Antiviral immune responses of fully allogeneic irradiation bone marrow mouse chimeras

In (B10.BR----B10) chimeras infected with lymphocytic choriomeningitis (LCM) virus higher titers were attained in spleens and livers than in organs of the mice used for their construction, and the subsequent elimination was retarded, but eventually the virus was cleared. The numbers of LCM virus-specific CTL and their precursors as quantitated with chromium-release assay and limiting dilution method, respectively, were lower in chimeras than in B10.BR or C57BL/10J mice, and fewer were restricted for the haplotypes of the donors than of the recipients. The same was true with regard to antiviral effector cells, which were determined by adoptive immunization. The numbers of spleen cells releasing IgM and IgG antiviral antibodies were virtually as high in chimeras as they were in C57BL/10J and B10.BR mice. Transfer of immune splenocytes from either B10.BR or C57BL/10J mice resulted in incomplete virus elimination from the spleens of infected chimeras, whereas injection of a mixture of the two types of immune cells led to efficient clearance. We conclude that in the chimeras cells of both donor and recipient haplotypes participate in the infection, which is terminated by H-2k- and H-2b-restricted T lymphocytes that these animals are capable of generating. We conclude, furthermore, that clearance of the LCM virus from the tissues requires contact between effector and target cells.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. VI. Migration and activity in vivo in acute and persistent infection

Cloned cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were adoptively transferred to syngeneic mice acutely or persistently (carrier mice) infected with LCMV. Although infectious virus was cleared from the spleens during acute LCMV infection begun 24 hr earlier and the spleens remained clear of virus for the 4 days of testing, there was no concomitant reduction of viral titers in lymph nodes. In contrast, adoptive transfer of cloned CTL into animals with persistent rather than acute LCMV infection resulted in deaths of syngeneic but not allogeneic recipients. LCMV-immune spleen cells taken 30 to 50 days after a primary immunization and activated by in vitro stimulation before transfer also caused death of syngeneic carrier mice. However, LCMV-immune spleen cell per se provoked no clinical manifestations when transferred but cleared infectious virus and viral nucleic acid sequences from syngeneic carrier mice. The migration of 51Cr-labeled, LCMV-specific, H-2-restricted cloned CTL was assessed in vivo. The circulation of these CTL clearly differed from that of spleen cells freshly isolated from uninfected mice and from non-LCMV-specific CTL clone. Further, the circulatory pattern of LCMV-specific, H-2-restricted, cloned CTL in carrier mice was markedly different than in uninfected animals; only 7% of the injected cells remained in the lungs of uninfected mice 8 hr after injection, whereas 30% had accumulated in the liver. However, 55% of the cells injected into carrier mice still remained in their lungs 8 to 16 hr later. Hence, LCMV-specific, H-2-restricted, cloned CTL have unique trafficking patterns in the presence of LCMV antigens and immune activities in vivo.     

Mechanism of recovery from acute virus infection. I. Role of T lymphocytes in the clearance of lymphocytic choriomeningitis virus from spleens of mice

Adult mice were infected by i.v. inoculation with 10(3) mouse infectious doses of lymphocytic choriomeningitis virus (LCM virus). Despite widespread replication of the agent, overt illness did not develop; histopathologic alterations were moderate. High virus concentrations were attained in the spleen, which was chosen for further study. Cytotoxic spleen T cell responses were found to vary among inbred mouse strains, and as a rule, these were correlated with other virus-specific cell-mediated immune phenomena. However, high- and low-responder mice eliminated the virus equally fast and already at times when spleen cytotoxic T lymphocytes were just beginning to appear (and before delayed-type hypersensitivity could be demonstrated). Adoptive transfer experiments showed that very few immune T lymphocytes were capable of reducing virus replication in the recipients' spleens and, furthermore, that protection was rapidly induced; when low numbers of cells were transferred, the effect was apparent 8 hr later, and with higher numbers diminished virus replication was evident after an interval as short as 6 hr. In fact, the data suggest that virus was actually inactivated. In spite of this marked efficiency, morphologic alterations in spleens of adoptively immunized mice were absent. Attempts to reveal expansion of immune cells in the recipients have failed, and the observation that adoptive transfer was as efficient in nude mice as in their furred counterparts makes it unlikely that the recipients' T lymphocytes participated to any extent. The low number of T lymphocytes causing reduction of virus, the short interval after which the effect became measurable, and the lack of histopathologic alterations has led to a working hypothesis in which it is assumed that immunologically activated T lymphocytes secrete lymphokines that directly interfere with virus replication in neighboring cells.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo.

Our data show that 1 X 10(7) to 1.5 X 10(7) lymphocytic choriomeningitis virus-specific, H-2-restricted cloned cytotoxic T lymphocytes (CTL) administered intravenously into acutely infected mice totally cleared virus from the spleens (10(4) to 10(5) PFU per spleen reduced to less than 50 PFU per spleen) by 24 h. This activity was genetically restricted in that cloned CTL could reduce titers of infectious virus in syngeneic C57BL/6 mice but not allogeneic BALB/c mice. Dose-response analysis indicated that at least 3 X 10(6) to 5 X 10(6) cloned CTL injected intravenously were needed to reduce significant amounts of infectious virus in the spleens. No infectious virus could be recovered from the spleens for at least 4 days after injection of cloned CTL. Hence, CTL play a major role in elimination of infectious virus from spleens during lymphocytic choriomeningitis virus infection. Our results also indicate that cloned CTL propagated in vitro for long periods of time can mediate a biologically relevant effect in vivo. These cells should be of considerable value in defining the precise manner in which CTL bring about control of viral infection, analyzing lymphocyte trafficking, and the potential use of cloned CTL in immunotherapy against viral disease.     

Enhanced virus replication and inhibition of lymphocytic choriomeningitis virus disease in anti-gamma interferon-treated mice.

The role of gamma interferon (IFN-gamma) induced during a viral infection in the ability of the host to acquire antiviral immunity was studied in mice. They were injected subcutaneously daily with an ammonium sulfate-precipitated sheep anti-IFN-gamma antibody preparation able to neutralize 10(4) U of IFN-gamma. Specificity of the anti-IFN-gamma antiserum was demonstrated by absence of detectable activity against natural IFN-alpha and -beta. Controls were treated with a similarly prepared normal sheep serum. Treatment with the IFN-gamma-specific antibody preparation had no influence on the ability of mice to generate anti-vaccinia virus- or anti-vesicular stomatitis virus (VSV)-specific cytotoxic T-cell (CTL) responses or T helper-dependent immunoglobulin G responses to VSV. In contrast, treatment of mice with sheep anti-IFN-gamma impaired CTL responses against lymphocytic choriomeningitis (LCM) virus (LCMV, Aggressive isolate); in addition, under the experimental conditions used, it prevented lethal LCM. Cytotoxic T-cell activity measured in the spleens of anti-IFN-gamma-treated mice was comparable to that found in mice initially infected with a 100-fold-larger dose of LCMV. Evaluation of the effects of treatment on the kinetics of virus replication revealed that in both euthymic and athymic nude C57BL/6 mice, anti-IFN-gamma treatment led to an increase of virus titers up to 100-fold compared with control mice. Therefore, IFN-gamma may play a role in controlling viruses with tropism for lymphocytes and monocytes/macrophages, such as LCMV.     

Suppression of virus-specific antibody production by CD8+ class I-restricted antiviral cytotoxic T cells in vivo.

The question of whether virus-induced immunosuppression includes the antibody response against the infecting virus itself was evaluated in a model situation. Transgenic mice expressing the T-cell receptor (TCR) specific for peptide 32-42 of lymphocytic choriomeningitis virus (LCMV) glycoprotein 1 presented by Db reacted with a strong transgenic cytotoxic T-lymphocyte (CTL) response starting on day 3 after infection with a high dose (10(6) PFU intravenously [i.v.]) of the WE strain of LCMV (LCMV-WE); LCMV-specific antibody production in the spleen was suppressed in these mice. Low-dose (10(2) PFU i.v.) infection resulted in an antiviral antibody response comparable to that of the transgene-negative littermates. The induction of suppression of LCMV-specific antibody responses was specifically mediated by CD8+ TCR transgenic CTLs, since the LCMV-8.7 variant virus (which is not recognized by transgenic TCR-expressing CTLs because of a point mutation) did not induce suppression. In addition, treatment with CD8 monoclonal antibody in vivo abrogated suppression. Once suppression had been established, it was found to be nonspecific. The abrogation of antibody responses depended on the relative kinetics of the antibody response involved and the kinetics of the anti-LCMV CTL response. Analysis of T- and B-cell subpopulations showed no significant changes, but immunohistochemical analysis of spleens revealed extensive destruction of follicular organization in lymphoid tissue by day 4 in transgenic mice infected with LCMV-WE but not in those infected with the CTL escape mutant LCMV-8.7. Impairment of antigen presentation rather than of T or B cells was also suggested by adoptive transfer experiments, showing that transferred infected macrophages may improve the anti-LCMV antibody response in LCMV-immunosuppressed transgenic recipients; also, T and B cells from suppressed transgenic mice did respond in irradiated and virus-infected nontransgenic mice with antibody formation to LCMV. Such virus-triggered, T-cell-mediated immunopathology causing the suppression of B cells and of protective antibody responses, including those against the infecting virus itself, may permit certain viruses to establish persistent infections.     

Retrovirus D/New England and its relation to Mason-Pfizer monkey virus.

We have generated lymphocytic choriomeningitis virus-specific, H-2-restricted cytotoxic thymus-derived lymphocyte (CTL) clones. By using these reagents in several in vitro assays with infected target cells, we show that CTLs by themselves prevent the release of infectious virus into culture fluids and significantly lower the titers of infectious virus previously produced. This ability of cloned CTLs is not influenced by monensin. However, monensin does abrogate the ability of CTLs from spleens of mice primed 6 to 8 days previously with virus to kill virus-infected syngeneic targets. When tested for the participation of lymphokines in this system, the CTLs proliferate when reacted with syngeneic lymphocytic choriomeningitis virus-infected macrophages but fail to make interleukin-2. These CTLs make gamma interferon when reacted with syngeneic virus-infected targets. However, the production of interferon does not directly correlate with CTL-mediated killing. The number of H-2K and D molecules expressed on the target cell surface is not altered during the course of lymphocytic choriomeningitis virus infection. Electron microscopy shows finger-like projections of the CTL clone thrust into the infected cell and lesions bearing an internal diameter of approximately 15 nm in those membranes, illustrating the lytic process.     

Effects of treatment with IL-2 receptor specific monoclonal antibody in mice. Inhibition of cytotoxic T cell responses but not of T help

Contribution of IL-2R-bearing activated lymphocytes to antiviral host defense was investigated in C57BL/6 mice by treatment in vivo with IL-2R-specific mAb PC61. When treated on days 0 and 1 with respect to infection with either vaccinia virus, lymphocytic choriomeningitis (LCM) virus (LCMV) or vesicular stomatitis virus, 6-day immune mice had low numbers of CD8+ T cells that were reduced to about 10% of the values found for infected but otherwise untreated controls. In contrast, the number of CD4+ T cells was within normal ranges. Correspondingly, induction of strictly T help-dependent antiviral neutralizing IgG antibody titers remained unaffected by the mAb treatment, whereas generation of antiviral cytotoxic T cell activity was abrogated. Anti-IL-2R treatment of thymectomized mice 14 and 15 days after infection prevented generation of secondary antiviral cytotoxic T cells in restimulation cultures in vitro initiated 24 days later. Treatment with IL-2R-specific mAb was comparable to treatment with CD8-specific mAb in preventing mice to eliminate virus. Because of the involvement of antiviral cytotoxic T cells in disease manifestations, treatment with IL-2R-specific mAb protected mice from lethal LCM after intracerebral infection with LCMV and inhibited the footpad swelling reaction caused by local infection with the same virus.     

Interleukin-4 causes delayed virus clearance in influenza virus-infected mice.

Two different subsets of T cells, Th1 and Th2 cells, have been demonstrated to secrete different profiles of cytokines and to influence various infections in different ways. Whereas cytokines secreted by Th1 cells, particularly gamma interferon, promote the generation of cell-mediated immunity, Th2 cells and their cytokines (interleukin-4 [IL-4], IL-5, IL-10, and IL-13) have been shown to function in recovery from parasitic infections and in antibody responses. In this study, we analyzed the effects of the dominant Th2 cytokine, IL-4, on immunity to virus infection. We assessed the effects of IL-4 on both secondary immune responses by an adoptive transfer assay and primary immune responses by in vivo treatment of influenza virus-infected mice with IL-4. The results demonstrated that IL-4 can function to inhibit antiviral immunity at both stages. We found that IL-4 treatment of sensitized cells during secondary stimulation in vitro had little effect on their ability to lyse virus-infected target cells in a 51Cr release assay. Nevertheless, the clearance of influenza A/PR/8/34 (H1N1) virus from the lungs of infected BALB/c mice was significantly delayed after the transfer of virus-specific T cells secondarily stimulated in the presence of IL-4 in comparison to virus clearance in recipients of cells stimulated in the absence of IL-4. In contrast to the adoptive transfer results, the treatment of PR8 virus-infected mice with IL-4 during primary infection greatly suppressed the generation of cytotoxic T-cell precursors, as assessed by secondary stimulation in vitro. In addition, culture supernatants of secondarily stimulated spleen cells from IL-4-treated mice contained significantly less gamma interferon and more IL-4 than did spleen cells from controls. More importantly, the treatment of mice with IL-4 resulted in an extremely significant delay in virus clearance. Thus, IL-4 can inhibit both primary and secondary antiviral immune responses.     

Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present an immunodominant viral cytotoxic T lymphocyte epitope.

For controlling infection of the mouse with the lymphocytic choriomeningitis (LCM) virus, CD8+ CTL are essential. In the infected BALB/c mouse the arising LCM virus-specific CTL are exclusively restricted by the class I MHC-encoded molecule L; K- or D-restricted antiviral CTL cannot be detected. Thus, the infected L-deficient BALB/c mutant C-H-2dm2 should not be capable of eliminating the virus. The experimental evidence proves the contrary, which is explained by K- and D-restricted CTL that this mouse generates. Why such cells remain undetectable in BALB/c mice is currently unexplained, because there is no lack of precursors and the corresponding virus Ag is presented. Despite the absence of lytic activity in vitro, other than the one associated with L, transfusion of day 8-immune spleen cells from BALB/c into infected C-H-2dm2 (L-deficient) mice results in accelerated virus elimination from the organs of the latter, which was manifest as soon as 8 h after cell transfer. Furthermore, lytic activity did not attain measurable levels in the recipients' spleens. Obviously, this infection can be terminated by CD8+ T lymphocytes even when these cells' lytic activity is below detectability.     

Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells.

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-gamma) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-gamma in control of the virus infection, mice lacking the cellular receptor for IFN-gamma (IFN-gamma R-/- mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-gamma R-/- mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection. In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-gamma R-/- mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4+ and CD8+ T cells occurred. These changes were accompanied by a 10- to 100-fold greater load of latently infected cells in IFN-gamma R-/- mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2'-deoxyl-5-ethyl-beta-4'-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8+ T cells prior to and during the primary infection. Depletion of CD4+ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-gamma receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed.     

Contributions of host and donor T cells to the inflammatory process in murine lymphocytic choriomeningitis

The severe inflammation characteristic of the infection of adult mice with murine lymphocytic choriomeningitis virus (LCMV) is induced earlier in unsuppressed, virus-infected recipients by the adoptive transfer of class I MHC-compatible, CD4- CD8+ LCMV-immune spleen cell populations. The time to onset of fatal LCM may also be slightly diminished, though not to the extent that would be expected from the enhanced kinetics of the extravasation of cells into cerebrospinal fluid. The development of symptoms is thus not solely related to the magnitude of the inflammatory process. The majority of the T lymphocytes in the inflammatory exudate are of host origin and have the size characteristics of resting cells, while the minority population of donor T cells show more of a lymphoblast morphology. The findings are consistent with the idea that relatively few CD8+ virus-immune effectors trigger an inflammatory process which consists largely of secondarily recruited host T cells and monocyte/macrophages.     

Rapid recruitment of virus-specific CD8 T cells restructures immunodominance during protective secondary responses

In this study we investigate the attributes of virus-specific memory CD8 T cells which most effectively control secondary infections. By rechallenging mice that had cleared primary lymphocytic choriomeningitis virus infections, we revealed that the secondary response is remarkably swift. Within 6 h following secondary infection, the production of gamma interferon becomes detectable directly ex vivo. During this protective phase of the secondary response, a very early elaboration of effector activities is preferentially exhibited by T cells specific for the viral NP396 epitope. This wave of activation contains the infection primarily before the initiation of the proliferative phase of the secondary response. Marked expansion is observed, but its magnitude differs depending on the epitope specificity of the responding cells; between 42 and 48 h following infection, approximately 70% of NP396-specific memory cells are in the S phase of the cell cycle, as assessed by bromodeoxyuridine incorporation studies. Epitope-dependent differences during the proliferative phase of the secondary response were confirmed by adoptive transfer studies with CFSE-labeled T cells. Although NP396-specific T cells typically dominate secondary responses, the broader multiepitope-specific population of antiviral T cells is beneficial for controlling a variant virus with an escape mutation in this epitope. These findings indicate that the induction and maintenance of a focused response contribute to the clearance of secondary infections; however, a more diverse pool of antiviral T cells facilitates long-term immunity to mutable pathogens.     

Mechanism of recovery from acute virus infection. IV. Questionable role of mononuclear phagocytes in the clearance of lymphocytic choriomeningitis virus from spleens of mice

After intravenous infection of mice with 10(3) infectious units (IU) the WE strain lymphocytic choriomeningitis (LCM) virus multiplied in the spleens (as in all other major organs), reaching more than 10(8) IU/g of tissue on days 4 to 5. Subsequently, the virus was quickly eliminated, being below detectability usually by day 10. During the time of virus clearance, the mononuclear phagocytes (MNP) of the spleen were activated as revealed by suppression of growth of Listeria monocytogenes and increase of cell-associated hydrolytic enzymes. In athymic nude mice, in whom the MNP system is assumed to be permanently activated, the virus replicated slightly but reproducibly less than in their euthymic counterparts. However, when the MNP were activated by Corynebacterium parvum, virus in spleens attained higher concentrations than in mice not so treated, and the rate of elimination was not altered. In mice whose MNP had been damaged by injection of dextran sulfate 500, the spleen virus titers were also increased, but the subsequent immune elimination was slightly delayed. Activation of spleen MNP was not evident at the time virus was rapidly cleared as a result of transfusion of LCM-immune T lymphocytes. Adoptive immunization was as successful in mice that had been pretreated with gamma-rays or cyclophosphamide, suggesting that replicating cells or their descendants, in particular monocytes, did not participate measurably in the process of elimination. Pretreatments of recipients with dextran sulfate 500 reduced the efficacy of transfused LCM-immune T lymphocytes, but this compound probably directly affected the cells. We interpret these findings to mean that the LCM virus in the mouse's spleen is controlled by a mechanism in which MNP do not play an essential role.     

Distinct CD8 T cell functions mediate susceptibility to histoplasmosis during chronic viral infection

It has long been recognized that some viral infections result in generalized immune suppression. In acute infections, this period of suppressed immunity is relatively short. However, chronic infections associated with a prolonged period of immune suppression present far greater risks. Here, we examined the role of CD8 T cell responses following viral infection in immunity to systemic histoplasmosis. Although wild-type mice with systemic histoplasmosis were able to control the infection, those simultaneously infected with lymphocytic choriomeningitis virus clone 13 showed reduced immunity with greater fungal burden and high mortality. The immune suppression was associated with loss of CD4 T cells and B cells, generalized splenic atrophy, and inability to mount a granulomatous response. Removing the anti-viral CD8 T cells in the coinfected mice enabled them to reduce the fungal burden and survive the infection. Their lymphoid organs were replenished with CD4 T and B cells. In contrast to wild-type mice, perforin-deficient mice infected with lymphocytic choriomeningitis virus clone 13 and Histoplasma showed an absence of immunopathology, but the animals still died. These results show that CD8 T cells can suppress immunity through different mechanisms; although immunopathology is perforin-dependent, lethality is perforin-independent.     

T-lymphocyte downregulation after acute viral infection is not dependent on CD95 (Fas) receptor-ligand interactions.

Infection of mice with lymphocytic choriomeningitis virus (LCMV) causes a major expansion of CD8+ T cells followed by a period of immune downregulation that coincides with the induction of lymphocyte apoptosis in the mouse spleen. CD95 (Fas) and its ligand are important for regulating peripheral T-lymphocyte numbers and can mediate apoptosis of mature T lymphocytes. We infected CD95- and CD95L-deficient mice (lpr and gld, respectively) with LCMV to determine if the immune downregulation that occurred following resolution of the LCMV infection was due to a CD95-dependent apoptotic mechanism. Lymphocytes from LCMV-infected lpr and gld mice were capable of normal T-cell expansion and cytolytic function but were, in contrast to activated cells from normal virus-infected mice, relatively more resistant to T-cell receptor-induced apoptosis in vitro. However, in vivo there were significant numbers of apoptotic cells in the spleens of lpr and gld mice recovering from the infection, and the T-cell number and cytolytic activity decreased to normal postinfection levels. Thus, CD95 is not required for the immune downregulation of the CD8+-T-lymphocyte response following acute LCMV infection.     

Antiviral immune responses of lymphocytic choriomeningitis virus-infected mice lacking CD8+ T lymphocytes because of disruption of the beta 2-microglobulin gene.

Mice infected intracerebrally with lymphocytic choriomeningitis virus (LCM virus) develop a characteristic central nervous system disease and usually die. If the intravenous or intraperitoneal route is used, the infection leads to less severe clinical signs and the virus is eliminated. Illness and virus clearance are immunological phenomena, which are assumed to be caused exclusively by CD8+ T lymphocytes. In contrast, of the two phases of a delayed-type hypersensitivity reaction caused by inoculation of the virus into the mouse's foot, only the first is mediated by CD8+ cells, whereas the second is mediated by CD4+ cells. We have examined LCM virus-specific immune responses in mice devoid of CD8+ T lymphocytes as a result of disruption of the beta 2-microglobulin gene. As expected, the virus persisted but footpad swelling did not occur, although intracerebral infection resulted in CD4+ T-lymphocyte-mediated illness and antiviral antibodies were produced. Different results had been obtained by Fung-Leung et al. (W.-P. Fung-Leung, T. M. Kündig, R. M. Zinkernagel, and T. W. Mak, J. Exp. Med. 174:1425-1429, 1991), who, is essentially identical experiments but with mice lacking CD8+ T lymphocytes as a result of disruption of the Lyt-2-encoding gene, recorded control of the infection and development of a local delayed-type hypersensitivity reaction. We consider these differences important, because they provide us with clues that may help to understand the mode of action of the CD8+ T cells in cell-mediated antiviral immunity.     

Persistent virus infection despite chronic cytotoxic T-lymphocyte activation in gamma interferon-deficient mice infected with lymphocytic choriomeningitis virus

The role of gamma interferon (IFN-gamma) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-gamma-deficient (IFN-gamma -/-) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-gamma -/- mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-gamma -/- mice. The prolonged effector phase in infected IFN-gamma -/- mice was associated with elevated numbers of CD8(+) T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8(+) T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-gamma -/- mice than in wild-type mice. These findings indicate that in the absence of IFN-gamma, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-gamma-mediated antiviral activity on the dynamics between virus replication and CTL activity.     

CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.     

Lymphocytic choriomeningitis virus-induced immune dysfunction: induction of and recovery from T-cell anergy in acutely infected mice.

Acute infection of immunocompetent mice by lymphocytic choriomeningitis virus induces a potent cytotoxic T-lymphocyte response that eliminates infectious virus. Concurrently and paradoxically, there is a general suppression of lymphocyte responses to mitogens and to other infectious agents. Splenocytes from infected mice released significant amounts of gamma interferon in response to mitogenic stimulation in vitro, but neither interleukin 2 nor interleukin 4 was similarly elevated relative to the amounts released by control cells. Early T-cell receptor-proximal signaling events were found to be intact, confirming that the cells were viable and had received the mitogenic stimuli in an appropriate manner. Acutely infected adult thymectomized mice regained concanavalin A responsiveness in parallel with euthymic mice, if T cells were left unmanipulated for several weeks after clearance of virus from the mice. Therefore, although acute lymphocytic choriomeningitis virus infection has the effect of disrupting proliferation when the T-cell receptor is ligated, this state is only temporary. In contrast, T cells from persistently infected adult mice reveal long-lasting alterations in concanavalin A responsiveness.