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Disruption of Innate-Mediated Proinflammatory Cytokine and Reactive Oxygen Species Third Signal Leads to Antigen-Specific Hyporesponsiveness¹

Diabetes Institute, Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA 15213

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interacting with MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflammatory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptive immune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innate immune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with a catalytic antioxidant effectively inhibited the generation of third signal components from the innate immune response (TNF-, IL-1, ROS). In this study, we demonstrate that innate immune-derived signals are necessary for adaptive immune effector function and disruption of these signals with in vivo CA treatment conferred Ag-specific hyporesponsiveness in BALB/c, NOD, DO11.10, and BDC-2.5 mice after immunization. Modulating redox balance led to decreased Ag-specific T cell proliferation and IFN- synthesis by diminishing ROS production in the APC, which affected TNF- levels produced by CD4⁺ T cells and impairing effector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as autoimmune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4⁺ T cells to transition from expansion to effector function.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The adaptive immune response is the result of an interaction of receptor-expressing lymphocytes and APCs to foreign immunogenic peptides. Efficient activation of the T cell adaptive immune response requires: 1) signal 1 consisting of TCR:MHC molecular interactions stabilized by a peptide bound to the MHC; 2) signal 2, the interaction of costimulatory molecules on T cells and APC (1); and 3) signal 3, which consists of proinflammatory signals mediated by the adjuvant properties of CFA, LPS, or other microbial products (2, 3). The proinflammatory third signal is important for inducing, enhancing, and prolonging the Ag-specific proliferative response in T cells (3, 4). Previous studies have demonstrated that immunization with a foreign Ag in the absence of a third signal results in unresponsiveness upon subsequent immunizations with Ag and adjuvant (5, 6). The responding T cells will proliferate in draining lymph nodes (LN)³ but will become anergic or tolerant and incapable of recognizing the immunogenic peptide (7). In the absence of this third signal, CD8⁺ T cells are unable to gain cytolytic effector function (8), and CD4⁺ T cells do not undergo clonal expansion or provide help for B cells to class switch to the IgG isotype Ab (3).

The third signal relies on the production of reactive oxygen species (ROS), which fuels the generation of the proinflammatory cytokines necessary for the linkage of innate to adaptive immunity and Ag-specific T cell activation (9, 10). ROS promotes proinflammatory cytokine production from APCs by the activation of redox-sensitive signal transduction pathways such as MAPK, AP-1, and NF-B (10, 11, 12, 13, 14), which control the expression of the innate proinflammatory immune response, cellular proliferation, and apoptosis (11, 15). Intracellular ROS levels are elevated in T cells and dendritic cells (DC) during Ag recognition (16). Stimulation of T cells with anti-CD3 and anti-CD28 Ab can rapidly generate superoxide and hydrogen peroxide for mediating activation-induced cell death and proliferation by activating Fas ligand and the ERK pathway, respectively (17, 18).

We hypothesized that strategies that inhibit the proinflammatory third signal would allow for the specific control of T cell transition from expansion to effector function. To achieve this goal we used the previously described catalytic antioxidants (CA), (Mn(III) mesotetrakis(di-N-diethylimidazole)porphyrin; MnTE₂) and (Mn(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin; MnTDE), to scavenge oxidants and decrease proinflammatory cytokine production allowing its use as an immunomodulatory reagent (19, 20, 21, 22). Our previous work has shown that these compounds decrease the innate immune response in LPS-stimulated bone marrow-derived macrophages and DCs due to their abilities to function as oxidoreductases (23). Depending on the redox environment, the CA are able to oxidize redox-sensitive transcription factors such as NF-B to inhibit DNA binding (23). Furthermore, CA treatment protected NOD.scid recipients from adoptive transfer with the diabetogenic BDC-2.5 T cell clone (24). We demonstrated that CA directly inhibited APC effector function (TNF- and ROS synthesis) and suppressed APC-dependent T cell proliferation and IFN- production of the BDC-2.5 T cell clone. The ability of CA to modulate redox function and suppress TNF- production demonstrates that this strategy of redox alteration inhibits the third signal-dependent linkage of the innate and adaptive immune response in a robust model of Type 1 diabetes. Given the role of ROS in mediating signaling events necessary for synergizing the innate with the adaptive immune response (25, 26), we hypothesized that CA directly or indirectly modulate redox-dependent signaling pathways that are essential for initiating the innate immune response (23). In this report, we demonstrate the efficacy of proinflammatory third signal disruption to markedly reduce Ag-specific T cell TNF- production, which appears to negatively impact responsiveness, expansion, and effector function of Ag-specific T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Materials

NOD, DO11.10 (27), OT-II (28), and BDC-2.5 TCR-transgenic (29) mice were bred and housed under specific pathogen-free conditions in the Animal Facility of the Rangos Research Center in the University of Pittsburgh (Pittsburgh, PA). BALB/c and C57BL/6 mice were purchased from The Jackson Laboratory. Female mice at 6 to 8 wk of age were used in all experiments. KJ1-26, the anticlonotypic mAb specific for DO11.10 TCR (30) was purchased from Caltag Laboratories. Ab pairs for IL-2 and IFN- ELISAs, CD4, CD90.2, and CD11b fluorochrome-conjugated Ab were purchased from BD Pharmingen. MnTDE and MnTE₂, generous gifts from Incara Pharmaceuticals, were resuspended in HBSS with Ca²⁺ and Mg²⁺ (Invitrogen Life Technologies) at a stock concentration of 2 mM and filter sterilized (0.2 µm) before use. The working concentration chosen for MnTDE and MnTE₂ in all in vitro experiments was 34 µM, after consulting with Dr. Joe McCord (Webb-Waring Institute, Denver, CO; unpublished observations) that this concentration of CA was necessary to recapitulate the native superoxide dismutase 2 antioxidant activity. Sustained release pellets of MnTDE (3.6 mg/kg/day) were synthesized from Innovative Research of America. Chicken OVA_323–339 (ISQAVHAAHAEINEAGR) and the BDC-2.5 mimotope (EKAHRPIWARMDAKK) were synthesized by Sigma Genosys. Hen egg lysozyme (HEL) and pigeon cytochrome c (PCC) were purchased from Sigma-Aldrich. Recombinant murine TNF- was purchased from R&D Systems.

Measurement of intracellular ROS

The determination of intracellular oxidant formation was based on the oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Molecular Probes). CM-H₂DCFDA was prepared in DMSO immediately before loading by making a 1 mM stock solution. The measurement of intracellular ROS was measured in a DO11.10 primary recall assay by making single-cell splenocyte suspensions and pretreating with or without 34 µM MnTDE for 1 h at 37°C. The splenocytes were washed three times with PBS, resuspended in FACS buffer (1% heat-inactivated FCS in PBS), and loaded with 10 µM CM-H₂DCFDA for 30 min at 37°C. The cells were washed twice and then stimulated with 1 µM OVA_323–339 for 60, 90, and 120 min at 37°C. Fifteen minutes before the end of the time point, the cells were stained with KJ1-26 APC-conjugated and CD11b PE-conjugated Abs for distinguishing between the T cell and APC populations. Cell acquisition (excitation 480 nm; emission 520 nm) was performed on a FACSCalibur (BD Biosciences) and data was analyzed with BD FACSDiva software version 4.0.1 (BD Biosciences).

Flow cytometric analysis

Cells were washed twice in FACS buffer, counted, and resuspended in a final concentration of 2 x 10⁷ cells/ml in FACS buffer. Then 10⁶ cells were stained with directly fluorochrome-conjugated Ab at the appropriate dilution (10 µl of each Ab) for 30 min in the dark at 4°C in FACS buffer, and fluorescence was measured.

Adoptive transfer of T cells

DO11.10 or OT-II T cells were negatively selected and purified from splenocytes by using a MACS mouse pan T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. The purity of T cells obtained from this kit was consistently >95% as determined by FACS analysis for KJ1-26⁺, CD4⁺, and CD3⁺ cell surface markers. To track adoptively transferred cells in vivo, T cells were labeled with CFSE by following a previously described protocol (31). Briefly, purified T cells were suspended in PBS, pH 7.0, at a concentration of 5 x 10⁷ cells/ml and labeled with 5 µM CFSE for 15 min in a 37°C water bath. The T cells were washed in HBSS and adjusted to a concentration of 5 x 10⁷ cells/ml, and 0.1 ml of cells (5 x 10⁶ cells) was transferred into the retro-orbital vein.

Antigenic immunization of mice

Several hours before immunization, mice were divided into two groups and treated i.p. with CA (MnTE₂ or MnTDE; 10 mg/kg of body weight) or HBSS or implanted at the nape of the neck with sustained release pellets of MnTDE (3.6 mg/kg of body weight/day; Innovative Research of America) or placebo using a trochar device. The mice were injected with 100 µg of HEL, 100 µg of PCC, 150 µg of OVA_323–339, or 25 µg of the BDC-2.5 mimotope in CFA s.c. at the base of the tail. After immunization, mice were injected i.p. daily for 7 days with CA (10 mg/kg) or HBSS. On day 8, mice were sacrificed, and the inguinal and periaortic LNs were harvested for in vitro Ag recall assays.

In vitro T cell proliferation and Ag recall assay

Ag recall and primary recall assays were set up with LN or splenocyte single-cell suspensions by seeding in 96-well flat-bottom plates with 25 µg of HEL, 25 µg of PCC, or (0.1 µM or 1 µM) OVA_323–339 in 200 µl total volume of DMEM supplemented with 10% heat-inactivated FCS, 10 mM HEPES buffer, 4 mM L-glutamine, 2x nonessential amino acids, 1 mM sodium pyruvate, 61.5 µM 2-ME, and 100 µg/ml Gentamicin (Invitrogen Life Technologies; complete DMEM). The DO11.10 or OT-II primary recall assays were performed with splenocyte single-cell suspensions in the presence or absence of 34 or 68 µM CA and 1 µM OVA_323–339 in complete DMEM. After incubation at 37°C in a 5% CO₂ humid air chamber for 2, 3, or 4 days, the cells were pulsed with 1 µCi of [³H]TdR for 18 h and then harvested onto glass fiber filters with a sample harvester. The amount of incorporated counts was determined using a beta scintillation counter.

Cytokine measurements by ELISA and intracellular cytokine staining

IL-2 and IFN- cytokines produced in the supernatants of the various Ag recall assays were measured using Ab pairs from BD Pharmingen. IL-12 p70, TNF-, and IL-1 were detected with DuoSet ELISA kits (R&D Systems). ELISA plates were read on a Benchmark microplate reader (Bio-Rad), and data was analyzed using DeltaSoft (Molecular Devices). Intracellular TNF- was measured with 1.2 x 10⁷ DO11.10 or OT-II splenocytes in a primary recall response in the presence or absence of 68 µM MnTE₂ for 4 h at 37°C in a 5% CO₂ humid air incubator with the aid of the murine BD intracellular cytokine staining kit (BD Biosciences). After stimulation, splenocytes were fixed in BD Cytofix/Cytoperm buffer, washed in BD Perm/Wash buffer, and then stained with R-PE-conjugated rat anti-TNF- (MP6-XT22; BD Biosciences) and isotype controls. Cells were washed twice in BD Perm/Wash buffer and resuspended in FACS buffer; stained cells were analyzed on a FACSCalibur.

Anti-TNF- and anti-IL-1 neutralization

Neutralization of TNF- and IL-1 in a DO11.10 and OT-II primary recall was performed with 5 µg/ml final concentration of purified rat anti-mouse TNF- (Clone MP6-XT3, BD Pharmingen) and/or purified hamster anti-mouse IL-1 (clone B122; BD Pharmingen) Abs. Splenocyte single-cell suspensions were plated at 2.5 x 10⁵ cells/well and stimulated with 1 µM OVA_323–339 in the presence of TNF-, IL-1 neutralization Abs, or isotype controls (rat IgG1 or hamster IgG; BD Pharmingen) in complete DMEM in 96-well flat-bottom plates. After incubation at 37°C in a 5% CO₂ humid air chamber for 2, 3, or 4 days, supernatants were harvested for cytokine analysis by ELISA, and cells were pulsed with [³H]TdR to assess proliferation as described above. The purified rat anti-mouse TNF- (clone MP6-XT3; BD Pharmingen) Ab did not interfere with TNF- detection when assayed with the R&D DuoSet TNF- ELISA, given that titrating concentrations (0, 0.625, 1.25, 2.5, 5, and 10 µg/ml) of neutralization Ab did not significantly affect the detection of a known TNF- standard concentration (data not shown).

Exogenous addition of recombinant murine TNF- to CA-treated DO11.10 primary recall assay

DO11.10 splenocyte single-cell suspensions were plated out at 2.5 x 10⁵ cells/well in a 96-well flat-bottom plate and stimulated with 1 µM OVA_323–339 in the presence or absence of 34 µM CA. Recombinant murine TNF- (R&D Systems) at a concentration of 1 or 2.5 ng/ml was exogenously added to the CA-treated splenocytes and incubated at 37°C in a 5% CO₂ humid air chamber for 3 days. Supernatants were collected for cytokine analysis by ELISA as described above in Cytokine measurements by ELISA and intracellular cytokine staining.

Statistical analysis

Determination of the difference between mean values for each experimental group was assessed by Student’s t test, with p < 0.05 considered significant. All experiments were performed at least three separate times with data obtained in triplicate wells in each experiment.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Catalytic antioxidants decrease TNF- synthesis in a DO11.10 and OT-II in vitro primary recall resulting in suppression of effector cytokine production

Recent evidence has demonstrated the importance of a third signal consisting of innate immune-derived proinflammatory cytokines such as TNF-, IL-1, and IL-12 p70 in maturing the adaptive immune effector response of CD4 and CD8 T cells for IFN- synthesis (2, 3, 4, 8, 32, 33). The levels of TNF-, IL-1, and IL-12 p70 proinflammatory third signal cytokines produced in a DO11.10 and OT-II primary recall response was analyzed by ELISA after OVA_323–339-stimulation. As shown in Fig. 1, TNF- was detected in both DO11.10 and OT-II primary recall responses. In the presence of CA, TNF- levels significantly decreased 2-fold. In all time points assayed, IL-1 was not detected in either the DO11.10 or OT-II primary recall (data not shown), and the levels of IL-12 p70 were not significantly different with the DO11.10 splenocytes when treated with or without CA (Fig. 1A). The OT-II primary recall did exhibit a 2-fold decrease in IL-12 p70 synthesis in the presence of CA (Fig. 1B). T cell proliferation and IFN- synthesis was significantly diminished after CA treatment of OVA peptide-stimulated DO11.10 and OT-II splenocytes (Fig. 1), but interestingly, IL-2 levels were not altered by redox modulation. In the presence of high levels of IL-2, CA-treated DO11.10 and OT-II T cells were refractory to the effects of IL-2 and exhibited a significant decrease in T cell proliferation.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. IFN- synthesis is inhibited in a DO11.10 and OT-II primary recall by modulation of the redox state with a catalytic antioxidant. DO11.10 (A) or OT-II (B) splenocytes (5 x 105 cells) were stimulated with 1 µM OVA323–339 in the presence or absence of 34 µM catalytic antioxidant for 48 and 72 h. Supernatants were harvested, and the levels of cytokines produced in the primary recall responses were measured with cytokine-specific ELISAs. Proliferation was determined by measuring [3H]TdR incorporation. , No Ag; , cells stimulated with Ag. Results are representative of the mean (±SEM) of four independent experiments done in triplicate. **, p < 0.05 vs the respective control group.

To demonstrate that during Ag recognition proinflammatory third signal ROS are generated and enhance the adaptive immune response, DO11.10 splenocytes were stimulated with OVA_323–339 and ROS formation was assessed by measuring the oxidation of the H₂O₂-specific fluorogenic probe CM-H₂DCFDA by FACS analysis (Fig. 2). Surface staining of DO11.10 splenocytes with T cell-specific (KJ1-26; Fig. 2A) and APC-specific (CD11b) Abs (Fig. 2B) demonstrated that both cell types synthesized ROS in the primary recall response. After OVA_323–339 stimulation, there was a 1.6-fold increase in double-positive KJ1-26⁺/CM-H₂DCFDA⁺ cells (Fig. 2A) and similarly, a 5-fold increase in the percentage of CD11b⁺/CM-H₂DCFDA⁺ double-positive cells (Fig. 2B). Interestingly, CA treatment of OVA peptide-stimulated DO11.10 splenocytes did not have a profound effect on the percentage of double-positive KJ1-26⁺/CM-H₂DCFDA⁺ cells because they still demonstrated a 1.6-fold increase after OVA peptide stimulation, but there was a significant decrease in the percentage of CD11b⁺/CM-H₂DCFDA⁺ double-positive cells as they only exhibited a 1.5-fold increase after stimulation. In addition to an increase in the percentage of CD11b⁺/CM-H₂DCFDA⁺ and KJ1-26⁺/CM-H₂DCFDA⁺ double-positive cells after OVA peptide stimulation, an increase in the mean fluorescence intensity (MFI) of CM-H₂DCFDA oxidation was also observed. The MFIs of CM-H₂DCFDA oxidation increased in KJ1-26⁺ cells by 7-fold (Fig. 2A) and in CD11b⁺ cells by 1.5-fold (Fig. 2B). Conversely, CA-treated CD11b⁺ cells demonstrated only a 1.15-fold increase in the MFI of CM-H₂DCFDA oxidation (Fig. 2B), and CA-treated KJ1-26⁺ cells exhibited MFIs that were barely above unstimulated samples (Fig. 2A).

View larger version (28K): [in this window] [in a new window]   FIGURE 2. DO11.10 splenocytes produce ROS upon stimulation with OVA323–339 and are suppressed by pretreatment with a CA. DO11.10 splenocytes were preloaded with 10 µM CM-H2DCFDA for 30 min at 37°C and then stimulated with 1 µM OVA323–339 for 60 min. The generation of ROS by these cells was determined by the presence of oxidized CM-H2DCFDA by FACS (excitation, 480 nm; emission, 520 nm) along with surface staining for KJ1-26 (A) or CD11b (B) expression with directly fluorochrome-labeled Abs. Results are representative of three independent experiments.

Catalytic antioxidants diminish TNF- intracellular cytokine levels in DO11.10 and OT-II primary recall responses

To determine whether reduced ROS levels in CA-treated APC and T cells was responsible for diminished TNF- synthesis in the primary recall response in Fig. 1, TNF- intracellular cytokine staining was measured with DO11.10 and OT-II splenocytes after OVA_323–339 stimulation in the presence or absence of CA treatment. As shown in Fig. 3A, the percentage of DO11.10 CD11b⁺TNF-⁺ and KJ1-26⁺TNF-⁺ double-positive cells was 2.4 and 7.8%, respectively, after OVA peptide stimulation. In the presence of CA, however, the percentage of DO11.10 CD11b⁺TNF-⁺ double-positive cells decreased slightly after peptide stimulation, whereas the percentage of DO11.10 KJ1-26⁺TNF-⁺ double-positive cells exhibited a 2-fold decrease (Fig. 3B). Interestingly, when we analyzed the MFI of DO11.10 CD11b⁺TNF-⁺ double-positive cells after OVA peptide stimulation, no significant differences were observed between the control (MFI 15,768) and CA (MFI 15,746)-treated APCs. However, there were stark contrasts observed with the MFI of DO11.10 KJ1-26⁺TNF-⁺ double-positive cells as the CA-treated T cells (MFI, 11,376) displayed a 2-fold increase in MFI as compared with the control-treated T cells (MFI 6271). Similar to the DO11.10 primary recall response, we also observed an increase in intracellular TNF- levels after OVA peptide stimulation of OT-II splenocytes. OT-II splenocytes demonstrated an increase in the percentage of double-positive CD11b⁺TNF-⁺ and CD4⁺TNF-⁺ cells of 3.8 and 6.4%, respectively (Fig. 3C), after OVA_323–339 stimulation. In the presence of the CA, there was a marginal decrease in the percentage of double-positive OT-II CD11b⁺TNF-⁺ cells and a 2-fold reduction of OT-II CD4⁺TNF-⁺ cells (Fig. 3D). Surprisingly, when the MFI of OT-II double-positive CD11b⁺TNF-⁺ and CD4⁺TNF-⁺ cells was analyzed, the CA-treated cells (Fig. 3D) all exhibited a 1.5-fold increase in MFI as compared with the control-treated cells (Fig. 3C). These results suggest that intracellular TNF- had accumulated in the CA-treated cells but was not released extracellularly like the control-treated cells (Fig. 1). Thus, blocking ROS production early during APC-T cell interactions (Fig. 2B) has a marked effect on the ability of the T cell to produce endogenous TNF-.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Intracellular TNF- levels are decreased in catalytic antioxidant-treated DO11.10 primary recall response. DO11.10 splenocytes were stimulated with 1 µM OVA323–339 for 4 h at 37°C in the absence (A) or presence of 34 µM CA (B). OT-II splenocytes were also stimulated similarly in the absence (C) or presence (D) of 34 µM CA. Intracellular TNF- FACS staining was performed according to the BD Pharmingen intracellular cytokine staining protocol. Results are representative of three independent experiments.

TNF- is necessary for enhancing effector cytokine synthesis in a DO11.10 and OT-II primary recall

To further delineate how CA-dependent reduction of TNF- expression mitigates the transition to effector function (i.e., IFN- production) in T cells, we recapitulated the inhibition of proinflammatory cytokine synthesis mediated by CA treatment with TNF- and IL-1 neutralization Abs in an OT-II and DO11.10 primary recall response. OT-II splenocytes stimulated with 1 µM OVA_323–339 in the presence of 5 µg/ml anti-TNF- Abs demonstrated a 3-fold decrease in IFN- synthesis (Fig. 4A). DO11.10 splenocytes also demonstrated a decrease in IFN- levels when treated with 5 µg/ml anti-TNF- Abs (Fig. 4B), but not nearly to the same extent as the OT-II splenocytes (Fig. 4A). Interestingly, IL-1 neutralization in both the OT-II and DO11.10 primary recall responses did not have any effect on the synthesis of IFN- (Fig. 4, A and B). Neutralization of both TNF- and IL-1 cytokines did not exhibit any additive effects on IFN- production in either the OT-II or DO11.10 primary recall responses (Fig. 4, A and B), suggesting that TNF- and not IL-1 is more important in maturing the effector response of both OT-II and DO11.10-transgenic T cells. Anti-TNF- Abs did not have any significant effects on proliferation (data not shown) or synthesis of IL-2 (Fig. 4, A and B) in OVA peptide-stimulated DO11.10 or OT-II splenocytes. As expected, the induction of TNF- in the DO11.10 or OT-II primary recall responses were ablated in the presence of TNF- neutralization Abs (Fig. 4, A and B).

View larger version (24K): [in this window] [in a new window]   FIGURE 4. TNF- neutralizing Abs diminishes IFN- synthesis in a DO11.10 and OT-II primary recall response. DO11.10 (A) and OT-II (B) primary recall with 2 x 105 splenocytes stimulated with 1 µM OVA323–339 in the presence or absence of 5 µg/ml isotype control, 5 µg/ml anti-TNF-, 5 µg/ml anti-IL-1, or both neutralization Abs. DO11.10 primary recall assay treated with 34 µM CA and stimulated with 1 µM OVA323–339 in the presence of 1 or 2.5 ng/ml of murine recombinant TNF- (C). Supernatants were harvested and the levels of cytokines produced in the primary recall responses were measured with cytokine-specific ELISAs. Results are representative of the mean (±SEM) of 3 independent experiments done in triplicate. **, p < 0.05 vs the respective control group.

In vivo redox modulation of mice with a catalytic antioxidant can generate Ag-specific hyporesponsiveness

The next question we addressed is whether in vivo redox modulation with CA treatment could affect the activation and expansion of a naive pool of TCR-transgenic T cells from DO11.10 and OT-II mice in an adoptive transfer model. As shown in Fig. 5A, an OT-II recall response from LN cells from CA-treated mice demonstrated a 2-fold decrease in IFN- synthesis without any significant effects on T cell proliferation or IL-2 synthesis. The decrease in IFN- synthesis mediated by CA treatment was not due to the absence of adoptively transferred OT-II T cells as CFSE-labeled T cells were readily identified in the LN (gated region P9; Fig. 5B) of C57BL/6 mice after immunization. Corroborating our in vitro results, OVA-immunized and CA-treated mice exhibited no significant difference in CFSE dilution profiles (compare gates P14–P22; (Fig. 5B) of adoptively transferred OT-II T cells in the LN after OVA peptide immunization as compared with HBSS-treated mice. Therefore, even though there were no differences in T cell proliferation after CA treatment in the draining LN, they did differ significantly in IFN- synthesis (Fig. 5A), suggesting that proliferation and effector function were uncoupled after CA treatment. The ability to induce Ag-specific hyporesponsiveness was not MHC specific given that CA were also effective in the DO11.10 TCR-transgenic mouse model. Following the same adoptive transfer protocol with purified DO11.10 T cells and BALB/c mice as recipients, the recall response of DO11.10 T cells after CA treatment differed from the OT-II recall response by displaying a significant decrease not only in IFN- synthesis but also in proliferation and IL-2 production (Fig. 5C).

View larger version (40K): [in this window] [in a new window]   FIGURE 5. Decrease in Ag-specific T cell proliferation and IFN- synthesis with in vivo CA treatment of adoptively transferred DO11.10 and OT-II T cells. Ag recall response of OT-II (A) purified T cells (5 x 106 cells) that were adoptively transferred into C57BL/6 mice and immunized with OVA323–339 in CFA in the presence or absence of CA (10 mg/kg of body weight). FACS of LN cells from C57BL/6 mice adoptively transferred with OT-II T cells after immunization with OVA323–339 in CFA with CD90.2-APC and CFSE (B). LN CFSE-dilution profiles were derived by gating CD90.2+ cells (as depicted in gate P9 in Fig. 5B) and then comparing CFSE intensity with forward scatter (FSC). Ag recall response of DO11.10 (C)-purified T cells (5 x 106 cells) that were adoptively transferred into BALB/c mice and immunized with OVA323–339 in CFA in the presence or absence of CA (10 mg/kg of body weight). Inguinal and periaortic LN cells were purified and used in an Ag recall assay to assess proliferation by [3H]TdR incorporation and cytokine synthesis by ELISA. , No Ag; , cells stimulated with Ag. Results are representative of the mean (±SEM) of four independent experiments done in triplicate. **, p < 0.05 vs the respective control group.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Decrease in Ag-specific T cell proliferation and IFN- synthesis with in vivo CA treatment of NOD and BALB/c mice. Ag recall response of NOD mice immunized with HEL (A) or BALB/c mice immunized with PCC (B) in CFA in the presence or absence of CA (10 mg/kg of body weight). Inguinal and periaortic LN cells were purified and used in an Ag recall assay to assess proliferation by [3H]TdR incorporation and cytokine synthesis by ELISA. , No Ag; , cells stimulated with Ag. Results are representative of the mean (±SEM) of four independent experiments done in triplicate. **, p < 0.05 vs the respective control group.

There is evidence that TNF- is important in mediating the pathogenic effects of diabetogenic T cell clones after adoptive transfer and the synthesis of this cytokine is notably absent in nonpathogenic T cell clones (34). TNF- neutralization in NOD neonatal mice resulted in a decrease in CD11c⁺CD11b⁺ DC maturation markers with a concomitant decrease in spontaneous diabetes, decrease in BDC-2.5 T cell proliferation after adoptive transfer, inactivation of islet-specific pancreatic LN T cells, increase in CD4⁺ CD25⁺ regulatory T cells, and the generation of immunological tolerance to islet cell proteins (35, 36, 37). These results suggest TNF- is important for mediating the pathogenic effects of the BDC-2.5 T cell clone; therefore, we postulated that inhibition of TNF- synthesis with a CA would inhibit efficient priming of BDC-2.5 T cells. As expected, immunization of BDC-2.5 TCR-transgenic mice with the BDC-2.5 mimotope in conjunction with CA treatment could also modulate a diabetogenic T cell response. The absence of the proinflammatory third signal mediated by CA treatment in vivo was sufficient in decreasing BDC-2.5 T cell proliferation by 3-fold and IFN- synthesis by 15-fold (Fig. 7).

View larger version (10K): [in this window] [in a new window]   FIGURE 7. CA treatment during BDC-2.5 mimotope immunization can decrease BDC-2.5 T cell proliferation and IFN- synthesis. Ag recall response of BDC-2.5 TCR-transgenic mice immunized with the BDC-2.5 mimotope in CFA in the presence or absence of CA (10 mg/kg of body weight). Inguinal and periaortic LN cells were purified and used in an Ag recall assay 7 days after immunization to assess proliferation by [3H]TdR incorporation and IFN- synthesis by ELISA. , No Ag; , cells stimulated with Ag. Results are representative of the mean (±SEM) of four independent experiments done in triplicate. **, p < 0.05 vs the respective control group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Regardless of which MAMP is used to activate the TLRs, a consequence of this activation is the induction of NF-B and MAPK signaling for immune response activation and the synthesis of proinflammatory cytokines such as TNF-, IL-1, IL-12 p70, and type I IFNs (40). Thus, TLR interaction with MAMP is critical for activating the innate immune response to provide the necessary third signal for coordinating a robust Ag-specific T cell response (26, 41), and in conjunction with Ag and IL-2, initiate the differentiation and effector function of naive T cells (2, 42). In addition to proinflammatory cytokines, ROS are also an important component of the third signal necessary for optimal activation of the adaptive immune system. ROS function as second messengers to induce proinflammatory cytokine synthesis (43, 44) by activating redox-sensitive signal transduction pathways such as NF-B and MAPK (9, 11, 45). The activation of innate immune-derived ROS and proinflammatory cytokine synthesis is critical for maturing the adaptive immune effector response and the synthesis of IFN-. Recognition of MAMP by pattern recognition receptors facilitates not only innate but also adaptive immune activation by promoting the maturation of immature dendritic cells due to TLR signaling (46).

This study significantly advances our understanding of the underlying mechanism of the proinflammatory third signal for mediating efficient CD4⁺ T cell effector function and specifically, the critical role of T cell-derived TNF- for transitioning from expansion to effector function. We hypothesized that diminishing the third signal (proinflammatory cytokines and ROS) during antigenic immunization in the presence of a potent adjuvant could efficiently hinder T cell priming and adaptive immune activation. To test this hypothesis, we used an Ag-specific immunization protocol that relied on modulating the redox state of the innate immune response through the use of CA (MnTE₂ and MnTDE) as an in vivo pharmacological tool during Ag immunization. Since we previously demonstrated the efficiency of CA in ablating the proinflammatory third signal (23), we explored the ability of these compounds to induce Ag-specific hyporesponsiveness in naive CD4⁺ T cells by disrupting the transition of T cell expansion to effector function.

Our results demonstrated Ag-specific hyporesponsiveness through the use of in vivo CA treatment to modulate the redox state in wild-type (BALB/c), autoimmune prone (NOD), TCR-transgenic (DO11.10, OT-II), and diabetogenic TCR-transgenic mice (BDC-2.5), with four different immunizing Ags in CFA (HEL, PCC, OVA_323–339, and BDC-2.5 mimotope, respectively). Modulating the redox state in all mice uncoupled proliferative capacity (IL-2 synthesis) from T cell effector function (IFN- synthesis) as assessed by significant decreases in T cell proliferation and IFN- synthesis in a secondary Ag recall response, much like depriving Ag-stimulated naive T cells of the proinflammatory third signal (8, 47). Flow cytometric analysis of a DO11.10 primary recall response with a H₂O₂-specific redox-sensitive fluorogenic probe (CM-H₂DCFDA) resulted in a 5-fold increase in the percentage of double-positive CD11b⁺CM-H₂DCFDA⁺ cells that was also corroborated with a 1.5-fold increase in the MFI of CM-H₂DCFDA oxidation in comparison with unstimulated cells. Conversely, the DO11.10 primary recall response in the presence of the CA only generated a 1.5-fold increase in the percentage of double-positive CD11b⁺CM-H₂DCFDA⁺ cells and a negligible increase in the MFI of CM-H₂DCFDA oxidation. Not only did APCs initiate a respiratory burst upon antigenic stimulation but our CM-H₂DCFDA studies also corroborated previous reports of T cells containing a functional NADPH oxidase complex (17, 18) because the T cells in the DO11.10 primary recall response were also capable of oxidizing CM-H₂DCFDA. Upon OVA peptide stimulation, there was 1.6-fold increase in the percentage of KJ1-26⁺CM-H₂DCFDA⁺ double-positive cells, but more importantly, the MFI of CM-H₂DCFDA oxidation had increased 6.5-fold in comparison with unstimulated T cells. CA treatment also inhibited T cell-derived ROS production in the DO11.10 recall response. The percentage of KJ1-26⁺/CM-H₂DCFDA⁺ double-positive cells only increased 0.5-fold, and the MFI of CM-H₂DCFDA oxidation was barely detected in comparison with unstimulated T cells.

We hypothesize that early in an immune response, APC and T cells undergo a respiratory burst to initiate APC-T interactions and/or to facilitate their engagement. Upon interaction, T cells produce TNF- to further shape the activation of the immune response by recruiting and arming adaptive immune effector cells by synthesizing chemokines and adhesion molecules. Evidence for this dependence on TNF- for T cell transition to effector function has been demonstrated in models of superantigen-induced expansion, where CD28^–/– animals stimulated with superantigens demonstrated an inability to expand and, more importantly, make the effector cytokine IFN- (48, 49). Administration of exogenous TNF- in vivo restored superantigen-induced IFN- synthesis, pointing to the need for the production of this cytokine by the responding T cells to transition to effector function. Our TNF- intracellular cytokine staining studies demonstrate that in both OVA peptide-stimulated DO11.10 and OT-II primary recall responses, the percentage of KJ1-26⁺TNF-⁺ and CD4⁺TNF-⁺ double-positive cells increased to 7.8 and 6.4%, respectively. Interestingly, CA-treated KJ1-26⁺TNF-⁺ and CD4⁺TNF-⁺ cells exhibited a 2-fold decrease in the percentage of double-positive cells but displayed a 2-fold increase in intracellular TNF- MFI. These results suggest the exciting possibility that CA-treatment may be affecting the activity of TNF- convertase (TACE), an enzyme necessary for TNF- secretion (50, 51). ROS have a role in activating TACE activity and the shedding of TNF p75 receptor in both lymphocytes and monocytic cells (52) by oxidizing the inhibitory prodomain of TACE at a critical cysteine residue causing the release of the prodomain from the catalytic domain. We are currently planning experimental studies to address whether CA treatment prevents the oxidation of the inhibitory prodomain of TACE and thereby retaining an elevated level of intracellular TNF-.

TNF- is a proinflammatory cytokine with an important function in immune development, activation, and inflammation. Our results confirm and extend earlier observations (35, 37) that for efficient effector function of diabetogenic T cells, a proinflammatory third signal is necessary for endogenous TNF- production by the responding T cell. The importance of TNF- in T1D development with diabetogenic T cell clones was recently described (34); Cantor and Haskins used an ex vivo protocol for measuring TNF- levels produced by T cells. The level of TNF- synthesis correlated with the extent of diabetogenicity for the T cell clones including BDC-2.5. T cell clones that did not express TNF- were incapable of inducing diabetes. The importance of TNF- for driving diabetogenesis may not be merely due to its conventional effects during inflammation, but more specifically as a proinflammatory third signal for transitioning CD4⁺ and CD8⁺ T cells to effector function (2, 3).

The current study further expands on the importance of TNF- inhibition mediated by CA treatment with naive antigenic T cell responses. The use of TNF- neutralization Abs in DO11.10 and OT-II T primary recall assays were capable of inhibiting T cell transition to effector function similar to CA treatment. Although neutralization of TNF- significantly decreased the ability of DO11.10 and OT-II T cells to synthesize IFN-, the treatment was unable to completely abrogate its production. We hypothesize that other third signal proinflammatory cytokines unaffected by CA treatment, like IL-12 p70, were able to compensate but not with the same efficiency for the loss in TNF- for adaptive immune maturation in these primary recall responses. Interestingly, neutralization of IL-1 did not interfere with the ability of DO11.10 or OT-II T cells to gain adaptive immune effector function, suggesting that at least in these two transgenic TCR T cells, IL-1 is not important for IFN- synthesis. These results are in slight disagreement with previous work demonstrating that a danger signal, such as IL-1 and IL-12, is required for CD4⁺ and CD8⁺ effector function, respectively (2). These earlier studies used IL-1 to stimulate CD4⁺ T cell activation with MHC protein-peptide complexes on microspheres as artificial APC (3, 32), whereas in our studies we focused on IL-1 only and used professional APC derived from OT-II or DO11.10 mice that were capable of undergoing a respiratory burst, unlike MHC protein-peptide microspheres. It is possible that other proinflammatory cytokines could provide the third signal for transitioning to adaptive immune effector function, such as IL-23, IL-27, or IL-18. We are currently looking at the role of these cytokines as potential third signals and whether CA treatment can also abolish their synthesis.

To further demonstrate the importance of CA-mediated suppression of TNF- synthesis in adaptive immune maturation, the reciprocal experiment was performed whereby recombinant TNF- was added back to CA-treated DO11.10 splenocytes and the ability of these T cells to synthesize IFN- was explored. rTNF- restored IFN- synthesis to CA-treated DO11.10 splenocytes, but the increase was only 2-fold when stimulated with 1 ng/ml TNF- and was still 3-fold less than with OVA peptide-stimulation alone. Exogenous addition of 2.5 ng/ml TNF- also restored IFN- synthesis in CA-treated T cells, but this increase was only 1.5-fold, and cells stimulated with this higher concentration appeared to display some cytotoxicity as the levels of IFN- from 2.5 ng/ml TNF- and OVA peptide costimulated samples were almost 2-fold lower than stimulation with OVA peptide only. The ability of rTNF- to only partially rescue the IFN- synthesis defect in CA-treated DO11.10 splenocytes suggests the importance of other third signal proinflammatory mediators still suppressed by CA treatment that are important in adaptive immune effector function. This experiment further illustrates the importance of ROS as a direct proinflammatory third signal and its role in temporally and kinetically activating proinflammatory cytokine synthesis. We would suggest that exogenously adding a robust proinflammatory cytokine such as TNF- is not a true physiological measure of how this cytokine functions as a proinflammatory third signal. The synthesis and interaction of this cytokine with its receptor is tightly regulated and must be synchronized for proper immune stimulation without causing cytotoxicity (53).

CA treatment had no effect on the levels of IL-2 in both the primary and secondary recall responses, suggesting that redox modulation of an immune response with CA treatment uncouples the expansion phase (IL-2 synthesis) from the effector phase (IFN- synthesis) of an Ag-specific T cell response, by affecting the proinflammatory third signal necessary for adaptive immune function. The uncoupling of expansion and effector function from Ag-specific T cells was also described by Pape et al. (3). Their work demonstrated that immunization of mice in the absence of a proinflammatory adjuvant resulted in a significant decrease in IFN- synthesis from adoptively transferred DO11.10 T cells into BALB/c mice even though they were capable of undergoing expansion in vivo. What is striking about our work is that in the presence of a very potent adjuvant, we are still capable of significantly inhibiting the transition to effector function by modulating the redox state during innate immune activation.

In addition to IL-2 synthesis, there was also no difference in IL-12 p70 levels after CA treatment with the DO11.10 primary recall suggesting that the decrease in IFN- synthesis was not due to suppression of IL-12 signaling, but downstream at the Th1-specific T box transcription factor (T-bet) and/or Stat4 signaling pathways. We have preliminary evidence demonstrating that CA treatment of DO11.10 splenocytes result in a decrease in Stat4 phosphorylation that may explain the decrease in IFN- synthesis (H. M. Tse and J. D. Piganelli, unpublished observations), but studies are ongoing to determine whether redox modulation may also affect the T-bet signaling pathway, another pathway necessary for IFN- synthesis (54, 55, 56). Interestingly, there was a difference in IL-12 p70 levels in the CA-treated OT-II splenocytes; they exhibited a 2-fold decrease in IL-12 p70 as compared with control-treated OT-II splenocytes. Whether or not the decrease in IL-12 p70 expression can explain the decrease in IFN- synthesis in OT-II mice is not known, but we are currently exploring any potential differences in the IL-12, Stat4, and T-bet signaling pathways demonstrated with the OT-II, but not the DO11.10 recall response.

Targeting the innate immune response by modulating the redox state as a means of suppressing the adaptive immune response may hold promise as a new avenue of immunotherapy because this strategy can be utilized in conjunction with Ag-specific treatment to efficiently decrease innate immune-derived proinflammatory mediators leading to ablation of Ag-responding T cell populations. The ability to induce Ag-specific hyporesponsiveness with various mouse strains by modulating the redox state suggests the importance of reduction-oxidation reactions in activating the immune response. Current immunosuppressive therapies to treat T1D such as anti-CD3 mAb (57) target the adaptive immune response and the ablation of T cells, but not without adverse effects (58). CA are novel anti-inflammatory agents that may be used in conjunction with other classical immunosuppressants at a significantly lower toxic dose for curtailing adaptive immune effector function. Our future studies will further characterize how redox modulation by CA treatment can suppress IFN- synthesis in T cells by affecting Stat4 and T-bet pathway activation and determine whether Ag-specific hyporesponsiveness mediated by CA-treatment is global or Ag-specific in models of type 1 diabetes.

	Acknowledgments

 
We thank Robert Lakomy and Alexis Styche for excellent technical assistance and Drs. Henry Dong and Nick Giannoukakis for critical reading of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by a Cochrane-Weber research award (to H.M.T.), a Research Advisory Council Postdoctoral Fellowship Award by Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center Health System (to H.M.T.), a Research Advisory Council Award (to J.P.), an American Diabetes Association Junior Faculty Award (to J.P.), and a Juvenile Diabetes Research Foundation Research Grant (to J.P.).

² Address correspondence and reprint requests to Dr. Jon D. Piganelli, Diabetes Institute, Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA 15213-3205. E-mail address: jdp51{at}pitt.edu

³ Abbreviations used in this paper: LN, lymph node; CA, catalytic antioxidant; ROS, reactive oxygen species; MnTDE, Mn(III) 5,10,15,20-tetrakis(N,N'-diethylimidazolium-2-yl)porphyrin; MnTE₂, Mn(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2- yl)porphyrin; DC, dendritic cell; HEL, hen egg lysozyme; PCC, pigeon cytochrome c; IHC, immunohistochemistry; RA, rheumatoid arthritis; CM-H₂DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; MAMP, microbial-associated molecular pattern; MFI, mean fluorescence intensity; TACE, TNF- convertase; T-bet, Th1-specific T box transcription factor.

Received for publication April 6, 2006. Accepted for publication October 27, 2006.

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