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Infection and Immunity, May 2009, p. 1894-1903, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01315-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Interleukin-18-Related Genes Are Induced during the Contraction Phase but Do Not Play Major Roles in Regulating the Dynamics or Function of the T-Cell Response to Listeria monocytogenes Infection

Jodie S. Haring¹ and John T. Harty^1,2*

Department of Microbiology,¹ Interdisciplinary Program in Immunology, University of Iowa, Iowa City, Iowa 52242²

Received 28 October 2008/ Returned for modification 4 January 2009/ Accepted 5 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Proinflammatory cytokines, such as gamma interferon (IFN-), impact aspects of T-cell responses after infection, including expansion, contraction, and memory formation. Interleukin-18 (IL-18) functions as a proinflammatory cytokine by stimulating the production of IFN- from multiple cell types and accentuating the development of Th1 CD4 T-cell responses. Focused microarray analyses revealed upregulation of IL-18 and IL-18 receptor genes in CD8 T cells during the contraction phase. Based on these findings we investigated if and how signaling through the IL-18 receptor influences the development and kinetics of antigen (Ag)-specific CD8 and CD4 T-cell responses following infection. IL-18R^–/– and IL-18^–/– mice developed frequencies and total numbers of Ag-specific CD8 T cells after Listeria monocytogenes infection that were similar to those of wild-type C57BL/6 mice. The kinetics of expansion, contraction, and memory CD8 T-cell maintenance were also similar. When IL-18R deficiency was isolated to Ag-specific CD8 T cells, the kinetics of the expansion and contraction phases were also normal. These basic findings were confirmed by examining the response to vaccinia virus infection. In contrast, the expansion of Ag-specific CD4 T cells was slightly curtailed by the absence of IL-18R; however, contraction and the maintenance of memory were not altered. Importantly, both memory Ag-specific CD8 and CD4 T cells generated in the absence of IL-18R expanded appropriately after secondary antigen exposure and were protective, indicating that signaling through the IL-18 receptor is not required for normal T-cell response kinetics and survival of immunized mice challenged with a lethal L. monocytogenes infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To function optimally, the immune system must be fine-tuned to generate an appropriate response, which includes induction of a specific set of cytokines that clear the infection with the least immunopathology. While it has been generally known for some time, the idea that cytokines play key roles in regulating each phase of antigen (Ag)-specific T-cell responses is now getting a tremendous amount of attention. The recently termed "signal 3" cytokines interleukin-12 (IL-12), alpha/beta interferon (IFN-/β), and IFN-, for example, have been demonstrated to be important for optimal T-cell expansion and survival after exposure to Ag in different model systems (17). Although first focused on as an important effector molecule employed by Ag-specific T cells to clear infection, IFN- has since been shown to be a crucial regulator of both CD4 and CD8 T-cell contraction after infection (5, 6, 19). IL-15 is required for the homeostatic maintenance of memory T cells, which is vital for their ability to provide long-term protection against future infections (8, 24, 43). It is not surprising that the importance of each cytokine to the regulation of Ag-specific T-cell kinetics and homeostasis has proven to be somewhat model specific, and therefore this field remains intensely investigated.

IL-18 was first identified as a novel protein that induced IFN- production in mice that had been serially challenged with Propionibacterium acnes and lipopolysaccharide (LPS) (34). Many types of cells, including nonimmune cells, express IL-18 at least in part due to the TATA box-less promoters located upstream of exons 3 to 7, which are the coding regions of the gene (29, 35). In contrast to the highly regulated nature of most cytokine production, IL-18 mRNA is constitutively expressed in a wide range of cells due to the lack of tight promoter regulation and the absence of mRNA-destabilizing elements. In addition to the basal, constitutive production, IL-18 mRNA synthesis can also be upregulated in response to proinflammatory stimuli, such as LPS and IFNs (30). The IL-18 protein is synthesized as a 24-kDa precursor protein, which requires cleavage by activated caspase-1 in most cells to its 18-kDa mature form before it is secreted from the cell (15). The release of biologically active IL-18 from cells such as macrophages or dendritic cells typically requires activation with stimuli such as LPS or proinflammatory cytokines (30, 47).

The IL-18 receptor is similar to the IL-1 receptor both in structure and in the associated downstream signaling components (30). IL-18R (also called IL-18R1 or IL-1R-related protein) is the major ligand-binding portion of the receptor (22, 45), and IL-18Rβ (also called IL-1R accessory protein-like) is required for the execution of downstream signaling events (11, 13, 14). As demonstrated by gene-targeting experiments, both chains of the receptor must be present for cells to respond to IL-18 (13, 22). The primary signaling cascade initiated by IL-18 binding to its receptor overlaps with Toll-like receptor and IL-1 signaling and involves MyD88, IL-1 receptor-associated kinases 1 and 4, and eventual activation of the transcription factors NF-B and JNK (1, 25, 44).

The biological effects of IL-18 are varied and depend in large part on cell type and what other cytokine signals the cells are receiving at the time they are exposed to IL-18. When combined with IL-12, IL-18 significantly contributes to Th1 polarization and is a potent stimulator of CD4 T cells, CD8 T cells, dendritic cells, and NK cells to make IFN- (7, 9, 16, 26, 30, 35, 39). This is augmented by the reciprocal upregulation of IL-18R by IL-12 and of IL-12β2 by IL-18 (12, 50). The ability of IL-12 and IL-18 to stimulate IFN- production by previously activated CD4 and CD8 T cells in an Ag-independent manner has been shown to be important in the early phases of defense against Salmonella and Listeria infections (9, 39). IL-18 does not function strictly as a proinflammatory cytokine. In the absence of IL-12, IL-18 pushes CD4 cells toward a Th2 phenotype, stimulates IL-4 and IL-13 production from T cells, mast cells, and basophils, and can induce immunoglobulin E antibody responses (23, 49, 51).

IL-18 has been shown to play a role in the defense against organisms that cause various infectious diseases, including Plasmodium berghei (40), Yersinia enterocolitica (10), Mycobacterium tuberculosis and Mycobacterium bovis BCG (41, 42), and Salmonella enterica serovar Typhimurium (28, 39). Protection against virulent Listeria monocytogenes infection is severely compromised in MyD88^–/– and caspase-1^–/– mice. MyD88 and caspase-1 are involved in IL-18 receptor signaling and pro-IL-18 processing, respectively, among other important biological functions (38, 46). In addition, a study by Neighbors et al. (31) demonstrated that anti-IL-18Rβ neutralizing antibody (14) treatment of mice infected with virulent L. monocytogenes resulted in uniform mortality that was not attributed simply to decreased IFN- production. Immunized mice treated with anti-IL-18Rβ during a secondary challenge were also significantly impaired in the ability to control bacterial replication, leading to the conclusion that IL-18 was required for protection during both primary and secondary infections. In this study, infected mice treated with anti-IL-18Rβ antibodies did not survive long enough to evaluate the development or kinetics of primary or secondary Ag-specific T-cell responses after infection (31).

In the experiments described here, Ag-specific CD4 and CD8 T-cell responses were evaluated in the absence of IL-18 or IL-18R to determine if and how this cytokine signaling pathway was involved in their development and/or homeostasis. These experiments were undertaken to address previous findings suggesting that IL-18 was required for resistance to primary and secondary L. monocytogenes infections and also because of preliminary data generated during microarray analyses conducted to identify candidate genes involved in Ag-specific CD8 T-cell contraction, which highlighted a potential role for IL-18 and/or IL-18 receptor signaling in regulating this important phase of the T-cell response. These experiments revealed that neither IL-18 nor IL-18R is required for the generation, contraction, or maintenance of memory Ag-specific CD4 or CD8 T cells after infection of mice with an attenuated strain of L. monocytogenes or vaccinia virus. Importantly, immunized IL-18R^–/– mice were protected as well as immunized wild-type (Wt) mice after challenge with a lethal dose of L. monocytogenes, indicating that signaling through the IL-18 receptor is, in fact, not required for resistance to secondary infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice. C57BL/6 (B6) control mice were purchased from the National Cancer Institute (Frederick, MD). Breeding pairs of B6 IL-18R^–/– mice (strain B6.129P2-Il18r1^tm1Aki/J; stock number 004131) and B6 IL-18^–/– mice (strain B6.129P2-Il18^tm1Aki/J; stock number 004130) were purchased from Jackson Laboratory (Bar Harbor, ME), bred, and maintained at the University of Iowa. IL-18R^–/– OT-1 (Thy^1.1/1.2) mice were created by mating IL-18R^–/– mice with OT-1 (Thy^1.1/1.2) mice from our existing colony. Both Wt OT-1 (Thy^1.1/1.1) mice from our breeding colony and IL-18R^–/– OT-1 (Thy^1.1/1.2) mice were used as adoptive transfer donors in some experiments. W+ B6 mice were used as the adoptive transfer recipients. All infected mice were housed in accordance with biosafety regulations. All animal experiments followed approved Institutional Animal Care and Use Committee protocols.

Infections. Strain LM-OVA, an L. monocytogenes strain that expresses ovalbumin (OVA), was a gift from Hao Shen (University of Pennsylvania) and Leo Lefrancois (University of Connecticut) (36). An attenuated version of this strain (designated actA-deficient LM-OVA) was created by introducing an in-frame deletion in the actA gene as previously described (48). For all primary bacterial infections Wt B6 mice, IL-18R^–/– mice, IL-18^–/– mice, and adoptive transfer recipients were given 5 x 10⁶ to 7 x 10⁶ CFU of actA-deficient LM-OVA intravenously. In some experiments, for the primary infection mice were inoculated intraperitoneally with a recombinant vaccinia virus expressing the SIINFEKL epitope as a mini-gene (VV-OVA_257-264) (32); the dose was 5 x 10⁶ VV-OVA_257-264 particles. Immunized mice were challenged with 7 x 10⁵ to 9 x 10⁵ virulent LM-OVA CFU, which is 7 to 9 50% lethal doses (LD₅₀) for this strain of bacteria in Wt B6 mice. Bacteria were grown and quantified as previously described (20, 21). The number of LM-OVA CFU present in the livers of challenged mice were determined on day 3 postinfection (p.i.) as previously described (21).

Microarray and real-time PCR. PIQOR cell death microarrays (Miltenyi Biotec, Auburn, CA) were used to compare gene expression in OT-1 transgenic (Tg) T cells purified from mice 8 days after infection with actA-deficient LM-OVA to the expression in naïve OT-1 T cells. For microarray analysis, OT-1 Tg T cells were purified from the day 8 p.i. mice using magnetic separation as previously described (18). Microarray data were confirmed using quantitative real-time PCR as previously described (18).

Antibodies. The following antibodies were used to stain for surface markers in combination with intracellular IFN- to identify Ag-specific T cells: peridinin-chlorophyll-protein complex (PerCP)-anti-mouse CD4 (clone RM4-5), PerCP-anti-mouse CD8 (clone Ly-2), fluorescein isothiocyanate- or allophycocyanin-anti-mouse Thy 1.2 (clone 53-2.1), and allophycocyanin-anti-mouse IFN- (clone XMG1.2; Biolegend, San Diego, CA). In some experiments adoptively transferred OT-1 T cells were identified by costaining with PerCP anti-mouse Thy1.1 (clone OX-7) in addition to the CD8 and Thy1.2 antibodies listed above. All antibodies were obtained from Pharmingen (San Diego, CA) unless indicated otherwise.

Detection and calculation of the number of Ag-specific CD4 and CD8 T cells. Ag-specific CD4 and CD8 T cells were detected by intracellular staining (ICS) for IFN- as previously described (3) after 5.5 h of stimulation with either 5 µM LLO_190-201 (NEKYAQAYPNVS) or 200 nM OVA_257-264 (SIINFEKL) peptide (Global Peptide, Fort Collins, CO) in the presence of brefeldin A (Biolegend). The total number of Ag-specific T cells per spleen was calculated by multiplying the frequency of CD4 or CD8⁺ Thy1.2⁺ IFN-⁺ cells after stimulation with a specific peptide by the total number of splenocytes. The number of cells producing cytokine in the absence of peptide was subtracted.

Adoptive transfer. In experiments performed to substantiate the microarray data, naïve OT-1 T cells (Thy^1.1/1.2) were enriched using negative selection (Stem Cell Technologies, Vancouver, Canada) from the spleens of donors, and 5 x 10⁴ cells were transferred into Wt B6 recipients. For mixed adoptive transfers approximately 100 µl of blood was obtained from prescreened Wt OT-1 (Thy^1.2/1.2) and IL-18R^–/– OT-1 (Thy^1.1/1.2) mice. Small samples were stained with CD8, Thy1.1, and Thy1.2 and analyzed by fluorescence-activated cell sorting to estimate the frequencies of OT-1 T cells in the blood samples. Based on calculations for the staining experiment, the samples were mixed, and 500 Wt OT-1 (Thy^1.2/1.2) T cells plus 500 IL-18R^–/– OT-1 (Thy^1.1/1.2) T cells were delivered to B6 Thy^1.1/1.1 recipients using a single 200-µl intravenous injection. Figure 2 shows examples of the gating strategy used to identify Wt and IL-18R^–/– OT-1 T cells in the same host.

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FIG. 2. The absence of IL-18 receptor signaling on Ag-specific CD8 T cells alone does not change their response kinetics after bacterial or viral infection. (A) IL-18R–/– OT-1 T cells (Thy1.1/1.2) were created and transferred together with Wt OT-1 T cells (Thy1.1/1.1) into the same adoptive transfer recipients (Thy1.2/1.2) to directly ascertain the effect(s) of IL-18 receptor deficiency on the response kinetics of Ag-specific CD8 T cells following actA-deficient LM-OVA infection. The left panel shows staining on day 7 p.i. (d7 p.i.), and the right panel shows staining on day 13 p.i. Wt and IL-18R–/– OT-1 T cells were identified based on Thy1.1 and Thy 1.2 staining, as indicated. The dot plots were first gated on CD8+ cells. The frequencies are the percentages of CD8+ cells that are present in each gate. (B) Total numbers of Wt and IL-18R–/– OT-1 T cells in the spleen on the days p.i. indicated on the x axis. Three mice were analyzed for each time point. (C and D) Adoptive transfer recipients of Wt and IL-18R–/– OT-1 T cells were infected with VV-OVA257-264, and the resulting OT-1 response was monitored by using the same method that was used for actA-deficient LM-OVA-infected recipients. Three mice were analyzed for each time point.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
IL-18-related genes were upregulated in Ag-specific CD8 T cells undergoing contraction. The response of low numbers of OT-1 T-cell receptor Tg T cells peaks in the spleen on day 6 or 7 after infection with actA-deficient LM-OVA, and then the numbers of OT-1 T cells decline precipitously during the contraction phase. To assist in identification of candidate genes involved in the contraction of Ag-specific CD8 T cells after infection, we performed a focused microarray analysis comparing gene expression in OT-1 Tg T cells purified from mice on day 8 after infection with actA-deficient LM-OVA to gene expression in naïve OT-1 cells. We performed two separate microarray analyses with OT-1 T cells purified from two different infected mice. The microarrays focused on 509 genes that were related to known death pathways. Listed in Table 1 are the 15 genes identified as the most upregulated in the Ag-specific CD8 T-cell population undergoing contraction. Among these genes was an interesting collection of related genes encoding IL-18, IL-18R, IL-18Rβ, and caspase-1. The upregulation of IL-18, IL-18R, and IL-18Rβ mRNA in OT-1 Tg T cells on day 8 p.i. was confirmed using probe-based quantitative real-time PCR (data not shown). Given that we had identified a group of related genes that were concurrently upregulated, we chose to examine the potential role of IL-18 in the contraction of Ag-specific T cells.

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TABLE 1. Top 15 upregulated genes during contraction

Kinetics of the Ag-specific CD8 T-cell response after bacterial infection was not altered in the absence of IL-18R or IL-18. In order to determine if IL-18 receptor signaling or IL-18 itself was required for the expansion, contraction, or memory generation of Ag-specific CD8 T cells, mice deficient in these molecules were infected with actA-deficient LM-OVA, and the response to OVA_257-264 was monitored using peptide-stimulated ICS for IFN-. No infected IL-18R^–/– mice died from infection with this attenuated strain of L. monocytogenes, unlike Wt mice treated with anti-IL-18Rβ antibody and infected with a virulent L. monocytogenes strain (31). We also observed no death of IL-18R1^–/– mice in a small experiment in which four mice received a primary infection consisting of 0.1 LD₅₀ (1 x 10⁴ CFU) of virulent LM-OVA (data not shown). Our results support a recent study in which the authors demonstrated equal or less susceptibility to virulent Listeria infection in IL-18^–/– mice or Wt mice treated with anti-IL-18 antibodies (27).

Figure 1A shows representative examples of OVA_257-264-stimulated ICS for IFN- on splenocytes harvested from Wt B6 and IL-18R^–/– mice on day 7 after infection with actA-deficient LM-OVA. OVA_257-264-specific CD8 T cells were present in IL-18R^–/– mice, indicating that signaling through this receptor is not required for generation of Ag-specific CD8 T cells following L. monocytogenes infection. Slightly higher frequencies of OVA_257-264-specific CD8 T cells were detected in Wt B6 mice; however, this did not translate into a statistically higher number of OVA_257-264-specific CD8 T cells in the spleen compared to the number in IL-18R^–/– mice on day 7 p.i. (Fig. 1B). The numbers of OVA_257-264-specific CD8 T cells declined from day 7 to day 14 p.i. at the same rate in Wt B6 and IL-18R^–/– mice; therefore, signaling through the IL-18 receptor was dispensable for contraction of Ag-specific CD8 T cells (Fig. 1B). The number of memory OVA_257-264-specific CD8 T cells was maintained over time until day 72 p.i., indicating that IL-18R is also not required for homeostatic maintenance of memory Ag-specific CD8 T cells (Fig. 1B).

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FIG. 1. Ag-specific CD8 T-cell kinetics were not altered in the absence of IL-18R or IL-18. (A) Wt B6 control and IL-18R–/– mice were infected with an attenuated strain of L. monocytogenes, actA-deficient LM-OVA, and evaluated for their abilities to generate an Ag-specific CD8 T-cell response to OVA257-264 on day 7 p.i. (d7 p.i.) using ICS for IFN-. The dot plots were first gated on CD8+ cells. The frequencies indicate the percentages of CD8+ Thy1.2+ IFN-+ cells in the spleens of the mice analyzed. (B) The total numbers of Ag-specific CD8 T cells were determined on the days p.i. indicated on the x axis to monitor the kinetics of the response. The total numbers of OVA257-264-specific CD8 T cells were calculated as described in Materials and Methods. Three to nine mice were analyzed for each time point. (C) Wt B6 control and IL-18–/– mice were infected with actA-deficient LM-OVA to compare their abilities to generate an OVA257-264-specific CD8 T-cell response to that of IL-18R–/– mice. Representative dot plots of ICS for IFN- performed on day 7 p.i. are shown. The dot plots were first gated on CD8+ cells, and the frequencies indicate the percentages of CD8+ Thy1.2+ IFN-+ cells in the spleens of the mice analyzed. (D) Total numbers of Ag-specific CD8 T cells on the days p.i. indicated on the x axis. Three mice were analyzed for each time point.

Even though IL-18R is the ligand-binding portion of the receptor and the absence of this receptor chain abolishes known biological activities of IL-18 (22), we felt that it was important to confirm our initial findings with IL-18^–/– mice. OVA_257-264-specific CD8 T cells were identified in the spleens of Wt B6 and IL-18^–/– mice on day 7 after infection with actA-deficient LM-OVA using ICS for IFN- (Fig. 1C). No IL-18^–/– mice died from this infection. The frequency of Ag-specific CD8 T cells detected in IL-18^–/– mice was slightly higher, but there was no statistical difference in the peak numbers of OVA_257-264-specific CD8 T cells detected in the spleens of infected mice (Fig. 1D). These data are consistent with the findings of a recent study, where nearly equal numbers of OVA_257-264 tetramer-positive CD8 T cells were detected on day 7 after infection with virulent LM-OVA in the spleens of Wt mice, IL-18^–/– mice, and Wt mice treated with anti-IL-18 antibodies (27). As we observed with IL-18R^–/– mice, OVA_257-264-specific CD8 T cells contracted normally in IL-18^–/– mice. Ag-specific CD8 T cells were also detected at a memory time point (day 28 p.i.) indicating that IL-18 was also not required for the maintenance of memory Ag-specific CD8 T cells.

IL-18R deficiency isolated in the Ag-specific CD8 T-cell population does not affect contraction following bacterial or viral infection. The absence of a molecule in an entire host does not address the importance of the deficiency for any one specific cell type or for any one biological process. In addition, there could be global compensation by a gene-deficient host that replaces the biological function of a missing molecule, which could mask the effect of the deficiency in a single cell type. In order to specifically address the requirement for IL-18R in the Ag-specific CD8 T-cell response, we generated OT-1 Tg mice lacking IL-18R. IL-18R^–/– OT-1 T cells (Thy^1.1/1.2) were enriched from these mice and adoptively transferred together with Wt OT-1 T cells (Thy^1.1/1.1) into Wt B6 mice (Thy^1.2/1.2) to directly compare contraction of these two groups of Ag-specific CD8 T cells. A small number of OT-1 T cells were transferred (500 IL-18R^–/– OT-1 T cells and 500 Wt OT-1 T cells) to ensure that we were analyzing an Ag-specific CD8 T-cell response that would closely mimic the behavior of an endogenous response (2). We also chose to infect some adoptive transfer recipients with actA-deficient LM-OVA and some recipients with VV-OVA_257-264 to determine if there was a differential requirement for IL-18R in contraction of Ag-specific CD8 T cells following a bacterial or viral infection.

Figure 2A shows the gating scheme used to detect the two OT-1 T-cell populations in the same host on days 7 and 13 after infection with actA-deficient LM-OVA. Both IL-18R^–/– and Wt OT-1 T cells expanded appropriately following infection and were distinctly identifiable. As shown in Fig. 2B, IL-18R^–/– OT-1 T cells contracted with the same kinetics as Wt OT-1 T cells, demonstrating that IL-18R signaling in Ag-specific CD8 T cells was not required for their death during the contraction phase after bacterial infection.

As discussed above, we also infected some adoptive transfer recipients with VV-OVA_257-264 to investigate the requirement for IL-18R in the contraction phase following a viral infection. IL-18R^–/– OT-1 T cells expanded to the same degree as Wt OT-1 T cells (Fig. 2C), indicating that IL-18 receptor signaling was not required for Ag-specific CD8 T-cell expansion in this infection model. As was observed following L. monocytogenes infection and in the directly infected gene-deficient mice, IL-18R was also not required for contraction of Ag-specific CD8 T cells following VV-OVA_257-264 infection. Taken together, these data demonstrate that IL-18 signaling does not play an essential role in Ag-specific CD8 T-cell contraction.

IL-18R and IL-18 are not required for generation or contraction of Ag-specific CD4 T cells. IL-18 is recognized as an important regulator of Th1/Th2 polarization and has been shown to be important in the stimulation of previously activated CD4 T cells to make IFN- (29, 30, 39). We infected IL-18R^–/– and Wt B6 mice with actA-deficient LM-OVA and followed the LLO_190-201 CD4 response using peptide-stimulated ICS for IFN-. The absence of IL-18R diminished the expansion of Ag-specific CD4 T cells both in frequency (Fig. 3A) and in total number (Fig. 3B) in the spleens of infected mice. Following the peak of expansion, IL-18R^–/– and Wt B6 LLO_190-201-specific CD4 T cells contracted similarly. Thus, IL-18R may play a small role in augmenting Ag-specific CD4 T-cell expansion and/or survival, but it is not required for Ag-specific CD4 T-cell contraction. Memory Ag-specific CD4 T cells were detected in IL-18R^–/– mice until day 72 p.i. (Fig. 3B), indicating that IL-18 receptor signaling is not required for the maintenance of memory CD4 T cells. Similar results were obtained when the LLO_190-201-specific CD4 T-cell response in IL-18^–/– mice was evaluated (Fig. 3). These findings are consistent with a recent report showing that there was slightly diminished expansion of LLO_190-201-specific CD4 T cells on day 7 p.i. for Wt mice, IL-18^–/– mice, and Wt mice treated with anti-IL-18 antibodies (27). Despite the slight expansion defect observed in the absence of IL-18, the contraction phase and numbers of LLO-specific memory CD4 T cells were very similar to what was detected in Wt B6 mice (Fig. 3C and D). These data suggest that signaling through the IL-18 receptor may augment the expansion of Ag-specific CD4 T cells after bacterial infection; however, neither IL-18 nor IL-18 receptor signaling is involved in CD4 T-cell contraction or the establishment of memory.

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FIG. 3. Ag-specific CD4 T-cell responses are not affected by the absence of IL-18 or IL-18R following attenuated L. monocytogenes infection. (A) Wt B6 control and IL-18R–/– mice were infected with actA-deficient LM-OVA and evaluated for their abilities to generate an Ag-specific CD4 T-cell response to LLO190-201 on day 7 p.i. (d7 p.i.) using ICS for IFN-. The dot plots were first gated on CD4+ cells. The frequencies are the percentages of CD4+ Thy1.2+ IFN-+ cells in the spleens of the mice analyzed. (B) Total numbers of Ag-specific CD4 T cells on the days p.i. indicated on the x axis. Three to nine mice were analyzed for each time point. (C) Wt B6 control and IL-18–/– mice were infected with actA-deficient LM-OVA to compare their abilities to generate a LLO190-201-specific CD4 T-cell response to that of IL-18R–/– mice. Representative dot plots for ICS for IFN- performed on day 7 p.i. are shown. The dot plots were first gated on CD4+ cells, and the frequencies are the percentages of CD4+ Thy1.2+ IFN-+ cells in the spleens of the mice analyzed. (D) Total numbers of Ag-specific CD4 T cells on the days p.i. indicated on the x axis. Three mice were analyzed for each time point.

IL-18R is not required for secondary Ag-specific T-cell expansion or protection against lethal challenge of immunized mice. The results of a previous study performed by another group demonstrated that mice treated with a neutralizing antibody to IL-18Rβ exhibited dramatically enhanced mortality during primary infection with virulent L. monocytogenes compared to isotype antibody-treated and anti-IL-12-treated mice. In addition, treatment of immunized mice with anti-IL-18Rβ during secondary challenge resulted in significantly increased bacterial loads in the spleens of infected mice 3 days p.i., leading to the conclusion that IL-18 was required for protection against both primary infection and secondary infection with L. monocytogenes (31). We observed no mortality of IL-18^–/– or IL-18R^–/– mice after infection with attenuated actA-deficient LM-OVA, and, as shown in Fig. 1 and 3, memory Ag-specific CD8 and CD4 T cells were generated and maintained in the absence of IL-18R or IL-18. We next wanted to determine if memory cells generated in the absence of these molecules were capable of secondary expansion and to formally resolve if IL-18 or IL-18R is required for protection of mice from a lethal secondary bacterial challenge.

First, the numbers of memory OVA_257-264-specific CD8 T cells were determined in IL-18R^–/–, IL-18^–/–, and Wt B6 mice that had been immunized with either actA-deficient LM-OVA (Fig. 4A) or VV-OVA_257-264 (Fig. 4B) 28 days previously. These mice were then infected with a high dose of virulent LM-OVA, and the degree of secondary expansion of T cells was ascertained on day 5 postchallenge. As shown in Fig. 4A and B, memory Ag-specific CD8 T cells in IL-18R^–/– and IL-18^–/– mice expanded in number similarly to Wt Ag-specific CD8 T cells whether they were primed during a bacterial infection (Fig. 4A) or during a viral infection (Fig. 4B), indicating that there was no defect in the ability to respond to a secondary challenge despite the absence of IL-18R or IL-18 during priming and during the challenge. These data are in support of a recent study that showed nearly identical expansion of memory Ag-specific CD4 and CD8 T cells on day 5 following virulent bacterial challenge in Wt, IL-18^–/–, and Wt mice treated with anti-IL-18 antibodies (27). The expansion in each case was related to the starting numbers of memory CD8 T cells (the expansion was greatest in mice with the lowest starting numbers of memory T cells), as has been observed in other model systems (4). None of the immunized IL-18^–/– or IL-18R^–/– mice died as a result of the virulent bacterial challenge.

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FIG. 4. Memory IL-18R–/– and IL-18–/– Ag-specific T cells expand after secondary challenge and protect against lethal bacterial challenge. (A and B) IL-18R–/–, IL-18–/–, and Wt B6 control mice were immunized with either actA-deficient LM-OVA (A) or VV-OVA257-264 (B). The numbers of memory OVA257-264-specific CD8 T cells were determined on day 28 p.i. (d28) using ICS for IFN-. Immunized mice were then challenged with 9 LD50 of virulent LM-OVA. The expansion of OVA257-264-specific CD8 T cells on day 5 postchallenge was determined. Three mice were evaluated for each time point. (C and D) IL-18R–/– and Wt B6 control mice that had been immunized with actA-deficient LM-OVA were challenged on day 54 p.i. with 7 LD50 of virulent LM-OVA. The secondary response kinetics of OVA257-264-specific CD8 (C) and LLO 190-201-specific CD4 (D) T cells were monitored on the days postchallenge indicated on the x axis. Three mice were analyzed for each time point. (E) CFU counts in the livers of immunized Wt B6 control (circles) and IL-18R–/– (squares) mice on day 3 postchallenge. Plus signs indicate death of the naïve Wt B6 mice included in the experiment, which occurred between days 2 and 3 p.i. None of the challenged immunized Wt B6 or IL-18R–/– mice died. Each symbol represents one mouse. The data are expressed as the number of CFU/g of liver. LOD, limit of detection.

In a separate experiment, IL-18R^–/– and Wt B6 mice were immunized with actA-deficient LM-OVA and challenged with a high dose of virulent LM-OVA 54 days later. The kinetics of the secondary Ag-specific CD8 (Fig. 4C) and CD4 (Fig. 4D) T-cell responses were monitored over time, and the ability of the memory cells to protect against this lethal infection was determined (Fig. 4E). Both memory IL-18R^–/– and Wt B6 OVA_257-264-specific CD8 T cells expanded and contracted to the same degree after this infection (Fig. 4C). Secondary memory CD8 T cells were detected in both types of mice as late as 100 days postchallenge, demonstrating that IL-18 receptor signaling was not required for expansion, contraction, or establishment of secondary memory Ag-specific CD8 T cells. We also observed expansion of memory IL-18R^–/– LLO_190-201-specific CD4 T cells after secondary challenge. The degrees of expansion, contraction, and maintenance of secondary memory Ag-specific CD4 T cells were similar in IL-18R^–/– and Wt B6 mice (Fig. 4D); thus, signaling through IL-18R is also not required for memory CD4 T cells to respond following exposure to a secondary antigen.

The most important test of the quality of memory cells is their ability to protect mice from a high-dose challenge. Again, we observed no mortality in the IL-18R^–/– groups of immunized mice after challenge with a high dose (7 LD₅₀) of virulent LM-OVA. To get a more precise determination of the requirement for IL-18R in the protection against lethal L. monocytogenes infection, the numbers of virulent LM-OVA cells in the livers of challenged IL-18R^–/– and Wt B6 mice were determined on day 3 postchallenge (Fig. 4E). The three naïve mice challenged with this dose of bacteria all succumbed to the infection by day 3 p.i. No LM-OVA could be detected in the livers of immunized IL-18R^–/– mice on day 3 p.i., and one immunized Wt B6 mouse had only a few detectable bacteria, demonstrating that IL-18R^–/– mice were at least as well protected as Wt B6 mice (Fig. 4E). Thus, signaling through IL-18R is not required for protection of immunized mice after challenge with a lethal dose of virulent bacteria.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cytokines are critical messengers for T cells, directing most stages of their existence, including development, survival, expansion, phenotypic evolution, and death. Dissecting when and how cytokines influence T cells will have a large impact on the direction of vaccine design and on the development of therapies for the maintenance of beneficial T cells and the elimination of harmful T cells. Recently, the impact of proinflammatory cytokines on the development and progression of Ag-specific T-cell responses following infection has become a field of intense investigation.

We identified genes encoding IL-18, both chains of the IL-18 receptor, and caspase-1 in a microarray analysis performed to identify candidate genes that are involved in the contraction of Ag-specific CD8 T cells. The data obtained generated considerable interest due to the coordinate upregulation of all of these related genes in Ag-specific CD8 T cells at day 8 p.i. and also due to the link between IL-18 and IFN-. As discussed previously, IL-18 can induce the production of IFN- from multiple types of cells (7, 9, 16, 26, 33, 39), and IFN- has already been shown to be a key regulator of the contraction of Ag-specific CD4 and CD8 T cells (5, 6, 19).

In creating a model for how contraction of Ag-specific T cells may be regulated, we considered the hypothesis that IL-18 might be upstream from IFN- and that perhaps the T cells themselves could participate in initiating their own death pathway by being capable of making and responding to IL-18. We also had previously generated somewhat surprising quantitative real-time PCR data demonstrating that Ag-specific CD8 T cells produce a large amount of IFN- mRNA at time points corresponding to the beginning of the contraction phase (data not shown) despite the absence of antigen at this time after infection (37). These data suggested to us that T cells may be stimulated to make IFN- via a T-cell receptor-independent mechanism, and the microarray findings highlighted the potential involvement of IL-18 in regulating contraction, perhaps through inducing autocrine production of IFN-.

However, the in vivo experiments with gene-deficient mice demonstrated that IL-18 and IL-18R are dispensable for the contraction phases of both Ag-specific CD4 and CD8 T cells following infection. When IL-18R^–/– Ag-specific CD8 T cells were transferred into Wt hosts to ascertain the importance of IL-18 receptor signaling directly in this specific cell type, we also observed no defect in contraction. This was the case after both bacterial and viral infections, indicating that that contraction can occur in Ag-specific CD8 T-cell populations responding to two different types of infection in the absence of IL-18 receptor signaling. The conclusion from these experiments is that, despite the coordinated upregulation of IL-18 and its receptor components during the onset of T-cell death, these genes are not required for contraction of Ag-specific T cells. Thus, the precise role of upregulated IL-18 and IL-18R mRNAs in T cells responding to infection remains to be determined.

We did observe a slight decrease in Ag-specific CD4 T-cell expansion in the absence of IL-18 and IL-18R compared to that in Wt mice. This finding was also reported by another group investigating expansion of Ag-specific CD4 T cells in IL-18^–/– mice (27). Perhaps after L. monocytogenes infection IL-18 may serve as a "signal 3" cytokine promoting optimal expansion; however, its contribution is limited since only a slight decrease in the total number of Ag-specific CD4 T cells was observed in the absence of this cytokine signaling pathway.

Despite the normal contraction phenotype in the absence of IL-18 or IL-18R, these experiments provided a detailed description of Ag-specific T-cell response kinetics in the absence of IL-18 or IL-18R, formally showed that these molecules are not required for the expansion or survival of Ag-specific T cells after two different types of infections, and showed that maintenance of memory T cells is not dependent on IL-18 receptor signaling. These studies also formally demonstrated that IL-18 is not required for protection of immunized mice against secondary challenge. This finding is in contrast to the findings of Neighbors et al., who showed that mice immunized with a low dose of L. monocytogenes and treated with anti-IL-18Rβ antibodies 1 day prior to challenge exhibited dramatically elevated numbers of spleen CFU on day 3 postchallenge (31). Our study demonstrated that immunized IL-18R^–/– mice cleared virulent LM-OVA as well as immunized Wt mice on day 3 postchallenge. The strains of L. monocytogenes and mice used in the two studies were different, which may partially explain the disparate results. The previous study used BALB/c mice and a virulent strain of L. monocytogenes with an LD₅₀ of approximately 1 x 10⁴ CFU for their control antibody-treated group of mice (31). In our study C57BL/6 background mice were challenged with L. monocytogenes expressing OVA, which has an LD₅₀ of approximately 1 x 10⁵ CFU for unimmunized mice. Despite the potential differences in the virulence of the bacterial strains used in these two studies, naïve control mice in our challenge experiments succumbed to virulent LM-OVA infection, which indicates that the immunized IL-18R^–/– mice should also have died from this infection if they had a defect in their memory recall response or if IL-18 receptor signaling was required in any way for protection against lethal bacterial challenge. An alternative explanation for the differences in the results could be that IL-18Rβ may contribute to an additional cytokine signaling pathway that has not been identified yet and that by neutralizing this receptor chain more than just IL-18 signaling was abrogated in the previous study of Neighbors et al. (31). Our findings also support the results of a very recent study that reported clearance of virulent LM-OVA on day 5 postchallenge in immunized IL-18^–/– and Wt mice treated with anti-IL-18 antibodies (27).

The search for cytokines that participate in regulating different phases of the T-cell response will continue to be crucial for developing our understanding of T-cell homeostasis. The requirement for particular cytokines is likely to differ from model to model and is expected to be directly related to which cytokines are normally elicited during individual in vivo infections. This is important to remember when designing vaccines to create the most favorable memory response to defend against a real infection. In addition to determining which cytokines are involved, it is also vital to determine when during an immune response T cells are receptive to these cytokines in order to optimize the effectiveness of cytokine exposure.

 
This work was supported by National Institutes of Health grants AI42767, AI46653, and AI50073 to J.T.H. and by American Cancer Society grant PF-05-142-01-LIBto J.S.H.

We thank Rebecca Podyminogin for excellent laboratory assistance.

 
* Corresponding author. Mailing address: Department of Microbiology, 3-512 BSB, University of Iowa, 51 Newton Road, Iowa City, IA 52242. Phone: (319) 335-9720. Fax: (319) 335-9006. E-mail: john-harty{at}uiowa.edu

Published ahead of print on 17 February 2009.

Editor: J. L. Flynn

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, May 2009, p. 1894-1903, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01315-08
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