INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Viruses use a number of strategies, including escape from immune recognition or induction of immunosuppression, to avoid immunological surveillance and thereby persist in the host (reviewed in references 1, 14, 35, 45, and 59). The immune response to viruses involves activation of both effector arms of the adaptive immune system, i.e., virus-specific CD8⁺ T cells and neutralizing antibody production, as well as components of the innate response, including type I (alpha/beta interferon [IFN-/]) and type II (IFN-) IFNs(27, 56, 69, 72). IFNs are an essential part of both the innate and adaptive cytokine responses to viral infection, having important functions in the regulation of the immune system (12, 24, 38, 49). In addition to inducing an antiviral state (24, 38), IFNs are noted for their function in many immunoregulatory processes, including up-regulation of major histocompatibility complex (MHC) class I and II molecules, activation of macrophages and natural killer cells (68), augmentation of dendritic cell responses, and promotion of proliferation and survival of activated lymphocytes (15, 36, 60).

Infection of mice with the relatively noncytopathic lymphocytic choriomeningitis virus (LCMV) results in an early and dramatic elevation of IFN-/, within day 2 to 3 of infection (18, 67). The adaptive T-cell immune response, characterized by profound CD8⁺-T-cell expansion and IFN- production, is elicited by day 7 to 9 after infection (11, 23, 73). The central concept derived from studies with this viral system is that in previously unexposed individuals a race occurs between the development of cell-mediated immunity and the extent of viral replication. Virus clearance or persistence is determined by a critical balance between the virus-specific immune response and the rate of virus replication. Consistent with this model, virus control and functional T-cell memory, or viral persistence and exhaustion of virus-specific CD8⁺ T cells, reflect the ends of the spectrum of the virus-host interaction. Thus, infection with invasive strains of LCMV that can rapidly replicate and produce a high viral load can drive the activation and vigorous expansion of antigen-specific CD8⁺ T cells, followed by their functional inactivation resulting in irreversible anergy and/or deletion (43, 71). This phenomenon, called clonal exhaustion, results in viral persistence. In contrast, infection with less invasive, slowly replicating LCMV strains induces virus-specific T cells capable of efficiently clearing the infection. Typically, a fraction of these cells persist as long-term memory cells after virus elimination. These distinct outcomes of LCMV infection are critically controlled by host factors, which determine the magnitude of the virus-specific cytotoxic-T-lymphocyte (CTL) response, and by the rapidity of spread of the virus, determined by the virus strain and the route and dose of infection (2, 40, 42). Thus, susceptibility to persistent infection by clonal exhaustion correlates with a quantitatively lower virus-specific CTL response from the host and with rapidly replicating LCMV strains.

We previously observed that the ability of individual virus strains to induce extensive spread of infection correlates with their relative resistance to IFN-/ and IFN- (39); hence, fast-growing IFN-resistant isolates, such as Docile and CL 13 Armstrong, readily induce persistent infection, whereas slow-growing IFN-sensitive strains, such as WE, Aggressive, and Armstrong, do not. Thus, it is of interest to further understand the roles played by IFNs during the establishment of persistent infections, and LCMV serves as a valuable model for such studies. It is particularly important to determine the impact of IFNs on viral dissemination during the onset of infection and to elucidate their role in initiation and regulation of the T-cell response and therefore the outcome of infection. Clonal expansion and differentiation of LCMV-specific CD8⁺ T cells reportedly proceed normally in the absence of IFN-, and the importance of this cytokine during acute LCMV infection is influenced by the invasiveness of the virus strains used (34, 39, 48, 58). However, complete inhibition of virus clearance following treatment with antibody specific to IFN- has been reported (61, 70). This correlated with a greatly reduced CTL response and suggested an essential role for IFN- in generation of virus-specific CD8⁺ T cells. Similarly, studies with mice deficient in the IFN-/ pathway revealed that LCMV-WE is able to initiate a persistent infection due to the absence of virus-specific CD8⁺ T cells, while clearance of LCMV-Armstrong proceeds but with slower kinetics (19, 46, 63). Together, these studies imply an essential role for IFN-/ or IFN- in LCMV infection, which can determine the outcome of acute infection. However, the mechanisms by which IFNs are operational in this respect (direct suppression of virus replication and/or regulation of adaptive antiviral responses) are not well understood.

In this work the kinetics of virus replication and the development of the virus-specific CTL response were studied in mice deficient in receptors for IFN-/, IFN-, or both IFN-/ and IFN- during infection with LCMV strains with different potentials for causing persistent infection. The specific CD8⁺-CTL response was examined by direct visualization with MHC class I tetramers complexed to LCMV epitopes and with stimulation of IFN- expression by viral peptides. The results reveal a critical role for both IFN-/ and IFN- in restricting LCMV spread at the onset of infection and thus preventing extinction of the antiviral T-cell response. Our study shows that production of IFN-/ and/or IFN- critically regulates the virus-host balance during the acute phase of infection, such that a high viral burden drives responding cells into different programs of exhaustion. This dampening of virus-specific CD8⁺-T-cell responses in the early phase of infection results in a protracted or permanent persistence of infection.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Mice. Mice deficient in IFN-/ receptor (IFN-/R^/), IFN- receptor (IFN-R^/), or both IFN-/ and IFN- receptors (IFN-/-R^/) on the 129/SvEv background (29, 46, 63), originally obtained from B&K Universal Limited (Hull, United Kingdom), were bred and maintained under specific-pathogen-free conditions. Age-matched 129/SvEv control mice were purchased from Jackson Laboratories (Bar Harbor, Maine). All mice used in this study had the H-2^b MHC, and animals were kept and experiments were performed in accordance with institutional animal welfare guidelines.

Viruses. LCMV-WE was originally obtained from F. Lehmann-Grube (Hamburg, Germany), and LCMV-Docile and LCMV-Aggressive (variants isolated from an LCMV-WE [UBC] carrier mouse) were obtained from C. J. Pfau (Troy, N.Y.) as plaque-purified second passage virus (52). LCMV-Armstrong and CL 13 Armstrong viruses were obtained originally from M. B. A. Oldstone (Scripps Clinic and Research Foundation, La Jolla, Calif.) (20) and Rafi Ahmed (Emory University Vaccine Center, Atlanta, Ga.) (2). CL 13 Armstrong is a variant isolated from the spleen cells of an adult BALB/WEHI mouse neonatally infected with LCMV-Armstrong.

Virus titers and neutralization assay. LCMV titers in the spleen and neutralizing antibody titers in serum were determined with an immunological focus assay (8). Neutralizing titers were defined as the dilution of serum causing half-maximal reduction of plaques of LCMV with the same amount of virus inoculated with control serum and measured as log₂, starting with a 1:10 dilution of serum.

Depletion of CD8⁺- or CD4⁺-T-cell subsets in vivo. Mice were depleted of CD8⁺- or CD4⁺-T-cell subsets by intraperitoneal injection of purified specific antibody (YTS169 anti-CD8 or YTS191 anti-CD4) as previously described (41). Antibody was administrated 1 day before and 2 days after infection as indicated. This treatment depleted >99% of the splenic CD4⁺-T-cell population for a 3- to 4-week period, but within 2 months these mice had reconstituted their T-cell subsets and contained normal levels of CD4⁺ T cells. However, although these mice recovered their T-cell populations, virus-specific CD4⁺-T-cell activity was not detectable in the periphery (due to persisting virus antigen in the thymus resulting in negative selection), as determined by measuring virus-specific antibody activity by enzyme-linked immunosorbent assay (ELISA) and plaque neutralization assay (10, 44). Alternatively, CD4⁺ T cells were eliminated on day 20 after infection by treatment with anti-CD4 antibody on day 20, on day 23, and continuing at monthly intervals throughout the experiment. Similarly, mice treated with antibody to CD8 depleted >90% of their CD8⁺ T cells, and while the CD8⁺-T-cell population eventually returned to normal levels, functional virus-specific CD8⁺ T cells were undetectable.

Viral peptides. Peptides were synthesized at the Medical College of Georgia Molecular Biology Core Facility (Augusta, Ga.), using a Perkin-Elmer Applied Biosystems (Berkeley, Calif.) 433A peptide synthesizer. The LCMV-specific CTL epitope peptides used in this study were the H-2D^b-binding peptides GP1_33-41 (KAVYNFATC), GP2_276-286 (SGVENPGGYCL), NP_396-404 (FQPQNGQFI), and GP1_92-101 (CSANNSHHYI) and the H-2K^b-binding peptides GP1_34-43 (AVYNFATCGI) and NP_205-212 (YTVKYPNL). Except for LCMV-Docile, which contains an amino acid change in the peptide GP2_276-286 (380_NS), all virus strains used in this study were conserved in epitopes recognized by virus-specific T cells. Note that the mutation (380_NS) in LCMV-Docile substantially reduces the ability of the GP2_276-286 peptide to bind H-2D^b.

CTL response. CTL precursor activity was determined in a bulk culture system as described previously (45). Briefly, splenocytes were prepared from LCMV-infected mice at the indicated time points. Cells were cultured for 5 days at densities of 4 × 10⁶, 2 × 10⁶, and 0.5 × 10⁶ cells/well together with peptide-pulsed (0.1 µg/ml) irradiated (30 Gy) splenocytes (4 × 10⁶ cells/well) or virus-infected peritoneal macrophages (5 × 10⁵ cells/well) in 2 ml of Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum and 10 U of recombinant murine interleukin-2 (IL-2) per ml. Restimulated cells were resuspended in 1 ml of medium per culture well, and serial threefold dilutions of effector cells were tested in a ⁵¹Cr release assay using MC57G (H-2^b) cells infected with virus or pulsed with 10 µg of the indicated peptide per ml as target cells.

Quantitative analysis of virus-specific CD8⁺ T cells in spleen. MHC-peptide tetramers for staining of epitope-specific T cells were prepared as previously described (3, 4, 21, 47). Soluble MHC class I (H-2D^b) with a specific biotinylation site and human 2-microglobulin were produced in large amounts as recombinant proteins by transforming Escherichia coli strain BL21(DE3) with the plasmid pET23-D^b-BSP, pET23-K^b-BSP, or pHN1-2m (kindly provided by J. D. Altman, Emory University, Atlanta, Ga.). Expression of the proteins was induced with isopropyl--thiogalactopyranoside as described previously (4). Folding, purification, and biotinylation of H2-D^b peptide complexes were performed as described previously (3). Finally, biotinylated MHC-peptide complexes were tetramerized by addition of phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, Oreg.). Experiments utilized H-2D^b tetramers complexed with LCMV GP1_33-41, GP2_276-286, or NP_396-404 peptide. Single-cell suspensions prepared from spleen were stained with H-2D^b tetramer along with anti-CD8 fluorescein isothiocyanate-conjugated rat monoclonal antibody (clone 53-6.7) (Caltag, Burlingame, Calif.) in fluorescence-activated cell sorter (FACS) buffer (phosphate-buffered saline [PBS] with 1% bovine serum albumin and 0.2% sodium azide). After staining for 1 h at 4°C, cells were fixed in PBS containing 0.1% paraformaldehyde and analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, Calif.).

Intracellular staining for IFN- following peptide stimulation. Splenocytes were cultured in 96-well U-bottom plates at 4 × 10⁶ cells/well in 200 µl of RPMI 1640 (Gibco) supplemented with 10% fetal calf serum, 10 U of murine IL-2 per well, and 1 µg of Brefeldin A (Pharmingen, San Diego, Calif.) per well in the presence or absence of CTL epitope peptide at a concentration of 1 µg/ml (21, 47). The peptides used were the H-2D^b-binding GP1_33-41, GP2_276-286, NP_396-404, or GP1_92-101 and H-2K^b-binding GP1_34-43 or NP_205-212. After 6 h of culture, cells were harvested, washed once in FACS buffer, and surface stained with phycoerythrin-conjugated monoclonal rat antibody specific to mouse CD8 (clone 53-6-72). After washing, cells were stained for intracellular IFN- using a Cytofix/Cytoperm kit (Pharmingen) according to the manufacturer's instructions. Fluorescein isothiocyanate-conjugated monoclonal rat antibody specific to murine IFN- (clone XMG1.2) and its isotype control antibody (rat immunoglobulin G1 [IgG1]) were used to identify cytokine-positive cells. Stained cells were washed a further time and fixed in PBS containing 0.1% paraformaldehyde. Samples were analyzed as described above.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Contributions of IFN-/ and IFN- to control of acute infection with the WE or Docile strain of LCMV. Having previously demonstrated that the capacity of LCMV strains to replicate vigorously and generate high viral burdens in vivo correlates with their ability to initiate viral persistence (39), we questioned whether antiviral cytokines such as IFN-/ and IFN- play a critical role in determining viral load and thus the outcome of infection in immune-competent adults. The purpose of these experiments was to define roles for IFN-/- and/or IFN--mediated pathways in establishing an effective defense against LCMV strains differing in their capacities to induce persistent infection.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Kinetics of LCMV-Docile and LCMV-WE replication in the spleens of mice deficient in type I, type II, or both type I and II IFN receptors compared to their 129/SvEv congenic controls. Virus titers in spleens were measured at the time points indicated following intravenous infection of IFN-/R/, IFN-R/, IFN-/-R/, or 129/SvEv mice either with 102 PFU (A) or 106 PFU (B) of Docile or with 102 PFU of WE (C). Data are means and standard errors of the means for three to five mice. Different symbols used in the columns represent values obtained from individual mice.

/

/

/

/

/

/

/

/

Contribution of IFN-/ and IFN- to control of acute infection with the CL 13 Armstrong or Armstrong strain of LCMV. Next, to assess whether the significant contribution of the IFN response towards viral clearance noted for Docile and WE infection can be generalized to other LCMV strains, kinetic studies of virus clearance were carried out with LCMV strain Armstrong and its variant CL 13 Armstrong. As expected, virus clearance proceeded efficiently in 129/SvEv mice infected with LCMV-Armstrong. Thus, studies of infection with 10² PFU (not shown) or 10⁵ PFU of Armstrong demonstrated an acute infection with viral burdens below the limit of detection by day 10 or 15 after infection, respectively (Fig. 2 and data not shown). Consistent with the experiments shown in Fig. 1, 10² PFU of CL 13 Armstrong was efficiently eliminated from the spleen by day 15; however, a relatively high dose of 10⁶ PFU persisted for several weeks. Interestingly, virus burdens declined over time and were below limits of detection by day 70 after infection.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Kinetics of LCMV CL 13 Armstrong and Armstrong replication in the spleens of mice deficient in type I, type II, or both type I and II IFN receptors compared to their 129/SvEv congenic controls. Virus titers in spleens were measured at the time points indicated following intravenous infection of IFN-/R/, IFN-R/, IFN-/-R/, or 129/SvEv mice either with 102 PFU (A) or 106 PFU (B) of CL 13 Armstrong or with 105 PFU of Armstrong (C). Data are means and standard errors of the means for three to five mice. The different symbols used in the columns represent values obtained from individual mice.

/

IFN-/- and IFN--mediated regulation of the virus-specific CD8⁺-CTL response during infection of mice with the WE or Docile strain of LCMV. As host CD8⁺ T cells are critical for efficient clearance of LCMV infection and their inactivation (via deletion and/or anergy) during the acute phase of infection results in persistence of infection, the development of the virus-specific CD8⁺-T-cell response was examined in IFN-/R^/, IFN-R^/, IFN-/-R^/, or 129/SvEv control mice infected with LCMV. The specificity and function of the CD8⁺-T-cell response were examined by direct visualization with binding of D^b MHC class I tetramer molecules complexed to LCMV peptides immunodominant for CD8⁺-T-cell responses (GP1_33-41, GP2_276-286, or NP_396-404) and by stimulation of expression of IFN- with viral peptides. The results revealed a distinct pattern of virus-specific CD8⁺-T-cell responses associated with an acute or chronic (protracted or permanent) course of infection based on the experimental findings in Fig. 1 and 2.

View larger version (81K): [in this window] [in a new window]   FIG. 3.   Kinetics of the virus-specific CD8+-T-cell response in the spleens of mice deficient in type I, type II, or both type I and II IFN receptors compared to their 129/SvEv congenic controls following infection with the Docile or WE LCMV strain. IFN-/R/, IFN-R/, IFN-/-R/, or 129/SvEv mice were infected with 106 PFU (A and B) or 102 PFU (C and D) of Docile or with 102 PFU of WE (E to G). Total numbers of GP133-41, GP2276-286, or NP396-404 peptide-specific CD8+ T cells were measured by staining with H-2Db tetramers (filled circles) or intracellular IFN- production (open circles) following stimulation of cells with viral epitope peptide to determine the functional responsiveness of these cells. Data are means and standard errors of the means for three to five mice. The limit of detection was 103 antigen-specific cells per spleen. These analyses were performed to correlate the kinetics of virus replication (Fig. 1) with the kinetics of virus-specific CD8+-T-cell responses (panels A, B, and C in Fig. 1 correspond to panels A and B, C and D, and E to G here, respectively).

/

/

/

/

/

/

/

IFN-/- and IFN--mediated regulation of virus-specific CD8⁺ T cells during infection of mice with the Armstrong or CL 13 Armstrong strain of LCMV. To further correlate the link between susceptibility of the host to chronic infection and the extent of virus-specific CD8⁺-T-cell exhaustion, the kinetics of the LCMV-specific T-cell response were observed in Armstrong- and CL 13 Armstrong-infected mice deficient in IFN pathways.

View larger version (116K): [in this window] [in a new window]   FIG. 4.   Kinetics of the virus-specific CD8+-T-cell response in the spleens of mice deficient in type I, type II, or both type I and II IFN receptors compared to their 129/SvEv congenic controls following infection with the CL 13 Armstrong or Armstrong LCMV strain. IFN-/R/, IFN-R/, IFN-/-R/, or 129/SvEv mice were infected with 106 PFU (A to C) or 102 PFU (D to F) of CL 13 Armstrong or with 105 PFU of Armstrong (G to I). Total numbers of GP133-41, GP2276-286, or NP396-404 peptide-specific CD8+ T cells were measured by staining with H-2Db tetramers (filled circles) or intracellular IFN- production (open circles) following stimulation of cells with viral epitope peptide to determine the functional responsiveness of these cells. Data are means and standard errors of the means for three to five mice. The limit of detection was 103 antigen-specific cells per spleen. These analyses were performed to correlate the kinetics of virus replication (Fig. 2) with the kinetics of virus-specific CD8+-T-cell responses (panels A, B, and C in Fig. 2 correspond to panels A to C, D to F, and G to I here, respectively).

/

/

/

/

/

/

/

/

/

CD8⁺-T-cell effector functions are lost in mice with a protracted persistent infection. The results described above revealed a close association between virus persistence and exhaustion (anergy and/or deletion) of the virus-specific CD8⁺-T-cell repertoire. However, under certain conditions of infection (e.g., 129/SvEv mice infected with 10⁶ PFU of LCMV-Docile), permanent persistence of infection was not observed and the virus was eventually cleared from the host. This occurred despite the absence of functional CD8⁺ T cells specific to dominant epitopes, as determined by intracellular IFN- staining of restimulated MHC-tetramer-binding cells. It is possible in this case that resolution of infection may be mediated by an altered physiological pattern of T-cell response, such as synthesis of alternate cytokines, which would not be detected in these experiments. In addition, the tetramer-MHC-peptide complex and IFN- secretion staining assay techniques are limited by their ability to resolve a signal over background staining by FACS (around 0.1% of CD8⁺ T cells). Thus, it is possible that functional CD8⁺ T cells specific to dominant or even subdominant epitopes, which escape T-cell exhaustion at the onset of infection and persist at low levels, are capable of subsequently mounting an effective antiviral response and go on to clear the viral infection.

/

/

/

Contribution of CD4⁺ T cells and neutralizing antibodies in clearance of a protracted infection in the absence of functional virus-specific CD8⁺ T cells. CD8⁺ T cells are critical for clearance of LCMV infection, and their depletion results in persistence of virus infection. However, under certain conditions, as described above, virus was eventually cleared from mice with protracted infection, although functional CD8⁺ T cells were undetectable. Evidence for CD4⁺ T cells and neutralizing antibody contributing to the resolution of LCMV infection has been obtained in several experimental settings and may provide an explanation for the eventual clearance of virus following a protracted infection. Note that CD4⁺-T-cell help is essential for virus-specific antibody (IgM or IgG) production and for maintaining specific CD8⁺-T-cell responses (41).

/

/

/

View larger version (37K): [in this window] [in a new window]   FIG. 5.   A critical role for CD4+ T cells in clearance of a protracted viral infection via regulation of virus-specific antibody response in the absence of functional virus-specific CD8+ T cells. Kinetics of virus replication and antigen-specific CD8+-T-cell responses in the spleens of 129/SvEv mice infected with 106 PFU of LCMV-Docile and depleted of CD4+ or CD8+ T cells are shown. 129/SvEv untreated infected mice were used as controls. (A) Virus titers in spleens were measured at the time points indicated following treatment with antibody (Ab) against CD4 () at the time of infection (day 0 [d0]) or on day 20 after infection (d20). In a separate group mice were treated with antibody against CD8 (). Data are means and standard errors of the means for three to five mice. (B and C) Total numbers of GP133-41 or NP396-404 epitope-specific CD8+ T cells in CD4+-T-cell-depleted or control animals were measured by staining with H-2Db-peptide tetramers () or intracellular IFN- production () following stimulation of cells with viral epitope peptide to determine the functional responsiveness of these cells. The kinetics of GP133-41 or NP396-404 peptide-specific CD8+ T cells in control mice are indicated as broken lines in the right-hand panels. Data are means and standard errors of the means for three to five mice.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this work, we studied the down-regulation of the virus-specific CD8⁺-T-cell response (clonal exhaustion) during persistent infection of adult mice, with particular emphasis on examining the contribution of IFNs, which play a large part in containment of virus growth in the early course of infection. The data obtained support the hypothesis that virus load in the early phase of infection is a critical factor in the effectiveness of the T-cell response and that the production of IFNs can critically regulate the delicate balance between viral replication and host immune control in favor of viral dissemination, resulting in exhaustion of virus-specific CD8⁺ T cells and persistent infection. The main conclusions of this study are that an inverse correlation exists between antigen persistence and responsiveness of virus-specific T cells and that distinct programs of activation or tolerance (functional unresponsiveness and/or physical elimination of antigen-specific cells) are operative during acute and chronic virus infections. (i) During the course of acute infection (which lasts only about 2 weeks), CD8⁺ T cells expand and differentiate into effector cells, which mediate viral clearance. Fully functional, antigen-specific memory CD8⁺ T cells are maintained throughout the life of the host at relatively high levels. (ii) During chronic infection with protracted persistence of virus, antigen-specific CD8⁺ T cells initially exhibiting antiviral function lose this function and persist in this state for several weeks. However, after virus levels decline below threshold levels, functional CD8⁺ T cells become detectable again. The origin of these cells is unknown, but they may be anergic virus-specific T cells, which have regained their function in the antigen-free environment, or newly selected virus-specific T cells emerging from the antigen-free thymus. (iii) During permanent persistence of infection, activated antigen-specific cells progressively lose their function and either persist indefinitely in this state or are deleted in the late stages of infection. A further distinction in the last two scenarios is that anergy followed by physical deletion of virus-specific CD8⁺ T cells can operate to silence certain epitope-specific CD8⁺ T cells irrespective of whether permanent or time-limited persistence of virus infection is observed. Thus, NP_396-406 tetramer-binding CD8⁺ T cells are more sensitive to deletion than GP2_276-286-specific cells, whereas GP1_33-41-specific T cells are relatively resistant and are deleted only under conditions of long-term virus persistence. Finally, the data concur with and provide further experimental evidence for the concept that an appropriate balance between cellular and humoral components of the immune system to avoid host disease caused by immunopathology and prevent immune escape variations of the pathogen is a feature of a successful immune response with definitive viral clearance. Thus, failure of CD8⁺ T cells to rapidly clear the virus due to their functional exhaustion may lead to a protracted viral persistence. In this situation, CD4⁺ helper T cells and neutralizing antibody produced by B cells can become the principal arm of antiviral defense, and viral clearance can be achieved at a later stage of infection. Long-term persistence of infection can occur if the effectiveness of both arms of the immune response is lost or drastically reduced. Furthermore, an imbalance between viral spread and the host immune response can lead to host death, due to excessive activation of CD8⁺ T cells resulting in lethal immune pathology. This has been observed in our experiments with IFN-R^/ mice under conditions of infection where rapid viral clearance or viral persistence cannot be readily achieved.

A vital question concerns the mechanisms and factors that determine the nature and kinetic patterns of functional inactivation and/or physical deletion of virus-specific CD8⁺ T cells during persistent infection. Down-regulation of the virus-specific CD8⁺-T-cell response is the first critical step for survival and persistence of the virus in the host, but CD4⁺ helper T cells are also susceptible to anergy or physical deletion as reported previously (51, 65). According to these earlier reports, antigen-specific CD4⁺ T cells persist for several weeks and retain a functional phenotype in C57BL/6 mice infected with a high dose of LCMV-Docile, which causes permanent viral persistence, but they progressively become unresponsive, and eventually a fraction are physically deleted. Furthermore, the process of exhaustion proceeds with much slower kinetics, in that anergic CD4⁺ T cells become detectable at around day 70, compared to virus-specific CD8⁺ T cells, which develop an anergic phenotype by day 15. As CD4⁺ helper T cells are central regulators for the B-cell response, exhaustion of virus-specific CD4⁺ T cells at this late time may affect the production of neutralizing antibody, which represents a critical component of the host immune system for control of persistent LCMV infection (6, 17, 53, 57). The concept that acute or persistent viral infection (protracted or permanent) is dictated by the degree and kinetics of down-regulation of the virus-specific immune response can explain the different outcomes of the viral infection in this study. As virus-specific CD8⁺ T cells in mice with LCMV persistence become functionally inactive or deleted in the early stages of infection, the eventual clearance of infection seen in mice with protracted viral persistence may involve intervention of antigen-specific CD4⁺ T cells and antibody production. Three roles for virus-specific CD4⁺ T cells can be considered. Either (i) virus-specific CD4⁺ T cells are critically involved in induction and differentiation of CD8⁺ T cells, for example, by secretion of cytokines (such as IL-2) or increasing expression of costimulatory molecules (e.g., B7-1 and B7-2) on antigen-presenting cells (32), (ii) CD4⁺ T cells are involved in the virus clearance process by regulating neutralizing antibody production, or (iii) they function by direct inhibition of virus replication via secretion of antiviral factors (e.g., IFN- or tumor necrosis factor alpha) (6, 25, 26, 53, 57). A major role for CD4⁺ T cells in the induction and differentiation of CD8⁺ T cells can be excluded, as our data indicate that depletion of CD4⁺ T cells at the time of infection has no significant effect on the kinetics of proliferation or functional inactivation of virus-specific CD8⁺ T cells during the acute phase of infection. However, it remains possible that CD4⁺ T cells do have a function in the maintenance of CD8⁺-T-cell activation and influence the longevity of the CD8⁺-T-cell population once it has become anergic. Our data most favor a role for CD4⁺ T cells in initiation of the virus-specific antibody response responsible for eventual clearance of LCMV infection, as elimination of CD4⁺ T cells inhibits antiviral antibody production. The fact that CD4⁺-T-cell depletion on day 20 after infection, when functional virus-specific CD8⁺ T cells are not detectable, does not prevent clearance of virus infection and can no longer affect virus-specific antibody production provides further support for an antibody-mediated role in the clearance of a protracted viral persistence. Note that the induction and differentiation of virus-specific B cells are advanced on day 20 after infection and may no longer be dependent on CD4⁺-T-cell help. Consistent with this, we have reproducibly observed moderate levels of neutralizing antibody in IFN-deficient mice infected with a high dose of LCMV-Docile, while 129/SvEv control mice exhibited significantly higher antibody levels, having cleared the infection. However, we should keep in mind that the virus-specific antibody response is auxiliary in the virus clearance process and that inactivation of CD8⁺ T cells is the crucial step for virus persistence. This view is consistent with studies by us and others (9, 16, 37, 66) showing that CD4⁺ T cells are critical only in threshold situations where complete exhaustion of CD8⁺ T cells normally does not occur and thus virus resolution proceeds with delayed kinetics. Finally, a vital question concerns the mechanisms allowing virus to reemerge in the spleens of infected mice that were depleted of CD4⁺ T cells by day 20 after infection. One plausible explanation for this could be that neutralizing antibody escape variants are selected at late times in the course of infection as a result of immune pressure and are able to persist in the host. Analyses to test this hypothesis are under way.

It is well recognized that the interaction between T cells and antigen-presenting cells can result in T-cell activation, anergy, or deletion, depending on the level of T-cell receptor (TCR) engagement (30, 33, 62). Likewise, the levels of TCR engagement can trigger a spectrum of biological responses, such as cytotoxicity or cytokine secretion (31). In this situation, different factors, such as the activation state of T cells, the dose of antigen, and the presence or absence of costimulation, have been shown to play a role in determining the outcome of the T-cell response. Clearly, the complexity of the molecular interactions between virus-infected antigen-presenting cells and T cells makes it difficult to analyze the relative contributions of these factors to the final outcome. However, the following scenarios, based on the central concept that the duration of antigen stimulation determines the fate of T cells during persistent LCMV infection, may help to shed light on the phenomenon of T-cell exhaustion. The extent of T-cell activation can be influenced by the abundance of viral antigen presented and by the affinity of the TCR for the given MHC-peptide complex. LCMV-specific CD8⁺ T cells recognize the dominant epitope peptide (GP1_33-41, GP2_276-286, or NP_396-404) presented by H-2D^b molecules on virus-infected cells. One possible scenario is that the density of the peptide-MHC complexes on infected cells in persistently infected mice differs for individual epitopes. Consequently, CD8⁺ T cells specific for individual peptides may not be similarly activated, and therefore the fate of these cells could be differentially regulated. Evidence against this is provided in a recent report, where estimation of peptide density on LCMV-infected MC57G cells revealed that GP1_33-41 was present at 1,000 copies/cell, NP_396-404 was present at 160 copies/cell, and GP2_276-286 was present at 90 copies/cell (22). Note that NP_396-406-specific CD8⁺ T cells are more sensitive to deletion than GP2_276-286-specific cells, whereas GP1_33-41-specific T cells are relatively resistant to deletion in chronically infected mice. However, the fact that differential regulation of viral gene expression may occur in different cell types or that different amounts of the viral glycoprotein and nucleoprotein are produced in different stages of the viral replication cycle leaves this possibility open. Consistent with this, it has been found that nucleoprotein accumulated in large amounts in different cell types in persistently infected mice (50). Alternatively, the range of T-cell responses may result from differences in TCR affinity and dissociation rates of epitope-specific T cells. In this case, even if viral peptides are presented in similar densities on infected cells, the responding T cells may undergo different activation programs and consequently succumb to different fates (anergy versus physical deletion). It is possible that sustained signaling from high levels of antigen in the absence of appropriate costimulation could explain the functionally inactive phenotype of T cells. Alternatively, it has been suggested that clonal exhaustion is a consequence of a targeted infection of professional antigen-presenting cells (dendritic cells), rendering these critical accessory cells targets for destruction by an antiviral immune response (13, 55). The facts that induction of virus-specific T cells (CD8⁺ and CD4⁺) proceeds normally and that some of these cells persist for many weeks while others are deleted might argue more for a TCR-specific regulated process rather than a deficit in stimulation of T cells by accessory cells. However, infection of dendritic cells in chronically infected mice may disrupt the production of cytokines critical for survival and activation of virus-specific T cells. This is an attractive possibility that will be addressed in detail in ongoing studies. Finally, despite large viral loads at the onset of infection of IFN-/R^/ mice with 10⁵ PFU of Armstrong and the resultant delay in clearance of virus, CD8⁺ T cells never entirely lost their antiviral function. This suggests that inactivation of IFN-mediated responses may differentially affect the pattern of infection, as determined by spread of the infection in different cell types and/or by the rate of virus replication per cell, depending on the virus isolate. Analyses to explor