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The Journal of Immunology, 2004, 173: 6873-6881.
Copyright © 2004 by The American Association of Immunologists

Activation of Virus-Specific CD8+ T Cells by Lipopolysaccharide-Induced IL-12 and IL-181

Hans-Peter Raué, James D. Brien, Erika Hammarlund and Mark K. Slifka2

Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-{gamma} production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8+ T cells directly ex vivo. Following acute viral infection, 70–80% of virus-specific T cells will produce IFN-{gamma} after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-{gamma} production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 and IL-18 are potent, inflammatory cytokines that are often secreted by professional APCs such as macrophages and dendritic cells in response to invading pathogens (1, 2, 3, 4, 5). In some circumstances, one or both of these cytokines may also be produced by nonprofessional APC such as astrocytes (6), keratinocytes (7, 8), epithelial cells (9, 10), and possibly even fibroblasts (9). IL-12 and IL-18 production can be triggered by infection or exposure to a wide variety of pathogens, including viruses, bacteria, parasites, and fungi (2, 5). Moreover, simple microbial products, such as LPS, peptidoglycan, unmethylated CpG DNA, and certain purified proteins from bacteria (Listeria monocytogenes listeriolysin-O) or protozoa (Leishmania major LeIF protein) can induce IL-12 and/or IL-18 in vitro (4, 11, 12, 13). This indicates that an active microbial infection is not absolutely necessary to initiate IL-12 and IL-18 synthesis, and that inert microbial components may be the minimal requirement for the production of these inflammatory factors. The importance of these cytokines in antiviral host defense may also be extrapolated from the knowledge that certain herpesviruses selectively reduce IL-12 production (14), and both poxviruses and papillomaviruses express IL-18-binding proteins that greatly suppress IL-18 activity (15, 16).

Although IL-12 and IL-18 are unable to stimulate naive (CD44low) T cells (17, 18, 19, 20), they readily induce IFN-{gamma} production by activated T cells and NK cells (1, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31), and this can contribute greatly to the lethal cytokine storm elicited during the course of septic shock (27, 32, 33). Several studies have indicated that CD44high T cells from uninfected, naive mice can be activated by LPS, IL-12, and/or IL-18 in vivo or in vitro (17, 18, 19, 20, 34, 35). How these CD44high memory T cells from uninfected animals relate functionally to conventional memory T cells induced during or after an acute viral infection has yet to be explored. In this current study we examined the functional responsiveness of CD8+ T cells from uninfected mice to stimulation by LPS, IL-12, and IL-18 and compared these directly to virus-specific CD8+ T cell populations induced during acute infection with a natural murine pathogen, lymphocytic choriomeningitis virus (LCMV).3 By examining T cells of defined Ag specificity, we have been able to compare and contrast T cell activation via stimulation through the TCR (i.e., peptide stimulation) or through cytokine receptors (i.e., IL-12 and/or IL-18 stimulation). We found that cytokine-mediated activation of virus-specific T cell populations differs substantially from that observed in naive, uninfected animals. Moreover, our results reveal a surprising dichotomy between the activation requirements of virus-specific effector T cells examined at 8 days postinfection and memory T cells examined during the convalescent stages of infection, and this may be an important factor in host susceptibility to endotoxic shock. Further analysis indicated that the synergy of IL-12 with IL-18 was not due to up-regulation of IL-18R{alpha}, because virus-specific T cells already express high levels of IL-18R{alpha} and maintain expression of this receptor for >1 year after recovery from an acute viral infection, but are largely unable to respond to this cytokine by itself. This represents a shift in our understanding of how T cell responses to innate activation signals are mediated through IL-18; it is not simply due to up- or down-regulation of the IL-18R, as assumed from previous studies in naive mice. Together, these results demonstrate that the immunological history of the host can have a profound impact on the phenotype and cytokine responsiveness of CD8+ T cells, representing a relevant, yet often overlooked, aspect of viral and bacterial sepsis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Virus, LPS, and mice

BALB/c mice were bred at Oregon Health and Science University or were purchased from The Jackson Laboratory (Bar Harbor, ME). At 6–16 wk of age, mice were infected i.p. with 2 x 105 PFU of LCMV-Armstrong (Arm-53b) and were used at the time points indicated. For LPS challenge experiments, age-matched BALB/c mice (10–11 wk of age; eight mice per group) were either uninfected (naive) or infected with LCMV for 8 or 30 days before i.p. injection of LPS (Escherichia coli O111:B4; List Biological Laboratories, Campbell, CA) in 500 µl of sterile saline. Survival was checked twice daily for 1 wk. We found that 100 µg of LPS was nearly 100% lethal for all three groups of mice, 10 µg of LPS/mouse caused 0% mortality, and 30 µg of LPS/mouse showed significant differences in survival rates between groups. All animal experiments were reviewed and approved by the Oregon Health and Science University institutional animal care and use committee.

Cytokines, peptides, and Abs

IL-12 was purchased from R&D Systems (Minneapolis, MN), and IL-18 was purchased from Medical and Biological Laboratories (Watertown, MA). HPLC-purified (>95% pure) LCMV nucleoprotein, NP118–126 was purchased from Alpha Diagnostic International (San Antonio, TX). Neutralizing Abs specific for IL-12 or IL-18 were purchased from R&D Systems and Medical and Biological Laboratories, respectively.

In vitro stimulation

Splenocytes (1–2 million/well) from naive or LCMV-infected mice were cultured at 37°C in 6% CO2 in the presence or the absence of LPS, peptide, or cytokines, as indicated in the figure legends, in 200 µl of RPMI 1640 supplemented with 5% FBS, 20 mM HEPES, L-glutamine, and antibiotics in 96-well, round-bottom plates; 20 µl of brefeldin A (Sigma-Aldrich, St. Louis, MO) was added to yield a final concentration of 2 µg/ml for the last 60 min of the incubation period. Except for experiments in which FACS-purified CD8+ T cells were used, all experiments involved direct ex vivo stimulation of unfractionated spleen cell cultures.

Intracellular cytokine staining and flow cytometry

After stimulation, cells were centrifuged, washed with ice-cold PBS with 1% FBS (HyClone, Logan, UT), and stored on ice until all stimulations were complete. For detection of IL-18R{alpha}, cells were first blocked at 4°C with Fc Block (clone 2.4G2), mouse IgG (Sigma-Aldrich), and rabbit IgG (Sigma-Aldrich) for 15 min, then stained with anti-CD8 (BD Pharmingen, San Diego, CA) and goat anti-mouse IL-18R{alpha} (R&D Systems) or normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) as an isotype control. Cells were washed, incubated with biotinylated mouse anti-goat IgG (H&L) (R&D Systems) washed, and incubated with streptavidin-allophycocyanin (Molecular Probes, Eugene, OR). For detection of virus-specific CD8+ T cells, samples were blocked with Fc Block and mouse IgG and were incubated with anti-CD8 and NP118 tetramers (1/100) at 4°C for 60 min. Tetramers were obtained from the National Institutes of Health Tetramer Core Facility (Atlanta, GA). Cells were then washed, fixed with 2% formaldehyde in PBS, and permeabilized with Permwash (0.1% saponin (Sigma-Aldrich), 0.1% NaN3 (Sigma-Aldrich), and 2% FBS in PBS). Samples were stained intracellularly for IFN-{gamma} (Caltag Laboratories, Burlingame, CA), washed with Permwash, followed by washing with PBS and 1% FBS, and resuspended in 2% formaldehyde in PBS. Samples were acquired on a FACSCalibur flow cytometer (105–106 events/sample) and analyzed using CellQuest software (BD Biosciences, San Jose, CA). In experiments in which the mean fluorescence intensity (MFI) of IFN-{gamma} staining was determined, at least 300–700 IFN-{gamma}+CD8+ events from naive animals and 1,500–30,000 events from LCMV-infected animals were analyzed and compared.

Statistics

The statistical significance of differences observed between samples was analyzed using two-tailed Student’s t test with unequal variance, and statistical analysis of mortality rates of mice due to endotoxic shock was performed using Fisher’s exact test (mid p). A value of p ≤ 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T cell activation by LPS, IL-12, and IL-18

Antiviral T cells perform many Ag-specific functions, but much less is known about the regulatory requirements for responding to non-Ag-specific activation via LPS or innate cytokines such as IL-12 or IL-18. In these studies we compared T cell responses from uninfected animals to those observed in LCMV-infected mice at different stages of activation. After direct ex vivo stimulation for 6 h, IFN-{gamma} production by naive CD8+ T cells was barely detectable following exposure to LPS, IL-12, or IL-18, but a weak IFN-{gamma} response was elicited by the combination of IL-12 and IL-18 (~1–2% IFN-{gamma}+; Fig. 1a). Interestingly, this result was not due to a broad spectrum deficiency in these animals, because a substantial number of CD8-negative cells responded to stimulation with IL-12 and IL-18, and these IFN-{gamma}+CD8– cells were predominantly NK cells (DX5+CD3–; data not shown). In contrast to T cells from naive animals, we observed a robust IFN-{gamma} response by effector CD8+ T cells from LCMV-infected mice at 8 days postinfection after stimulation with LPS, IL-12, or IL-18. The IFN-{gamma} response induced by LPS was not due to contaminating cross-reactive proteins, because the response to LPS was completely abrogated by pretreatment with polymyxin B, an antibiotic that binds LPS and neutralizes its activity, but which in parallel experiments had no effect on peptide-specific T cell responses (data not shown). As expected, the combination of IL-12 and IL-18 was synergistic and resulted in much stronger IFN-{gamma} responses than stimulation with either cytokine by itself. A larger number of CD8+ T cells from LCMV-immune mice examined at 100 days postvaccination were responsive to LPS, IL-12, IL-18, and IL-12 plus IL-18 than T cells from naive mice, indicating a substantial degree of skewing in innate T cell function after recovery from a single acute viral infection. T cell reactivity to LPS, IL-12, IL-18, or IL-12 plus IL-18 was examined as a function of time post-LCMV infection (Fig. 1, b–e). Interestingly, the percentage of CD8+ T cells that could respond to these individual inflammatory factors decreased gradually over time after resolving the acute viral infection, but the responsiveness to the combination of IL-12 and IL-18 was much more stable and corresponded well with sustained levels of LCMV-specific T cell memory (36, 37, 38, 39).



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  		FIGURE 1. CD8+ T cell-mediated IFN-{gamma} production after stimulation with LPS, cytokines, or peptide. Spleen cells containing CD8+ T cells from naive or LCMV-infected mice at 8 or 100 days after infection were cultured in medium alone, LPS (10 µg/ml), IL-12 (1 µg/ml), IL-18 (1 µg/ml), IL-12 plus IL-18 (10 ng/ml each), or NP118 peptide (0.1 µg/ml) for 6 h directly ex vivo and assayed for intracellular IFN-{gamma} production. a, Representative dot plots indicating the total IFN-{gamma} response after in vitro stimulation. Numbers in the upper right quadrant of each dot plot indicate the percentage of IFN-{gamma}+CD8+ T cells after subtracting the percentage of IFN-{gamma}+CD8+ T cells in the medium-only control (shown in parentheses). The lower panels show the percentage of CD8+ T cells (average ± SD) from naive and LCMV-infected mice that produce IFN-{gamma} in response to IL-12 (b), IL-18 (c), IL-12 plus IL-18 (d), or LPS (e). The data show the average and SD of T cell responses from three to eight mice per group under each of the stimulation conditions described above.

 
To determine whether the cytokine-responsive T cells in each of these groups demonstrated a memory phenotype, we examined CD11a and Ly6C expression on IFN-{gamma}+ CD8+ T cells after 6 h of direct ex vivo stimulation with IL-12 and IL-18 (Fig. 2 and Table I). As expected, only CD11ahigh T cells (similar to CD44high T cells in C57BL/6 mice) were able to respond to IL-12 and IL-18. However, only a small proportion of memory phenotype CD11ahighCD8+ T cells from naive mice could respond to IL-12 and IL-18 (~6% IFN-{gamma}+), whereas the majority of CD11ahigh T cells from LCMV-infected animals were activated by exposure to these cytokines (65–85% IFN-{gamma}+; Table I). Ly6C is another commonly used activation/memory marker on T cells (40), and we found that the majority of IFN-{gamma}+ CD8+ T cells activated by IL-12 plus IL-18 were Ly6Cint/high, again indicating an activated/memory phenotype. Compared with LCMV-infected mice, we again noted that there were fewer IL-12- plus IL-18-responsive CD8+ T cells in naive mice than would be expected based on the total number of memory phenotype T cells present in the spleen (Fig. 2). Although LCMV infection induced substantial splenomegaly and an increase in the total number of CD8+ T cells in the spleen at 8 days postinfection, after clearing the viral infection, the total number of CD8+ T cells was no longer substantially different from that in naive mice. In contrast, the number of cytokine-responsive CD8+ T cells was increased by >600-fold at 8 days postinfection, and even after resolving the infection, the number of cytokine-responsive CD8+ T cells remained significantly higher than that in naive, age-matched controls (38-fold increase; p = 0.002). To determine whether these differences between naive and LCMV-infected mice were simply due to differences in the frequency of Ag-experienced T cells or might also be indicative of more important fundamental differences in T cell function, we examined the levels of IFN-{gamma} production induced by IL-12 and IL-18 stimulation by flow cytometry (Fig. 3a). We found that activated CD8+ T cells examined at 8 days postinfection produced significantly more IFN-{gamma} than memory T cells at 100 or 400 days postinfection (p < 0.01). Interestingly, the maximum level of IFN-{gamma} produced by effector T cells and memory T cells appeared to be "hard-wired" by their state of activation, because peptide stimulation (Fig. 3b) resulted in cytokine expression levels in each population that were similar to those observed after stimulation with IL-12 and IL-18. CD8+ T cells at 8, 100, or 400 days postinfection also produced significantly more IFN-{gamma} in response to IL-12 and IL-18 than T cells from naive (uninfected) animals (p < 0.05). To determine whether the observed differences in IFN-{gamma} production might be a function of age, we examined the levels of IFN-{gamma} produced from CD8+ T cells obtained from uninfected naive animals at 8–9 wk of age vs naive animals at 22–23 wk of age, and we found no significant difference in IFN-{gamma} MFI (p = 0.7), whereas the IFN-{gamma} levels observed in IL-12- plus IL-18-activated T cells from 22- to 23-wk-old, LCMV-infected mice were again much higher (Fig. 3c). These results demonstrate that a smaller proportion of memory-phenotype CD8+ T cells from naive mice are responsive to IL-12 and/or IL-18 stimulation, as are CD8+ T cells obtained from LCMV-immune mice (Fig. 2 and Table I), and they produce substantially less IFN-{gamma} after cytokine-mediated activation (Fig. 3).



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  		FIGURE 2. Phenotype of IL-12- plus IL-18-responsive CD8+ T cells in naive and LCMV-infected mice. CD11a and Ly6C expression levels were determined on IFN-{gamma}+CD8+ T cells from naive or LCMV-infected mice after direct ex vivo stimulation with IL-12 and IL-18 (10 ng/ml each). Two groups of naive mice were assayed, young (8–9 wk of age) and old (22–23 wk of age) mice, and the surface phenotype of cytokine-responsive T cells from these mice was compared with that of LCMV-infected mice at 8 or 92 days postinfection (note that LCMV-infected mice were also 22–23 wk of age). The numbers in the upper right and lower right quadrants indicate the percentage of cells within each quadrant, and the data are representative of four animals per group from two independent experiments.

 

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  		Table I. Relationship between acute viral infection and cytokine-responsive CD8+ T cell numbers

 


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  		FIGURE 3. IFN-{gamma} production differs according to the activation state of the responding T cell population. To determine whether there were significant differences in the magnitude of IFN-{gamma} production after stimulation by cytokines or peptide, unfractionated CD8+ T cells from naive or LCMV-infected mice at 8, 100, or 400 days postinfection were stimulated with IL-12 and IL-18 (10 ng/ml each; a) or NP118 peptide (NP118; 0.1 µg/ml; b) for 6 h directly ex vivo before being analyzed for intracellular IFN-{gamma} production. c, To determine whether the age of an animal effected the level of IFN-{gamma} produced after exposure to IL-12 plus IL-18 (10 ng/ml each), we compared the CD8+ T cell responses from young (8–9 wk of age) and old (22–23 wk of age) naive animals to age-matched animals on day 8 or days 86–92 post-LCMV infection (22–23 wk of age). The MFI of IFN-{gamma} production by CD8+ T cells is shown as the average and SD using data collected from four to eight mice per group.

 
LPS-induced IFN-{gamma} production

LPS and other microbial products stimulate robust production of IL-12 and IL-18 by macrophages and dendritic cells (1, 2, 3, 4, 5), and several studies indicate that this is probably the underlying cause of T cell-mediated IFN-{gamma} production (17, 19, 27, 41). Evidence in one study suggests that LPS may stimulate purified T cell clones directly (42), whereas other studies indicate that T cells do not respond directly to LPS, but, rather, respond through indirect mechanisms (17, 34). To determine whether LPS directly stimulates virus-specific T cells into IFN-{gamma} production, we used FACS to purify T cells before direct ex vivo stimulation (Fig. 4). We found that there was little difference in the responses of purified CD8+ T cells (>98% pure) or unsorted CD8+ T cells when stimulated with either NP118 peptide or IL-12 and IL-18. In sharp contrast, purified CD8+ T cells were unable to respond effectively to LPS even though it stimulated high levels of IFN-{gamma} production by unsorted CD8+ T cells cultured in the presence of other splenic APC. To determine what cytokines were important in mediating indirect LPS-mediated T cell activation, we stimulated unpurified splenic CD8+ T cells with LPS in the presence or the absence of neutralizing Abs against IL-12, IL-18, or both (Fig. 4b). Each of these reagents reduced LPS-induced IFN-{gamma} production by activated T cells (day 8 postinfection) and memory T cells (day 100 postinfection), and simultaneous neutralization of both IL-12 and IL-18 reduced T cell-mediated IFN-{gamma} production by >90%. Together, this indicates that virus-specific T cells do not respond directly to LPS stimulation, but instead respond to intermediate cytokines produced by resident splenic APC. Moreover, these data show that IL-12 and IL-18 are indeed the major cytokines responsible for in vitro LPS-induced (but indirect) stimulation of virus-specific T cells.



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  		FIGURE 4. IFN-{gamma} responses to LPS are triggered indirectly through the production of IL-12 and IL-18. To determine whether LPS stimulates CD8+ T cells to produce IFN-{gamma} directly or indirectly through the production of other intermediate cytokines, samples were stimulated for 6 h as described and then stained for intracellular IFN-{gamma} production. a, Unsorted and FACS-purified CD8+ T cells (>98% pure) from mice at 8 days post-LCMV infection were cultured with medium alone, peptide (NP118; 0.1 µg/ml), IL-12 plus IL-18 (10 ng/ml each), or LPS (10 µg/ml). The number in the upper right quadrant of each dot plot indicates the percentage of IFN-{gamma}+CD8+ T cells after subtracting the IFN-{gamma} response observed in the medium-only control. b, Unpurified CD8+ T cells from BALB/c mice at 8 or 100 days post-LCMV infection were stimulated with LPS (10 µg/ml) in the presence or the absence of neutralizing Abs specific for IL-12 (10 µg/ml) or IL-18 (10 µg/ml) or Abs against both IL-12 and IL-18 (10 µg/ml each). Data are representative of four mice per group, analyzed in two independent experiments.

 
Kinetic differences in T cell activation by cytokines

We found significant differences in the levels of IFN-{gamma} produced by effector and memory T cells after stimulation with IL-12 and IL-18 (Fig. 3a). To determine whether this is the only difference between effector and memory T cell functions after cytokine-mediated activation or whether other functional differences might exist, we compared the on-rate kinetics of IFN-{gamma} production by virus-specific CD8+ T cells at 8 or 100 days postinfection after stimulation with peptide or IL-12 plus IL-18 (Fig. 5). Peptide stimulation resulted in rapid up-regulation of IFN-{gamma} synthesis by both effector and memory T cell populations, which peaked within 6 h poststimulation (Fig. 5b) and resulted in nearly identical rates of IFN-{gamma} production (Fig. 5d), as previously described (43). IL-12 and IL-18 stimulate Ag-experienced T cells to produce IFN-{gamma} regardless of their peptide specificity, but to ensure that we were directly comparing T cells of the same relative lineage(s), we stained CD8+ T cells with NP118 tetramers immediately before performing intracellular cyctokine staining analysis (Fig. 5a). After gating on NP118 tetramer+ CD8+ T cells, we found that IFN-{gamma} production in response to IL-12 and IL-18 peaked in primary T cells by 4–6 h poststimulation, whereas IFN-{gamma} production by memory T cells peaked much later, at ~8 h poststimulation (Fig. 5c). Remarkably, although ~70–80% of NP118 tetramer+ T cells at either time point were eventually capable of responding to IL-12 and IL-18 stimulation, the on-rate kinetics of IFN-{gamma} synthesis by memory T cells were substantially slower than those observed in activated T cells examined 8 days postinfection (Fig. 5e). Similar results were observed even when activated (Thy1.1+) and memory (Thy1.2+) T cells were mixed together at a 1:1 ratio in the same wells before direct ex vivo stimulation with peptide or cytokines (data not shown). This demonstrates an intriguing and previously unrecognized dichotomy in which effector and memory T cells show equally rapid rates of IFN-{gamma} production after direct peptide stimulation, but sharply dissimilar rates of IFN-{gamma} synthesis after non-Ag-specific stimulation by IL-12 and IL-18.



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  		FIGURE 5. Kinetics of CD8+ T cell-mediated IFN-{gamma} production induced by peptide or IL-12 plus IL-18. The speed of IFN-{gamma} production by virus-specific CD8+ T cells at different stages of activation was examined directly ex vivo after stimulation with NP118 peptide or IL-12 plus IL-18. a, Representative dot plots (gated on CD8+ cells) show the up-regulation of IFN-{gamma} production in virus-specific, NP118 tetramer+CD8+ T cells from a mouse at 100 days post-LCMV infection as a function of time poststimulation with IL-12 plus IL-18. b, Kinetics of IFN-{gamma} production after NP118 peptide stimulation of CD8+ T cells from mice at 8 or 100 days post-LCMV infection. c, Kinetics of IFN-{gamma} production by NP118 tetramer+CD8+ T cells after stimulation with IL-12 and IL-18. Rates of IFN-{gamma} production were determined after stimulation with peptide (d) or after stimulation with IL-12 and IL-18 (e) after normalizing the IFN-{gamma} response to the maximum response observed for each population of cells. Data include four mice per group and are representative of four independent experiments.

 
IL-12 decreases the threshold of T cell activation by IL-18

To further characterize cytokine-mediated activation of effector and memory T cell populations, we prepared a matrix in which CD8+ T cells were exposed to >60 different combinations of IL-12 and/or IL-18 ranging from 0.001–1000 ng/ml of each cytokine, and the percentage of responding T cells was determined by IFN-{gamma} production (Fig. 6). Activated CD8+ T cells examined at 8 days postinfection were more responsive to a greater range in individual cytokine levels than memory T cells examined at 400 days postinfection. This indicates that highly activated CD8+ T cells may be more promiscuous in terms of their activation requirements than memory T cells, which mainly produce IFN-{gamma} only in the presence of both IL-12 and IL-18.



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  		FIGURE 6. Synergy between IL-12 and IL-18 results in enhanced cytokine-mediated T cell activation. To investigate the synergy between IL-12 and IL-18, CD8+ T cells from LCMV-infected mice at 8 or 400 days after infection were stimulated for 6 h with varying amounts of IL-12 and/or IL-18 as indicated. Cytokine-mediated IFN-{gamma} production by CD8+ T cells was examined at 8 days postinfection (a) or 400 days postinfection (b). c, At 8 days postinfection, the functional responsiveness of CD8+ T cells to graded doses of IL-18 was determined in the presence or the absence of IL-12. In each case, the IFN-{gamma} response to IL-12 alone was subtracted before determining the dose-response curve to IL-18, and the results were expressed as a percentage of the maximum response obtained with the highest concentration of IL-18 (shown in a). The p values were determined by two-tailed Student’s t test, comparing the percentage of IFN-{gamma}+ T cells in the presence of IL-18 alone to the percentage of IFN-{gamma}+ T cells observed at each concentration of IL-18 and IL-12. Data are representative of two independent experiments and four mice per group.

 
Although the synergistic effects of IL-12 and IL-18 are well established, we wondered whether this synergy would also influence the relative threshold of cytokine-mediated T cell activation. To address this question, we compared the dose-response curve of IL-18-stimulated CD8+ T cells at 8 days postinfection to the dose-response curves of the same T cells cultured in the presence of different doses of IL-12 (Fig. 6c). After subtracting the percentage of IFN-{gamma}+ T cells induced by IL-12 alone, we still observed a significant improvement in CD8+ T cell responsiveness to suboptimal levels of IL-18. This occurred even at low IL-12 concentrations that, by themselves, were barely able to induce a detectable degree of T cell activation (Fig. 6a). This indicates that not only does IL-12 function to increase the number of IL-18-responsive (IFN-{gamma}+) T cells (Fig. 1) and their levels of IFN-{gamma} production (data not shown), but IL-12 also increases the sensitivity of T cells to further stimulation by normally suboptimal levels of IL-18.

IL-12/IL-18 synergy does not require up-regulation of IL-18R{alpha}

It is thought that the synergy observed between IL-12 and IL-18 is due at least in part to the up-regulation of IL-18R{alpha} by IL-12 stimulation (24, 25, 44), and we were curious to learn whether this might be the underlying mechanism of IL-12-enhanced T cell sensitivity to IL-18 stimulation (Fig. 6). Moreover, down-regulation of IL-18R{alpha} expression might explain why memory T cells were less reactive to IL-18 stimulation (Fig. 1), produced lower amounts IFN-{gamma} (Fig. 3), and were substantially slower at mounting IFN-{gamma} responses (Fig. 5) after cytokine-mediated activation. To investigate this hypothesis, we examined CD8+ T cells after stimulation with IL-12, IL-18, IL-12 plus IL-18, or NP118 peptide and assessed the ensuing IFN-{gamma} response in relation to the level of IL-18R{alpha} expression (Fig. 7a). CD8+ T cells from naive, uninfected animals expressed mainly low/intermediate levels of IL-18R{alpha}. However, there was a remarkable increase in IL-18R{alpha} expression, from only ~3–6% IL-18R{alpha}high T cells before infection to ~80% IL-18R{alpha}high by 8 days postinfection, and skewing of IL-18R{alpha} expression remained apparent out to at least 500 days postinfection. Virtually all virus-specific T cells remain IL-18R{alpha}high (Fig. 7a; peptide-stimulated IFN-{gamma}+ T cells), thus ruling out direct up-regulation of IL-18R{alpha} as a mechanism of IL-12/IL-18 synergy in virus-specific T cells, because the T cells are already IL-18R{alpha}high. More importantly, these results indicate that IL-18R{alpha} down-regulation is not the mechanism for decreased responsiveness of memory T cells to IL-18 stimulation (Figs. 1, 6, and 7a), an observation not previously described in the functional analysis of memory T cells. Instead, our results show that the synergistic effects of IL-12 are driven by an underlying mechanism that does not require the modulation of surface receptor expression levels.



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  		FIGURE 7. Synergy between IL-12 and IL-18 does not depend on increased IL-18R expression. The phenotype and activation requirements for effector and memory T cells were determined and compared with the in vivo outcome of a model of endotoxic shock. a, To determine the levels of IL-18R{alpha} expression, CD8+ T cells from naive and LCMV-infected mice at 8, 30, or 500 days after LCMV infection were cultured for 6 h with medium alone, IL-12 (1 µg/ml), IL-18 (1 µg/ml), IL-12 plus IL-18 (10 ng/ml each), or NP118 peptide (0.1 µg/ml). After stimulation, the cells were stained for CD8, IL-18R{alpha}, and IFN-{gamma} as described. b, To determine whether mice with a high proportion of activated or memory T cells are more susceptible to endotoxic shock, naive mice or mice at 8 or 30 days post-LCMV infection were challenged with 30 µg of LPS i.p., and survival was monitored for 7 days. Data in a are representative of two independent experiments including four mice per group; data in b show the results of two experiments using a total of eight mice per group. Statistical significance was determined using Fisher’s exact test (mid p).

 
T cell maturation state and susceptibility to endotoxic shock

It is well established that the greatest risk for septic shock occurs during the acute stages of an active primary infection (45, 46). Murine models of endotoxic shock mimic human sepsis, and in this regard, mice acutely infected with LCMV are far more susceptible to endotoxic shock than uninfected mice (27, 33). In Fig. 7b, we challenged naive, uninfected BALB/c mice or age/sex-matched BALB/c mice at 8 or 30 days post-LCMV infection with LPS to compare their susceptibilities to endotoxic shock. As expected, mice challenged with LPS at 8 days postinfection (the peak of the primary T cell response) were significantly more susceptible to endotoxic shock than uninfected animals (p = 0.04), a result shown to be mediated primarily through T cells (27, 33). Interestingly, shortly after mice recovered from an acute infection (day 30 postinfection), their susceptibility to endotoxic shock was not significantly different from that observed in uninfected animals (p = 0.3). This indicates that even though LCMV-immune mice have nearly 40-fold more cytokine-responsive memory T cells than uninfected animals (Table I), the innate memory T cell response in vivo is tempered so that severe immunopathology and symptoms of septic shock are avoided.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that a CD8+ T cell’s antigenic history and pre-existing state of activation play critical roles in determining its degree of responsiveness to non-Ag-specific stimulation by inflammatory cytokines such as IL-12 and/or IL-18. Highly activated T cells examined at the peak of a primary virus-specific CD8+ T cell response produced significantly more IFN-{gamma} on a per cell basis (Fig. 3) and were surprisingly faster at responding to IL-12 and IL-18 than resting memory T cells examined after recovery from acute viral infection, even though these two T cell populations had identical rates of peptide-induced IFN-{gamma} production (Fig. 5). Both primary and memory T cell subsets expressed similarly high levels of IL-18R{alpha}, but only a subpopulation of primary T cells could respond directly to IL-18 stimulation without IL-12 costimulation, and the majority of memory T cells were almost completely unresponsive to IL-18 unless they were also costimulated through the IL-12 pathway (Figs. 6 and 7). This suggests that in contrast to stable, high efficiency TCR-mediated T cell activation, memory T cells preferentially decrease their responsiveness to stimulation through the IL-18R, resulting in an increased threshold of activation and a decreased risk of lethal endotoxic shock (Fig. 7b). This may be one of many important mechanisms explaining why septic shock (which results in >100,000 deaths annually in the Unites States alone (47, 48, 49)) is far more likely to occur during the acute stages of disease when T cells are in a high state of activation than during the convalescent stages of infection when the host’s memory T cells are in a resting state of homeostasis and less likely to participate in cytokine-induced immunopathology.

Several studies have demonstrated that a small number of CD8+ T cells from uninfected mice are capable of responding to stimulation by LPS, IL-12, and/or IL-18 (17, 18, 19, 20, 34). Interestingly, if the T cells are first gated on CD44high subpopulation, only 10–30% of these memory T cells appear to respond to cytokine-mediated activation (19, 20). In BALB/c mice, only ~6% of memory phenotype CD11ahigh CD8+ T cells respond to IL-12 and IL-18 (Table I), whereas 70–80% of Ag-experienced, NP118 tetramer+ CD8+ T cells produce IFN-{gamma} under these conditions (31) (Fig. 5). Previous studies have suggested that some naive T cells will masquerade as memory T cells (40, 50) and advised caution in considering CD44high T cells from uninfected animals to be true memory T cells based on the expression of CD44 alone, especially because this may be up-regulated simply due to homeostatic proliferation (40). In our study we directly compared the cytokine responsiveness of naive T cells from uninfected mice to conventional, virus-specific T cells examined at different stages of activation after acute LCMV infection. We found several differences between T cells from uninfected and virus-infected mice, with significant changes in 1) the total number of cytokine-responsive T cells in the spleen (Table I), 2) the overall frequency of cytokine-responsive T cells (Fig. 1), and 3) the magnitude of IFN-{gamma} production induced by IL-12 and IL-18 stimulation (Fig. 3). The results of our study indicate that although a subpopulation of memory phenotype T cells exists in specific pathogen-free animals, their biological functions, at least in terms of responsiveness to inflammatory factors such as IL-12 and IL-18, may not necessarily be equivalent to those observed in conventional virus-specific T cells with a defined antigenic history. This has important implications in future studies of septic shock in animal models; the outcome of septic shock is significantly altered during acute viral infections (27, 32, 33). We have also found significantly higher susceptibility to endotoxic shock on day 8 vs day 30 postinfection (p = 0.04; Fig. 7b), which confirms these previous findings. Together, this indicates that analysis of this phenomenon in naive, uninfected animals may not provide a true representation of the in vivo phenomenon observed during human septic shock, which often occurs during viral infections complicated by secondary bacterial infections (46, 47, 48, 49).

We examined IL-18R expression as a plausible mechanism explaining the functional differences observed between activated T cells and memory T cells after cytokine-mediated stimulation. Surprisingly, we found that although essentially all virus-specific CD8+ T cells expressed very high levels of IL-18R{alpha} (the receptor subunit directly involved with binding IL-18) directly ex vivo, these T cells were largely unresponsive to stimulation by IL-18 unless they were exposed to IL-12 as well. The addition of IL-12 had no measurable effect on the percentage of T cells that expressed IL-18R{alpha}, nor did it influence the relative levels of IL-18R{alpha} expression. Unlike previous observations made using naive T cell populations from uninfected mice (24, 25, 44), this indicates that in virus-specific T cells, further up-regulation of the IL-18R{alpha} is not the mechanism by which IL-12/IL-18 synergy is mediated. Future studies are aimed at determining the underlying cause of this transformation, including whether these effects are provided by better cytokine receptor complex formation, or whether improvements in the internal signal transduction machinery are also involved. Although many other factors are involved, the biological relevance of the changes in cytokine responsiveness during the maturation process from effector to memory T cell status may be extrapolated from the significant differences in susceptibility to endotoxic shock during the acute and convalescent stages of viral infection (27, 32, 33) (Fig. 7b). These results indicate that memory T cells, although fully capable of responding to LPS (Fig. 1) through intermediate cytokines (IL-12 and IL-18; Fig. 4), do not represent an immediate hazard to the host when confronted with LPS in vivo (Fig. 7b). This demonstrates one example of how regulation of innate memory T cell effector functions might be controlled better than that observed in activated T cells at the peak of the immune response (day 8 postinfection) and represents an important aspect of T cell activation that may be useful in developing better therapeutic strategies for combating sepsis in a clinical setting.

This study provides quantitative analysis of cytokine-mediated T cell activation both before (i.e., naive mice) and after acute viral infection and shows that not all T cells are equal in their responsiveness to these non-TCR-mediated signals of inflammation. A more thorough understanding of these non-Ag-specific mechanisms of T cell activation will provide a greater knowledge of the role pre-existing T cell memory plays during the early stages of specific or unrelated microbial infections and may provide important clues for future therapeutic interventions aimed at thwarting cytokine-induced, T cell-mediated immunopathology.


   Acknowledgments
 
H-2Ld NP118–126 tetramers were generously prepared by the National Institutes of Health Tetramer Core Facility (Atlanta, GA).


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI54458 (to M.K.S.) and Oregon National Primate Research Center Grant RR00163 (to M.K.S.). Back

2 Address correspondence and reprint requests to Dr. Mark K. Slifka, Vaccine and Gene Therapy Institute, Oregon Health and Sciences University, 505 NW 185th Avenue, Beaverton, OR 97006. E-mail address: slifkam{at}ohsu.edu Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; NP, nuclear protein. Back

Received for publication June 1, 2004. Accepted for publication September 7, 2004.


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