Disruption of Innate-Mediated Proinflammatory Cytokineand Reactive Oxygen Species Third Signal Leads toAntigen-Specific Hyporesponsiveness1

Hubert M. Tse, Martha J. Milton, Sheila Schreiner, Jennifer L. Profozich, Massimo Trucco, andJon D. Piganelli2

Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interactingwith MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflam-matory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptiveimmune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innateimmune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with acatalytic 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 effectorfunction 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 andIFN-� synthesis by diminishing ROS production in the APC, which affected TNF-� levels produced by CD4� T cells and impairingeffector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as auto-immune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4� T cells totransition from expansion to effector function. The Journal of Immunology, 2007, 178: 908–917.

T he adaptive immune response is the result of an interac-tion of receptor-expressing lymphocytes and APCs to for-eign immunogenic peptides. Efficient activation of the T

cell adaptive immune response requires: 1) signal 1 consisting ofTCR:MHC molecular interactions stabilized by a peptide bound tothe MHC; 2) signal 2, the interaction of costimulatory moleculeson T cells and APC (1); and 3) signal 3, which consists of proin-flammatory signals mediated by the adjuvant properties of CFA,LPS, or other microbial products (2, 3). The proinflammatory thirdsignal is important for inducing, enhancing, and prolonging theAg-specific proliferative response in T cells (3, 4). Previous stud-ies have demonstrated that immunization with a foreign Ag in theabsence of a third signal results in unresponsiveness upon subse-quent immunizations with Ag and adjuvant (5, 6). The respondingT cells will proliferate in draining lymph nodes (LN)3 but will

become anergic or tolerant and incapable of recognizing the im-munogenic 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 cellsto class switch to the IgG isotype Ab (3).

The third signal relies on the production of reactive oxygenspecies (ROS), which fuels the generation of the proinflammatorycytokines necessary for the linkage of innate to adaptive immunityand Ag-specific T cell activation (9, 10). ROS promotes proin-flammatory cytokine production from APCs by the activation ofredox-sensitive signal transduction pathways such as MAPK,AP-1, and NF-�B (10–14), which control the expression of theinnate proinflammatory immune response, cellular proliferation,and apoptosis (11, 15). Intracellular ROS levels are elevated in Tcells and dendritic cells (DC) during Ag recognition (16). Stimu-lation of T cells with anti-CD3 and anti-CD28 Ab can rapidlygenerate superoxide and hydrogen peroxide for mediating activa-tion-induced cell death and proliferation by activating Fas ligandand the ERK pathway, respectively (17, 18).

We hypothesized that strategies that inhibit the proinflammatorythird signal would allow for the specific control of T cell transitionfrom expansion to effector function. To achieve this goal we usedthe previously described catalytic antioxidants (CA), (Mn(III) me-sotetrakis(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 pro-duction allowing its use as an immunomodulatory reagent (19–22). Our previous work has shown that these compounds decrease

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

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

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 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 Re-search Advisory Council Postdoctoral Fellowship Award by Children’s Hospital ofPittsburgh of the University of Pittsburgh Medical Center Health System (to H.M.T.),a Research Advisory Council Award (to J.P.), an American Diabetes AssociationJunior Faculty Award (to J.P.), and a Juvenile Diabetes Research Foundation Re-search Grant (to J.P.).2 Address correspondence and reprint requests to Dr. Jon D. Piganelli, Diabetes In-stitute, Division of Immunogenetics, Department of Pediatrics, Children’s Hospital ofPittsburgh, University of Pittsburgh, Pittsburgh, PA 15213-3205. E-mail address:jdp51@pitt.edu3 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�-diethylimidazo-lium-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 cytochromec; IHC, immunohistochemistry; RA, rheumatoid arthritis; CM-H2DCFDA, 5-(and-6)-

chloromethyl-2�,7�-dichlorodihydrofluorescein diacetate; MAMP, microbial-associ-ated molecular pattern; MFI, mean fluorescence intensity; TACE, TNF-� convertase;T-bet, Th1-specific T box transcription factor.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

The Journal of Immunology

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the innate immune response in LPS-stimulated bone marrow-de-rived macrophages and DCs due to their abilities to function asoxidoreductases (23). Depending on the redox environment, theCA are able to oxidize redox-sensitive transcription factors such asNF-�B to inhibit DNA binding (23). Furthermore, CA treatmentprotected NOD.scid recipients from adoptive transfer with the di-abetogenic BDC-2.5 T cell clone (24). We demonstrated that CAdirectly inhibited APC effector function (TNF-� and ROS synthe-sis) and suppressed APC-dependent T cell proliferation and IFN-�production of the BDC-2.5 T cell clone. The ability of CA tomodulate redox function and suppress TNF-� production demon-strates that this strategy of redox alteration inhibits the third signal-dependent linkage of the innate and adaptive immune response ina robust model of Type 1 diabetes. Given the role of ROS inmediating signaling events necessary for synergizing the innatewith the adaptive immune response (25, 26), we hypothesized thatCA directly or indirectly modulate redox-dependent signalingpathways that are essential for initiating the innate immune re-sponse (23). In this report, we demonstrate the efficacy of proin-flammatory third signal disruption to markedly reduce Ag-specificT cell TNF-� production, which appears to negatively impact re-sponsiveness, expansion, and effector function of Ag-specific Tcells.

Materials and MethodsMaterials

NOD, DO11.10 (27), OT-II (28), and BDC-2.5 TCR-transgenic (29) micewere bred and housed under specific pathogen-free conditions in the An-imal Facility of the Rangos Research Center in the University of Pittsburgh(Pittsburgh, PA). BALB/c and C57BL/6 mice were purchased from TheJackson Laboratory. Female mice at 6 to 8 wk of age were used in allexperiments. 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 werepurchased from BD Pharmingen. MnTDE and MnTE2, generous gifts fromIncara Pharmaceuticals, were resuspended in HBSS with Ca2� and Mg2�

(Invitrogen Life Technologies) at a stock concentration of 2 mM and filtersterilized (0.2 �m) before use. The working concentration chosen forMnTDE and MnTE2 in all in vitro experiments was 34 �M, after consult-ing with Dr. Joe McCord (Webb-Waring Institute, Denver, CO; unpub-lished observations) that this concentration of CA was necessary to reca-pitulate the native superoxide dismutase 2 antioxidant activity. Sustainedrelease pellets of MnTDE (3.6 mg/kg/day) were synthesized from Innova-tive Research of America. Chicken OVA323–339 (ISQAVHAAHAEINEAGR) and the BDC-2.5 mimotope (EKAHRPIWARMDAKK)were synthesized by Sigma Genosys. Hen egg lysozyme (HEL) and pigeoncytochrome c (PCC) were purchased from Sigma-Aldrich. Recombinantmurine TNF-� was purchased from R&D Systems.

Measurement of intracellular ROS

The determination of intracellular oxidant formation was based on the ox-idation of 5-(and-6)-chloromethyl-2�,7�-dichlorodihydrofluorescein diac-etate (CM-H2DCFDA; Molecular Probes). CM-H2DCFDA was preparedin DMSO immediately before loading by making a 1 mM stock solution.The measurement of intracellular ROS was measured in a DO11.10 pri-mary recall assay by making single-cell splenocyte suspensions and pre-treating with or without 34 �M MnTDE for 1 h at 37°C. The splenocyteswere washed three times with PBS, resuspended in FACS buffer (1% heat-inactivated FCS in PBS), and loaded with 10 �M CM-H2DCFDA for 30min at 37°C. The cells were washed twice and then stimulated with 1 �MOVA323–339 for 60, 90, and 120 min at 37°C. Fifteen minutes before theend of the time point, the cells were stained with KJ1-26 APC-conjugatedand CD11b PE-conjugated Abs for distinguishing between the T cell andAPC populations. Cell acquisition (excitation 480 nm; emission 520 nm)was performed on a FACSCalibur (BD Biosciences) and data was analyzedwith BD FACSDiva software version 4.0.1 (BD Biosciences).

Flow cytometric analysis

Cells were washed twice in FACS buffer, counted, and resuspended in afinal concentration of 2 � 107 cells/ml in FACS buffer. Then 106 cells werestained with directly fluorochrome-conjugated Ab at the appropriate dilu-

tion (10 �l of each Ab) for 30 min in the dark at 4°C in FACS buffer, andfluorescence was measured.

Adoptive transfer of T cells

DO11.10 or OT-II T cells were negatively selected and purified fromsplenocytes by using a MACS mouse pan T cell isolation kit (MiltenyiBiotec) according to the manufacturer’s protocol. The purity of T cellsobtained from this kit was consistently �95% as determined by FACSanalysis for KJ1-26�, CD4�, and CD3� cell surface markers. To trackadoptively transferred cells in vivo, T cells were labeled with CFSE byfollowing a previously described protocol (31). Briefly, purified T cellswere suspended in PBS, pH 7.0, at a concentration of 5 � 107 cells/ml andlabeled with 5 �M CFSE for 15 min in a 37°C water bath. The T cells werewashed in HBSS and adjusted to a concentration of 5 � 107 cells/ml, and0.1 ml of cells (5 � 106 cells) was transferred into the retro-orbital vein.

Antigenic immunization of mice

Several hours before immunization, mice were divided into two groups andtreated i.p. with CA (MnTE2 or MnTDE; 10 mg/kg of body weight) orHBSS or implanted at the nape of the neck with sustained release pellets ofMnTDE (3.6 mg/kg of body weight/day; Innovative Research of America)or placebo using a trochar device. The mice were injected with 100 �g ofHEL, 100 �g of PCC, 150 �g of OVA323–339, or 25 �g of the BDC-2.5mimotope in CFA s.c. at the base of the tail. After immunization, micewere injected i.p. daily for 7 days with CA (10 mg/kg) or HBSS. On day8, mice were sacrificed, and the inguinal and periaortic LNs were harvestedfor 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 splenocytesingle-cell suspensions by seeding in 96-well flat-bottom plates with 25 �gof HEL, 25 �g of PCC, or (0.1 �M or 1 �M) OVA323–339 in 200 �l totalvolume of DMEM supplemented with 10% heat-inactivated FCS, 10 mMHEPES buffer, 4 mM L-glutamine, 2� nonessential amino acids, 1 mMsodium pyruvate, 61.5 �M 2-ME, and 100 �g/ml Gentamicin (InvitrogenLife Technologies; complete DMEM). The DO11.10 or OT-II primary re-call assays were performed with splenocyte single-cell suspensions in thepresence or absence of 34 or 68 �M CA and 1 �M OVA323–339 in completeDMEM. After incubation at 37°C in a 5% CO2 humid air chamber for 2,3, or 4 days, the cells were pulsed with 1 �Ci of [3H]TdR for 18 h and thenharvested onto glass fiber filters with a sample harvester. The amount ofincorporated counts was determined using a beta scintillation counter.

Cytokine measurements by ELISA and intracellularcytokine staining

IL-2 and IFN-� cytokines produced in the supernatants of the various Agrecall assays were measured using Ab pairs from BD Pharmingen. IL-12p70, TNF-�, and IL-1� were detected with DuoSet ELISA kits (R&DSystems). ELISA plates were read on a Benchmark microplate reader (Bio-Rad), and data was analyzed using DeltaSoft (Molecular Devices). Intra-cellular TNF-� was measured with 1.2 � 107 DO11.10 or OT-II spleno-cytes in a primary recall response in the presence or absence of 68 �MMnTE2 for 4 h at 37°C in a 5% CO2 humid air incubator with the aid ofthe murine BD intracellular cytokine staining kit (BD Biosciences). Afterstimulation, splenocytes were fixed in BD Cytofix/Cytoperm buffer,washed in BD Perm/Wash buffer, and then stained with R-PE-conjugatedrat anti-TNF-� (MP6-XT22; BD Biosciences) and isotype controls. Cellswere washed twice in BD Perm/Wash buffer and resuspended in FACSbuffer; 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 recallwas performed with 5 �g/ml final concentration of purified rat anti-mouseTNF-� (Clone MP6-XT3, BD Pharmingen) and/or purified hamster anti-mouse IL-1� (clone B122; BD Pharmingen) Abs. Splenocyte single-cellsuspensions were plated at 2.5 � 105 cells/well and stimulated with 1 �MOVA323–339 in the presence of TNF-�, IL-1� neutralization Abs, or isotypecontrols (rat IgG1 or hamster IgG; BD Pharmingen) in complete DMEM in96-well flat-bottom plates. After incubation at 37°C in a 5% CO2 humid airchamber for 2, 3, or 4 days, supernatants were harvested for cytokineanalysis by ELISA, and cells were pulsed with [3H]TdR to assess prolif-eration as described above. The purified rat anti-mouse TNF-� (cloneMP6-XT3; BD Pharmingen) Ab did not interfere with TNF-� detectionwhen assayed with the R&D DuoSet TNF-� ELISA, given that titratingconcentrations (0, 0.625, 1.25, 2.5, 5, and 10 �g/ml) of neutralization Ab

909The Journal of Immunology

did not significantly affect the detection of a known TNF-� standard con-centration (data not shown).

Exogenous addition of recombinant murine TNF-� toCA-treated DO11.10 primary recall assay

DO11.10 splenocyte single-cell suspensions were plated out at 2.5 � 105

cells/well in a 96-well flat-bottom plate and stimulated with 1 �MOVA323–339 in the presence or absence of 34 �M CA. Recombinant murineTNF-� (R&D Systems) at a concentration of 1 or 2.5 ng/ml was exog-enously 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 cyto-kine analysis by ELISA as described above in Cytokine measurements byELISA and intracellular cytokine staining.

Statistical analysis

Determination of the difference between mean values for each experimen-tal group was assessed by Student’s t test, with p � 0.05 considered sig-nificant. All experiments were performed at least three separate times withdata obtained in triplicate wells in each experiment.

ResultsCatalytic antioxidants decrease TNF-� synthesis in a DO11.10and OT-II in vitro primary recall resulting in suppression ofeffector cytokine production

Recent evidence has demonstrated the importance of a third signalconsisting of innate immune-derived proinflammatory cytokinessuch as TNF-�, IL-1�, and IL-12 p70 in maturing the adaptiveimmune effector response of CD4 and CD8 T cells for IFN-� syn-thesis (2–4, 8, 32, 33). The levels of TNF-�, IL-1�, and IL-12 p70proinflammatory third signal cytokines produced in a DO11.10 andOT-II primary recall response was analyzed by ELISA afterOVA323–339-stimulation. As shown in Fig. 1, TNF-� was detectedin both DO11.10 and OT-II primary recall responses. In the pres-ence of CA, TNF-� levels significantly decreased 2-fold. In alltime points assayed, IL-1� was not detected in either the DO11.10or OT-II primary recall (data not shown), and the levels of IL-12p70 were not significantly different with the DO11.10 splenocyteswhen treated with or without CA (Fig. 1A). The OT-II primaryrecall did exhibit a 2-fold decrease in IL-12 p70 synthesis in thepresence of CA (Fig. 1B). T cell proliferation and IFN-� synthesiswas significantly diminished after CA treatment of OVA peptide-stimulated DO11.10 and OT-II splenocytes (Fig. 1), but interest-ingly, IL-2 levels were not altered by redox modulation. In thepresence of high levels of IL-2, CA-treated DO11.10 and OT-II Tcells were refractory to the effects of IL-2 and exhibited a signif-icant decrease in T cell proliferation.

Proinflammatory third signal ROS are derived from APCs andT cells during antigenic stimulation of DO11.10 splenocytes

To demonstrate that during Ag recognition proinflammatory thirdsignal ROS are generated and enhance the adaptive immune re-sponse, DO11.10 splenocytes were stimulated with OVA323–339

and ROS formation was assessed by measuring the oxidation ofthe H2O2-specific fluorogenic probe CM-H2DCFDA by FACSanalysis (Fig. 2). Surface staining of DO11.10 splenocytes with Tcell-specific (KJ1-26; Fig. 2A) and APC-specific (CD11b) Abs(Fig. 2B) demonstrated that both cell types synthesized ROS in theprimary recall response. After OVA323–339 stimulation, there wasa 1.6-fold increase in double-positive KJ1-26�/CM-H2DCFDA�

cells (Fig. 2A) and similarly, a 5-fold increase in the percentage ofCD11b�/CM-H2DCFDA� double-positive cells (Fig. 2B). Inter-estingly, CA treatment of OVA peptide-stimulated DO11.10splenocytes did not have a profound effect on the percentage ofdouble-positive KJ1-26�/CM-H2DCFDA� cells because they stilldemonstrated 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 a1.5-fold increase after stimulation. In addition to an increase in thepercentage of CD11b�/CM-H2DCFDA� and KJ1-26�/CM-H2DCFDA� double-positive cells after OVA peptide stimulation,

FIGURE 1. IFN-� synthesis is inhibited in a DO11.10 and OT-II pri-mary recall by modulation of the redox state with a catalytic antioxidant.DO11.10 (A) or OT-II (B) splenocytes (5 � 105 cells) were stimulated with1 �M OVA323–339 in the presence or absence of 34 �M catalytic antiox-idant for 48 and 72 h. Supernatants were harvested, and the levels ofcytokines produced in the primary recall responses were measured withcytokine-specific ELISAs. Proliferation was determined by measuring[3H]TdR incorporation. f, No Ag; �, cells stimulated with Ag. Results arerepresentative of the mean (�SEM) of four independent experiments donein triplicate. ��, p � 0.05 vs the respective control group.

910 REDOX MODULATION OF INNATE IMMUNE PROINFLAMMATORY SIGNAL 3

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 theMFI of CM-H2DCFDA oxidation (Fig. 2B), and CA-treated KJ1-26� cells exhibited MFIs that were barely above unstimulatedsamples (Fig. 2A).

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

To determine whether reduced ROS levels in CA-treated APC andT cells was responsible for diminished TNF-� synthesis in theprimary recall response in Fig. 1, TNF-� intracellular cytokinestaining was measured with DO11.10 and OT-II splenocytes afterOVA323–339 stimulation in the presence or absence of CAtreatment. As shown in Fig. 3A, the percentage of DO11.10

CD11b�TNF-�� and KJ1-26�TNF-�� double-positive cells was2.4 and 7.8%, respectively, after OVA peptide stimulation. In thepresence of CA, however, the percentage of DO11.10CD11b�TNF-�� double-positive cells decreased slightly afterpeptide 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 DO11.10CD11b�TNF-�� double-positive cells after OVA peptide stimu-lation, no significant differences were observed between the con-trol (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 thecontrol-treated T cells (MFI 6271). Similar to the DO11.10 pri-mary recall response, we also observed an increase in intracellularTNF-� levels after OVA peptide stimulation of OT-II splenocytes.OT-II splenocytes demonstrated an increase in the percentage ofdouble-positive CD11b�TNF-�� and CD4�TNF-�� cells of 3.8and 6.4%, respectively (Fig. 3C), after OVA323–339 stimulation. Inthe presence of the CA, there was a marginal decrease in the per-centage of double-positive OT-II CD11b�TNF-�� cells and a

FIGURE 2. DO11.10 splenocytes produce ROS upon stimulation withOVA323–339 and are suppressed by pretreatment with a CA. DO11.10splenocytes were preloaded with 10 �M CM-H2DCFDA for 30 min at37°C and then stimulated with 1 �M OVA323–339 for 60 min. The gener-ation of ROS by these cells was determined by the presence of oxidizedCM-H2DCFDA by FACS (excitation, 480 nm; emission, 520 nm) alongwith surface staining for KJ1-26 (A) or CD11b (B) expression with directlyfluorochrome-labeled Abs. Results are representative of three independentexperiments.

FIGURE 3. Intracellular TNF-� levels are decreased in catalytic anti-oxidant-treated DO11.10 primary recall response. DO11.10 splenocyteswere stimulated with 1 �M OVA323–339 for 4 h at 37°C in the absence (A)or presence of 34 �M CA (B). OT-II splenocytes were also stimulatedsimilarly in the absence (C) or presence (D) of 34 �M CA. IntracellularTNF-� FACS staining was performed according to the BD Pharmingenintracellular cytokine staining protocol. Results are representative of threeindependent experiments.

911The Journal of Immunology

2-fold reduction of OT-II CD4�TNF-�� cells (Fig. 3D). Surpris-ingly, 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 thecontrol-treated cells (Fig. 3C). These results suggest that intracel-lular TNF-� had accumulated in the CA-treated cells but was notreleased 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 produceendogenous TNF-�.

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

To further delineate how CA-dependent reduction of TNF-� ex-pression mitigates the transition to effector function (i.e., IFN-�production) in T cells, we recapitulated the inhibition of proin-flammatory cytokine synthesis mediated by CA treatment withTNF-� and IL-1� neutralization Abs in an OT-II and DO11.10primary recall response. OT-II splenocytes stimulated with 1 �MOVA323–339 in the presence of 5 �g/ml anti-TNF-� Abs demon-strated a 3-fold decrease in IFN-� synthesis (Fig. 4A). DO11.10splenocytes also demonstrated a decrease in IFN-� levels whentreated with 5 �g/ml anti-TNF-� Abs (Fig. 4B), but not nearly tothe same extent as the OT-II splenocytes (Fig. 4A). Interestingly,IL-1� neutralization in both the OT-II and DO11.10 primary recallresponses did not have any effect on the synthesis of IFN-� (Fig.4, A and B). Neutralization of both TNF-� and IL-1� cytokines didnot exhibit any additive effects on IFN-� production in either theOT-II or DO11.10 primary recall responses (Fig. 4, A and B),suggesting that TNF-� and not IL-1� is more important in matur-ing the effector response of both OT-II and DO11.10-transgenic Tcells. Anti-TNF-� Abs did not have any significant effects on pro-liferation (data not shown) or synthesis of IL-2 (Fig. 4, A and B) inOVA peptide-stimulated DO11.10 or OT-II splenocytes. As ex-pected, the induction of TNF-� in the DO11.10 or OT-II primaryrecall responses were ablated in the presence of TNF-� neutral-ization Abs (Fig. 4, A and B).

To further demonstrate that CA-mediated suppression of TNF-�prevents efficient IFN-� synthesis, murine recombinant TNF-�was titrated into a CA-treated DO11.10 primary recall response inhopes of rescuing the adaptive immune effector response. As dem-onstrated in Fig. 4C, CA-treated DO11.10 splenocytes exhibited asignificant suppression in IFN-� production as compared withOVA peptide-stimulated cells alone. The addition of 1 and 2.5ng/ml TNF-� to CA-treated DO11.10 splenocytes increased IFN-�levels by 2- and 1.5-fold, respectively, but not to the extent ofOVA peptide stimulation alone. Costimulation with 1 ng/mlTNF-� and OVA peptide did not exhibit any defects in IFN-�synthesis given that the levels were similar to OVA peptide stim-ulation alone. However, costimulation with 2.5 ng/ml TNF-�and OVA peptide generated IFN-� levels that were almost2-fold lower than OVA peptide stimulation alone. Interestingly,the addition of TNF-� did not affect IL-2 synthesis in theDO11.10 primary recall response, but not surprisingly, TNF-�levels were increased (Fig. 4C).

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

The next question we addressed is whether in vivo redox modu-lation with CA treatment could affect the activation and expansionof a naive pool of TCR-transgenic T cells from DO11.10 andOT-II mice in an adoptive transfer model. As shown in Fig. 5A,an OT-II recall response from LN cells from CA-treated micedemonstrated a 2-fold decrease in IFN-� synthesis without any

significant effects on T cell proliferation or IL-2 synthesis. Thedecrease in IFN-� synthesis mediated by CA treatment was notdue to the absence of adoptively transferred OT-II T cells asCFSE-labeled T cells were readily identified in the LN (gated re-gion P9; Fig. 5B) of C57BL/6 mice after immunization. Corrob-orating our in vitro results, OVA-immunized and CA-treated miceexhibited no significant difference in CFSE dilution profiles (com-pare gates P14–P22; (Fig. 5B) of adoptively transferred OT-II Tcells in the LN after OVA peptide immunization as compared withHBSS-treated mice. Therefore, even though there were no differ-ences in T cell proliferation after CA treatment in the draining LN,

FIGURE 4. TNF-� neutralizing Abs diminishes IFN-� synthesis in aDO11.10 and OT-II primary recall response. DO11.10 (A) and OT-II (B)primary recall with 2 � 105 splenocytes stimulated with 1 �M OVA323–339

in the presence or absence of 5 �g/ml isotype control, 5 �g/ml anti-TNF-�,5 �g/ml anti-IL-1�, or both neutralization Abs. DO11.10 primary recallassay treated with 34 �M CA and stimulated with 1 �M OVA323–339 in thepresence of 1 or 2.5 ng/ml of murine recombinant TNF-� (C). Supernatantswere harvested and the levels of cytokines produced in the primary recallresponses were measured with cytokine-specific ELISAs. Results are rep-resentative of the mean (�SEM) of 3 independent experiments done intriplicate. ��, p � 0.05 vs the respective control group.

912 REDOX MODULATION OF INNATE IMMUNE PROINFLAMMATORY SIGNAL 3

they did differ significantly in IFN-� synthesis (Fig. 5A), suggest-ing that proliferation and effector function were uncoupled afterCA treatment. The ability to induce Ag-specific hyporesponsive-ness was not MHC specific given that CA were also effective in theDO11.10 TCR-transgenic mouse model. Following the same adop-tive transfer protocol with purified DO11.10 T cells and BALB/cmice as recipients, the recall response of DO11.10 T cells after CAtreatment differed from the OT-II recall response by displaying a

significant decrease not only in IFN-� synthesis but also in pro-liferation and IL-2 production (Fig. 5C).

Because CA generated an Ag-specific hyporesponse withDO11.10 and OT-II T cells, the ability to induce Ag-specific im-munosuppression with a naive pool of T cells from non-TCR-transgenic mice like NOD and BALB/c was performed. NOD micewere immunized with the nominal Ag HEL emulsified in thepotent proinflammatory adjuvant CFA in the presence or absence

FIGURE 5. Decrease in Ag-spe-cific T cell proliferation and IFN-�synthesis with in vivo CA treatmentof adoptively transferred DO11.10and OT-II T cells. Ag recall responseof OT-II (A) purified T cells (5 � 106

cells) that were adoptively transferredinto C57BL/6 mice and immunizedwith OVA323–339 in CFA in the pres-ence or absence of CA (10 mg/kg ofbody weight). FACS of LN cells fromC57BL/6 mice adoptively transferredwith OT-II T cells after immuniza-tion with OVA323–339 in CFA withCD90.2-APC and CFSE (B). LNCFSE-dilution profiles were derivedby gating CD90.2� cells (as depictedin gate P9 in Fig. 5B) and then com-paring CFSE intensity with forwardscatter (FSC). Ag recall response ofDO11.10 (C)-purified T cells (5 �106 cells) that were adoptively trans-ferred into BALB/c mice and immu-nized with OVA323–339 in CFA in thepresence or absence of CA (10 mg/kgof body weight). Inguinal and peri-aortic LN cells were purified and usedin an Ag recall assay to assess prolif-eration by [3H]TdR incorporation andcytokine synthesis by ELISA. f, NoAg; �, cells stimulated with Ag. Re-sults are representative of the mean(�SEM) of four independent experi-ments done in triplicate. ��, p � 0.05vs the respective control group.

913The Journal of Immunology

of a CA (10 mg/kg of body weight). Seven days after immuniza-tion, an in vitro recall response with CA-treated LN cells restim-ulated with HEL demonstrated a decrease ( p � 0.05) in T cellproliferation (Fig. 6A) and a 10-fold decrease in IFN-� synthesis incomparison with mice immunized with HEL alone (Fig. 6A). In-terestingly, the decrease in IFN-� synthesis was not dependent onthe synthesis of IL-2, given that CA-treated cells still retained theircapacity to produce IL-2 (Fig. 6A), which underscores the impor-tance of the early production of TNF-� for effector function tran-sition. To ensure that the results obtained with NOD mice were notstrain or Ag related, Ag-specific hyporesponsiveness was also gen-erated with BALB/c mice immunized with another nominal Ag,PCC, in the presence of CA (10 mg/kg; Fig. 6B). LN cells fromCA-treated mice displayed a 3-fold decrease ( p � 0.05) in Ag-specific proliferation, but more importantly, a 2-fold decrease( p � 0.05) in IFN-� synthesis (Fig. 6B) that was independent ofIL-2 levels (Fig. 6B).

In vivo redox modulation of BDC-2.5 TCR-transgenic mice witha catalytic antioxidant can inhibit effector function

There is evidence that TNF-� is important in mediating the patho-genic effects of diabetogenic T cell clones after adoptive transferand the synthesis of this cytokine is notably absent in nonpatho-genic T cell clones (34). TNF-� neutralization in NOD neonatalmice resulted in a decrease in CD11c�CD11b� DC maturationmarkers with a concomitant decrease in spontaneous diabetes, de-crease in BDC-2.5 T cell proliferation after adoptive transfer, in-activation of islet-specific pancreatic LN T cells, increase in CD4�

CD25� regulatory T cells, and the generation of immunologicaltolerance to islet cell proteins (35–37). These results suggestTNF-� is important for mediating the pathogenic effects of theBDC-2.5 T cell clone; therefore, we postulated that inhibition ofTNF-� synthesis with a CA would inhibit efficient priming ofBDC-2.5 T cells. As expected, immunization of BDC-2.5 TCR-transgenic mice with the BDC-2.5 mimotope in conjunction withCA treatment could also modulate a diabetogenic T cell response.The absence of the proinflammatory third signal mediated by CAtreatment in vivo was sufficient in decreasing BDC-2.5 T cell pro-liferation by 3-fold and IFN-� synthesis by 15-fold (Fig. 7).

DiscussionTo optimally activate the adaptive immune response, sufficient sig-nals must be generated by the innate arm of the immune system tocoordinate the interaction between APC and T cells (38). The eventsof Ag recognition leading to APC activation can occur through patternrecognition receptors (38, 39) called TLR on APC that recognize var-ious unique microbial-associated molecular patterns (MAMP) such asLPS (TLR4), CpG-rich DNA (TLR9), peptidoglycan/lipoprotein(TLR2), and flagellin (TLR5) (38).

Regardless of which MAMP is used to activate the TLRs, aconsequence of this activation is the induction of NF-�B andMAPK signaling for immune response activation and the synthesisof proinflammatory cytokines such as TNF-�, IL-1�, IL-12 p70,and type I IFNs (40). Thus, TLR interaction with MAMP is criticalfor activating the innate immune response to provide the necessarythird signal for coordinating a robust Ag-specific T cell response(26, 41), and in conjunction with Ag and IL-2, initiate the differ-entiation and effector function of naive T cells (2, 42). In additionto proinflammatory cytokines, ROS are also an important com-ponent of the third signal necessary for optimal activation of the

FIGURE 6. Decrease in Ag-specific T cell proliferation and IFN-� syn-thesis with in vivo CA treatment of NOD and BALB/c mice. Ag recallresponse of NOD mice immunized with HEL (A) or BALB/c mice immu-nized with PCC (B) in CFA in the presence or absence of CA (10 mg/kgof body weight). Inguinal and periaortic LN cells were purified and usedin an Ag recall assay to assess proliferation by [3H]TdR incorporationand cytokine synthesis by ELISA. f, No Ag; �, cells stimulated withAg. Results are representative of the mean (�SEM) of four independentexperiments done in triplicate. ��, p � 0.05 vs the respective controlgroup.

FIGURE 7. CA treatment during BDC-2.5 mimotope immunization candecrease BDC-2.5 T cell proliferation and IFN-� synthesis. Ag recall re-sponse of BDC-2.5 TCR-transgenic mice immunized with the BDC-2.5mimotope in CFA in the presence or absence of CA (10 mg/kg of bodyweight). Inguinal and periaortic LN cells were purified and used in an Agrecall assay 7 days after immunization to assess proliferation by [3H]TdRincorporation and IFN-� synthesis by ELISA. f, No Ag; �, cells stimu-lated with Ag. Results are representative of the mean (�SEM) of fourindependent experiments done in triplicate. ��, p � 0.05 vs the respectivecontrol group.

914 REDOX MODULATION OF INNATE IMMUNE PROINFLAMMATORY SIGNAL 3

adaptive immune system. ROS function as second messengersto induce proinflammatory cytokine synthesis (43, 44) by acti-vating redox-sensitive signal transduction pathways such asNF-�B and MAPK (9, 11, 45). The activation of innate im-mune-derived ROS and proinflammatory cytokine synthesis iscritical for maturing the adaptive immune effector response andthe synthesis of IFN-�. Recognition of MAMP by pattern rec-ognition receptors facilitates not only innate but also adaptiveimmune activation by promoting the maturation of immaturedendritic cells due to TLR signaling (46).

This study significantly advances our understanding of the un-derlying mechanism of the proinflammatory third signal for me-diating efficient CD4� T cell effector function and specifically, thecritical role of T cell-derived TNF-� for transitioning from expan-sion to effector function. We hypothesized that diminishing thethird signal (proinflammatory cytokines and ROS) during anti-genic immunization in the presence of a potent adjuvant couldefficiently hinder T cell priming and adaptive immune activation.To test this hypothesis, we used an Ag-specific immunization pro-tocol that relied on modulating the redox state of the innate im-mune response through the use of CA (MnTE2 and MnTDE) as anin vivo pharmacological tool during Ag immunization. Since wepreviously demonstrated the efficiency of CA in ablating the proin-flammatory third signal (23), we explored the ability of these com-pounds to induce Ag-specific hyporesponsiveness in naive CD4�

T cells by disrupting the transition of T cell expansion to effectorfunction.

Our results demonstrated Ag-specific hyporesponsivenessthrough the use of in vivo CA treatment to modulate the redoxstate in wild-type (BALB/c), autoimmune prone (NOD), TCR-transgenic (DO11.10, OT-II), and diabetogenic TCR-transgenicmice (BDC-2.5), with four different immunizing Ags in CFA(HEL, PCC, OVA323–339, and BDC-2.5 mimotope, respectively).Modulating the redox state in all mice uncoupled proliferative ca-pacity (IL-2 synthesis) from T cell effector function (IFN-� syn-thesis) as assessed by significant decreases in T cell proliferationand IFN-� synthesis in a secondary Ag recall response, much likedepriving Ag-stimulated naive T cells of the proinflammatory thirdsignal (8, 47). Flow cytometric analysis of a DO11.10 primaryrecall response with a H2O2-specific redox-sensitive fluorogenicprobe (CM-H2DCFDA) resulted in a 5-fold increase in the per-centage of double-positive CD11b�CM-H2DCFDA� cells thatwas also corroborated with a 1.5-fold increase in the MFI of CM-H2DCFDA oxidation in comparison with unstimulated cells. Con-versely, the DO11.10 primary recall response in the presence ofthe CA only generated a 1.5-fold increase in the percentage ofdouble-positive CD11b�CM-H2DCFDA� cells and a negligibleincrease in the MFI of CM-H2DCFDA oxidation. Not only didAPCs initiate a respiratory burst upon antigenic stimulation but ourCM-H2DCFDA studies also corroborated previous reports of Tcells containing a functional NADPH oxidase complex (17, 18)because the T cells in the DO11.10 primary recall response werealso capable of oxidizing CM-H2DCFDA. Upon OVA peptidestimulation, 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 incomparison with unstimulated T cells. CA treatment also inhibitedT cell-derived ROS production in the DO11.10 recall response.The percentage of KJ1-26�/CM-H2DCFDA� double-positivecells only increased 0.5-fold, and the MFI of CM-H2DCFDA ox-idation was barely detected in comparison with unstimulated Tcells.

We hypothesize that early in an immune response, APC and Tcells undergo a respiratory burst to initiate APC-T interactions

and/or to facilitate their engagement. Upon interaction, T cells pro-duce TNF-� to further shape the activation of the immune re-sponse by recruiting and arming adaptive immune effector cells bysynthesizing chemokines and adhesion molecules. Evidence forthis dependence on TNF-� for T cell transition to effector functionhas been demonstrated in models of superantigen-induced expan-sion, where CD28�/� animals stimulated with superantigens dem-onstrated an inability to expand and, more importantly, make theeffector cytokine IFN-� (48, 49). Administration of exogenousTNF-� in vivo restored superantigen-induced IFN-� synthesis,pointing to the need for the production of this cytokine by theresponding T cells to transition to effector function. Our TNF-�intracellular cytokine staining studies demonstrate that in bothOVA peptide-stimulated DO11.10 and OT-II primary recall re-sponses, the percentage of KJ1-26�TNF-�� and CD4�TNF-��

double-positive cells increased to 7.8 and 6.4%, respectively. In-terestingly, CA-treated KJ1-26�TNF-�� and CD4�TNF-�� cellsexhibited a 2-fold decrease in the percentage of double-positivecells but displayed a 2-fold increase in intracellular TNF-� MFI.These results suggest the exciting possibility that CA-treatmentmay be affecting the activity of TNF-� convertase (TACE), anenzyme necessary for TNF-� secretion (50, 51). ROS have a rolein activating TACE activity and the shedding of TNF p75 receptorin both lymphocytes and monocytic cells (52) by oxidizing theinhibitory prodomain of TACE at a critical cysteine residue caus-ing the release of the prodomain from the catalytic domain. We arecurrently planning experimental studies to address whether CAtreatment prevents the oxidation of the inhibitory prodomain ofTACE and thereby retaining an elevated level of intracellularTNF-�.

TNF-� is a proinflammatory cytokine with an important func-tion in immune development, activation, and inflammation. Ourresults confirm and extend earlier observations (35, 37) that forefficient effector function of diabetogenic T cells, a proinflamma-tory third signal is necessary for endogenous TNF-� production bythe responding T cell. The importance of TNF-� in T1D develop-ment 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 corre-lated with the extent of diabetogenicity for the T cell clones in-cluding BDC-2.5. T cell clones that did not express TNF-� wereincapable of inducing diabetes. The importance of TNF-� for driv-ing diabetogenesis may not be merely due to its conventional ef-fects during inflammation, but more specifically as a proinflam-matory third signal for transitioning CD4� and CD8� T cells toeffector function (2, 3).

The current study further expands on the importance of TNF-�inhibition mediated by CA treatment with naive antigenic T cellresponses. The use of TNF-� neutralization Abs in DO11.10 andOT-II T primary recall assays were capable of inhibiting T celltransition to effector function similar to CA treatment. Althoughneutralization of TNF-� significantly decreased the ability ofDO11.10 and OT-II T cells to synthesize IFN-�, the treatment wasunable to completely abrogate its production. We hypothesize thatother third signal proinflammatory cytokines unaffected by CAtreatment, like IL-12 p70, were able to compensate but not with thesame efficiency for the loss in TNF-� for adaptive immune mat-uration in these primary recall responses. Interestingly, neutraliza-tion of IL-1� did not interfere with the ability of DO11.10 or OT-IIT cells to gain adaptive immune effector function, suggesting thatat least in these two transgenic TCR T cells, IL-1� is not importantfor IFN-� synthesis. These results are in slight disagreement withprevious work demonstrating that a danger signal, such as IL-1 and

915The Journal of Immunology

IL-12, is required for CD4� and CD8� effector function, respec-tively (2). These earlier studies used IL-1� to stimulate CD4� Tcell activation with MHC protein-peptide complexes on micro-spheres as artificial APC (3, 32), whereas in our studies we focusedon IL-1� only and used professional APC derived from OT-II orDO11.10 mice that were capable of undergoing a respiratory burst,unlike MHC protein-peptide microspheres. It is possible that otherproinflammatory cytokines could provide the third signal for tran-sitioning to adaptive immune effector function, such as IL-23, IL-27, or IL-18. We are currently looking at the role of these cyto-kines as potential third signals and whether CA treatment can alsoabolish their synthesis.

To further demonstrate the importance of CA-mediated suppres-sion of TNF-� synthesis in adaptive immune maturation, the re-ciprocal experiment was performed whereby recombinant TNF-�was added back to CA-treated DO11.10 splenocytes and the abilityof these T cells to synthesize IFN-� was explored. rTNF-� restoredIFN-� synthesis to CA-treated DO11.10 splenocytes, but the in-crease was only 2-fold when stimulated with 1 ng/ml TNF-� andwas still 3-fold less than with OVA peptide-stimulation alone. Ex-ogenous addition of 2.5 ng/ml TNF-� also restored IFN-� synthe-sis in CA-treated T cells, but this increase was only 1.5-fold, andcells stimulated with this higher concentration appeared to displaysome cytotoxicity as the levels of IFN-� from 2.5 ng/ml TNF-�and OVA peptide costimulated samples were almost 2-fold lowerthan stimulation with OVA peptide only. The ability of rTNF-� toonly partially rescue the IFN-� synthesis defect in CA-treatedDO11.10 splenocytes suggests the importance of other third signalproinflammatory mediators still suppressed by CA treatment thatare important in adaptive immune effector function. This experi-ment further illustrates the importance of ROS as a direct proin-flammatory third signal and its role in temporally and kineticallyactivating proinflammatory cytokine synthesis. We would suggestthat exogenously adding a robust proinflammatory cytokine suchas TNF-� is not a true physiological measure of how this cytokinefunctions as a proinflammatory third signal. The synthesis and in-teraction of this cytokine with its receptor is tightly regulated andmust be synchronized for proper immune stimulation withoutcausing cytotoxicity (53).

CA treatment had no effect on the levels of IL-2 in both theprimary and secondary recall responses, suggesting that redoxmodulation of an immune response with CA treatment uncouplesthe expansion phase (IL-2 synthesis) from the effector phase(IFN-� synthesis) of an Ag-specific T cell response, by affectingthe proinflammatory third signal necessary for adaptive immunefunction. The uncoupling of expansion and effector function fromAg-specific T cells was also described by Pape et al. (3). Theirwork demonstrated that immunization of mice in the absence of aproinflammatory adjuvant resulted in a significant decrease inIFN-� synthesis from adoptively transferred DO11.10 T cells intoBALB/c mice even though they were capable of undergoing ex-pansion in vivo. What is striking about our work is that in thepresence of a very potent adjuvant, we are still capable of signif-icantly inhibiting the transition to effector function by modulatingthe redox state during innate immune activation.

In addition to IL-2 synthesis, there was also no difference inIL-12 p70 levels after CA treatment with the DO11.10 primaryrecall suggesting that the decrease in IFN-� synthesis was not dueto suppression of IL-12 signaling, but downstream at the Th1-specific T box transcription factor (T-bet) and/or Stat4 signalingpathways. We have preliminary evidence demonstrating that CAtreatment of DO11.10 splenocytes result in a decrease in Stat4phosphorylation 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 mayalso affect the T-bet signaling pathway, another pathway necessaryfor IFN-� synthesis (54–56). Interestingly, there was a differencein IL-12 p70 levels in the CA-treated OT-II splenocytes; they ex-hibited a 2-fold decrease in IL-12 p70 as compared with control-treated OT-II splenocytes. Whether or not the decrease in IL-12p70 expression can explain the decrease in IFN-� synthesis inOT-II mice is not known, but we are currently exploring any po-tential differences in the IL-12, Stat4, and T-bet signaling path-ways demonstrated with the OT-II, but not the DO11.10 recallresponse.

Targeting the innate immune response by modulating the redoxstate as a means of suppressing the adaptive immune response mayhold promise as a new avenue of immunotherapy because thisstrategy can be utilized in conjunction with Ag-specific treatmentto efficiently decrease innate immune-derived proinflammatorymediators leading to ablation of Ag-responding T cell populations.The ability to induce Ag-specific hyporesponsiveness with variousmouse strains by modulating the redox state suggests the impor-tance of reduction-oxidation reactions in activating the immuneresponse. Current immunosuppressive therapies to treat T1D suchas anti-CD3 mAb (57) target the adaptive immune response andthe ablation of T cells, but not without adverse effects (58). CA arenovel anti-inflammatory agents that may be used in conjunctionwith other classical immunosuppressants at a significantly lowertoxic dose for curtailing adaptive immune effector function. Ourfuture studies will further characterize how redox modulation byCA treatment can suppress IFN-� synthesis in T cells by affectingStat4 and T-bet pathway activation and determine whether Ag-specific hyporesponsiveness mediated by CA-treatment is globalor Ag-specific in models of type 1 diabetes.

AcknowledgmentsWe thank Robert Lakomy and Alexis Styche for excellent technical assis-tance and Drs. Henry Dong and Nick Giannoukakis for critical reading ofthe manuscript.

DisclosuresThe authors have no financial conflict of interest.

References1. Jenkins, M. K., and J. G. Johnson. 1993. Molecules involved in T-cell costimu-

lation. Curr. Opin. Immunol. 5: 361–367.2. Curtsinger, J. M., C. S. Schmidt, A. Mondino, D. C. Lins, R. M. Kedl,

M. K. Jenkins, and M. F. Mescher. 1999. Inflammatory cytokines provide a thirdsignal for activation of naive CD4� and CD8� T cells. J. Immunol. 162:3256–3262.

3. Pape, K. A., A. Khoruts, A. Mondino, and M. K. Jenkins. 1997. Inflammatorycytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4� T cells. J. Immunol. 159: 591–598.

4. Khoruts, A., A. Mondino, K. A. Pape, S. L. Reiner, and M. K. Jenkins. 1998. Anatural immunological adjuvant enhances T cell clonal expansion through aCD28-dependent, interleukin (IL)-2-independent mechanism. J. Exp. Med. 187:225–236.

5. Chiller, J. M., G. S. Habicht, and W. O. Weigle. 1971. Kinetic differences inunresponsiveness of thymus and bone marrow cells. Science 171: 813–815.

6. Dresser, D. W. 1962. Specific inhibition of antibody production. II. Paralysisinduced in adult mice by small quantities of protein antigen. Immunology 5:378–388.

7. Pape, K. A., E. R. Kearney, A. Khoruts, A. Mondino, R. Merica, Z. M. Chen,E. Ingulli, J. White, J. G. Johnson, and M. K. Jenkins. 1997. Use of adoptivetransfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell acti-vation in vivo. Immunol. Rev. 156: 67–78.

8. Curtsinger, J. M., D. C. Lins, and M. F. Mescher. 2003. Signal 3 determinestolerance versus full activation of naive CD8 T cells: dissociating proliferationand development of effector function. J. Exp. Med. 197: 1141–1151.

9. Karin, M. 1998. The NF-�B activation pathway: its regulation and role in in-flammation and cell survival. Cancer J. Sci. Am. 4(Suppl.1): S92–S99.

10. Suzuki, Y. J., H. J. Forman, and A. Sevanian. 1997. Oxidants as stimulators ofsignal transduction. Free Radic. Biol. Med. 22: 269–285.

11. Lander, H. M., J. S. Ogiste, K. K. Teng, and A. Novogrodsky. 1995. p21ras as acommon signaling target of reactive free radicals and cellular redox stress.J. Biol. Chem. 270: 21195–21198.

916 REDOX MODULATION OF INNATE IMMUNE PROINFLAMMATORY SIGNAL 3

12. Matsuzawa, A., K. Saegusa, T. Noguchi, C. Sadamitsu, H. Nishitoh, S. Nagai,S. Koyasu, K. Matsumoto, K. Takeda, and H. Ichijo. 2005. ROS-dependent ac-tivation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat. Immunol. 6: 587–592.

13. Rao, K. M. 2001. MAP kinase activation in macrophages. J. Leukocyte Biol. 69:3–10.

14. Ryan, K. A., M. F. Smith, Jr., M. K. Sanders, and P. B. Ernst. 2004. Reactiveoxygen and nitrogen species differentially regulate Toll-like receptor 4-mediatedactivation of NF-�B and interleukin-8 expression. Infect. Immun. 72: 2123–2130.

15. Klotz, L. O., S. M. Schieke, H. Sies, and N. J. Holbrook. 2000. Peroxynitriteactivates the phosphoinositide 3-kinase/Akt pathway in human skin primary fi-broblasts. Biochem. J. 352(Part 1): 219–225.

16. Matsue, H., D. Edelbaum, D. Shalhevet, N. Mizumoto, C. Yang, M. E. Mummert,J. Oeda, H. Masayasu, and A. Takashima. 2003. Generation and function ofreactive oxygen species in dendritic cells during antigen presentation. J. Immu-nol. 171: 3010–3018.

17. Devadas, S., L. Zaritskaya, S. G. Rhee, L. Oberley, and M. S. Williams. 2002.Discrete generation of superoxide and hydrogen peroxide by T cell receptor stim-ulation: selective regulation of mitogen-activated protein kinase activation andfas ligand expression. J. Exp. Med. 195: 59–70.

18. Jackson, S. H., S. Devadas, J. Kwon, L. A. Pinto, and M. S. Williams. 2004. Tcells express a phagocyte-type NADPH oxidase that is activated after T cellreceptor stimulation. Nat. Immunol. 5: 818–827.

19. Day, B. J., S. Shawen, S. I. Liochev, and J. D. Crapo. 1995. A metalloporphyrinsuperoxide dismutase mimetic protects against paraquat-induced endothelial cellinjury, in vitro. J. Pharmacol. Exp. Ther. 275: 1227–1232.

20. Day, B. J., I. Fridovich, and J. D. Crapo. 1997. Manganic porphyrins possesscatalase activity and protect endothelial cells against hydrogen peroxide-mediatedinjury. Arch. Biochem. Biophys. 347: 256–262.

21. Day, B. J., I. Batinic-Haberle, and J. D. Crapo. 1999. Metalloporphyrins arepotent inhibitors of lipid peroxidation. Free Radic. Biol. Med. 26: 730–736.

22. Batinic-Haberle, I., L. Benov, I. Spasojevic, and I. Fridovich. 1998. The orthoeffect makes manganese(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin apowerful and potentially useful superoxide dismutase mimic. J. Biol. Chem. 273:24521–24528.

23. Tse, H. M., M. J. Milton, and J. D. Piganelli. 2004. Mechanistic analysis of theimmunomodulatory effects of a catalytic antioxidant on antigen-presenting cells:implication for their use in targeting oxidation-reduction reactions in innate im-munity. Free Radic. Biol. Med. 36: 233–247.

24. Piganelli, J. D., S. C. Flores, C. Cruz, J. Koepp, I. Batinic-Haberle, J. Crapo,B. Day, R. Kachadourian, R. Young, B. Bradley, and K. Haskins. 2002. A met-alloporphyrin-based superoxide dismutase mimic inhibits adoptive transfer ofautoimmune diabetes by a diabetogenic T-cell clone. Diabetes 51: 347–355.

25. Lander, H. M., A. J. Milbank, J. M. Tauras, D. P. Hajjar, B. L. Hempstead,G. D. Schwartz, R. T. Kraemer, U. A. Mirza, B. T. Chait, S. C. Burk, andL. A. Quilliam. 1996. Redox regulation of cell signalling. Nature 381: 380–381.

26. Trevejo, J. M., M. W. Marino, N. Philpott, R. Josien, E. C. Richards, K. B. Elkon,and E. Falck-Pedersen. 2001. TNF-�-dependent maturation of local dendriticcells is critical for activating the adaptive immune response to virus infection.Proc. Natl. Acad. Sci. USA 98: 12162–12167.

27. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigen ofintrathymic apoptosis of CD4�CD8�TCRlo thymocytes in vivo. Science 250:1720–1723.

28. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCRexpression in transgenic mice constructed using cDNA-based �- and �-chaingenes under the control of heterologous regulatory elements. Immunol. Cell Biol.76: 34–40.

29. Katz, J. D., B. Wang, K. Haskins, C. Benoist, and D. Mathis. 1993. Following adiabetogenic T cell from genesis through pathogenesis. Cell 74: 1089–1100.

30. Haskins, K., R. Kubo, J. White, M. Pigeon, J. Kappler, and P. Marrack. 1983. Themajor histocompatibility complex-restricted antigen receptor on T cells. I. Isola-tion with a monoclonal antibody. J. Exp. Med. 157: 1149–1169.

31. Kearney, E. R., K. A. Pape, D. Y. Loh, and M. K. Jenkins. 1994. Visualizationof peptide-specific T cell immunity and peripheral tolerance induction in vivo.Immunity 1: 327–339.

32. Khoruts, A., R. E. Osness, and M. K. Jenkins. 2004. IL-1 acts on antigen-pre-senting cells to enhance the in vivo proliferation of antigen-stimulated naive CD4T cells via a CD28-dependent mechanism that does not involve increased ex-pression of CD28 ligands. Eur. J. Immunol. 34: 1085–1090.

33. Pape, K. A., and M. K. Jenkins. 1998. A role for inflammatory cytokines in theproductive activation of antigen-specific CD4� T-cells. Agents Actions Suppl. 49:23–31.

34. Cantor, J., and K. Haskins. 2005. Effector function of diabetogenic CD4 Th1 Tcell clones: a central role for TNF-�. J. Immunol. 175: 7738–7745.

35. Lee, L. F., B. Xu, S. A. Michie, G. F. Beilhack, T. Warganich, S. Turley, andH. O. McDevitt. 2005. The role of TNF-� in the pathogenesis of type 1 diabetesin the nonobese diabetic mouse: analysis of dendritic cell maturation. Proc. Natl.Acad. Sci. USA 102: 15995–16000.

36. Wu, A. J., H. Hua, S. H. Munson, and H. O. McDevitt. 2002. Tumor necrosisfactor-� regulation of CD4�CD25� T cell levels in NOD mice. Proc. Natl. Acad.Sci. USA 99: 12287–12292.

37. Yang, X. D., R. Tisch, S. M. Singer, Z. A. Cao, R. S. Liblau, R. D. Schreiber, andH. O. McDevitt. 1994. Effect of tumor necrosis factor � on insulin-dependentdiabetes mellitus in NOD mice. I. The early development of autoimmunity andthe diabetogenic process. J. Exp. Med. 180: 995–1004.

38. Takeda, K., and S. Akira. 2001. Roles of Toll-like receptors in innate immuneresponses. Genes Cells 6: 733–742.

39. Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptiveimmune responses. Nat. Immunol. 5: 987–995.

40. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Im-munol. 21: 335–376.

41. Swain, S. D., T. T. Rohn, and M. T. Quinn. 2002. Neutrophil priming in hostdefense: role of oxidants as priming agents. Antioxid. Redox Signal. 4: 69–83.

42. Vella, A. T., T. Mitchell, B. Groth, P. S. Linsley, J. M. Green, C. B. Thompson,J. W. Kappler, and P. Marrack. 1997. CD28 engagement and proinflammatorycytokines contribute to T cell expansion and long-term survival in vivo. J. Im-munol. 158: 4714–4720.

43. Forman, H. J., M. Torres, and J. Fukuto. 2002. Redox signaling. Mol. Cell. Bio-chem. 234–235: 49–62.

44. Remick, D. G., and L. Villarete. 1996. Regulation of cytokine gene expression byreactive oxygen and reactive nitrogen intermediates. J. Leukocyte Biol. 59:471–475.

45. Torres, M., and H. J. Forman. 2003. Redox signaling and the MAP kinase path-ways. Biofactors 17: 287–296.

46. Mazzoni, A., and D. M. Segal. 2004. Controlling the Toll road to dendritic cellpolarization. J. Leukocyte Biol. 75: 721–730.

47. Hernandez, J., S. Aung, K. Marquardt, and L. A. Sherman. 2002. Uncoupling ofproliferative potential and gain of effector function by CD8� T cells respondingto self-antigens. J. Exp. Med. 196: 323–333.

48. Mittrucker, H. W., A. Shahinian, D. Bouchard, T. M. Kundig, and T. W. Mak.1996. Induction of unresponsiveness and impaired T cell expansion by staphy-lococcal enterotoxin B in CD28-deficient mice. J. Exp. Med. 183: 2481–2488.

49. Saha, B., D. M. Harlan, K. P. Lee, C. H. June, and R. Abe. 1996. Protectionagainst lethal toxic shock by targeted disruption of the CD28 gene. J. Exp. Med.183: 2675–2680.

50. Black, R. A., C. T. Rauch, C. J. Kozlosky, J. J. Peschon, J. L. Slack,M. F. Wolfson, B. J. Castner, K. L. Stocking, P. Reddy, S. Srinivasan, et al. 1997.A metalloproteinase disintegrin that releases tumour-necrosis factor-� from cells.Nature 385: 729–733.

51. Moss, M. L., S. L. Jin, M. E. Milla, D. M. Bickett, W. Burkhart, H. L. Carter,W. J. Chen, W. C. Clay, J. R. Didsbury, D. Hassler, et al. 1997. Cloning of adisintegrin metalloproteinase that processes precursor tumour-necrosis factor-�.Nature 385: 733–736.

52. Zhang, Z., P. Oliver, J. J. Lancaster, P. O. Schwarzenberger, M. S. Joshi, J. Cork,and J. K. Kolls. 2001. Reactive oxygen species mediate tumor necrosis factor�-converting, enzyme-dependent ectodomain shedding induced by phorbol my-ristate acetate. FASEB J. 15: 303–305.

53. Smyth, M. J., and R. W. Johnstone. 2000. Role of TNF in lymphocyte-mediatedcytotoxicity. Microsc. Res. Tech. 50: 196–208.

54. Mullen, A. C., F. A. High, A. S. Hutchins, H. W. Lee, A. V. Villarino, D. M.Livingston, A. L. Kung, N. Cereb, T. P. Yao, S. Y. Yang, and S. L. Reiner. 2001.Role of T-bet in commitment of TH1 cells before IL-12-dependent selection.Science 292: 1907–1910.

55. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H.Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commit-ment. Cell 100: 655–669.

56. Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, andL. H. Glimcher. 2002. Distinct effects of T-bet in TH1 lineage commitment andIFN-� production in CD4 and CD8 T cells. Science 295: 338–342.

57. Smith, J. A., and J. A. Bluestone. 1997. T cell inactivation and cytokine deviationpromoted by anti-CD3 mAbs. Curr. Opin. Immunol. 9: 648–654.

58. Wiendl, H., and R. Hohlfeld. 2002. Therapeutic approaches in multiple sclerosis:lessons from failed and interrupted treatment trials. BioDrugs 16: 183–200.

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