View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Virology 305, 1–9 (2003) doi:10.1006/viro.2002.1754 Control of Lymphocytic Choriomeningitis Virus Infection in Granzyme B Deficient Mice Allan J. Zajac,*,† John M. Dye,* and Daniel G. Quinn*,1 *Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois 60153; and †Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294 Received July 11, 2001; returned to author for revision October 5, 2001; accepted August 30, 2002 We have investigated whether granzyme B (GzmB)is required for effective cytotoxic T lymphocyte (CTL)mediated control of lymphocytic choriomeningitis virus (LCMV)infection. Clearance of LCMV from tissues of GzmB-deficient (GzmB Ϫ)mice following intraperitoneal infection with LCMV was impaired compared with control mice; however, the virus was ultimately eliminated. The impaired clearance of LCMV in GzmBϪ mice was not due to a deficiency in the generation of LCMV-specific T cells. In addition, CTL from LCMV-infected GzmBϪ mice efficiently lysed virus-infected cells in vitro, but were deficient in their ability to induce rapid DNA fragmentation in target cells. We examined whether the development of protective immunity against intracranial (i.c.)rechallenge with LCMV was compromised in GzmB Ϫ mice. We found that clearance of LCMV from the brain following secondary i.c. infection also was slower in the absence of GzmB; however, the virus was ultimately eliminated and the mice survived. Our data indicate that clearance of LCMV is delayed in the absence of GzmB expression, but that other CTL effector molecules can compensate for the absence of this granule constituent in vivo. © 2002 Elsevier Science (USA) Key Words: granzyme B; LCMV; cytotoxic T lymphocyte. INTRODUCTION pathway of target cell killing is frequently used by CD4 CTL (Graubert et al., 1997; Zajac et al., 1996) but can be T cells play a critical role in the immune response also utilized by CD8 CTL (Ka¨gi et al., 1994b; Lowin et al., against viral infections. In general, the antiviral effects of 1994; Kojima et al., 1994). The major pathway of cytotox- T cells are mediated through the release of antiviral icity utilized by CD8 CTL, however, is the secretory path- cytokines or via direct killing of virus-infected cells by way. In this pathway, the release of perforin from gran- cytotoxic T lymphocytes (CTL) (reviewed by Doherty and ules contained within activated CTL leads to the forma- Ahmed, 1997; Zinkernagel, 1996). Control of infection with tion of polyperforin pores in the membrane of the target noncytopathic viruses, such as lymphocytic choriomen- ingitis virus (LCMV), is crucially dependent on CD8 CTL. cell, resulting in loss of membrane integrity. The CTL Infection of normal mice with LCMV induces high levels granules also contain enzymes called granzymes. The of virus-specific CD8 CTL activity and the virus is cleared major granzymes released by CD8 CTL are granzyme A within 8–10 days following infection. By contrast, mice (GzmA) and GzmB. Of these, GzmB has been shown to deficient in CD8 T cells fail to control LCMV and become bind to a specific receptor on target cell membranes persistently infected (Leist et al., 1987; Moskophidis et (Motyka et al., 2000) and to induce rapid apoptosis of al., 1987; Zajac et al., 1995). Since clearance of acute target cells in vitro (Simon et al., 1997). Upon entering the infection with LCMV is solely dependent on CD8 CTL target cell, GzmB is thought to proteolytically activate (Leist et al., 1987; Moskophidis et al., 1987), infection with several caspases culminating in DNA fragmentation and this virus provides a useful system with which to study in the rapid apoptotic death of the target cell (Darmon et the effector mechanisms of CD8 CTL in vivo. al., 1995; Duan et al., 1996; Gu et al., 1996; Quan et al., Two major pathways of CTL-mediated cytotoxicity 1996; Talanian et al., 1997). In addition, GzmB has been have been described. These are the secretory and non- reported to directly induce apoptosis independent of secretory pathways (Ka¨gi et al., 1994a,b; Lowin et al., caspase activation (Andrade et al., 1998; Talanian et al., 1994; Kojima et al., 1994). In the nonsecretory pathway, 1997). the interaction between Fas ligand (FasL) on the surface The primacy of the secretory pathway of CTL-medi- of the activated CTL and Fas on the surface of the target ated killing for the elimination of LCMV infection has cell results in apoptotic death of the target cell. This been conclusively demonstrated in studies using mice deficient in perforin (Ka¨gi et al., 1994a, 1995b; Walsh et al., 1994). Perforin-deficient mice do not have detectable 1 To whom correspondence and reprint requests should be ad- Fas-independent CTL and, consequently, fail to control dressed. Fax: (708) 216-9574. infection with LCMV. It is unclear, however, whether 0042-6822/02 $35.00 1 © 2002 Elsevier Science (USA) All rights reserved. 2 ZAJAC, DYE, AND QUINN mice (B6129SF2/J mice). We found no difference in the kinetics of clearance of LCMV from tissues of B6 mice and from tissues of B6129SF2/J mice. The control mice indicated in Fig. 1 represent three B6 mice and three B6129SF2/J mice at each time point. The levels of virus in both spleens and livers of GzmBϪ mice were similar to the levels in these tissues of control mice up to 4 days following infection. In contrast to control mice, however, peak viral titers are maintained in GzmBϪ mice until day 6 following intraperitoneal (ip) infection. By day 8 after infection splenic viral titers are substantially reduced in GzmBϪ mice, but the levels of virus are still significantly higher than the levels detected in the spleens of control mice at this time point. By contrast, although the viral titers in livers seem somewhat higher in GzmBϪ mice than B6 mice at this time point, this difference was not significant. By 10 days after infection, infectious virus is undetectable in spleens and livers of GzmBϪ mice and these animals remain virus-free for greater than 120 days following infection (data not shown). Expansion of virus-specific T cells in GzmBϪ mice Since acute infection with LCMV is controlled by CTL, we reasoned that the delayed clearance of LCMV by FIG. 1. Kinetics of clearance of LCMV from spleens and livers of GzmBϪ CTL could be due to either of two possibilities. Ϫ ϫ 5 GzmB mice. Mice were infected with 2 10 PFU of LCMV. Spleens First, the effector function of GzmBϪ cells could be suf- (A) and livers (B) were removed at the indicated times following infec- tion and viral titers were determined by plaque assay on Vero cell ficiently compromised such that they function less effi- Ϫ monolayers. ■, wild-type mice, n ϭ 6 at each time point (three B6 and ciently in vivo. Second, it is possible that the GzmB mice three B6129S2/J); Œ, GzmBϪ mice, n ϭ 5 at each time point; F, PfpϪ used in this study generated fewer numbers of virus- mice, n ϭ 3 at each time point. Data shown are the means Ϯ SEM of specific T cells following infection with LCMV. We first Ͻ Ͻ the viral titers in spleens at each time point. *P 0.01; **P 0.05. addressed the possibility that the delayed clearance of LCMV in GzmBϪ mice is due to the expansion of fewer virus-specific T cells compared with normal mice. We perforin-mediated loss of membrane integrity is suffi- enumerated T cells specific for the immunodominant cient for elimination of LCMV-infected cells in vivo or if viral CD4 and CD8 epitopes at various times following ip rapid induction of target cell apoptosis is also required. infection with LCMV using ELISPOT to detect IFN-␥- In this study we have investigated whether GzmB ex- secreting cells. There is a good correlation between the pression and, consequently, the rapid GzmB-dependent numbers of IFN-␥ producing CD8 cells and the total induction of DNA fragmentation in target cells are critical numbers of virus-specific cells (Murali-Krishna et al., for CTL-mediated control of LCMV infection of mice. 1998). As previously demonstrated (Butz and Bevan, 1998; Kamperschroer and Quinn, 1999; Murali-Krishna et RESULTS al., 1998), the numbers of virus-specific CD8 cells peak at Ϫ 8 days postinfection with LCMV (Figs. 2A–2C). At this Kinetics of virus clearance in GzmB mice 7 time point there are, combined, approximately 5 ϫ 10 We initially examined whether GzmBϪ mice could con- cells in the spleen that are specific for the three domi- trol infection with the noncytopathic virus, LCMV. Infec- nant class I restricted epitopes of LCMV. The numbers of tion of normal mice with LCMV results in peak viral titers virus-specific cells subsequently decline with approxi- in spleens and livers at day 4 postinfection, following mately 2 ϫ 10 6 cells being maintained for greater than 90 which virus is controlled and eliminated by 8–10 days days following infection. As seen in Fig. 2, the numbers postinfection (Fig. 1). That this control is dependent on of virus-specific CD8 cells that are generated and main- CTL is illustrated by the persistent infection that occurs tained in GzmBϪ mice are indistinguishable from the in PfpϪ mice (Fig. 1) (Ka¨gi et al., 1994a; Walsh et al., 1994). numbers of these cells in B6 mice. Since CD4 cells are Because the GzmBϪ mice used in this study were on a important in promoting and maintaining the function of mixed B6x129 genetic background, we also assessed CD8 cells (Battegay et al., 1994; Matloubian et al., 1994; virus clearance from spleens and livers of control B6x129 von Herrath et al., 1996; Zajac et al., 1998), we examined LCMV INFECTION OF GzmB-DEFICIENT MICE 3 FIG.