubly depleted mice had died by day 78. (B) MHV-68-specific serum IgG titres. Each bar shows the mean and SD antibody titre from three to six mice. (C) MHV-68-specific cytotoxicity from spleen cell bulk cultures. Net specific lysis l (percentage specific lysis of MHV-68-infected targets)k(percentage specific lysis of uninfected targets). Each bar shows the meanpSD of bulk cultures from three mice, with effector : target ratio of 30 : 1. (D) Analysis of T cell function by ELISpot. Spleens of surviving mice 78 and 103 days after infection were assayed against MHV-68-infected (CD4+ response) or peptide p56-pulsed (CD8+ response) feeder cells (see Methods). Reciprocal frequencies are expressed as input cells/ELISpot.

virus-specific CTL were undetectable in the CD8-depleted mice (Fig. 3C) and no virus-specific serum antibody could be demonstrated in the CD4-depleted mice (Fig. 3B). Thus, either CD4+ or CD8+ T cells could protect against acute MHV-68 infection when the numbers and function of the other T cell subset were greatly diminished. In agreement with previous data (Ehtisham et al., 1993), a high dose i.n. infection (10' p.f.u.) was lethal in CD8-depleted

BALB\c mice (100 % mortality ; n l 6). However, the same challenge was resisted by CD8-depleted B6 mice (0 % mortality ; n l 6), while a low-dose infection (6i10# p.f.u.) did not kill CD8-depleted mice of either strain (0 % mortality ; n l 6). Thus, CD8+ T cell-dependent protection against acute infection is both virus dose- and mouse strain-dependent. The numbers of latently infected spleen cells were not significantly higher in CD8-depleted mice at 16–19 days after EHJ


Fig. 4. Latent virus in spleens after T cell subset depletion and MHV-68 infection. Each bar shows meanpSD infective centres for three to six mice. Titres were significantly increased in the spleens of CD8-depleted mice at days 78 and 103 (P 0n02), but not at earlier time-points (P 0n5). Spleens of CD4-depleted mice had significantly lower titres at days 16–19 (P 0n03) but significantly higher titres after this time (P 0n02). Infectious virus was not detectable by plaque assay in any sample. Infective centre assays of mediastinal and cervical lymph node cells displayed similar trends (not shown).

infection, but evidence of increased prevalence was found 78 and 103 days after infection (Fig. 4). Significantly higher levels of latent virus were also found at a later stage in the spleens of CD4-depleted mice (Fig. 4), perhaps indicating the onset of a virus recrudescence similar to that seen in MHC class IIdeficient mice (Cardin et al., 1996). Thus, although T cell numbers (Fig. 3 A) and function (Fig. 3 B, D) had both recovered to some extent at the later time-points, these lymphocytes were apparently unable to completely control the infection. Low-dose i.n. MHV-68 infection of CD8+ T cell-deficient β M−/− mice also led to a more protracted pulmonary infection # and to an increase in detectable latent virus (Fig. 5). However, these mice again suffered no obvious adverse consequences in the long term (0 % mortality at 9 months ; n l 6). T cell depletion after MHV-68 infection

The ATx mice were infected i.n. with MHV-68, then treated as before with a 10 day course of T cell subset-specific MAbs, this time commencing 1 month after the initial virus challenge. Fig. 6 shows assays of virus-specific immune function with time after depletion. Again, the T cell numbers recovered to some degree with time after depletion (Fig. 6 A), EIA

Fig. 5. Virus in the lungs (A) and spleens (B) of β2M−/− and congenic B6 mice after i.n. MHV-68 infection. Each bar shows meanpSD log10 titres from two to five mice. (A) Levels of infectious virus in the lung [horizontal dashed line shows limit of detection (1n7)] were significantly higher in the β2M−/− mice at days 9 (P 0n02) and 22 (P 0n001), but not at day 32 (P l 0n14). (B) Numbers of latently infected cells in the spleens of β2M−/− mice were significantly higher than in the β2M+/+ B6 controls 22 and 32 days after infection (P 0n01). Infectious virus was detectable by plaque assay of freeze–thawed spleen cells in the β2M−/− but not the B6 mice, and was 1 % of the levels detectable by infective centre assay (not shown).

but 90 % numerical depletion of the CD8+ T cells was maintained for more than 3 months, as was substantial functional depletion (Fig. 6 C). The CD4+ T cell depletion, in turn, led to a prolonged reduction in virus-specific serum IgG (Fig. 6 B). The virological consequences of T cell subset depletion were surprisingly few. There was a significant, although not dramatic, rise in latent virus recoverable from the spleen and lymph nodes of CD8-depleted mice (Fig. 7). This was evident soon after depletion, but failed to increase further and eventually declined as a consequence of the partial recovery of CD8+ T cell immunity (Fig. 6 C). In contrast to the increase in infective centres, recrudescent pulmonary infection was not detected at any time in these chronic experiments ; lung tissue from the mice shown in Fig. 7 was consistently negative by plaque assay.

Discussion Virus clearance from the lung was significantly delayed in B6 mice depleted of either the CD4+ or the CD8+ T cell subset, but the infection was lethal only in mice lacking both T cell subsets. Thus, both CD4+ and CD8+ T effectors make significant contributions to acute protection and can operate effectively when the numbers of the other T cell subset are greatly diminished. Virus-specific antibody may be sufficient to control the persistent phase of the infection, with the mice

Gammaherpesvirus cell-mediated immunity

Fig. 6. Analysis of T cell subsets following MAb depletion subsequent to the control of MHV-68 infection. (A) Efficacy of T cell depletion. The residual CD4+ T cells in CD4-depleted and doubly depleted mice, and the residual CD8+ T cells in CD8depleted and doubly depleted mice, were determined by flow cytometry and are expressed as a percentage of the undepleted values. Each point shows the mean value from two to eight mice. (B) MHV-68-specific serum IgG titres. Each bar shows the meanpSEM of titres from two to eight mice. Comparing titres after the completion of depletion (day 40), t-tests showed significantly lower antibody titres in the CD4-depleted (P 0n001) and double-depleted (P 0n01) mice than in the undepleted controls. (C) MHV-68-specific cytotoxicity from spleen cell bulk cultures. Net specific lysis l (percentage specific lysis of MHV-68-infected targets)k(percentage specific lysis of uninfected targets). Each bar shows the meanpSD of bulk cultures from three mice, with effector : target ratio of 30 : 1.

suffering little effect from the elimination of most of the CD4+ and CD8+ T cells. A central role for antibody is also supported by recent experiments with immunocompromised B6 and µMT mice (Stewart et al., 1998). It is not clear whether CD8+ T cells are essential for the control of the latently infected B cell pool. Acutely, the CD4dependent increase (Cardin et al., 1996 ; Usherwood et al., 1996) in latent virus load in the spleen occurred (Fig. 4) despite the presence (Stevenson & Doherty, 1998) of virus-specific CTL (Fig. 3 C) and was not further enhanced in CD8-depleted mice (Fig. 4). In the long term, mice with impaired CTL function showed a consistent rise in detectable latent virus, but

this increase remained relatively small and the mice were clinically unaffected. Also, no significant incidence of tumours (Sunil-Chandra et al., 1994) was observed in the CD8-depleted or β M−/− mice over a period of 1 year. Since β M−/− mice are # # also deficient in NK cell function, NK1.1+ CD4+ T cells and serum IgG responses (Raulet, 1994), their increase in latent virus could not be attributed solely to a lack of CTL. Although the CD8+ T cell subset cannot be totally ablated by MAb treatment of ATx mice in the long term, and CD8+ T cells are not completely absent from β M−/− mice, there is # clearly no absolute requirement for CTL to limit this infection. Even BALB\c mice, which die after infection with a high dose EIB


Fig. 7. Virus titres in lymphoid tissue following late T cell subset depletion. The results of infective centre assays from spleens (meanpSD of two to eight individuals) and lymph nodes pooled from two to eight individuals (open symbols, mediastinal ; closed symbols, cervical) are shown, commencing after the 10 day MAb treatment. Infectious virus was not detectable by plaque assay of freeze–thawed spleen cells at any time. Comparing data from all time-points by t-tests, CD8 depletion led to a significant increase in latent virus in the spleen (P 0n01), mediastinal lymph nodes (P 0n001) and cervical lymph nodes (P 0n05), while neither CD4 depletion nor CD4jCD8 double depletion had a significant effect.

of MHV-68 and CD8+ T cell depletion, can tolerate a low-level challenge in the absence of CTL effectors. Overall, the immune control of MHV-68 has parallels to that of murine cytomegalovirus, where control is maintained in the absence of CD8+ T cells (Jonjic et al., 1990) and antibody limits the dissemination of recurrent infection (Jonjic et al., 1994). In each case, multiple mechanisms of host control and virus evasion probably contribute to the final outcome of low-level virus persistence. We wish to thank Vicki Henderson for her assistance with the manuscript and Medhi Mehrpooya for his assistance with the virus titrations. This work was supported by Public Health Service grants AI38359 and CA21765, and by the American Lebanese–Syrian Associated Charities. P. G. S. is the recipient of an MRC (UK) Travelling fellowship, J. P. C. is the recipient of a fellowship from the Alfred Benson Foundation, Denmark.

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