IL-18 Bridges Innate and Adaptive Immunity Through IFN- Γ and the CD134 Pathway Joseph R

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IL-18 Bridges Innate and Adaptive Immunity through IFN- γ and the CD134 Pathway Joseph R. Maxwell, Rajwardhan Yadav, Robert J. Rossi, Carl E. Ruby, Andrew D. Weinberg, Hector L. Aguila and This information is current as Anthony T. Vella of September 23, 2021. J Immunol 2006; 177:234-245; ; doi: 10.4049/jimmunol.177.1.234 http://www.jimmunol.org/content/177/1/234 Downloaded from

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IL-18 Bridges Innate and Adaptive Immunity through IFN-␥ and the CD134 Pathway1

Joseph R. Maxwell,2* Rajwardhan Yadav,* Robert J. Rossi,* Carl E. Ruby,† Andrew D. Weinberg,† Hector L. Aguila,* and Anthony T. Vella3*

IL-18 induces inflammation resulting in either enhanced protection from pathogens or exacerbation of autoimmunity, and T cells are profoundly activated during these responses. How IL-18 influences T cell activation is unknown, but this study in mice shows that IL-18 boosted Ag-specific T cell clonal expansion of effector T cells and induced a subpopulation of IFN-␥ superproducing T cells. Commitment to IFN-␥ production through IL-18 was independent of NK cells and IL-12 but dependent on host-derived IFN-␥. To determine how expansion of these effectors occurred, IL-18 was shown to induce OX40L on dendritic cells, whereas peptide stimulation induced CD134 (OX40) on specific T cells. CD134 blockade inhibited T cell effector expansion thereby re- ducing the number of IFN-␥ superproducers by 12-fold. Thus, independent of IL-12, IL-18 impacts T cell immunity throughout Downloaded from lymphoid and nonlymphoid tissue by bridging the innate and adaptive arms of the immune system through IFN-␥ and the CD134 costimulatory pathway. The Journal of Immunology, 2006, 177: 234–245.

L-18 is a multifunctional cytokine produced by macrophages direct responsiveness to IL-18 (26, 27). Interestingly, IL-18 can act and dendritic cells (DCs)4 (1, 2) with profound effects on the directly on effector and memory T cells by inducing migration I innate and adaptive immune systems (3–5). Activation by (28), proliferation, and IFN-␥ secretion even in the absence of http://www.jimmunol.org/ IL-18 through the IL-18R is supported by MyD88 (6) and IRAK-4 recall Ag (13–15, 29). Likewise, studies demonstrate enhanced (7) adapter molecules, which ultimately stimulate NF-␬B and memory T cell responses after signaling through the IL-18R (30). NFAT transcriptional activity (8). A predominant feature of stim- Ultimately, IL-18 mediates protection against Salmonella and Shi- ulation by IL-18 is IFN-␥ synthesis (9). Many cells, such as NK gella (31, 32), viral infections such as HIV and HSV (33–35), and cells (10), DCs (11), macrophages (12), and both CD4 (13) and in various tumor models (36–39). These responses are largely T CD8 (14, 15) T cells respond in this manner, although the presence cell dependent, but a unifying mechanism linking IL-18, the in- of IL-12 is usually required for optimal IFN-␥ secretion. Mice nate, and adaptive immune systems is undefined. deficient in either IL-18 (16) or the IL-18R (17) show defective In this in vivo study, we show how IL-18 bestows its effects on NK cell and Th1 responses in vivo, confirming the importance of adoptively transferred CD4 T cells. After immunization, IL-18 by guest on September 23, 2021 IL-18 during inflammatory responses. Specifically, IL-18 has been profoundly improved Ag-specific T cell clonal expansion of effec- shown to directly or indirectly induce TNF, IL-1, IL-8, IL-4, and tor cells and recall potential of IFN-␥ and IL-4 production. Spe- IL-13 (18–20). Importantly, experimental models of sepsis (21), cifically, we detected an IL-18-conditioned subpopulation of arthritis (22), and colitis (23) in mice are treatable by IL-18 block- IFN-␥ superproducing CD4 effector T cells. In targeting the mech- ade. Thus, IL-18 is a powerful inflammatory stimulant that can anism of IL-18, we investigated commitment to IFN-␥ production induce severe pathological damage, and it has been suggested that and expansion of these effector cells. IL-18 acted independently of a better understanding of this process will fuel much-needed im- NK cells and IL-12 to induce IFN-␥ commitment; however, IFN-␥ munotherapeutic intervention (24). production by the host was required for commitment to IFN-␥, but Conversely, IL-18 has potential as an efficacious adjuvant (25). not for effector cell expansion. To pursue the effector expansion Although IL-18 potently stimulates cells of the innate immune mechanism, IL-18 was shown to induce OX40L on DCs, and pep- system, it also affects T cells. T cells from unstimulated mice do tide-stimulated T cells up-regulated CD134 (OX40). We found not respond to IL-18 because they lack the IL-18R; however, upon that CD134 blockade inhibited expansion of the effector cells. stimulation with IL-12, surface IL-18R appears, thereby conferring Thus, IL-18 bridges the innate and adaptive immune systems through induction of IFN-␥ and the CD134 pathway.

*Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030; and †Providence Medical Center, Portland, OR 97213 Materials and Methods Received for publication January 17, 2006. Accepted for publication April 10, 2006. Mice The costs of publication of this article were defrayed in part by the payment of page C57BL/6, IL-18R-deficient (IL-18RϪ/Ϫ), p35Ϫ/Ϫ, p40Ϫ/Ϫ, and IFN-␥Ϫ/Ϫ charges. This article must therefore be hereby marked advertisement in accordance mice were purchased from either the National Cancer Institute (Frederick, with 18 U.S.C. Section 1734 solely to indicate this fact. ϩ 1 MD) or The Jackson Laboratory. SM1 TCR transgenic mice (CD4 , This work was supported by National Institutes of Health Grants RO1-AI42858, Thy1.1ϩ, RAGϪ/Ϫ) were bred by our laboratory, and all mice were housed RO1-AI52108, PO1-AI56172 Project 3 (to A.T.V.) and RO1-CA102577 (to A.D.W.). in the animal facility at the University of Connecticut Health Center (Farm- 2 Current address: Department of Inflammation, Amgen, 1201 Amgen Court West, ington, CT) under specific pathogen-free conditions according to National Seattle WA 98119. Institutes of Health guidelines. 3 Address correspondence and reprint requests to Dr. Anthony T. Vella, Department of Immunology, University of Connecticut Health Center, 263 Farmington Avenue, Injection schedules Farmington, CT 06030. E-mail address: [email protected] 5 4 Abbreviations used in this paper: DC, dendritic cell; PLN, peripheral lymph node; For the adoptive transfer studies, ϳ5 ϫ 10 bulk cells from the lymph MLN, mesenteric LN; CTM, complete tumor medium; WT, wild type. nodes and spleen of SM1 TCR transgenic mice were injected i.v. on day

Ϫ1. We found it unnecessary to purify CD4 T cells from the bulk popu- from RM134L hybridoma supernatant (42) and purified over a protein G lation before transfer because this did not affect the outcome of the T cell column. Rat IgG (Sigma-Aldrich) was injected as a control for both of the response (data not shown). On day 0, 100 ␮g of flagellin peptide (40) above mentioned mAbs. All mAbs were diluted into PBS for injection. (residues 427–441; Invitrogen Life Technologies), was injected i.p., and 30 min later, 5 ␮g of IL-18 (MBL International) was injected i.p. IL-18 injections (5 ␮g each) were given on the subsequent 2 days (days 1 and 2). Cell isolation and processing For peptide alone-treated mice, balanced salt solution medium was injected in the place of IL-18. The 5-␮g dose of IL-18 was based on titration Spleens, peripheral lymph nodes (PLNs) (inguinal, axillary, or brachial) studies, which showed that 5 ␮g yielded a maximal effect at the lowest and mesenteric LNs (MLNs) were crushed through nylon mesh cell strain- dose (data not shown). ers (Falcon/BD Biosciences), and spleens were treated with ammonium Protein G (Invitrogen Life Technologies) purified agonistic mAb spe- chloride to lyse RBC. In Fig. 1e, the tissues were digested with collagenase cific to CD134 (41) was injected at 50 ␮g immediately after flagellin pep- D (Roche) for 30 min at 37°C before passing through the cell strainers. tide. The anti-OX40L blocking mAb was injected on days 0, 1 and 2 before Liver and lung lymphocytes were obtained as described (43). For isolation each IL-18 injection at a dose of 100 to 400 ␮g. This mAb was purified of liver lymphocytes, perfused livers were crushed through cell strainers, Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. IL-18 enhances clonal expansion and effector function of peptide-stimulated CD4 T cells. a, Bulk SM1 spleen and LN cells were transferred into C57BL/6 mice (day Ϫ1). On day 0, flagellin peptide was injected. IL-18 was injected on days 0, 1, or 2; on all three days (0, 1, 2); or not at all (None). On day 5, spleens were removed, and the average percentages (left graph) and numbers (right graph) Ϯ SEM were determined. Data are pooled results from two separate experiments, with a total of six mice per group, but are representative of more than three similar experiments. b–d, SM1 cells were transferred into C57BL/6 mice on day Ϫ1 and stimulated with flagellin peptide on day 0. Mice were further treated without IL-18 (u) or with IL-18 on days 0, 1, and 2 (f). b, On day 5, bulk splenocytes from each group were restimulated in vitro with flagellin peptide, and 24 or 48 h later, IFN-␥ levels were determined by ELISA. Bars show average IFN-␥ (ng/ml) Ϯ SEM from nine wells pooled from triplicate cultures from three separate mice. c, Day 5 splenocytes were restimulated for5hinvitro and then stained for intracellular IFN-␥. Representative autoscaled histograms of Thy1.1-gated cells are shown from each treatment group. A control IgG analysis region for each mouse was set to ϳ0.5% to determine the percentage of IFN-␥ producers. The superproducer analysis region was determined by setting the percent of Thy1.1 cells to ϳ2% in the peptide alone group and applying this region to all samples. Data in the table show the percentage Ϯ SEM of total IFN-␥ producer or superproducer Thy1.1ϩ cells from three mice per group from one experiment but are representative of more than three similar experiments. d, Splenocytes taken from mice treated 5 days earlier with peptide Ϯ IL-18 were restimulated with peptide in vitro, and the resulting [3H]thymidine incorporation was measured. Each point is the average cpm Ϯ SEM from nine wells pooled from triplicate wells from each of three mice per group. Data are representative of more than three similar experiments. e, SM1 cells were transferred into C57BL/6 mice (day Ϫ1) and stimulated with flagellin peptide on day 0. Mice were further treated with (f) or without (u) one shot of IL-18 on day 0. Forty-eight hours after peptide injection, PLN and MLN tissues were collagenase treated, and the recovered cells were labeled with CFSE. Bulk cells (1 ϫ 106) were cultured in vitro without restimulation. After 36 h in vitro, CD4 Thy1.1ϩ cells were gated and analyzed for survival by CFSE dilution and cell counts. Histograms overlayed on the same scale show representative CFSE profiles from each treatment group. Graphs (inset) show the percentage of Thy1.1 SM1 CD4 T cells at the beginning and after 36 h of culture. Data are representative of three experiments. 236 IL-18 LINKS INNATE AND T CELL IMMUNITY

and the cells partitioned on a 35% Percoll (Sigma-Aldrich) gradient. Per- (Fig. 1a, right panel). The majority of this effect was attributable fused lungs were cut into pieces and incubated at 37°C in 1.3 mM EDTA to the day 0 injection which yielded a large but smaller increase in for 30 min and then collagenase (Invitrogen Life Technologies) for 1 h. clonal expansion, compared with the triple injection. After peptide Low-density lymphocytes were obtained from the interface lying between 44 and 67% Percoll (Amersham Biosciences). injection, administration of IL-18 on days 1 or 2 induced much less DCs and macrophages were enriched as described (44). Briefly, LN or expansion based on percentages and numbers (Fig. 1a). To expand spleen tissues were digested with collagenase D (Roche) for 30 min at this observation, we tested the effects of IL-18 in a superantigen 37°C. After passing through a cell strainer and washing, enriched DC pop- model and demonstrated that IL-18 boosted not only superantigen- ulations were obtained from the low-density fraction on a BSA gradient. specific CD4 T cells, but also CD8 T cells (data not shown). Cell culturing We next examined whether IL-18 could improve CD4 T cell In vitro restimulation of isolated cells was performed in three ways. Pro- effector cytokine production. Bulk SM1 spleen and LN cells were liferation assays were set up by culturing 3-fold dilutions of whole spleen transferred into C57BL/6 mice (day Ϫ1), stimulated with peptide cells in 200 ␮l of complete tumor medium (CTM). CTM consists of MEM on day 0 and given IL-18 (days 0, 1, and 2) or not. On day 5, with FBS, amino acids, salts, and antibiotics. The cells were cultured with splenocytes were isolated from each group and restimulated in ␮ ϳ ␮ 3 or without flagellin peptide (5 g/ml). After 72 h, 1 Ci of [ H]thymi- vitro with flagellin peptide for 24 h. IFN-␥ in supernatants was dine (PerkinElmer) was added for the last8hofculture. Incorporation of 3H was analyzed with a Harvester 96 (Tomtec) and a 1450 Microbeta measured by ELISA and the data showed a 33-fold increase of Trilux scintillation counter (PerkinElmer). IFN-␥ by cells taken from mice treated with peptide/IL-18, com- For ELISA cultures, a total of 2 ϫ 106 splenocytes was cultured in 200 pared with cells taken from peptide-alone mice (Fig. 1b). Even ␮ ␮ l of CTM with or without flagellin peptide (5 g/ml). After 24 or 48 h, supernatants from 48 h showed a 7-fold increase in IFN-␥ levels, the supernatants were analyzed with IFN-␥ or IL-4 ELISA kits from BD Biosciences. and the same trend also was observed in a superantigen model Downloaded from Intracellular cytokine cultures were set up by culturing 1 ϫ 106 bulk (data not shown). spleen cells in 200 ␮l of CTM without any prior cell purification or frac- Because ELISA data does not measure specific cell cytokine tionation. Flagellin peptide was added to some of the wells, while brefeldin production, we used intracellular cytokine staining to test whether ␮ A (Calbiochem) was added to all wells, both at a concentration of 5 g/ml. Ag-specific CD4 T cells from peptide/IL-18-treated mice synthe- Cultures were left for5hat37°C before staining. sized more IFN-␥ than those from peptide alone-treated mice. In

Cell staining and flow cytometry vitro peptide restimulation of day 5 splenocytes revealed that http://www.jimmunol.org/ ϩ The following mAbs were purchased from eBioscience: FITC-conjugated ϳ50% of Thy1.1 cells from peptide/IL-18-treated mice made anti-CD44, anti-F4/80, anti-IFN-␥ and control rat IgG1, allophycocyanin- IFN-␥, whereas only 22% were IFN-␥ϩ from peptide alone-treated conjugated anti-Thy1.1 and streptavidin, and biotinylated anti-OX40L (rat mice (Fig. 1c). Based on mean fluorescence intensity analysis, we IgG2b control). The following mAbs were purchased from BD Bio- also observed that IL-18-treated cells produced greater quantities sciences: FITC-conjugated anti-Thy1.1, PE-conjugated CD45R, anti-CD80 ␥ (hamster IgG2 control), anti-CD86 (rat IgG2a control) and streptavidin, of IFN- . To examine this subpopulation of super producers, an ϩ PerCP-conjugated anti-CD4, and biotinylated anti-CD134 (rat IgG1 analysis region was set on peptide alone-treated Thy1.1 cells at control). ϳ2% of the top IFN-␥ producing cells. This exact region was then Surface and intracellular staining was performed as described previously applied to all treatment groups. As shown in the table, there was a (45). Briefly, nonspecific binding was blocked by a solution containing Ͼ ␥ by guest on September 23, 2021 mouse serum, human IgG, and the anti-Fc mAb 2.4G2 (46). Staining buffer 5.5-fold increase of IFN- superproducers in the peptide/IL-18 consisted of balanced salt solution, 3% FBS, and 0.1% sodium azide. For group, compared with the peptide alone group. Thus, in vivo in- intracellular staining, surface staining was performed first, followed by jection of IL-18 substantially boosts IFN-␥ production by CD4 fixation with 2% formaldehyde, permeabilization with 0.25% saponin, and effector T cells responding to recall Ag. then incubation at room temperature with the anti-cytokine mAb. All flow Although cytokine production was enhanced, [3H]thymidine in- cytometry was conducted on a BD FACSCalibur flow cytometer and the data analyzed using CellQuest (BD Biosciences) or FlowJo software (Tree corporation was comparable between IL-18-treated or untreated Star). groups. Based on total cells in culture, peptide/IL-18-treated cells proliferated better than peptide alone cells upon restimulation (Fig. NK depletion 1d); however, if normalized for actual numbers of CD4 Thy1.1 SM1 TCR transgenic T cells were transferred into C57BL/6 mice. Six cells in culture, both groups proliferated comparably (data not hours later, anti-NK1.1 mAb clone PK136 (47) ascites fluid or mouse IgG shown). was injected. Two days after injection, the depletion was verified by staining blood samples with anti-CD94-FITC and anti-CD49b (DX5)-PE (eBio- Lastly, we tested if IL-18 adjuvanticity intrinsically conditioned science). Flagellin peptide and IL-18 were injected as described above, and Ag-specific T cells to survive. Bulk SM1 spleen and LN cells were T cell responses were measured on day 5. transferred into C57BL/6 mice, and the next day (day 0) stimulated with peptide with or without IL-18. On day 2, the PLN and MLN Results were collagenase digested to improve recovery of Ag-specific T IL-18 as an adjuvant boosts Ag-specific CD4 T cell responses in cells (45), and the cells labeled with CFSE and cultured in vitro vivo without any restimulation. Before culture, the percentage of To understand the basis of IL-18 adjuvanticity, our initial studies CD4 Thy1.1 cells was comparable between both groups (Fig. sought to examine what effect administration of exogenous IL-18 1e, see inset graphs), but by 36 h, very few viable specific cells would have on developing T cell responses in vivo. SM1 TCR from the peptide-alone group were detected in culture. This is transgenic mice produce Thy1.1 CD4 T cells specific to a Salmo- in contrast to Thy1.1 SM1 T cells taken from the peptide/IL- nella flagellin peptide (48). A total of 5 ϫ 105 bulk SM1 spleen 18-treated mice, which survived extremely well and actually and LN cells were transferred into Thy1.2 C57BL/6 mice (day Ϫ1) increased in number due in part to proliferation coupled with and the following day (day 0) immunized with 100 ␮g of flagellin survival (Fig. 1e, inset). After 36 h, CFSE dilution by the peptide. As shown in Fig. 1a, 5 days after peptide injection without Thy1.1ϩ cells taken from the peptide/IL-18-treated mice was IL-18 (None), an increase in specific T cells in the spleen was initiating in PLN cultures, whereas in MLN cultures, nearly observed, compared with no peptide (ϳ0.05% CD4 Thy1.1; data every cell had gone through at least one round of division (Fig. not shown). Injection of IL-18 on days 0, 1, and 2 led to a 7.5-fold 1e). Thy1.1ϩ cells from the peptide-alone mice did not survive increase in the percent of splenic CD4 Thy1.1 cells, compared with culture as indicated by the decrease of CFSE-labeled cells. peptide alone, as well as an 8.5-fold increase based on numbers Taken together, these data show that exposing Ag-stimulated The Journal of Immunology 237

CD4 T cells to IL-18 enhances clonal expansion, effector func- tantly, Thy1.1ϩ SM1 CD4 T cells expanded in response to IL-18 tion, and at least short-term survival. in the absence of NK cells (Fig. 2b). Compared with peptide treatment alone, IL-18 boosted CD4 Thy1.1 percentages 13-fold in the IL-18 initiates T cell responses through an indirect mechanism spleen, 10-fold in the MLN, and 9-fold in the PLN, whereas num- Numerous reports show that NK cells respond to IL-18 by prolif- bers increased by 14.5-, 8.5-, and 23-fold, respectively. SM1 T erating and secreting cytokines (10, 16, 49). To study the role of cells efﬁciently produced IFN-␥, because 2.5-fold more made NK cells in this system, we transferred bulk SM1 spleen and LN IFN-␥, compared with cells from peptide alone-treated mice (Fig. cells into C57BL/6 mice, and 6 h later, injected anti-NK1.1 mAb 2c). Taken together, these data show that IL-18 boosts T cell im- ascites. Depletion was conﬁrmed 2 days later by staining blood munity in a NK cell-independent manner. cells with anti-DX5 and CD94 mAbs (Fig. 2a, day 0). Very few Another possibility was that T cells were directly responding to double-positive cells were present, although they were readily de- IL-18 regardless of whether the APCs could respond to IL-18. tected in control IgG-treated mice. All mice were injected with Bulk SM1 spleen and LN cells were transferred into C57BL/6 or Ϫ Ϫ peptide, with or without IL-18, and on day 5, NK cells in the IL-18R / mice and stimulated with peptide alone or peptide/ spleen and other tissues were still absent (Fig. 2a, day 5). Impor- IL-18 (Fig. 3a). SM1 T cells exposed to peptide alone reached peak expansion on day 4 and began to decline thereafter (Fig. 3b). In C57BL/6 recipients, IL-18 enhanced clonal expansion in all tissues examined. By day 4, the percentage (upper panels)ofCD4 Thy1.1 cells increased by 4-fold in the spleen and MLN, and by

3-fold in the PLN and blood, compared with mice treated with Downloaded from peptide alone. Based on numbers (lower panels), CD4 Thy1.1 cells in the spleen and PLN increased by ϳ6.5-fold, whereas those in the MLN increased by ϳ2.5-fold. By day 6, contraction and/or migration out of the PLN and blood was becoming apparent. IL-18 had no effect in IL-18RϪ/Ϫ mice even though the speciﬁc

Thy1.1 SM1 CD4 T cells were IL-18R sufficient. This trend per- http://www.jimmunol.org/ sisted to day 10 where very few SM1 cells were detected in IL- 18RϪ/Ϫ recipients but were still present in C57BL/6 recipients (Fig. 3b: bar graph at lower right, compare upper with lower bars). In accordance with the lack of expansion, the SM1 cells in IL18RϪ/Ϫ recipients made small amounts of IFN-␥ after IL-18 treatment as determined by ELISA and intracellular cytokine staining (data not shown). Nevertheless, SM1 cells in IL-18RϪ/Ϫ recipients responded to peptide alone similar to SM1 cells in C57BL/6 mice, suggesting that there is no defect in basal activa- by guest on September 23, 2021 tion or Ag presentation. These data show that IL-18R must be on the host to initiate the T cell response, but it does not preclude IL-18 binding to the T cell later in the response. IL-18 requires IFN-␥, but not IL-12, for T helper IFN-␥ production Many reports have demonstrated induction of IL-18R on T cells by IL-12, and thus IL-18 and IL-12 are often thought to work in synergy (14, 15, 26, 27). We tested whether the IL-18 effects were dependent on IL-12 by transferring SM1 cells into p35Ϫ/Ϫ and p40Ϫ/Ϫ mice (Fig. 4). Compared with peptide alone, injection of peptide/IL-18 boosted clonal expansion of SM1 T cells comparably in the PLN and spleen of wild-type (WT), p35Ϫ/Ϫ,orp40Ϫ/Ϫ mice. For example, peptide/IL-18 enhanced the percentage of SM1 T cells in the spleen 8- to 12-fold and the number 10- to 15-fold (Fig. 4a). In vitro restimulation of spleen cells taken from primed ␥ FIGURE 2. NK cells are not required for the adjuvant effect of IL-18. WT or knockout mice showed no significant differences in IFN- Six hours after adoptive transfer of SM1 cells, NK cells were depleted from production, as measured by total IFN-␥ producers or superproduc- C57BL/6 host mice by injection of PK136 ascites (ϳ200 ␮g of mAb). a, ers (Fig. 4b). Furthermore, ELISAs performed on supernatants Two days later, CD94ϩ and DX5ϩ blood cells were analyzed (day 0). After taken from peptide-restimulated cultures found that IL-18 also en- depletion, on day 0, mice were injected with flagellin peptide Ϯ IL-18, and hanced IL-4 production from cells taken from WT mice and that residual NK populations in the spleen on day 5 are shown. b, Bar graphs this was not altered in either of the knockout mice (Fig. 4c). A show the day 5 average percentage and number Ϯ SEM from five mice per second direct test of IL-18 bypassing an IL-12 requirement was f u group of SM1 cells from NK-depleted mice treated with ( ) or without ( ) examined by stimulating endogenous T cells in p35Ϫ/Ϫ mice with IL-18. c, Day 5 splenocytes taken from mice treated with peptide alone superantigen plus IL-18 or no IL-18, and the data showed en- (gray histogram) or peptide/IL-18 (black histogram) were restimulated in ␥ vitro to measure intracellular IFN-␥ synthesis. Representative autoscaled hanced expansion and a substantial increase in IFN- synthesis in histograms of gated Thy1.1 cells are shown. The positioning of the analysis the IL-18 group but not in the superantigen alone group (data not line is set against a negative control IgG stain. Percentages represent the shown). average IFN-␥ production Ϯ SEM from five mice per group. All data are Knowing that IL-12 was not required for the adjuvant effect, we from one of two similar experiments. next studied IFN-␥, as this is a key mediator of IL-18 effects. To 238 IL-18 LINKS INNATE AND T CELL IMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. IL-18 initiates Ag-specific T cell clonal expansion indirectly. a, Bulk SM1 cells were adoptively transferred into C57BL/6 or IL-18RϪ/Ϫ mice. Recipients were given flagellin peptide with (f) or without (u) IL-18. b, After peptide injection, T cells from the spleen, MLN, PLN, and blood were analyzed. Line graphs show the percentages and numbers Ϯ SEM of SM1 cells in each tissue, with at least three mice per group. Bar graphs (lower right) show the numbers Ϯ SEM of SM1 cells in the MLN and PLN of IL-18-treated mice 10 days after peptide injection. examine a role for IFN-␥, bulk SM1 cells were transferred into IL-18 uses the CD134 pathway to boost T cell immunity ␥Ϫ/Ϫ either WT or IFN- mice and stimulated with peptide and Because NK cells and IL-12 were not required for adjuvanticity, IL-18 as before. Clonal expansion of the SM1 T cells was unaf- but IL-18R was, we reasoned that DCs were central. DCs likely fected by the absence of IFN-␥, with 7- to 8-fold boosts in T cell present peptide to the Ag-specific SM1 T cells and thus may be accumulation in the spleen and MLN by day 5 (Fig. 5a). In con- properly positioned to influence T cell activation in response to trast, there was a profound reduction of IFN-␥ producers after Ϫ/Ϫ IL-18. To examine this possibility in vivo, bulk SM1 spleen and recall in the SM1 population taken from IFN-␥ mice, compared with those removed from WT mice (Fig. 5, b and c). This LN cells were transferred into C57BL/6 mice and treated with was the case as measured by intracellular cytokine staining (2-fold nothing, IL-18, peptide, or peptide/IL-18. After 18 h, DC-enriched less IFN-␥ after5hofrestimulation), or by ELISA (8-fold less LN cells were stained with a panel of mAbs recognizing activa- ϩ IFN-␥ after 24 h in culture). Importantly, recall IL-4 production tion/costimulatory molecules. In response to IL-18 alone, CD11c was unaffected, which speaks to the idea that there continued to DCs increased CD80, CD86, CD40, CD54, and MHC class II be production of effectors, just very few committed to IFN-␥ expression (data not shown). However, we also observed (Fig. 5c). OX40L induction in response to IL-18 alone (Fig. 6a), a finding The Journal of Immunology 239

FIGURE 4. IL-18 does not require IL-12 for IFN-␥ commitment. Bulk SM1 cells pooled from LNs and spleen were transferred into C57BL/6 (WT), p35Ϫ/Ϫ,or p40Ϫ/Ϫ mice. The next day, mice were treated with peptide alone (u) or peptide/IL-18 (f). a, Five days after peptide injection, T cells from the LNs and spleen were enumerated. Data show the percentages and numbers Ϯ SEM pooled from three or four separate experiments, and each bar contains either 5–10 mice (peptide alone) or 7–12 mice (peptide/IL-18). b, From each treated mouse, bulk splenocytes without any fractionation were Downloaded from restimulated in vitro for 5 h with ﬂagellin peptide, and intracellular IFN-␥ synthesis was analyzed. The total percentage of IFN-␥ producers (f) was determined as described in the legend for Fig. 1. The percentage of superproducers (s) was determined by setting the WT percentage to ϳ7% and using the same region to ana- lyze all mice. Bars represent peptide/IL-18-treated mice http://www.jimmunol.org/ only and are average values Ϯ SEM from three or four experiments with 7–12 mice per group. c, Splenocytes were restimulated in vitro with ﬂagellin peptide for 48 h, and the resulting supernatants were analyzed for the presence of IL-4 by ELISA. Each data point represents the concentration of IL-4 in pg/ml from individual wells of triplicate cultures set up from 5 to 12 mice over three or four separate experiments. by guest on September 23, 2021

that is consistent with our initial data showing that stimulation would restore SM1 T cell expansion and effector function in of purified bone marrow-derived DCs with IL-18 directly in- IL-18RϪ/Ϫ host mice. SM1 cells were transferred into IL- duced OX40L (data not shown). Further, induction of OX40L 18RϪ/Ϫ mice, and the next day stimulated with peptide and was dependent on the DCs expressing IL-18R, because no in- anti-CD134 agonist mAb. Five days later, the numbers of crease of OX40L expression was observed on IL-18RϪ/Ϫ DCs. Thy1.1 SM1 CD4 T cells increased by 2.4-fold in the spleen, Nevertheless, macrophages, but not T or B cells, also increased 3.9-fold in the MLN, and 2.8-fold in the PLN, compared with OX40L expression in response to IL-18 (data not shown). mice treated with control IgG, and similar data were obtained Next, it was determined whether the receptor for OX40L, by analyzing percentages (Fig. 6c). This was the case, even CD134, was expressed by the Thy1.1 SM1 CD4 T cells in our though both treatments induced comparable levels of CD44high system. After 18 h of in vivo peptide stimulation, we detected high SM1 T cells (Fig. 6d), which were at least 3-fold higher than in levels of CD134 on gated peptide-specific CD4 Thy1.1 LN cells untreated mice (data not shown). Additionally, IFN-␥ was se- (Fig. 6b). Induction was completely dependent upon peptide in- creted in large quantities after in vitro restimulation of popu- jection, because CD134 was not observed on SM1 T cells with lations that were costimulated through CD134 (Fig. 6e). These IL-18 alone (Fig. 6b), nor did IL-18 enhance CD134 expression on results are comparable with those observed in C57BL/6 mice peptide-stimulated T cells (data not shown). Thus, IL-18 increased (data not shown). Other costimulators, such as anti-CD137 (4- the expression of OX40L on DCs and peptide stimulation-induced 1BB) and anti-CD40 agonistic mAbs, had little effect in boost- CD134 on the specific T cells. Based on this data, we hypothesized ing SM1 T cell clonal expansion and IFN-␥ production, com- that the CD134 costimulatory pathway may be important for the pared with the anti-CD134 mAb (data not shown). Furthermore, adjuvant effect induced by IL-18. dual injection of IL-18 and anti-CD134 mAb did not synergize Because SM1 T cells expressed CD134 after peptide treatment, to boost T cell responses (data not shown) like we have previ- we tested whether stimulation of CD134 with an agonistic mAb ously observed with LPS and anti-CD134 (50). 240 IL-18 LINKS INNATE AND T CELL IMMUNITY

FIGURE 5. IFN-␥ is important for effector IFN-␥ production but not expansion. Bulk SM1 cells were injected into C57BL/6 (WT) or IFN-␥Ϫ/Ϫ mice. The next day, mice were treated with peptide alone (u) or peptide/IL-18 (f). a, Clonal expansion on day 5 in the spleen and MLN is shown as the percentage or number Ϯ SEM. Data are pooled from four separate experiments and contain 6–13 mice per group. b, Bulk splenocytes from day 5 peptide/IL-18-treated mice were restimulated with peptide in vitro for 5 h, and intracel- Downloaded from lular IFN-␥ was analyzed. Histograms show representative plots of IFN-␥ staining from Thy1.1-gated SM1 cells. The bar graph (right) shows the mean percentage Ϯ SEM of IFN-␥ producing SM1 cells pooled from three separate experiments, with a total of 11 mice per group. c, Day 5 bulk splenocytes taken from peptide/ IL-18-treated mice were restimulated with peptide in http://www.jimmunol.org/ vitro for 24 h, and the accumulation of IFN-␥ or IL-4 in the culture supernatants was measured by ELISA. Bars show the mean Ϯ SEM concentration of each cytokine in culture from one of two separate experiments and contain 10 measurements each. by guest on September 23, 2021

To fully test whether CD134 played a dominant in vivo role total number of IFN-␥ superproducers was dramatically re- in this system, we blocked OX40L. SM1 cells were transferred duced from 12.2 Ϯ 2.1 ϫ 10Ϫ4 to 1.0 Ϯ 0.2 ϫ 10Ϫ4,aϾ12-fold into C57BL/6 mice and stimulated with peptide/IL-18 in the decrease. These data were also confirmed by ELISA and are presence of a nondepleting antagonistic anti-OX40L mAb consistent with data obtained from liver (data not shown). IL-4 (CD134 Blockade) (42, 51, 52) or with a control IgG. On day 5, production also was substantially reduced in culture superna- analysis of peripheral and lymphoid tissues showed that the tants from the CD134 blockade group, suggesting that the re- CD4 Thy1.1 populations expanded in the liver, lung, and spleen duction in IFN-␥ did not skew toward a Th2 response (data not in response to peptide/IL-18, reaching ϳ2% or higher in each shown). tissue (Fig. 7a). However, CD134 blockade inhibited accumu- Nevertheless, CD134 blockade did not prevent increases in lation of CD4 Thy1.1 T cells in these tissues. The greatest in- CD44 levels confirming that the specific T cells were stimulated hibition was in liver and lung where 2.5- to 3-fold decreases (Fig. 7c). Also, in vitro recall with flagellin peptide induced were observed, while T cell expansion also was inhibited by similar levels of [3H]thymidine incorporation, further confirm- ϳ2-fold in the spleen. Interestingly, expansion was not inhib- ing specific stimulation (Fig. 7d). Lastly, analysis of DC surface ited in the PLN (data not shown), suggesting that the CD134 phenotype after peptide/IL-18 and CD134 blockade showed pathway may exert itself most effectively in peripheral tissues similar induction of CD80 and CD86, compared with control where the largest effector populations are detected (43, 53). mice (Fig. 8). These data suggest that, when CD134 is blocked, CD134 blockade reduced IFN-␥ production (Fig. 7b) because these costimulators cannot make up the difference. Nevertheless the percentage of Thy1.1 cells making IFN-␥ was 47.0 Ϯ 2.0% it is possible that other costimulators may contribute. in control mice but dropped to 31.7 Ϯ 2.5% in CD134 blockade mice (Fig. 7b, top panels). The total number of IFN-␥ producers Discussion in the spleen decreased from 58.5 Ϯ 6.6 ϫ 10Ϫ4 in the control Previous reports show that IL-18 is a powerful adjuvant (9, 24, 25, to 19.4 Ϯ 3.9 ϫ 10Ϫ4 in the CD134 blockade group, while the 30). Although its adjuvant effects influence T cell responses, little is The Journal of Immunology 241 Downloaded from http://www.jimmunol.org/

FIGURE 6. IL-18 induces OX40L expression on DCs that can boost T cell responses. a, C57BL/6 (left plot) or IL-18RϪ/Ϫ (right plot) mice adoptively transferred with SM1 cells were injected with medium alone (blue line) or IL-18 (red histogram). After 18 h, pooled PLN and MLN cells were enriched for DCs, and CD11cϩ cells were gated to examine OX40L expression. Data from C57BL/6 mice are representative of 12 separate experiments, whereas the data from IL-18RϪ/Ϫ mice are from one of four similar experiments. In both plots, isotype control mAbs stained at background levels. b, SM1 cells by guest on September 23, 2021 transferred into C57BL/6 mice were stimulated with IL-18 or peptide alone. After 18 h, pooled PLN and MLN tissues were collagenase treated, and T cells were stained for expression of CD134. The left plot shows the CD4 Thy1.1 gate, and the right plot overlays CD134 expression by gated cells taken from IL-18-treated (red) or peptide-treated (gray) mice and an isotype control stain on cells from peptide-treated mice (black). c–e, SM1 cells were transferred into IL-18RϪ/Ϫ mice and the next day stimulated with flagellin peptide and an agonistic anti-CD134 mAb (Ⅺ) or control IgG (u). c, Five days after peptide injection, the lymphoid tissues were removed from each mouse, and the percentage and number Ϯ SEM of SM1 cells were determined. Bars represent pooled and averaged data from three separate experiments, with at least seven mice per group. Data from C57BL/6 control mice were comparable (data not shown). d, Day 5 cells from anti-CD134 or control IgG-treated mice were gated on CD4, and CD44 expression by Ag-specific Thy1.1 cells was determined. The number in parentheses represents the percentage of Thy1.1 cells that are CD44high. e, Day 5 bulk splenocytes were restimulated with flagellin peptide for 24 h and assayed for IFN-␥ production by ELISA. Data show IFN-␥ concentrations Ϯ SEM pooled from triplicate cultures and averaged from at least three mice from one of two similar experiments. known about the underlying mechanism. We clarify this issue by ducing subpopulation was detected after IL-18 stimulation. In addi- showing that Ag and IL-18 treatment mediates Ag-induced clonal tion to CD4 T cells, we have found that specific CD8 T cells also expansion of CD4 effector T cells with the appearance of a subpopu- clonally expand much better when exposed to IL-18 (data not shown). lation that produces large amounts of IFN-␥. It is demonstrated that Interestingly, IL-18 directly induces division and cytokine secretion IL-18 induces IFN-␥ commitment through IFN-␥ and expansion of by effector or memory T cells in the absence of recall Ag (29, 58). these effector cells is triggered by CD134 costimulation. Thus, IL-18 However, naive T cells do not express the IL-18R (27), and therefore bridges innate and adaptive immunity through the CD134 pathway. it was surprising that IL-18 boosted the CD4 T cells in our model. The As an adjuvant, IL-18 has been incorporated into DNA vaccines to initiation of the IL-18 response was shown to involve cells from the improve antitumor and antiviral responses (35, 39, 54, 55). DCs and recipient host mice, but not the transferred Thy1.1 SM1 CD4 T cells. fibroblasts transfected with IL-18 become effective tumor vaccines Thus, IL-18Rϩ/ϩ SM1 CD4 T cells were not boosted by IL-18 after (36, 38). Even injection of rIL-18 alone can combat fungal (56), bac- transfer into IL-18RϪ/Ϫ hosts (Fig. 3). These data show that the ini- terial (31, 32), and viral infections (34, 57). Importantly, in all of these tiation of the response requires IL-18R on cells other than the SM1 but studies, IL-18 enhanced Th1 or CTL responses in vivo. Our data does not speak to the possibility that IL-18 may work on the specific complement these observations by explaining how protective T cell T cells later in the response. responses develop after IL-18 administration. IL-18 induces massive Because IL-18 did not act directly on the Ag-specific T cells to accumulation of Ag-specific effector CD4 T cells by enhancing clonal initiate the response, we studied which cells directly responded. expansion and facilitating at least short-term survival (Fig. 1). Fur- IL-18 injection has been shown to rapidly stimulate NK cells to thermore, these cells disperse into lymphoid and peripheral tissues produce IFN-␥ (10). NK-derived IFN-␥ induces changes in circu- and become robust producers of IFN-␥. In fact, a super IFN-␥ pro- lating leukocyte levels in mice (59) and is crucial for directing 242 IL-18 LINKS INNATE AND T CELL IMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. IL-18 adjuvanticity depends on CD134 costimulation. Bulk SM1 cells were transferred into C57BL/6 mice and stimulated with ﬂagellin peptide and three injections of IL-18 as shown in Fig. 3a. Before each injection of IL-18, antagonistic anti-OX40L mAb was injected to block CD134 ligation (Ⅺ), or a control IgG was injected for no blockade (f). a, On day 5, lymphoid and peripheral tissues were analyzed for T cell clonal expansion. Percentages and numbers Ϯ SEM of SM1 cells from the liver, lung, and spleen are shown. Arrows represent the fold decrease observed upon CD134 blockade, compared with control. Data are pooled and averaged from three separate experiments and contain 12 mice per group. b, Day 5 splenocytes recovered from control (left panel) or CD134 blockade-treated (right panel) mice were restimulated in vitro and analyzed for intracellular IFN-␥ production. The central number represents the percentage of IFN-␥ producers in the Thy1.1 SM1 population (determined by setting a control IgG stain to ϳ0.5%). The number to the right of the dotted line represents the percentage of IFN-␥ superproducers within the Thy1.1 population. This was determined by setting the CD134 blockade group to ϳ2% and keeping the same gate for all mice. Percentages listed in the plots are representative of the average percentages of all 12 mice. In the lower panels, individual numbers of splenic IFN-␥ producers or superproducers from all 12 mice are shown in scatter plots with average values denoted by thin lines. c, CD4-gated cells from each treatment group were analyzed for CD44 expression by Ag-speciﬁc Thy1.1 SM1 cells. Number in parentheses denotes the percentage of Thy1.1 CD44high cells in the control (left panel) or with CD134 blockade (right panel). d, Splenocytes from each treatment group were restimulated with peptide in vitro and [3H]thymidine incorporation was measured. Data show the cpm from 12 wells pooled from triplicate cultures from four separate mice and are representative of one of three experiments.

Th1-mediated autoimmunity (60). However, NK depletion had no (Fig. 5). Perhaps IFN-␥ commitment is mediated by IFN-␥-pro- effect on the ability of SM1 cells to clonally expand or secrete ducing DCs (61–63) or other cells of the immune system. Thus, IFN-␥ in response to IL-18 (Fig. 2). To extend this data, we dem- IL-18 can condition peptide-speciﬁc Th effectors for IFN-␥ and onstrated that IL-12 was not required for the IL-18-initiated re- IL-4 production. sponses. We also tested whether the p35Ϫ/Ϫ and p40Ϫ/Ϫ (IL- Because B cells and macrophages have limited access to naive 12Ϫ/Ϫ) recipients conditioned the SM1 response toward the Th2 T cells in vivo, we focused our attention on DCs as they are the direction but found that the WT mice induced as much IL-4 as the premier APC (64). Numerous studies have shown that DCs re- IL-12Ϫ/Ϫ mice. Ultimately, we demonstrated that host-derived spond to IL-18 by inducing CD80, CD86, CD40, CD54, and MHC IFN-␥ was required for IFN-␥ commitment, but not for expansion class II, and even by synthesizing IFN-␥ (11, 25, 65, 66). We The Journal of Immunology 243

ducers was reduced, while the superproducer population was al- most abolished, even though CD40 (data not shown), CD80, and CD86 expression was not inhibited during CD134 blockade (Fig. 8). This demonstrates that IL-18 boosts expansion of IFN-␥-producing T cells via CD134, and perhaps CD80 and CD86 contribute to expansion of peptide-stimulated naive T cells. Also, these data extend the known role for CD134 and IL-18 in supporting Th2 responses (20, 77). Because high levels of IFN-␥ were detected with IL-18 through the CD134 pathway, it may be possible to inﬂuence the balance of Th1 or Th2 differentiation. Ultimately, the expansion afforded by IL-18 through CD134 on either the Th1 or Th2 cells would dramatically increase potential responsiveness to a pathogen or to self. Therapeutically, this suggests that under inﬂammatory conditions which contain IL-18, inhibition of CD134 signaling may help to reduce the number of autoreactive cells, and there is substantial evidence that CD134 plays an important role in the generation and maintenance of autoimmunity (78). IL-18 has a relatively long half-life (ϳ18 h) in vivo (59), and

thus one or two injections may be a reasonable and practical ther- Downloaded from apeutic means of inducing rapid immune responses in the short- term, without contributing to memory or pathogenic sequelae. However, this potent immune boost also serves to underscore how harmful persistent IL-18 can be in autoimmune and inﬂammatory conditions. IL-18 injection can exacerbate diabetes (79), joint de-

struction (80), and intestinal inflammation (81) and can promote http://www.jimmunol.org/ myocardial dysfunction (82). Thus, IL-18 may be useful as a T cell adjuvant, but its risks and safety must be carefully considered. Disclosures FIGURE 8. CD134 blockade does not affect the activation phenotype of The authors have no financial conflict of interest. DCs. a, C57BL/6 mice were transferred with SM1 cells, and the next day treated with peptide/IL-18 in the presence (CD134 blockade) or absence References (control IgG) of an antagonistic anti-OX40L mAb. Two days later, PLNs 1. Matsui, K., T. Yoshimoto, H. Tsutsui, Y. Hyodo, N. Hayashi, K. Hiroishi,

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