key: cord-0823967-3pzyprbh
authors: Heemskerk, Mirjam H. M.; Schilham, Marco W.; Schoemaker, Henriette M.; Spierenburg, Gerrit; Spaan, Willy J. M.; Boog, Claire J. P.
title: Activation of virus‐specific major histocompatibility complex class II‐restricted CD8(+) cytotoxic T cells in CD4‐deficient mice
date: 2005-11-17
journal: Eur J Immunol
DOI: 10.1002/eji.1830250438
sha: 492c9c2fc002357be9a352688e3e77c0c94ffb91
doc_id: 823967
cord_uid: 3pzyprbh

Acute enteritic or respiratory disease is a consequence of coronavirus infection in man and rodents. Mouse hepatitis virus, stain A59 (MHV‐A59) causes acute hepatitis in mice and rats and induces a response of major histocompatibility complex (MHC) class II‐restricted CD4(+) cytotoxic T cells, protecting mice against acute infection. In the present study we show that MHV‐A59 infection of mice that lack a functional CD4 gene activates effector cells of the CD8(+) phenotype. These cytotoxic T cells lyse virus‐infected target cells in a MHC class II‐restricted fashion. The results indicate that CD8(+) T cells have the potential to utilize MHC class II as restriction element, illustrating that the immune system can effectively deal with evading microorganisms, such as viruses which down‐regulate MHC class I.

A rather strict association exists between the expression of CD4/CD8 accessory molecules and the MHC restriction of mature TcR ap-expressingT cells [l] . CD8+ T lymphocytes recognize antigen in the context of MHC class I moleculeis, whereas CD4+ T cells recognize antigen associated with MHC class I1 molecules. Exceptions of the strict association between expression of the co-receptor and MHC class I or I1 restriction have been previously described [2-71.

Here we describe another exception of mismatch between co-receptor and MHC restriction. We have analyzed mouse hepatitis virus strain A59 (MHV-A59)-infected CD4deficient mice for the induction of cytotoxic activity. Earlier studies showed that intraperitoneal infection with MHV-A59 causes acute hepatitis in mice and rats [8] , and induces a response of MHC class 11-restricted CD4+ cytotoxic T cells (CTL) [9, 101 . Transfer studies using virus-specific CD4+ CTL clones have shown that these cells are able to protect mice against a lethal virus challenge. In order tlo study the role of CD4 in the protection against MHV-ASS', mice that lack a functional CD4 gene (CD4-'-mice) were infected with MHV-A59.The CD4-deficient mice were able to generate cytotoxic T cells that were able to lyse the MHV-A59-infected target cells in a MHC class 11-restricted fashion. In contrast to the cells of the wild-type mice (CD4) the cells from the mutant mice were of the CD8 phenotype.

Thus, MHV-A59 infection induces the activation of virusspecific MHC class 11-restricted CD8+ T cells in CD4deficient mice. The characteristics and role of these cells in protection against an acute infection is determined and discussed. Furthermore, we discuss the thymic selection of these cells and the possible existence of a subset of class 11-restricted CD8+ T cells in normal mice.

Specific pathogen-free (including seronegative for MHV) mice used in this study were homozygous or heterozygous for the disrupted CD4 gene, and have been previously described [ll] . All mice were used between 6 and 10 weeks of age.

The virulent hepatotropic MHV strain A59 and the avirulent temperature-sensitive mutant ts342 of MHV-AS9,were propagated on Sac( -) cells and virus stocks were prepared as described [12, 131. For immunization experiments, mice were injected intraperitoneally with lo4 PFU ts342 and boosted 10 days later with 5 x lo4 PFU wild-type MHV-A59. 

Spleen cells (1 x los) from immunized mice were isolated and stimulated in bulk culture with 5 x lo7 irradiated (3000 rad) MHV-A59-infected syngeneic spleen cells (MOI of 0.3) in 50 ml IMDM (Gibco Laboratories, Grand Island, NY) supplemented with 10% FCS, 2 mM glutamine, antibiotics and 2-ME (2 X M) for 5 days. To separate CD4-CD8-from CD4-CD8+ effector cells, the cultures of in vitro stimulated spleen cells derived from MHV-A59 primed CD4-'-mice, were stained with FITC-conjugated anti-CD8 mAb (53-6.7) followed by cell sorting.

CTL assays were performed as described 1141. In short, 2.5 x lo3 51Cr-labeled target cells were added to varying numbers of effector cells and incubated for 5-6 h at 37 "C and 5 % COz. As target cells were used LB15. 13 , an H-2b-expressing tumor cell line (MHC class I+II+) and G4

[lo], a transfectant of the MHV-non-permissive H-2bT cell lymphoma EL4 expressing the receptor for MHV-A59 [15] (MHC class I+II-). Virus-infected target cells were prepared by infection of cells with MHV-A59 at a multiplicity of infection (MOI) of 50 for 5 h at 37°C prior to the 1-h labeling with 51Cr. The percentage of specific lysis was calculated as (cpm experimental release-cpm spontaneous release)/(cpm total release-cpm spontaneous release) X 100. Spontaneous release was below 20 YO of total release; standard deviations were below 10 % .

Eight-week-old, pathogen-free, C57BL/6 mice were To study the role of CD4 in the protection against are first inoculated with a temperature-sensitive mutant (ts342) of MHV-A59 (data not shown). Spleen cells of MHV-A59-infected CD4-I-mice proliferate specifically against inactivated MHV-A59 (stimulation index = 6.8 k 1.7), although the response is lower than that of CD4+/-mice (stimulation index = 29.7 k 8.7). In addition, the sera of CD4-I-mice contain virus-specific neu-MHV-A59, although these antibody titers are about tenfold lower than those of control mice (data not shown). Together these data show that a protective immunity is ; eflec!orRarge! ratio r tralizing antibodies 3 weeks after boosting with wild-type F

iniuced in CD4-/-mice.

In order to investigate the nature of this protective immunity in more detail, the cytotoxic T cell activity of MHV-A59-primed CD6deficient mice was tested. Spleen cells from primed CD4-I-mice, stimulated for 6 days in vitro with irradiated infected splenocytes, lyse MHV-A59-infected MHC class 11-positive LB15.13 target cells, but not virus-infected MHC class 11-negative G4 targets (Fig. 1A) . The cytotoxic activity of heterozygous mice did result in a similar pattern (Fig. lB) , showing that both CD4-'-as well as CD4+/-mice respond to MHV-A59 infection in a MHC class 11-restricted fashion. Since The flow cytometric analysis of the unseparated and the sorted effectors are indicated below the figures; they show that the separated populations are > 99 YO pure. Sorting was performed on clear CD8and CD8+ cells, therefore, two dotted lines are present in the flow cytometric analysis of the total effector population. responsible for the MHC class 11-restricted cytotoxici ty, sorting experiments were performed. After labeling with FITC-anti-CD8, CD8-and CD8+ cells were sorted. As can be seen in Fig. 2 , none of the two sorted populations lysed MHV-A59-infected G4 (class I+ IT-) targets. However, the CD8+ population was able to lyse infected LBlS.13 (class I+ II+) cells. These data show that the CD8+ T cells of the CD4-/-mice mediate the cytolytic activity. The fact that only the MHC class 11-positive targets were lysed demonstrates that the CD8+ cells utilize the MHC class I1 molecule as a restriction element. To test this more directly, we performed antibody inhibition experiments. During the cytotoxic assay, anti-MHCclass I1 mAb (M5/114, I-Ab) [19] were added and a complete inhibition of the cytotoxic activity of the sorted CD8+ effector cells against MHVinfected LB15.13 was observed (Fig. 3) . Anti-MHC class, I antibodies R1-21-2 [20] had no effect. In conclusioa, MHV-AS9-infected CD4-deficient mice generate CD8+ CTL, which are not MHC class I-, but MHC class IIrestricted.

Adoptive transfer of the polyclonal MHV-A59-specific MHC class 11-restricted CD8+ T cells (> 95 % C D F ) showed that three out of four mice were protected against a lethal challenge of MHV-A59 (Table I) . Polyclonal CD4+ CTL derived from CD4+/-mice (> 85 % CD4+) protected two out of four mice against a challenge with MHV-A59. Although we cannot exclude the contribution of thLe double-negative cells, the data suggest that MHC class Itrestricted CD8+ T cells play a pivotal role in the protection against MHV-A59 infection.

Infection of mice with the coronavirus MHV-A59 does not lead to a normal MHC class I-restricted cytotoxic response as observed with other viruses [21-231. In normal mice CD4+ class 11-restricted cells are the main effector cells against this virus [9] . In this study we show that CD4-Imice respond with MHC class 11-restricted CD8+ CTL.We However, we cannot exclude the presence of such cells, since CD8+ class 11-restricted T cells might be overruled by large populations of CD4+ T cells, and therefore would be hard to detect using the standard assays for CTL activity.

Regardless of whether the observed class I1 restriction is physiological or a consequence of the use of CD4-I-mice, it is striking that the immune system does not employ virus-specific class I-restricted cells. This supports our hypothesis that the virus interferes with presentation of its antigens on class I molecules. Activation of the class IIrestricted CTL, even in the CD4-/-mice, illustrates, the plasticity of the immune response when dealing with evasive mechanisms employed by microorganisms. This suggests that during infection with viruses that either down-regulate MHC class I or reduce the amount of CD4+ T cells, such as HIV, CD8+ class 11-restricted T cells may play a significant role.

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