CD8+ T Cell–Derived IFN-γ Prevents Infection by a Second Heterologous Virus

Laura Valentine, Rashaun Potts and Mary Premenko-Lanier
J Immunol December 15, 2012, 189 (12) 5841-5848; DOI: https://doi.org/10.4049/jimmunol.1201679

Abstract

Persistent viral infection is often associated with dysfunctional immune responses against unrelated pathogens. Lymphocytic choriomeningitis virus (LCMV) can establish acute or chronic infections in mice and is widely used as a model for persistent virus infections in humans. Mice infected with LCMV develop a transient defect in Ag-specific immunity against heterologous viral infection. Although it has been proposed that LCMV infection induces an immunosuppressed state within the host, our data show that infected mice successfully clear vaccinia virus through a mechanism that involves CD8+ T cell–derived IFN-γ. This observation demonstrates that chronic LCMV infection does not impair protective immunity against heterologous viral challenge. Rather, a natural sterilizing immunity is induced following a primary infection that prevents a secondary infection. Our findings suggest a need to re-evaluate current thoughts about the immune suppression that might occur during a persistent infection.

Introduction

Human immunodeficiency virus and hepatitis C virus–infected patients develop dysfunctional immune responses through mechanisms that remain unclear but that may be related to the chronic nature of the diseases (1–8). Understanding how chronic viral infections disrupt immune system function is a major goal in the treatment of infectious diseases. Lymphocytic choriomeningitis virus (LCMV) is widely used to understand immune suppression and dysfunction during a persistent viral infection (9–18). Many studies showed that chronic LCMV infection causes a transient defect in immune responses directed against subsequent infection with heterologous replicating or nonreplicating viruses (14, 19–22). As such, there has been intense interest in understanding the mechanisms of LCMV-induced immune suppression.

There are two LCMV clones that result in dramatically different infection outcomes in mice (12, 13). Although the Armstrong clone causes acute infection that resolves within 7 d, clone 13 causes a chronic infection in which the virus replicates in some tissues for the life of the mouse. Comparison of the immune responses that develop against these LCMV clones has brought much insight to our understanding of immunological memory and immune dysfunction (12, 14, 23–26). Less well understood is how coinfection of LCMV with other viruses affects the ability of the host to respond to either the primary or the secondary viral challenge (14, 19, 21, 22). Most of the studies in this area showed a decrease or lack of cell-mediated immunity against a second viral infection after a primary persistent LCMV infection. One report showed that neutralizing IFN-α/β during polyinosinic-polycytidylic acid treatment neutralized the inhibition of a primary vaccinia virus (VV) response (22). Alternatively, LCMV infection may dysregulate APC function to thwart immunity against the second infection (19–21).

Given the central role of types I and II IFNs in antiviral immunity, LCMV infection has been widely used to study IFN responses in vivo (14, 27, 28). Type I IFNs (IFN-I) include IFN-β and multiple subtypes of IFN-α (29). These all bind to and signal through the same receptors (IFNAR1 and IFNAR2) (30). Studies with IFNR-knockout mice demonstrate the importance of the IFN response following a virus infection in slowing virus replication and dissemination (31), and IFN has an important role in the activation of acquired immunity (reviewed in Refs. 32, 33).

Like previous investigations, it was our goal to study the dynamics of coinfection between LCMV and VV. We found that persistent LCMV infection (clone 13) in mice transiently impairs an Ag-specific immune response directed against a second pathogen. This transient immune suppression during LCMV also occurs in mice infected with the acute Armstrong strain of the virus. During this transient immune suppression, the mice are surprisingly immune-competent and are able to clear the second virus nonspecifically. Our data clearly suggest a need to re-evaluate the natural in vivo antiviral state and what defines and drives immune suppression during a virus infection.

Materials and Methods

Mice and infections

Female C57BL/6 mice from Charles River Laboratories (Wilmington, MA) and C57BL/6J mice from The Jackson Laboratory (Bar Harbor, ME) were used in these experiments. IFNAR mice were kindly provided by Mehrdad Matloubian (University of California San Francisco [UCSF]), and Charlie Kim (UCSF) provided the male perforin-knockout mice. Mice were 6–8 wk old at the time of the primary infection. All mice were housed in specific-pathogen free conditions at the UCSF San Francisco General Hospital animal facility and were used in accordance with university animal welfare guidelines (Institutional Animal Care and Use Committee). LCMV Armstrong and LCMV Clone 13 were gifts from Dr. Rafi Ahmed (Emory University, Atlanta, GA); they were propagated on BHK cells, aliquoted, and stored at −80°C. Mice were infected with LCMV by tail vein injection in a volume of 200 μl. VV Western Reserve was a gift from Joshy Jacob’s laboratory (Emory University, Atlanta, GA). Vaccinia was propagated on HeLa cells. Briefly, HeLa cells were infected with vaccinia at a low multiplicity of infection, removed from the flasks when they showed cytopathic effects, resuspended in PBS + 1% FCS, freeze-thawed three times, centrifuged to remove debris, aliquoted, and stored at −80°C. Mice were infected with vaccinia by i.p. injection in a volume of 200 μl. Virus stocks were titered on Vero cells, as described below.

Viral titers

Organs were harvested and frozen at −80°C in DMEM + 1% FCS. Organs were homogenized in a volume of 1 ml using a Polytron homogenizer (Kinematica). Samples were then serially diluted in DMEM/1% FCS, and 200 μl was added to confluent monolayers of Vero cells in six-well plates. The plates were overlaid with a 1:1 mixture of 2× Medium 199 (Invitrogen), 1% agarose (Lonza) 60–90 min later. Four days later, the cells were overlaid with additional Medium 199/agarose supplemented with Neutral Red dye. Plaques were read the next day. Vaccinia and LCMV plaques have clearly distinct morphology and were counted separately to calculate the PFU/ovary. For quantitative PCR (qPCR), viral DNA was isolated from individual ovaries using the QIAGEN All-Prep kit. Ten-fold dilutions of a plasmid standard containing the VV-HA gene were used to generate a standard curve in each qPCR run. Invitrogen platinum SYBR Green SuperMix was used. The qPCR primers for VV are OPHA-F89, 5′-GAT GAT GCA ACT CTA TCA TGT A-3′ and OPHA-R219, 5′-GTA TAA TTA TCA AAA TAC CCG ACG TC-3′, as described (34). Samples were run on an ABI Step One PCR machine.

Intracellular cytokine staining

Single-cell suspensions of splenocytes were prepared by mashing the spleen through a 70-μm strainer and washing with RPMI 1640. RBCs were lysed using ACK Buffer (Sigma-Aldrich, St Louis, MO). For intracellular cytokine-staining assays, splenocytes were resuspended in RPMI 1640 supplemented with 10% FCS (HyClone) and stimulated with 5 μg of the indicated peptides for 5–6 h at 37°C in the presence of GolgiPlug (BD Biosciences) and then stored at 4°C overnight. Cells were then stained for surface markers for 15–30 min at room temperature, washed twice with FACS buffer (PBS + 2% FCS), and fixed with 1% formaldehyde in PBS. Samples were then washed, permeabilized (FACS buffer + 0.1% saponin), stained for 15–30 min with Abs for intracellular Ags, washed twice with permeabilization buffer, and fixed in 1% formaldehyde. Monomers (B8R) were synthesized by the Microchemical Facility Core (Emory University, Atlanta, GA) and conjugated into tetramers using Biotin allophycocyanin (Invitrogen). Samples were read on an LSRII flow cytometer (BD Biosciences), and results were analyzed using FlowJo software (TreeStar, Ashland, OR). For FACS plots, CD8+CD4−CD19− cells are shown.

Abs and peptides

In vivo–depleting Abs for CD4 depletion (clone GK1.5), CD8 depletion (clone 2.43), NK depletion (clone PK136), and IFN-γ neutralization (clone XMG1.2) were purchased from the UCSF mAb Core; 250 μg each of these Abs was administered i.p. at the indicated times. The IFN-α/β receptor–blocking Ab (clone MAR1-5A3) was from Leinco Technologies (St Louis, MO). A total of 2.5 mg/mouse was administered i.p. to block IFN-I signaling (35). Abs used in flow cytometry analysis were CD8 Pacific Blue and CD4 Alexa Fluor 700 (Invitrogen), IFN-γ FITC (eBiosciences), and IL-2–allophycocyanin (eBioscience). AnaSpec (Fremont, CA) synthesized peptides for the immunodominant vaccinia epitope B8R (TSYKFESV) and LCMV GP34 (AVYNFATC).

IFN-I bioassay

IFN-I bioassay was used to measure IFN-I, as described (36). The murine IFN standard (12100-1; Biomedical Laboratories PBL), at a final concentration of 200 U ml−1, was used as a control.

IFN-γ ELISA

The mouse IFN-γ DuoSet (DY485E) was purchased from R&D Systems. Levels of IFN-γ in the serum were measured according to the manufacturer's instructions.

Results

The kinetics of LCMV and VV CD8 T cell responses in coinfected mice

Mice infected i.v. with 2 × 106 PFU of either LCMV Armstrong (acute) or LCMV clone 13 (chronic) were coinfected at various times points with 1 × 106 PFU VV i.p. To study how the CD8+ T cell response to vaccinia was altered at different stages of the underlying LCMV infections, mice were sacrificed at 6–7 d after secondary VV infection (Fig. 1). Splenocytes from the coinfected mice were harvested, and cytokine production was measured after in vitro stimulation with an immunodominant CD8+ T cell epitope from vaccinia B8R (TSYKFESV) (Fig. 2A) and LCMV gp33 peptide (Fig. 2B). Coinfection with vaccinia early after the primary LCMV infection (days 1 and 3) resulted in a total lack of T cell responses to VV, although mice had normal, robust anti-LCMV T cell responses (Fig. 2A, 2B). Furthermore, mice infected with LCMV clone 13 had undetectable or very low–frequency VV CD8+ T cell responses during the first 21 d after LCMV infection. By 4 wk, mice infected with LCMV clone 13 could respond to the VV coinfection, resulting in normal T cell responses to both viruses (Fig. 2, representative dot plot Fig. 2C).

FIGURE 1.
FIGURE 1.

Experimental design. Naive mice were infected i.v. with LCMV clone 13 or clone Armstrong (2 × 106 PFU). Mice were then are coinfected i.p. with VV (1 × 106 PFU) at different times (n). Mice were sacrificed 6 d following the secondary vaccinia infection for analysis.

FIGURE 2.
FIGURE 2.

The kinetics of the nonspecific antiviral state during LCMV infection in mice. Mice were infected i.v. with LCMV clone 13 (C) or clone Armstrong (A) (2 × 106 PFU) at various time points following i.p. infection with VV (V) (1 × 106). Virus-specific responses were evaluated 6 d following vaccinia infection. In vitro peptide stimulation was used to determine (22) the percentage of CD8+ IFN-γ+ cells in response to the vaccinia B8R epitope (A) and the LCMV gp34 epitope (B). (C) Representative dot plot of cytokine production. A standard plaque assay (D) or quantitative real-time PCR (E) was used to measure VV titers in the ovaries. D3, D5, and D7 indicate the time following LCMV infection when mice were coinfected with VV. Three independent experiments with similar results were performed. Error bars represent mean + SD. All statistical comparisons were made using the two-tailed Student t test.

Clearance of VV occurs in LCMV-infected mice that lack an Ag-specific vaccinia-immune response

With the lack of a vaccinia-specific T cell response during the first few weeks following the LCMV infection, we measured VV replication in the coinfected mice. LCMV-infected mice that were coinfected with vaccinia during the period of 1–22 d following clone 13 infection clear the VV infection (Fig. 2D). At 5 d, Armstrong-infected mice become susceptible to VV coinfection (Fig. 2D), and this correlates with a return of their ability to respond to the second infection immunologically (Fig. 2A). By 4 wk, clone 13–infected mice make immune responses to VV similar to VV-infected control mice (Fig. 2A) and harbor high levels of VV titers, similar to control infected mice (Fig. 2D). For the most part, immune responses against the primary LCMV infection were unaltered in the coinfected mice (Fig. 2B). We detected low levels of VV in the coinfected mice by PCR (Fig. 2E), although all mice at these time points following VV coinfection were negative for VV replication, as measured by a standard plaque assay (Fig. 2D).

LCMV-infected type I IFNR-knockout mice are resistant to VV coinfection after 72 h

We hypothesized that IFN-I was responsible for the resistance to VV coinfection during LCMV, because IFN-I is known as an innate antiviral cytokine, and VV is sensitive to IFN-I (37, 38). To test this, we infected IFN-IR–knockout mice (IFNAR) with clone 13 and coinfected them with VV. Importantly, IFNAR mice infected with 1 × 106 PFU of VV alone succumbed to the infection. Therefore, the infectious dose of VV was reduced to 1 × 104 so that the IFNAR mice would survive. We found that LCMV-infected IFNAR mice were susceptible to VV during the first 2 d following a primary LCMV infection (Fig. 3A). Surprisingly, when the clone 13–infected IFNAR mice are coinfected on day 3, they are able to clear the VV infection (Fig. 3B) without mounting an anti-VV immune response (Fig 3A, 3D). Therefore, by 72 h following a primary LCMV systemic virus infection, IFN-I signaling was not needed to prevent the secondary VV infection.

FIGURE 3.
FIGURE 3.

IFNAR mice can clear a secondary VV infection nonspecifically by 72 h following a primary LCMV infection. Type I IFNR–knockout mice (IFNAR) were infected with LCMV clone 13 (C) and coinfected either on day 2 or 3 (indicated in parentheses) with 1 × 104 PFU of VV (V). Vaccinia-specific B8R responses were measured in the spleen 30 d following coinfection. (A) Percentage of IFN-γ+ CD8 cells. (B) IFNAR mice were infected with LCMV clone 13 and infected with VV 3 d later. Mice were sacrificed 6 d following VV infection; VV titers in the ovaries and VV-specific B8R responses were measured in the spleen. (C) Percentage of IFN-γ+ CD8 cells. (D) Representative dot plot of B8R-specific IFN-γ–producing CD8 cells. Data are representative of four or five mice/group and of two or three individual experiments with similar results. Error bars represent mean + SD. Data were analyzed using the two-tailed Student t test.

The IFN response during an LCMV infection

Because IFNAR mice are capable of clearing VV nonspecifically within 72 h of an LCMV infection (Fig. 3C, 3D), we postulated that IFN-γ might play a role. We measured the IFN-I and IFN-γ responses during LCMV. Similar to published data (14, 39), we found that LCMV-infected mice had a robust IFN-I response beginning on the first day following infection that slowly wanes over time (Fig. 4A). The IFN-I response in IFNAR mice was reduced significantly on day 1 following an LCMV clone 13 infection (p = 0.0001, two-tailed Student t test, Fig. 4A) and is not detected by 3 d of infection. Therefore, IFNAR mice can produce IFN-I but not respond to it. We also could detect IFN-γ responses as early as day 3 (Fig. 4B). By day 5 following infection, there was a significant increase in IFN-γ levels in mice infected with LCMV clone 13 compared with LCMV Armstrong (Fig. 4B, p = 0.01, two-tailed Student t test). The levels of IFN-γ were sustained in LCMV-infected mice regardless of their susceptibility to VV coinfection (Fig. 1). LCMV clone 13–infected IFNAR mice have a significant reduction in IFN-γ production compared with clone 13–infected wild-type (WT) mice on days 3 and 5 following infection (Fig. 4B, p < 0.0001 at both time points, nonparametric two-tailed Student t test).

FIGURE 4.
FIGURE 4.

Type I and type II IFN production following LCMV infection. Wild-type (WT) mice were infected i.v. with LCMV clone 13 or clone Armstrong at 2 × 106 PFU (n = 5–10/group). IFNAR mice were infected i.v. with LCMV clone 13 at 2 × 106 PFU (n = 5–10/group). WT mice were bled on days 1, 2, 3, 5, 14, and 22 following infection. IFNAR mice were bled out to 5 d postinfection. (A) Type I IFN responses were measured using a standard bioassay. (B) IFN-γ responses were measured by a standard ELISA. Day-1 IFN-I levels are decreased significantly in IFNAR mice compared with all other groups. Day-5 WT LCMV clone 13 IFN-γ levels are increased significantly compared with day-5 Armstrong levels. Day-5 clone 13–infected IFNAR IFN-γ levels are decreased significantly compared with day-5 WT clone 13 IFN-γ levels. Error bars represent mean + SD. Data were analyzed using the two-tailed Student t test.

The nonspecific clearance of vaccinia is dependent on IFN-γ

Because LCMV-infected IFNAR mice are susceptible to a VV coinfection during the first 2 d, but are resistant by day 3, and because we can detect IFN-γ in the serum of LCMV-infected mice on day 3 (Fig. 4B), we postulated that IFN-γ might play a role. We administered an IFN-γ–neutralizing Ab to WT mice at days 1 and 3 after LCMV and infected them with VV on day 3. Surprisingly, these mice were highly susceptible to vaccinia infection, harboring very high titers of VV (Fig. 5A, p = 0.0004, two-tailed Student t test). These mice also do not have detectable vaccinia-specific CD8+ T cells (Fig. 5B, p = 0.0001, two-tailed Student t test), suggesting that the transient immune suppression is independent of the in vivo antiviral state. The IFN-I response in these WT mice was intact (Fig. 5C). In addition, if we block IFN-γ in LCMV-infected IFNAR mice, the mice are susceptible to a day-3 VV coinfection (data not shown). Therefore, by 72 h of an LCMV infection, IFN-I was insufficient to render mice resistant to a secondary infection without IFN-γ.

FIGURE 5.
FIGURE 5.

The nonspecific clearance of vaccinia is dependent on IFN-γ. WT mice were infected i.v. with LCMV clone 13 at 2 × 106 PFU. Mice were coinfected with vaccinia on day 3. Mice were sacrificed 6 d after coinfection. (A) Levels of vaccinia in ovaries was assessed by standard plaque assay. (B) Percentage of vaccinia B8R IFN-γ–producing cells in the spleen. (C) Day-1 and day-2 following clone 13 infection IFN-I bioactivity, as measured using the standard IFN-I bioassay. Error bars represent mean + SD. Data from two independent experiments were combined (n = 8–10/group). Data were analyzed using the two-tailed Student t test.

Type I IFN signaling is required for an IFN-γ response capable of clearing high doses of VV in LCMV-infected mice

Because the resistance of VV in IFNAR mice was with a lower dose of vaccinia, we wanted to test infection with the high dose of VV by blocking the IFN signaling in WT mice. We blocked IFN-I signaling by administering 2.5 mg of an IFNR-blocking Ab 1 d before infection of WT mice with LCMV clone 13. WT LCMV-infected mice were then injected with vaccinia at 1 × 106 PFU 3 d later and sacrificed 6 d following VV. These results are consistent with our original hypothesis that IFN-I was required for the in vivo resistance to vaccinia; these mice had high vaccinia titers and normal-frequency vaccinia-specific CD8+ T cell responses at the time of sacrifice (Fig. 6A, 6B, respectively). However, it is known that IFN-I signaling is necessary for a robust CD8+ T cell response (40), and these anti-IFNAR–treated mice have a significant reduction in their LCMV-specific CD8 T cell responses (Fig. 6C, p < 0.0001, t test). Therefore, we hypothesize that low levels of IFN-γ produced in the absence of IFN-I signaling can resist low-dose VV infection (Fig. 2). Although IFN-I signaling is required to generate an IFN-γ response necessary to resist high-dose VV (Fig. 6), and this dose-response effect was suggested previously (21).

FIGURE 6.
FIGURE 6.

Type I IFN signaling is required for an IFN-γ response capable of clearing high-dose VV infection. WT mice were treated with 2.5 mg of an anti-IFNAR Ab. The mice were infected i.v. 1 d later with LCMV clone 13 at 2 × 106 PFU. Mice were coinfected with VV 3 d after the LCMV infection and were analyzed 6 d following vaccinia infection. (A) Vaccinia titers in the ovary. Splenocytes were harvested, and Ag-specific responses to vaccinia B8R (B) and LCMV gp34 (C) were measured. Two independent experiments were combined (n = 8–10/group). Error bars represent mean + SD. Data were analyzed using the two-tailed Student t test.

The in vivo antiviral state is dependent on CD8+ cells

Having observed that IFN-γ is required for LCMV-induced resistance to vaccinia infection, we next determined what particular subsets of lymphocytes mediated this resistance. Subsets of cells that produce IFN-γ include NK, CD4+, and CD8+ T cells (41). NK cells are activated and produce IFN-γ very early after LCMV infection in an IFN-I–dependent mechanism (28), so we expected these cells to be crucial for vaccinia resistance. However, depletion of NK cells at the time of LCMV infection did not affect whether animals were susceptible to vaccinia infection 3 d later (Fig. 7A, 7B). Similar to intact mice, coinfected NK1.1-depleted mice did not have detectable vaccinia-specific CD8+ T cell responses (Fig. 7A), and they had very low vaccinia titers (Fig. 7B). Likewise, depletion of CD4+ cells had no effect on susceptibility to the second infection. CD4-depleted animals had negligible CD8+ T cell responses to vaccinia (Fig. 7C), although they were still able to clear VV from the ovaries by day 6 after vaccinia (Fig. 7D). In contrast, depletion of CD8+ cells showed a dramatic effect. Mice depleted of CD8+ cells at the time of LCMV infection were susceptible to vaccinia, having high titers at the time of sacrifice (Fig. 7E). There was no significant change in the levels of IFN-I in these anti–IFN-γ–treated mice (Fig. 7F).

FIGURE 7.
FIGURE 7.

The in vivo antiviral state is dependent on CD8+ T cells. WT mice were infected with LCMV clone 13 (C). Some mice were coinfected with VV (C+V) 3 d later. Some mice were treated with the NK-depleting Ab (PK136) on days 0 and 3 post-LCMV infection. (A) VV titers in the ovaries. (B) Percentage of CD8+ T cell response to the dominant vaccinia epitope B8R. (C) Some infected mice were treated at the time of LCMV infection with anti-CD4 Ab (GK1.5). VV titers in the ovaries. (D) CD8+ T cell response to the vaccinia epitope B8R. (E) Some mice were treated with anti-CD8 Ab (2.43) on days 0 and 3 post-LCMV infection, and VV titers in the ovary were measured. (F) Type I IFN bioactivity was measured on day 2 following clone 13 infection. Plots represent the combined data from at least two independent experiments. Error bars represent mean + SD. Data were compared using the nonparametric two-tailed Student t test.

The IFN-γ antiviral state is independent of perforin, subdominant, or cross-reactive T cell responses

CD8 T cytotoxic T cells have a variety of effector functions. To determine whether other CD8 functions are required for this nonspecific antiviral state, we infected perforin knockout mice with LCMV clone 13 and coinfected the mice with vaccinia 3 d later. We found that perforin knockout mice are also resistant to the vaccinia coinfection (Fig. 8A) and do not mount as high an Ag-specific immune response to the secondary vaccinia infection (Fig. 8B). We also measured subdominant VV-specific responses to the A47L epitope (Fig. 8C) and to published cross-reactive CD8 T cell epitopes in the coinfected mice (42). Some of these cells were detected 21 d following an Armstrong infection but not at any time during the LCMV clone 13 infection (Fig. 8D, data not shown). Therefore, perforin, subdominant vaccinia-specific response, or known cross-reactive T cells do not have a role in the CD8-mediated IFN-γ nonspecific antiviral state.

FIGURE 8.
FIGURE 8.

The IFN-γ antiviral state is independent of perforin, subdominant, or cross-reactive T cell responses. Perforin-knockout mice were infected with LCMV clone 13 (C), and some were coinfected with vaccinia (C+V) 3 d later. (A) VV titers in the ovaries. (B) Percentage of CD8+ T cell response to the dominant vaccinia epitope B8R. (C) WT mice were infected with LCMV clone 13 (C) and coinfected with VV on day 8 postprimary LCMV infection. Mice were sacrificed 6 d after VV coinfection, and VV-specific A47L responses were measured. (D) WT mice were infected with either LCMV Armstrong (A) or clone 13 (C) and coinfected with VV 21 d later. Percentage of IFN-γ produced by cross-reactive a11r CD8 T cell responses were measured in uninfected controls, singly infected mice, or coinfected mice. VV titers represent the data from one of at least two independent experiments with similar results. Error bars represent mean + SD. Population distributions were compared using the nonparametric two-tailed Student t test.

Discussion

We described the susceptibility and T cell responses to a secondary viral infection following infection of mice with either acute or chronic LCMV. We found that mice infected with either strain of LCMV were transiently resistant to subsequent infection with VV for different periods of time. This resistance required IFN-I signaling but rapidly became dependent on IFN-γ and CD8+ cells. We do not believe that the resistance observed is due to cross-recognition of VV epitopes by LCMV-specific CD8+ T cells or by the activation of subdominant epitopes that were not detected in the coinfected mice. Rather, the mechanism for preventing a secondary virus infection during a primary LCMV systemic viral infection appears to be the nonspecific establishment of a potent antiviral state. This state results in sterilizing immunity against the second VV infection, because neither VV virus replication nor VV specific immunity is detected up to 27 d post-VV coinfection in IFNAR mice (Fig. 3A, data not shown).

Several laboratories reported diminished cell-mediated immune responses toward a second infection in LCMV-infected mice (14, 19–22). These studies concluded that the inability to respond to the second infection is due to the immune-suppressive qualities of the strain of LCMV used. However, even mice that are infected with an acute strain of LCMV do not mount an Ag-specific response to the second VV infection. This suggests this transient suppression is independent of disease outcome. Additionally, mice infected with either acute or persistent strains of LCMV are immune competent and clear the secondary VV infection nonspecifically. Further, we found that, in vivo, this nonspecific clearance is dependent on IFN-γ production by the acquired immune response.

We found that, when IFN-γ is blocked, the LCMV-infected mice become susceptible to the VV coinfection. However, in these susceptible mice, an immune response to the second VV infection is still prevented. Treating mice with anti–IFN-γ, followed by infection with VV alone, had no effect on the VV immune response (43). This suggests that the IFN-γ nonspecific clearance of the secondary VV infection and the inability to mount a specific immune response to the secondary VV infection are independent. Therefore, we propose that the inability of the LCMV-infected mice to mount an Ag-specific response to the VV secondary infection is a partial immune suppression. The purpose of the partial immune suppression may reflect defects in APC function, and this has been studied extensively (44). We conclude that the partial immune suppression occurs during both acute and persistent LCMV infections as soon as 24 h following infection and is independent of the final outcome of the infection (i.e., clearance or persistence).

In our model, the exact role of the IFN-I response, which is potent during the first few days of the LCMV infection, is not clear. VV is both sensitive and resistant to IFN-I and IFN-γ (reviewed Refs. 37, 45). Treating mice with exogenous IFN-I can prevent VV infection (38). In our studies, we found that IFN-I signaling, without IFN-γ signaling, was not sufficient to prevent VV. Type I IFN was recently shown to stimulate direct IFN-γ production by NK cells (28), although we found that depletion of NK cells had no effect on the IFN-γ clearance of VV. In addition, IFN-I signaling is important to activate dendritic cells and T cells (46, 53), and it can promote IFN-γ production by T cells (54). Further, neutralizing IFN-α/β during polyinosinic-polycytidylic acid treatment in mice neutralized the inhibition of a primary VV immune response (22). We found that WT mice given an anti-IFNAR–blocking Ab prior to the LCMV infection were susceptible to the VV coinfection. These mice had a significant reduction in their LCMV-specific responses. Our findings suggest that IFN-I is required during the first few days of infection, but after 72 h it supports IFN-γ production through direct or indirect activation of CD8 T cells that is required for the extended in vivo antiviral state.

The downmodulation of the IFN-γ antiviral state occurred 5 d following LCMV Armstrong infection, although high levels of IFN-γ are still detected in the serum of the mice. In addition, the in vivo antiviral state is still in place in mice 15 d following infection with LCMV clone 13, although IFN-γ is not detected in the serum at this time. Therefore, the presence of IFN-γ alone is not sufficient for the in vivo antiviral state. Other factors must be important in maintaining the in vivo antiviral state, such as the expression of IFNRs, the translation of IFN response genes, or a threshold of virus present. The in vivo bioactive levels of IFN-γ may also be below the level of detection of our assays.

The antiviral properties of IFN-I and IFN-γ are well characterized and similar to many virus infections; VV is sensitive to IFN. IFN-I and IFN-γ were shown to be key to clear a poxvirus infection independently (38, 55, 56). Using the LCMV system, we showed that there is a close interplay between the innate and acquired immune response during the antiviral state. Very early, the innate response seems to pass this role on to the acquired immune response, which may reflect the evolution of IFN-γ produced by activated CD8 T cells as a mechanism to sustain the antiviral state following more complex infections. Our results do not support a model in which animals experiencing a chronic LCMV infection are immune-compromised with regard to secondary infection. Rather, this result highlights the effectiveness of the nonspecific antiviral response at protecting against infection, even in diseased hosts. Additionally, we found that the in vivo antiviral state is ineffective without IFN-γ signaling. Further analysis will be required to carefully understand this early interplay between the innate and adaptive immune response and whether the host, in response to the LCMV infection, initiates the partial immune suppression.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Mehrdad Matloubian for providing the type I IFNR–knockout mice and Charlie Kim (UCSF) for providing the perforin-knockout mice used in these studies. We thank members of the Department of Experimental Medicine at UCSF for comments and suggestions on the experiments done in this study and for review of the manuscript.

Footnotes

  • This work was supported by the Harvey V. Berneking Living Trust; the generous support of the Hellman Fellows Fund, established by Warren and Chris Hellman; and in part by National Institutes of Health Grant R00 AI076346-01. L.V. was supported in part by National Institutes of Health Grant T32AI007334. R.P. was supported by National Institutes of Health (National Institute of General Medical Sciences) Minority Biomedical Research Support and Research Initiative for Scientific Enhancement Grant R25-GM059298.

  • Abbreviations used in this article:

    IFN-I
    type I IFN
    LCMV
    lymphocytic choriomeningitis virus
    qPCR
    quantitative PCR
    UCSF
    University of California San Francisco
    VV
    vaccinia virus
    WT
    wild-type.

  • Received June 18, 2012.
  • Accepted October 9, 2012.

References

  1. ↵
    1. Golden-Mason L.,
    2. B. Palmer,
    3. J. Klarquist,
    4. J. A. Mengshol,
    5. N. Castelblanco,
    6. H. R. Rosen
    . 2007. Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction. J. Virol. 81: 9249–9258.
    OpenUrlAbstract/FREE Full Text
    1. Gruener N. H.,
    2. F. Lechner,
    3. M. C. Jung,
    4. H. Diepolder,
    5. T. Gerlach,
    6. G. Lauer,
    7. B. Walker,
    8. J. Sullivan,
    9. R. Phillips,
    10. G. R. Pape,
    11. P. Klenerman
    . 2001. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J. Virol. 75: 5550–5558.
    OpenUrlAbstract/FREE Full Text
    1. Wedemeyer H.,
    2. X. S. He,
    3. M. Nascimbeni,
    4. A. R. Davis,
    5. H. B. Greenberg,
    6. J. H. Hoofnagle,
    7. T. J. Liang,
    8. H. Alter,
    9. B. Rehermann
    . 2002. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J. Immunol. 169: 3447–3458.
    OpenUrlAbstract/FREE Full Text
    1. van Grevenynghe J.,
    2. R. A. Cubas,
    3. S. Dafonseca,
    4. T. Metcalf,
    5. C. L. Tremblay,
    6. L. Trautmann,
    7. R. P. Sekaly,
    8. J. Schatzle,
    9. E. K. Haddad
    . 2012. Foxo3a: An integrator of immune dysfunction during HIV infection. Cytokine Growth Factor Rev. 23: 215–221.
    OpenUrlCrossRefPubMed
  5. Nixon, D. E., and A. L. Landay. 2010. Biomarkers of immune dysfunction in HIV. Curr. Opin. HIV AIDS 5: 498–503.
    1. Epeldegui M.,
    2. E. Vendrame,
    3. O. Martínez-Maza
    . 2010. HIV-associated immune dysfunction and viral infection: role in the pathogenesis of AIDS-related lymphoma. Immunol. Res. 48: 72–83.
    OpenUrlCrossRefPubMed
    1. Kottilil S.,
    2. K. Shin,
    3. J. O. Jackson,
    4. K. N. Reitano,
    5. M. A. O’Shea,
    6. J. Yang,
    7. C. W. Hallahan,
    8. R. Lempicki,
    9. J. Arthos,
    10. A. S. Fauci
    . 2006. Innate immune dysfunction in HIV infection: effect of HIV envelope-NK cell interactions. J. Immunol. 176: 1107–1114.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Westby M.,
    2. J. B. Marriott,
    3. M. Guckian,
    4. S. Cookson,
    5. P. Hay,
    6. A. G. Dalgleish
    . 1998. Abnormal intracellular IL-2 and interferon-gamma (IFN-gamma) production as HIV-1-assocated markers of immune dysfunction. Clin. Exp. Immunol. 111: 257–263.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Mueller S. N.,
    2. R. Ahmed
    . 2009. High antigen levels are the cause of T cell exhaustion during chronic viral infection. Proc. Natl. Acad. Sci. USA 106: 8623–8628.
    OpenUrlAbstract/FREE Full Text
    1. Sarkar S.,
    2. V. Kalia,
    3. W. N. Haining,
    4. B. T. Konieczny,
    5. S. Subramaniam,
    6. R. Ahmed
    . 2008. Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates. J. Exp. Med. 205: 625–640.
    OpenUrlAbstract/FREE Full Text
    1. Wherry E. J.,
    2. V. Teichgräber,
    3. T. C. Becker,
    4. D. Masopust,
    5. S. M. Kaech,
    6. R. Antia,
    7. U. H. von Andrian,
    8. R. Ahmed
    . 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4: 225–234.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Wherry E. J.,
    2. J. N. Blattman,
    3. K. Murali-Krishna,
    4. R. van der Most,
    5. R. Ahmed
    . 2003. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77: 4911–4927.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Ahmed R.,
    2. A. Salmi,
    3. L. D. Butler,
    4. J. M. Chiller,
    5. M. B. Oldstone
    . 1984. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160: 521–540.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Zuniga E. I.,
    2. L. Y. Liou,
    3. L. Mack,
    4. M. Mendoza,
    5. M. B. Oldstone
    . 2008. Persistent virus infection inhibits type I interferon production by plasmacytoid dendritic cells to facilitate opportunistic infections. Cell Host Microbe 4: 374–386.
    OpenUrlCrossRefPubMed
    1. Brooks D. G.,
    2. L. Teyton,
    3. M. B. Oldstone,
    4. D. B. McGavern
    . 2005. Intrinsic functional dysregulation of CD4 T cells occurs rapidly following persistent viral infection. J. Virol. 79: 10514–10527.
    OpenUrlAbstract/FREE Full Text
    1. Sevilla N.,
    2. D. B. McGavern,
    3. C. Teng,
    4. S. Kunz,
    5. M. B. Oldstone
    . 2004. Viral targeting of hematopoietic progenitors and inhibition of DC maturation as a dual strategy for immune subversion. J. Clin. Invest. 113: 737–745.
    OpenUrlCrossRefPubMed
    1. Naniche D.,
    2. M. B. Oldstone
    . 2000. Generalized immunosuppression: how viruses undermine the immune response. Cell. Mol. Life Sci. 57: 1399–1407.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Tishon A.,
    2. H. Lewicki,
    3. G. Rall,
    4. M. Von Herrath,
    5. M. B. Oldstone
    . 1995. An essential role for type 1 interferon-gamma in terminating persistent viral infection. Virology 212: 244–250.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Althage A.,
    2. B. Odermatt,
    3. D. Moskophidis,
    4. T. Kündig,
    5. U. Hoffman-Rohrer,
    6. H. Hengartner,
    7. R. M. Zinkernagel
    . 1992. Immunosuppression by lymphocytic choriomeningitis virus infection: competent effector T and B cells but impaired antigen presentation. Eur. J. Immunol. 22: 1803–1812.
    OpenUrlCrossRefPubMed
    1. Borrow P.,
    2. C. F. Evans,
    3. M. B. Oldstone
    . 1995. Virus-induced immunosuppression: immune system-mediated destruction of virus-infected dendritic cells results in generalized immune suppression. J. Virol. 69: 1059–1070.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Roost H.,
    2. S. Charan,
    3. R. Gobet,
    4. E. Rüedi,
    5. H. Hengartner,
    6. A. Althage,
    7. R. M. Zinkernagel
    . 1988. An acquired immune suppression in mice caused by infection with lymphocytic choriomeningitis virus. Eur. J. Immunol. 18: 511–518.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Brenan M.,
    2. R. M. Zinkernagel
    . 1983. Influence of one virus infection on a second concurrent primary in vivo antiviral cytotoxic T-cell response. Infect. Immun. 41: 470–475.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Shin H.,
    2. E. J. Wherry
    . 2007. CD8 T cell dysfunction during chronic viral infection. Curr. Opin. Immunol. 19: 408–415.
    OpenUrlCrossRefPubMed
    1. Intlekofer A. M.,
    2. N. Takemoto,
    3. C. Kao,
    4. A. Banerjee,
    5. F. Schambach,
    6. J. K. Northrop,
    7. H. Shen,
    8. E. J. Wherry,
    9. S. L. Reiner
    . 2007. Requirement for T-bet in the aberrant differentiation of unhelped memory CD8+ T cells. J. Exp. Med. 204: 2015–2021.
    OpenUrlAbstract/FREE Full Text
    1. Blackburn S. D.,
    2. E. J. Wherry
    . 2007. IL-10, T cell exhaustion and viral persistence. Trends Microbiol. 15: 143–146.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Oldstone M. B.
    2007. A suspenseful game of ‘hide and seek’ between virus and host. Nat. Immunol. 8: 325–327.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Lee L. N.,
    2. S. Burke,
    3. M. Montoya,
    4. P. Borrow
    . 2009. Multiple mechanisms contribute to impairment of type 1 interferon production during chronic lymphocytic choriomeningitis virus infection of mice. J. Immunol. 182: 7178–7189.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Mack, E. A., L. E. Kallal, D. A. Demers, and C. A. Biron. 2011. Type 1 interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection. MBio. 2: e00169-11.
  29. ↵
    1. Nagata S.,
    2. N. Mantei,
    3. C. Weissmann
    . 1980. The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha. Nature 287: 401–408.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Mogensen K. E.,
    2. G. Uzé,
    3. P. Eid
    . 1989. The cellular receptor of the alpha-beta interferons. Experientia 45: 500–508.
    OpenUrlPubMed
  31. ↵
    1. Müller U.,
    2. U. Steinhoff,
    3. L. F. Reis,
    4. S. Hemmi,
    5. J. Pavlovic,
    6. R. M. Zinkernagel,
    7. M. Aguet
    . 1994. Functional role of type I and type II interferons in antiviral defense. Science 264: 1918–1921.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. González-Navajas J. M.,
    2. J. Lee,
    3. M. David,
    4. E. Raz
    . 2012. Immunomodulatory functions of type I interferons. Nat. Rev. Immunol. 12: 125–135.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Welsh R. M.,
    2. K. Bahl,
    3. H. D. Marshall,
    4. S. L. Urban
    . 2012. Type 1 interferons and antiviral CD8 T-cell responses. PLoS Pathog. 8: e1002352.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Kulesh D. A.,
    2. R. O. Baker,
    3. B. M. Loveless,
    4. D. Norwood,
    5. S. H. Zwiers,
    6. E. Mucker,
    7. C. Hartmann,
    8. R. Herrera,
    9. D. Miller,
    10. D. Christensen,
    11. et al
    . 2004. Smallpox and pan-orthopox virus detection by real-time 3′-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms. J. Clin. Microbiol. 42: 601–609.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Sheehan, K. C., K. S. Lai, G. P. Dunn, A. T. Bruce, M. S. Diamond, J. D. Heutel, C. Dungo-Arthur, J. A. Carrero, J. M. White, P. J. Hertzog, and R. D. Schreiber. 2006. Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hydrodynamic transfection. J. Interferon Cytokine Res. 26: 804–819.
  36. ↵
    1. Cembrzyńska-Nowak M.
    1989. Different antiviral activity and cell specificity of interferon preparations produced by mouse peritoneal cells at 37 degrees C and at 26 degrees C. Arch. Immunol. Ther. Exp. (Warsz.) 37: 499–502.
    OpenUrlPubMed
  37. ↵
    1. Esteban M.,
    2. J. Benavente,
    3. E. Paez
    . 1986. Mode of sensitivity and resistance of vaccinia virus replication to interferon. J. Gen. Virol. 67: 801–808.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Rodriguez J. R.,
    2. D. Rodriguez,
    3. M. Esteban
    . 1991. Interferon treatment inhibits early events in vaccinia virus gene expression in infected mice. Virology 185: 929–933.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Stamm A.,
    2. L. Valentine,
    3. R. Potts,
    4. M. Premenko-Lanier
    . 2012. An intermediate dose of LCMV clone 13 causes prolonged morbidity that is maintained by CD4+ T cells. Virology 425: 122–132.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Biron C. A.
    1998. Role of early cytokines, including alpha and beta interferons (IFN-alpha/beta), in innate and adaptive immune responses to viral infections. Semin. Immunol. 10: 383–390.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Schoenborn J. R.,
    2. C. B. Wilson
    . 2007. Regulation of interferon-gamma during innate and adaptive immune responses. Adv. Immunol. 96: 41–101.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Shen Z. T.,
    2. M. A. Brehm,
    3. K. A. Daniels,
    4. A. B. Sigalov,
    5. L. K. Selin,
    6. R. M. Welsh,
    7. L. J. Stern
    . 2010. Bi-specific MHC heterodimers for characterization of cross-reactive T cells. J. Biol. Chem. 285: 33144–33153.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Leist T. P.,
    2. M. Eppler,
    3. R. M. Zinkernagel
    . 1989. Enhanced virus replication and inhibition of lymphocytic choriomeningitis virus disease in anti-gamma interferon-treated mice. J. Virol. 63: 2813–2819.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Sullivan B. M.,
    2. S. F. Emonet,
    3. M. J. Welch,
    4. A. M. Lee,
    5. K. P. Campbell,
    6. J. C. de la Torre,
    7. M. B. Oldstone
    . 2011. Point mutation in the glycoprotein of lymphocytic choriomeningitis virus is necessary for receptor binding, dendritic cell infection, and long-term persistence. Proc. Natl. Acad. Sci. USA 108: 2969–2974.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Perdiguero B.,
    2. M. Esteban
    . 2009. The interferon system and vaccinia virus evasion mechanisms. J. Interferon Cytokine Res. 29: 581–598.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Bogdan C.,
    2. J. Mattner,
    3. U. Schleicher
    . 2004. The role of type I interferons in non-viral infections. Immunol. Rev. 202: 33–48.
    OpenUrlCrossRefPubMed
    1. Gallucci S.,
    2. M. Lolkema,
    3. P. Matzinger
    . 1999. Natural adjuvants: endogenous activators of dendritic cells. Nat. Med. 5: 1249–1255.
    OpenUrlCrossRefPubMed
    1. Ito T.,
    2. R. Amakawa,
    3. M. Inaba,
    4. S. Ikehara,
    5. K. Inaba,
    6. S. Fukuhara
    . 2001. Differential regulation of human blood dendritic cell subsets by IFNs. J. Immunol. 166: 2961–2969.
    OpenUrlAbstract/FREE Full Text
    1. Le Bon A.,
    2. N. Etchart,
    3. C. Rossmann,
    4. M. Ashton,
    5. S. Hou,
    6. D. Gewert,
    7. P. Borrow,
    8. D. F. Tough
    . 2003. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat. Immunol. 4: 1009–1015.
    OpenUrlCrossRefPubMed
    1. Le Bon A.,
    2. G. Schiavoni,
    3. G. D’Agostino,
    4. I. Gresser,
    5. F. Belardelli,
    6. D. F. Tough
    . 2001. Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity 14: 461–470.
    OpenUrlCrossRefPubMed
    1. Montoya M.,
    2. G. Schiavoni,
    3. F. Mattei,
    4. I. Gresser,
    5. F. Belardelli,
    6. P. Borrow,
    7. D. F. Tough
    . 2002. Type I interferons produced by dendritic cells promote their phenotypic and functional activation. Blood 99: 3263–3271.
    OpenUrlAbstract/FREE Full Text
    1. Trinchieri G.,
    2. D. Santoli
    . 1978. Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Enhancement of human natural killer cell activity by interferon and antagonistic inhibition of susceptibility of target cells to lysis. J. Exp. Med. 147: 1314–1333.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Hervas-Stubbs, S., J. L. Perez-Gracia, A. Rouzaut, M. F. Sanmamed, A. Le Bon, and I. Melero. 2011. Direct effects of type I interferons on cells of the immune system. Clin. Cancer Res. 17: 2619–2627.
  54. ↵
    1. Cousens L. P.,
    2. R. Peterson,
    3. S. Hsu,
    4. A. Dorner,
    5. J. D. Altman,
    6. R. Ahmed,
    7. C. A. Biron
    . 1999. Two roads diverged: interferon alpha/beta- and interleukin 12-mediated pathways in promoting T cell interferon gamma responses during viral infection. J. Exp. Med. 189: 1315–1328.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Ruby J.,
    2. I. Ramshaw
    . 1991. The antiviral activity of immune CD8+ T cells is dependent on interferon-gamma. Lymphokine Cytokine Res. 10: 353–358.
    OpenUrlPubMed
  56. ↵
    1. Atrasheuskaya A. V.,
    2. E. K. Bukin,
    3. T. M. Fredeking,
    4. G. M. Ignatyev
    . 2004. Protective effect of exogenous recombinant mouse interferon-gamma and tumour necrosis factor-alpha on ectromelia virus infection in susceptible BALB/c mice. Clin. Exp. Immunol. 136: 207–214.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section