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Strong TCR Signaling, TLR Ligands, and Cytokine Redundancies Ensure Robust Development of Type 1 Effector T Cells¹

Institute of Integrative Biology, Molecular Biomedicine, Swiss Federal Institute of Technology, Zürich-Schlieren, Switzerland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
T cell effector function is a central mechanism of adaptive immunity, and accordingly, protection of the host against pathogens. One of the primary effector molecules produced by T cells in response to such pathogens is the cytokine, IFN-. Although the signaling pathways associated with the production of IFN- are well established, disparate in vivo and in vitro results indicate that distinct pathways may become more prominent dependent upon the nature of the infection, inflammatory milieu and tissue localization. We have examined the roles and requirements of the major IFN--inducing pathways in vivo and in vitro, specifically: strength of TCR signal; paracrine release of IL-12, IL-23, and IL-18; and autocrine production of IFN-. Our data show a dynamic interaction between these activation pathways, which allows the host a degree of flexibility and redundancy in the induction of IFN-. Upon strong signaling through the TCR, IL-12, IL-18, and IL-23 play negligible roles in the induction of IFN-, whereas autocrine IFN- is an important component in sustaining its own secretion. However, the absence of any one of these factors during a weaker TCR signal, results in strikingly impaired T cell IFN- production. Of note, TLR-activated dendritic cells (DCs) were capable of overcoming the absence of a strong TCR signal, IL-12, IL-23, or IL-18 revealing an important additional mechanism for ensuring a robust IFN- response. Our findings clarify the hierarchical requirements of the major IFN- inducing pathways and highlight the important role TLR ligand-activated DCs have to preserve them.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The cytokine IFN- is the prototypic CD4⁺ Th1 cell and CD8⁺ cytotoxic T cell cytokine, and plays an integral role in host defense against intracellular pathogens such as bacteria and viruses. To date, two independent pathways have been identified that lead to IFN- promoter activation in both CD4⁺ and CD8⁺ T lymphocytes. The first pathway is Ag-dependent and initiated via the engagement of the TCR in the presence of IL-12, whereas the second pathway is Ag-independent, and is induced by the synergistic effect of IL-12 and IL-18 (1, 2). In both pathways IL-12 is believed to orchestrate the induction of IFN-, a role that is further highlighted by the impaired clearance of infection by Leishmania major in IL-12-deficient mice (3, 4) and enhanced clearance upon administration of IL-12 (5).

IL-12 is produced by macrophages and dendritic cell (DCs)³ in response to microbial stimulation and can induce IFN- production through binding to the IL-12R on T cells leading to STAT4 activation (6). Because of its ability to induce IFN- production by T cells (7), IL-12 has been considered as the main factor responsible for initiating Th1 differentiation (8). Yet, Th1 development and IFN- production have been shown to occur in the absence of IL-12 (9, 10); Mullen et al. (11) postulated that T-bet can induce Th1 cell commitment independently of IL-12 and IL-12-deficient mice can induce normal Th1 responses and IFN- production during viral infections (9, 12, 13). IL-23 is a cytokine closely related to IL-12, being a heterodimer composed of p19 and the IL-12p40 subunit. Similar to IL-12, it is produced by activated DCs and macrophages, and has been shown to induce IFN- production by human T cells (14). However, the relative importance of IL-23 and IL-12 in the induction of IFN- production by T cells is not clear. IL-18, a cytokine of the IL-1 family (originally known as IFN- inducing factor), is mainly produced by activated macrophages and DCs and can induce IFN- production from effector and memory T cells (15). It has been shown that IL-18 alone is not essential for Th1 differentiation but in synergy with IL-12 can facilitate Th1 development by optimizing IFN- production (1, 16) and activate memory CD8⁺ cells in the absence of specific Ag (17).

In the current investigation, we sought to clarify which factors were the primary mediators of T cell IFN- production, and in particular, the hierarchical importance of these factors in driving this response. We demonstrate that IL-12, IL-23, and IL-18 are not required for the development and maintenance of IFN--producing CD4⁺ and CD8⁺ effector T cells in vitro and in vivo, but rather TCR strength of signal and autocrine stimulation by IFN- are the primary mediators of IFN- production. Furthermore, we show that IL-12 and IL-18 become requisite in the absence of a strong TCR signal in both primary and secondary responses, highlighting their importance for supporting the differentiation of IFN--producing effector T cells. Of note, the defect in IFN- production found in the absence of strong TCR signaling and IL-12 or IL-18 was restored when T cells were activated by TLR licensed mature DCs. This finding reveals a further level of complexity in T cell-DC interactions whereby TLR ligand matured DCs ensure that T cells produce IFN-, even in the absence of polarizing cytokines and a strong TCR signal. Taken together, this study highlights the individual roles of TCR signal strength, IL-12, IL-23, IFN-, and IL-18 in the induction of effective IFN- responses by T cells, providing a platform for the interpretation of both in vivo and in vitro IFN- responses.

	Materials and Methods

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Mice

C57BL/6 wild-type (WT) mice were obtained from Charles River Breeding Laboratories. IL-12p35- and IL-12p40-deficient mice were backcrossed over eight times onto a C57BL/6 background and bred in BioSupport animal facility. IL-18-deficient mice were obtained from The Max Planck Institute for Immunobiology. SMARTA-2, lymphocytic choriomeningitis virus (LCMV) GP13 TCR transgenic mice (GLNGPDIYKGVYQFKSVEFD) (18) and TCR7, LCMV GP33 TCR transgenic mice (KAVYNFATM) (19), were backcrossed onto C57BL/6 background over six generations. Mice were maintained under specific pathogen-free at BioSupport in isolated ventilated cages. Animal used in experiments were between ages 8 and 10 wk. All experiments were performed with permission from the Zürich Animal Ethics Committee.

Peptides, virus-like particles (VLPs), and oligonucleotides

LCMV glycoprotein peptides GP13, GP33, and V4Y have been described previously (20, 21). The production and purification of recombinant p13-VLP is described previously (20). Phosphorothioate-modified CpG-containing oligonucleotide was synthesized by Microsynth. The following oligonucleotide sequence was used 1668pt (5'-TCC ATG ACG TTC CTG AAT AAT-3').

TCR transgenic T cell-DC coculture

Naive SMARTA-2 and TCR7 mice were sacrificed and spleens were removed. CD4⁺ and CD8⁺ T cells, respectively, were isolated by MACS bead separation following manufacturers instructions (Miltenyi Biotec) and were found to be 90% CD4⁺CD62L^high or CD8⁺CD62L^high by subsequent FACS analysis. DCs were isolated from spleens of naive WT, IL-12p35-, IL-12p40-, or IL-18-deficient mice as described previously (22). Isolated T cells (6.5 x 10⁴ cells/well) and DCs (1.4 x 10⁴ cells/well) were cultured in 96-well plates in the presence of GP13, GP33, or V4Y peptide at the indicated concentrations. In the indicated cultures, anti-IFN- Ab (clone AN-18) was added to reach the final concentration of 10 µg/ml. For activated DCs, CpG (1 µM) was added to the culture for 2 h, followed by washing in medium and Ag-pulsing. Alternatively, bone marrow-derived DCs were generated as previously described (23), activated overnight with 1 µM CpG and supernatant was added to the culture. The same experimental set up was applied on splenic DCs. On day 3 of culture, cells were activated in the presence of PMA and ionomycin for 4 h. IFN- and IL-4 production was determined by flow cytometry or ELISA as described previously. Alternatively, for day 5 cultures, cells were restimulated at day 3 with freshly isolated DCs and GP13 or GP33 peptide at the same concentration used in the primary stimulation. In the indicated cultures, cyclosporin A (Sigma-Aldrich) was added on day 3 at a final concentration of 100 ng/ml. On day 5 of culture, cells were activated with PMA and ionomycin for 4 h and cytokine production was determined by flow cytometry or ELISA. For long cultures (10 days) cells were harvested at day 4 after primary stimulation, rested for 3 days in medium containing IL-2 and restimulated for additional 3 days with freshly isolated DCs and GP13 or GP33 peptide at the same concentration used at the onset of the culture. Finally, cells were activated in the presence of PMA and ionomycin as described previously.

Intracellular cytokine staining and FACS analysis

Cells from in vitro culture were stimulated with 10^–7 M PMA and 1 µg/ml ionomycin for 4 h at 37°C in IMDM. For the final 2 h, 10 µg/ml brefeldin A was added to the cultures to retain cytokines in the cytoplasm. Thereafter, cells were washed with PBS/0.1% BSA and CD8⁺ T cells were surface stained with PE-labeled anti-CD8 mAb or FITC-labeled anti-V2 mAb (eBioscience). Next, cells were washed with PBS/0.1% BSA, and then again in PBS and fixed with 2% paraformaldehyde for 20 min at room temperature. Fixed cells were incubated in permeabilization buffer (0.5% saponin/PBS/0.5% BSA) containing PE-labeled anti-IL-4 mAb and allophycocyanin-labeled anti-IFN- mAb for CD4⁺ T cells or only allophycocyanin-labeled anti-IFN- mAb for CD8⁺ T cells (eBioscience) for 30 min at room temperature. Cells were washed twice in permeabilization buffer, and then resuspended in PBS/0.1% BSA and analyzed by flow cytometry (FACSCalibur; BD Biosciences) and FlowJo software (Tree Star). Lymph node cells from immunized mice were first surface-stained with PE-labeled anti-Ly5.1 mAb (BD Biosciences), fixed and subsequently incubated with permeabilization buffer containing allophycocyanin-labeled anti-IFN- mAb for 30 min at room temperature. After extensive washing, cells were resuspended in PBS/0.1% BSA and analyzed by FACS.

ELISA measurement of IFN-

Supernatant was collected on days 3, 5, or 10 of T cell-DC coculture and analyzed for IFN-. The 96-well plates (Maxisorp; Nunc) were coated with anti-IFN- (BD Biosciences) at 5 µg/ml in 50 µl PBS overnight at 4°C. Between each of the following steps, plates were washed five times with PBS. Coated plates were blocked with PBS/1% BSA for 2 h at room temperature. Samples from individual cultures were serially diluted in PBS/0.1% BSA, followed by incubation at room temperature for 2 h. Thereafter, alkaline phosphatase-labeled goat anti-IFN- (eBioscience) was added at room temperature for 2 h, followed by the addition of the substrate p-nitrophenyl phosphate (Sigma-Aldrich). OD was determined at 405 nm using a SpectraMax spectrophotometer (Bucher Biotech).

Adoptive transfer and in vivo activation of transgenic T cells

Ly5.1⁺ TCR transgenic CD4⁺ and CD8⁺ T cells were purified by magnetic separation (MACS; Miltenyi Biotec) and incubated with a final concentration of 2.5 mM CFSE (Molecular Probes) for 7 min, followed by extensive washing in medium before transfer. Labeled T cells (5 x 10⁶) were resuspended in 200 µl of PBS and injected into the tail vein of C57BL/6, IL-12p35-, IL-12p40-, or IL-18-deficient mice. After 24 h, recipients were immunized s.c. in the hind-leg flank with p13-VLPs and CpG oligonucleotides or p33 peptide with CpG oligonucleotides as described previously (20). On day 3 after immunization, total cell suspensions from inguinal lymph nodes were prepared and cells (3 x 10⁶ per well) were restimulated with 1 µM GP13 or GP33 peptide for 6 h at 37°C in IMDM. For the final 3 h, 10 µg/ml brefeldin A was added to the cultures to retain cytokines in the cytoplasm. Thereafter, cells were stained and analyzed by flow cytometry as described previously.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IL-12 and IL-18 are not required for IFN- production by CD4⁺ and CD8⁺ T cells in vitro

IL-12 can clearly induce Th1 cell differentiation and IFN- production, yet whether it is strictly required remains controversial. In addition, the closely related cytokine, IL-23, has been shown to induce IFN- production by human T cells, although recent publications question these findings in mice, instead postulating a role for IL-23 in inducing IL-17 production (24, 25). The cytokine IL-18 can also drive IFN- production, although its role appears to be limited to stimulation of previously differentiated cells. We sought to clarify the respective roles of these cytokines in CD4⁺ and CD8⁺ T cell differentiation using an in vitro TCR transgenic T cell-DC coculture system. CD11c⁺ DCs were isolated from spleens of naive WT or IL-12p35-deficient mice, and cultured in the presence of either naive CD4⁺ TCR transgenic SMARTA2 (Fig. 1A) or CD8⁺ TCR transgenic TCR7 T cells (Fig. 1B) and their corresponding specific peptides GP13 or GP33 of the LCMV glycoprotein, respectively (18, 19). After 3 days, cells were restimulated with PMA/ionomycin and intracellular cytokine staining for IFN- was performed. Alternatively, cells were harvested after 4 days of primary antigenic stimulation, rested in medium with IL-2 for 3 days before secondary stimulation with freshly isolated DCs. Restimulation with PMA/ionomycin was performed after an additional 3 days. Our results show that IFN- production, both at day 3 and 10 of culture, was comparable irrespective of whether the CD4⁺ or CD8⁺ T cells were activated by WT or IL-12p35-deficient DCs, indicating that in this system, IL-12 was superfluous for development and maintenance of type I effector responses (Fig. 1, A and B). Because IL-12p35-deficient DCs were still capable of producing IL-23, we isolated splenic CD11c⁺ DCs from mice deficient in IL-12p40 (which lack both IL-12 and IL-23), and cultured them with the transgenic T cells and peptide. Similar to the response driven by IL-12p35-deficient DCs, the development of IFN- producing CD4⁺ and CD8⁺ cells was normal in the combined absence of both IL-12 and IL-23 (Fig. 1, A and B), indicating that IL-23 is not involved in triggering Th1 responses, nor does it compensate for the absence of IL-12 in inducing IFN- production in CD4⁺ and CD8⁺ T cells. Of note, we also found that supplementing the cultures with rIL-23 had no influence upon T cell IFN- production (data not shown).

View larger version (47K): [in this window] [in a new window]   FIGURE 1. IL-12, IL-23, and IL-18 are not required for effector T cell differentiation and IFN- production in vitro. A, CD4+ TCR transgenic T cells were sorted from the spleen of SMARTA2 mice and cultured for 3 (top row) or 10 days (bottom row) with CD11c+ DCs from naive WT, IL-12p35-, IL-12p40-, or IL-18-deficient mice in the presence of specific peptide GP13 (1000 for 3-day culture or 100 nM for 10-day culture). On days 3 or 10, cells were restimulated with PMA/ionomycin and the proportion of IFN-- and IL-4-producing cells was determined by intracellular cytokine staining and flow cytometry. B, CD8+ TCR transgenic T cells from TCR7 mice were isolated and cultured with the indicated DCs in the presence of specific peptide, GP33 (1000 nM for 3-day culture or 100 nM for 10-day culture). On days 3 or 10, cells were restimulated with PMA/ionomycin, and the proportion of IFN--producing cells was determined by intracellular cytokine staining and flow cytometry. Values represent percentage of positive cells in each quadrant. C, Supernatant from CD4+ (left) or CD8+ (right) T cell-DC cultures shown in A and B was collected at days 3 or 10, and ELISA for IFN- was performed. Data are from representative experiments, each repeated three to five times.

We considered it plausible that any role for IL-12, IL-23, or IL-18 in the induction of IFN- production may be overcome through autocrine activation by IFN-. We therefore neutralized IFN- activity during the 3 days of culture and then determined the proportion of IFN--producing cells by flow cytometry. Neutralization of IFN- during the culture period reduced the proportion of IFN--producing CD4⁺ (Fig. 2A) and CD8⁺ T cells (Fig. 2B), regardless of whether the activating DCs produced IL-12, IL-23, or IL-18 (Fig. 2 and data not shown). Taken together, these data show that activation of T cells via the TCR, in addition to autocrine production of IFN-, is sufficient for differentiation into IFN--producing cells. However, these data did not clarify why IL-12, IL-23, and IL-18 production is important in other model systems (26, 27, 28, 29, 30, 31).

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Autocrine IFN- stimulation plays an important role in supporting strong IFN- production by CD4+ and CD8+ T cells. A, CD4+ transgenic T cells or (B) CD8+ transgenic T cells were cultured with WT, IL-12p35-, or IL-18-deficient DCs and 1000 nM GP13 or GP33 peptide in the presence of neutralizing anti-IFN- Abs. On day 3, cells were restimulated with PMA/ionomycin, and the proportion of IFN--producing cells was determined by intracellular cytokine staining and flow cytometry. Values represent percentage of positive cells in each quadrant. Data are from representative experiments, each repeated three times.

CD4⁺ and CD8⁺ transgenic T cells display normal proliferation and IFN- production in the absence of IL-12 or IL-18 in vivo

To establish whether these cytokines were essential for in vivo T cell differentiation, we next performed adoptive transfer studies using CD4⁺ and CD8⁺ transgenic T cells injected into IL-12p35-, IL-12p40-, or IL-18-deficient recipients. CD4⁺ or CD8⁺ T cells specific for the GP13 or GP33 peptide, respectively, were labeled with CFSE and injected i.v. into Ly5.2⁺ WT or cytokine-deficient recipients. On day 1 after transfer, mice were immunized s.c. with GP13 coupled to VLP or GP33 peptides (20). Three days after immunization, total cell suspensions were prepared from draining lymph nodes, and after peptide-specific restimulation, IFN- production at each cell division was assessed by flow cytometry. The transferred CD4⁺ transgenic T cells, identified through Ly5.1⁺ staining, underwent several rounds of proliferation as shown by CFSE dilution, and no difference was seen between WT, IL-12p35-, IL-12p40-, or IL-18-deficient recipient mice (Fig. 3A). IFN- production was detectable in CD4⁺ T cells that had proliferated for at least four rounds, and was comparable in both WT and deficient mice. Comparatively, Ly5.1⁺ CD8⁺ T cells underwent six rounds of divisions within this timeframe and IFN- production was detectable in every cycle at comparable frequencies between WT and deficient mice (Fig. 3, B and C). Taken together, these data demonstrated that CD4⁺ and CD8⁺ T cell proliferation and differentiation into IFN- producing effector cells in this in vivo system was independent of IL-12, IL-23, and IL-18.

View larger version (51K): [in this window] [in a new window]   FIGURE 3. IL-12, IL-23, and IL-18 are not required for CD4+ or CD8+ T cell proliferation and IFN- production in vivo. A, CD4+ T cells were isolated from Ly5.1+ SMARTA2 transgenic splenocytes, CFSE labeled and injected i.v. into naive recipients of the indicated genotype. After 24 h, mice were immunized s.c. in the hind-leg flank with 150 µg of GP13-VLPs mixed with 20 nmol CpG in PBS. On day 3 after immunization, cells were isolated from inguinal lymph nodes and restimulated for 6 h with 1000 nM GP13 peptide. Proliferation and the proportion of IFN--producing cells were determined by intracellular cytokine staining and flow cytometry of Ly5.1+ gated cells. B, CD8+ T cells were isolated from Ly5.1+ TCR7 transgenic splenocytes, labeled with CFSE and injected i.v. into naive recipients of the indicated genotype. Recipient mice were immunized on day 1 after transfer with 20 µg of GP33 peptide mixed with 20 nmol CpG in PBS, and draining lymph nodes were isolated on day 3 after immunization. Total lymph node cells were restimulated with 1000 nM GP33 peptide, and proliferation and the proportion of IFN--producing cells were determined by intracellular cytokine staining and flow cytometry of Ly5.1+ gated cells. C, Percentage of CD8+ IFN--producing cells for each CFSE peak as assessed by flow cytometry. Values in FACS plots represent the percentage of cells detected in the indicated gates. All experiments were performed twice with three mice per group; representative data from one mouse per group are shown.

IL-12 and/or IL-18 can drive IFN- production in the absence of TCR signaling

Considering two different pathways for IFN- promoter activation have been described: one mediated by signaling through the TCR; the other mediated by the synergistic effect of IL-12 and IL-18 in an Ag-independent manner (1), we hypothesized that one pathway might act to compensate for the other. To address this question we used the immunosuppressor cyclosporin A, which inhibits signaling downstream of the TCR (32). CD8⁺ TCR transgenic T cells were incubated for 3 days with specific-peptide pulsed-DCs as described in the prior section. On day 3, T cells were restimulated with specific peptide and freshly isolated WT or deficient DCs, in the presence or absence of cyclosporin A. After a further 2 day culture, we assessed IFN- secretion in culture supernatant by ELISA and intracellular cytokine staining for IFN- was performed. IFN- production by CD8⁺ T cells activated by WT DCs in the presence of cyclosporin A was reduced, but still present, as compared with nontreated effector T cells (Fig. 4). Comparatively, IFN- production was no longer detectable from CD8⁺ T cells stimulated with IL-12p35- or IL-18-deficient DCs in the presence of cyclosporin A (Fig. 4). Under the same conditions, CD4⁺ T cell IFN- production was abrogated after stimulation with DCs from each mouse strain, indicating that CD4⁺ T cells have a greater requirement for TCR signaling (data not shown). These results show that TCR engagement plays the major role in driving effector T cell differentiation and IFN- secretion, and additionally provide evidence that IL-12 and IL-18 are important factors in promoting and sustaining IFN- production by CD8⁺ effector cells when TCR signaling is impaired.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. IL-12 and/or IL-18 are critical for IFN- production upon impaired TCR signaling. A, CD11c+ DCs were isolated from the spleen of naive WT, IL-12p35-, and IL-18-deficient mice and cultured with CD8+ TCR transgenic T cells and GP33 peptide at 1000 nM. On day 3 of culture, cells were restimulated with freshly isolated DCs and peptide, in the presence or absence of cyclosporin A. After a further 2 days of culture, cells were restimulated for 4 h with PMA/ionomycin and assessed for IFN- production by intracellular cytokine staining and flow cytometry. Values represent percentage of positive cells in each quadrant. B, Additionally, supernatant from the same cultures was collected at day 5, and ELISA for IFN- was performed. Data are representative of two repeat experiments.

IFN- production in the absence of IL-12 or IL-18 is not regulated by Ag concentration

Because TCR signaling appears to be the primary mediator of IFN- responses, we next assessed the influence of Ag concentration. The 3 day coculture experiments shown in Fig. 1 were performed at the Ag concentration of 1 µM. In the CD4⁺ transgenic T cell-DC coculture system, this concentration of Ag leads to the development of Th1, IFN--producing cells, whereas a lower Ag concentration of 1 nM results in the differentiation of IL-4-producing Th2 cells (22, 33). In the CD8⁺ TCR transgenic T cell-DC coculture system, lower concentrations of Ag lead to reduced IFN- production, but no secretion of IL-4. We sought to determine whether the concentration of Ag itself regulated the requirement for IL-12 and IL-18 in the induction of IFN--producing cells. Thus, we reduced the amount of antigenic stimulation by titrating down the concentration of peptide in our coculture system. There was no significant difference in IFN- production between CD4⁺ T cells stimulated by WT, IL-12p35- or IL-18-deficient DCs (Fig. 5 A) regardless of the Ag concentration used, indicating that these cytokines are not important for Th1 effector cell differentiation even at low Ag concentration. We also investigated the role of IL-12 and IL-18 in inducing IFN- production by CD8⁺ T cells at low Ag concentrations. Similar to the results found for CD4⁺ T cells, the frequency and total number of CD8⁺ IFN--producing cells were decreased, as compared with higher concentrations of Ag; however, there was no difference in IFN- production in the absence of either IL-12 or IL-18 (Fig. 5B). A similar result was obtained by measuring IFN- secretion in the culture supernatant (Fig. 5C).

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Ag concentration does not influence the requirement of IL-12 or IL-18 for IFN- production. A, CD11c+ DCs were isolated from the spleen of naive WT, IL-12p35-, and IL-18-deficient mice and cultured with CD4+ TCR transgenic T cells and GP13 peptide at the indicated concentrations. On day 3 of culture, cells were restimulated for 4 h with PMA/ionomycin and assessed for IFN- and IL-4 production by intracellular cytokine staining and flow cytometry. B, CD8+ T cells were isolated from spleens of TCR7 transgenic mice and cultured with splenic CD11c+ cells from WT, IL-12p35-, or IL-18-deficient mice and GP33 peptide at the indicated concentrations. On day 3 of culture, cells were restimulated for 4 h with PMA/ionomycin and assessed for IFN- production by intracellular cytokine staining and flow cytometry. Values in FACS plots represent percentage of positive cells in each quadrant. C, Alternatively, culture supernatant was collected, and ELISA for IFN- was performed. Data are representative of four to five repeat experiments.

IL-12 and IL-18 are required for IFN- production by CD8⁺ T cells when signaling through the TCR is mediated by a weak agonist peptide

It was surprising that although TCR signaling was crucial for IFN- production in the absence of polarizing cytokines, titrating the concentration of peptide did not influence T cell differentiation. We hypothesized that the quality, rather than the quantity of TCR signaling might be crucial. We thus used altered peptide ligands so that a high- or low-affinity signal could be delivered in the same TCR transgenic T cell-DC coculture system (34). Altered peptide ligands for the CD4⁺ GP13-transgenic T cells have yet to be identified, so we performed a CD8⁺ transgenic T cell-DC coculture with WT, IL-12p35- or IL-18-deficient DCs and the weak agonist peptide, V4Y. This modified peptide interacts with the TCR with a much lower affinity than the full agonist peptide GP33 (21). After 3 days of culture with GP33 or V4Y, supernatants were collected and an IFN- ELISA was conducted. As expected, comparable amounts of IFN- were produced in the WT, IL-12p35-, or IL-18-deficient DC-T cell cultures upon stimulation with GP33 peptide (Fig. 6A). However, IFN- production by CD8⁺ transgenic T cells was dramatically reduced, as compared with the WT control, when these cells were stimulated by IL-12p35- or IL-18-deficient DCs presenting the weak agonist V4Y (Fig. 6A). This result was confirmed by intracellular cytokine staining (Fig. 6B). Notably, addition of exogenous IL-12 was sufficient to fully restore IFN- production by T cells under these conditions (data not shown). Our data support the hypothesis that signaling via the TCR alone drives IFN- production in the absence of IL-12 and IL-18. However, these cytokines become important when the strength of TCR signaling is weak. Taken together with the results shown in Fig. 5, these data show that the strength of TCR signaling that compensates for the absence of IL-12 and IL-18 depends more on the "quality" of the TCR signal rather than the "quantity."

View larger version (32K): [in this window] [in a new window]   FIGURE 6. IL-12 and IL-18 are necessary for IFN- production in the absence of strong TCR signaling or TLR ligands. A, Splenic CD11c+ DCs from WT, IL-12p35-, and IL-18-deficient mice were incubated with CD8+ T cells and 1000 nM final concentration of either GP33 (left) or V4Y (right) peptide. Supernatant was collected on day 3, and IFN- production was assessed by ELISA. B, Cells from the V4Y cultures were restimulated with PMA/ionomycin, and the proportion of IFN--producing cells was assessed by intracellular cytokines staining and flow cytometry. C, Before Ag-pulsing, CD11c+ DCs of the indicated genotypes were incubated for 2 h in the presence of 1 µM CpG in IMDM. After washing, DCs were cultured with CD8+ T cells in the presence of 1000 nM V4Y peptide. On day 3 of culture, cells were restimulated for 4 h with PMA/ionomycin and assessed for IFN- production by intracellular cytokine staining and flow cytometry. D, A CD8+ T cell-DC coculture was performed as described in A in the presence of supernatant from resting (top row) or CpG-activated DCs (bottom row). After 3 days of culture, cells were restimulated with PMA/ionomycin, and the proportion of IFN--producing cells was assessed by intracellular cytokine staining and flow cytometry. Values represent percentage of positive cells in each gate. Data in A represent combined data from two independent experiments. Data are representative of two to three repeat experiments.

TLR ligand licensed DCs provide innate signals which ensure IFN- production by T cells

An important characteristic of pathogens, known to modulate the quality of adaptive immune responses, is the binding of pathogen-associated molecular patterns to TLRs (35). We thus examined whether TLR ligand mediated DC activation influenced the requirement for the IFN--inducing pathways described. We performed a coculture with WT and IL-12p35- or IL-18-deficient DCs that were activated with the TLR ligand CpG for 2 h before incubation with CD8⁺ T cells and V4Y. Upon stimulation with TLR-matured DCs, IFN- production was comparable between T cells irrespective of the weak TCR signal and the absence of IL-12 or IL-18 (Fig. 6C). We next assessed whether the TLR ligand-activated DCs influenced T cell IFN- production through the production of a soluble factor. Accordingly, the CD8⁺ T cell coculture was performed as described above, in the presence of supernatant from CpG-activated DCs. IFN- production by CD8⁺ T cells was restored by supernatant from CpG-activated DCs, but not "resting" DCs that had not received a TLR stimulus (Fig. 6D) (for review on DC maturation states see Ref. 36). Notably, conditioned supernatant from IL-12p35-, or IL-18-deficient DCs similarly restored IFN- production (data not shown), indicating an additional soluble factor was capable of supporting T cell IFN- production. Addition of neutralizing Abs against IL-12 or IL-18 in the cultures with CpG activated DCs confirmed these cytokines were not responsible for TLR-induced IFN- production, although supplementing the cultures with IL-18 and IL-12 did restore the response (data not shown). Taken together, these data show that activation of DCs by TLR ligands provides an important additional safeguard for the production of IFN- by T cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IFN- plays a central role in adaptive immune responses against intracellular pathogens and tumors. Both Ag-dependent and Ag-independent pathways to induce IFN- production have been identified, however it remains unclear exactly how these pathways interact and their respective importance. In an attempt to clarify their roles, we have assessed the differential requirements for strength of TCR signaling together with autocrine IFN- stimulation, IL-12, IL-23, and IL-18 in promoting IFN- production. We show that provided TCR-Ag binding occurs with high affinity (strong TCR signal), IL-12, IL-23, and IL-18 are dispensable for the development of IFN--producing effector cells in vitro and in vivo. These cytokines are only required for IFN- production in primary or secondary responses in which TCR-Ag affinity is weak (weak TCR signal) (Fig. 6). Considering supplementation of cultures with IL-23 did not restore IFN- production (data not shown), the defect in IFN- production upon weak TCR stimulation in the IL-12p40-deficient mice likely results from the absence of IL-12, not IL-23. Of note, autocrine IFN- production played a central role in promoting IFN- production by both CD4⁺ and CD8⁺ T cells, implicating it high in the hierarchy of IFN--inducing factors.

Our findings reveal a clear hierarchy in the importance of the two different pathways for IFN- promoter activation, with TCR signaling playing the dominant role. Such redundancies, or fail-safe mechanisms, are in line with the important role IFN- plays in protective immunity. Depending on the nature of the infection, these different pathways are likely to be important to a greater or lesser extent, potentially underlying the disparate results regarding the role of IL-12 and IL-18 in vivo and in vitro (6, 12, 13, 16, 27, 30). For example, IL-12 was shown to be dispensable for Th1 development and IFN- production during LCMV, vesicular stomatitis virus, and mouse hepatitis virus infection (12, 13). However, in contrast to immune responses against viruses, it is well recognized that IL-12 is required for protection against the intracellular protozoan parasites L. major (3, 26) and Toxoplasma gondii (37). IL-12-deficient mice are highly susceptible to infection with L. major, and in line with this finding, the administration of exogenous IL-12 to IL-12p40 mice has been shown to restore primary effective immune responses against L. major (4). Considering our data, the differential requirements for IL-12 in antiviral versus antiparasite responses would suggest that viral infections may lead to stronger signaling through the TCR. Indeed, both GP13 and GP33 peptides derived from LCMV drive strong Th1/Tc1 responses in cocultures (Fig. 1, A and B), whereas cocultures with LACK-specific TCR transgenic T cells drive stronger Th2 responses as compared with Th1 responses (G. Iezzi and M. Kopf, unpublished observations). Furthermore, LACK-specific cells from L. major-susceptible BALB/c mice exhibit a lower affinity for LACK and develop into Th2 cells, as compared with LACK-specific cells from resistant B10.D2 mice, which exhibit a high affinity toward LACK and become IFN--producing Th1 cells (38). Although multiple mechanisms are sure to influence resistance or susceptibility to L. major infection, strength of TCR signal is likely to play a role in the requirement for IL-12 production.

In addition to the high Ag load present during viral infections, the presence of TLR ligands also influences the requirement for IL-12 production. We found that IL-12 and IL-18 were no longer required upon weak antigenic stimulus, when Ag-presenting DCs had previously been activated with a TLR ligand. The apparent mechanism involved a soluble factor distinct from IL-12, IL-23, and IL-18. Thus, the licensing of DCs by TLR ligands (for example during viral infections) appears to compensate for weaker TCR signals, helping to ensure an effective immune response develops. IL-12 may play a more prominent role in environments lacking sufficient pathogen associated molecular patterns or other inflammatory stimuli, for example in antitumor (6, 39) or some antiparasite responses.

The IL-12-induced transcription factor, T-bet, has been identified as being critical for driving transcription of the ifn gene in CD4⁺ T cells, whereas CD8⁺ T cells do not appear to require it to produce IFN- (40). Similarly, signal transducer and activator of transcription (STAT4) has been shown to be important for TCR-induced IFN- production by CD4⁺ but not CD8⁺ T cells (2). Considering we found TCR strength of signal was the initial defining factor leading to IFN- production, and that CD4⁺ and CD8⁺ T cells appear to have different thresholds for activation upon TCR stimulation (41, 42) (Fig. 3), it is plausible that the differential importance of T-bet and STAT4 described in these T cell subsets, may rather reflect their respective responsiveness to the anti-CD3 used to stimulate them. Indeed, we found that when the partial agonist, V4Y, was used to stimulate specific CD8⁺ T cells, IL-12 was required for full IFN- production. Thus, careful consideration of the nature of the TCR stimulus is important for the interpretation of differences between CD4⁺ and CD8⁺ T cells.

IL-12 can act in synergy with IL-18 to induce IFN- production by differentiated CD4⁺ and CD8⁺ effector cells. Although IL-18-deficient mice display impaired IFN- production in response to LPS (27), previous publications show that IL-18 alone is not sufficient to induce Th1 differentiation, but rather supports IL-12 in this respect (16). Our data showed that CD4⁺ and CD8⁺ effector T cell differentiation and IFN- production occurred in the absence of IL-18 in vitro and in vivo. However, similar to the IL-12-deficient system, T cells activated by IL-18-deficient DCs exhibited impaired IFN- production in the absence of strong TCR signaling. Our results indicate that IL-18 plays an equally important role as IL-12, in driving primary CD8⁺ T cell IFN- production when the TCR signal is suboptimal. However, it remains unclear whether IL-18 and IL-12 can substitute for each other when the TCR signal is weak, or whether their synergistic action is required.

Overall, our data are supportive of a hierarchical model whereby upon strong TCR stimulation IFN--producing effector T cells develop in the absence of IL-12 and IL-18, and are sustained through autocrine activation by IFN-. Upon weak TCR stimulation IL-12 and IL-18 become requisite for optimum IFN- production. Another facet of this system comes from TLR-activated DCs, which are capable of overcoming both the absence of IL-12/IL-18, and weak TCR stimulation (Fig. 7). In summary, our data indicate that TCR stimulation is the primary inducer of IFN- production in T cells, and that TLR-licensed DCs and the cytokines IL-12 and IL-18 play secondary supporting roles to ensure effective production of IFN-.

	Acknowledgment

 
We thank Marina Freudenberg for providing the IL-18-deficient mice.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. The hierarchy of IFN--inducing signals. High-affinity interactions between the MHC-peptide complex and the specific TCR lead to a "strong strength of signal," which can induce IFN- production by T cells in the absence of IL-12 or IL-18 (left). Low-affinity interactions between the MHC-peptide complex and the specific TCR lead to a "weak strength of signal," which then requires additional signaling through the IL-12 and/or IL-18 receptors to induce full IFN- production in both primary and secondary responses (middle). DCs activated by TLR ligands can overcome a weak signal and induce IFN- production by T cells in the absence of IL-12 and/or IL-18 (right).

	Footnotes

¹ This work was supported by Grant 3100A0-100233/1 from the Swiss National Foundation.

² Address correspondence and reprint requests to Dr. Benjamin J. Marsland, Molecular Biomedicine, Swiss Federal Institute of Technology, Wagistrasse 27, CH8952 Zürich-Schlieren, Switzerland; E-mail address: marsland{at}env.ethz.ch or Dr. Manfred Kopf, Molecular Biomedicine, Swiss Federal Institute of Technology, Wagistrasse 27, CH8952 Zürich-Schlieren, Switzerland; E-mail address: Manfred.Kopf{at}ethz.ch

³ Abbreviations used in this paper: DC, dendritic cell; WT, wild type; VLP, virus-like particle; LCMV, lymphocytic choriomeningitis virus.

Received for publication November 23, 2005. Accepted for publication March 24, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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