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• W1965575960 abstract "Activation and maturation of dendritic cells (DC) are crucial for the establishment of delayed-type hypersensitivity (DTH). However, antigen presentation by immature DC (iDC) might lead to antigen-specific peripheral tolerance. NF-κB plays significant roles in upregulation of co-stimulatory molecules and cytokines in DC and therefore we investigated whether NF-κB decoy oligodeoxynucleotide (ODN) might induce tolerance to DTH. NF-κB decoy ODN suppressed ovalbumin (OVA)-induced DTH responses not only in naïve but also in presensitized mice. The suppressive effect was found to be antigen-specific. NF-κB decoy ODN-induced tolerance involved CD4+CD25+ regulatory T cells (Treg), because in vivo depletion of CD25+ T cells abrogated the tolerance, whereas adoptive transfer of such T cell population from tolerant mice induced tolerance. Furthermore, the induction of Treg was related to insufficient migration and/or maturation of DC, because a sizable DC population still remained in peripheral tissue even after exposure to exogenous antigen in NF-κB decoy ODN-treated mice. Even if they migrated into lymph nodes, they showed insufficient upregulation of co-stimulatory molecules and impaired antigen-specific activation of T cells. Topical application of NF-κB decoy ODN might thus be a new approach to induce antigen-specific peripheral tolerance. Activation and maturation of dendritic cells (DC) are crucial for the establishment of delayed-type hypersensitivity (DTH). However, antigen presentation by immature DC (iDC) might lead to antigen-specific peripheral tolerance. NF-κB plays significant roles in upregulation of co-stimulatory molecules and cytokines in DC and therefore we investigated whether NF-κB decoy oligodeoxynucleotide (ODN) might induce tolerance to DTH. NF-κB decoy ODN suppressed ovalbumin (OVA)-induced DTH responses not only in naïve but also in presensitized mice. The suppressive effect was found to be antigen-specific. NF-κB decoy ODN-induced tolerance involved CD4+CD25+ regulatory T cells (Treg), because in vivo depletion of CD25+ T cells abrogated the tolerance, whereas adoptive transfer of such T cell population from tolerant mice induced tolerance. Furthermore, the induction of Treg was related to insufficient migration and/or maturation of DC, because a sizable DC population still remained in peripheral tissue even after exposure to exogenous antigen in NF-κB decoy ODN-treated mice. Even if they migrated into lymph nodes, they showed insufficient upregulation of co-stimulatory molecules and impaired antigen-specific activation of T cells. Topical application of NF-κB decoy ODN might thus be a new approach to induce antigen-specific peripheral tolerance. dendritic cells delayed-type hypersensitivity hen egg lysozyme immature DC lymph node major histocompatibility complex II oligodeoxynucleotide ovalbumin phosphate buffered saline phycoerythrin CD4+CD25+ regulatory T cells The induction of antigen-specific immune tolerance has long been targeted to controlling allergic or autoimmune disease activity without immunosuppressive reagents. For analysis of tolerance induction, we have focused on delayed-type hypersensitivity (DTH) because this can be induced by various antigens and applied to assess immune response and/or tolerance in animal models and human studies. Activation and maturation of dendritic cells (DC) are crucial for the establishment of DTH (Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (11820) Google Scholar). However, an increasing number of studies have demonstrated that antigen presentation by immature dendritic cells (iDC) and certain subsets of mature DC induce antigen-specific peripheral tolerance (Dhodapkar et al., 2001Dhodapkar M.V. Steinman R.M. Krasovsky J. Munz C. Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells.J Exp Med. 2001; 193: 233-238Crossref PubMed Scopus (1188) Google Scholar; Hawiger et al., 2001Hawiger D. Inaba K. Dorsett Y. Guo M. Mahnke K. Rivera M. et al.Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo.J Exp Med. 2001; 194: 769-779Crossref PubMed Scopus (1459) Google Scholar; O'Connell et al., 2002O'Connell P.J. Li W. Wang Z. Specht S.M. Logar A.J. Thomson A.W. Immature and mature CD8α+ dendritic cells prolong the survival of vascularized heart allografts.J Immunol. 2002; 168: 143-154Crossref PubMed Scopus (115) Google Scholar; Sato et al., 2003Sato K. Yamashita N. Yamashita N. Baba M. Matsuyama T. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse.Immunity. 2003; 18: 367-379Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). iDC are characterized by low surface expression of co-stimulatory molecules such as CD80 and CD86. Meanwhile, evidence is accumulating that CD4+CD25+ regulatory T cells (Treg) play significant roles in the maintenance of self-tolerance, and several reports have indicated that induction of Treg is required for tolerance to allogeneic and exogenous antigens and for prevention of autoimmune disorders (Takahashi et al., 1998Takahashi T. Kuniyasu Y. Toda M. Sakaguchi N. Itoh M. Iwata M. et al.Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state.Int Immunol. 1998; 10: 1969-1980Crossref PubMed Scopus (1276) Google Scholar; Kuniyasu et al., 2000Kuniyasu Y. Takahashi T. Itoh M. Shimizu J. Toda G. Sakaguchi S. Naturally anergic and suppressive CD25+CD4+ T cells as a functionally and phenotypically distinct immunoregulatory subpopulation.Int Immunol. 2000; 12: 1145-1155Crossref PubMed Scopus (249) Google Scholar; Shevach, 2001Shevach E.M. Certified professionals: CD4+CD25+ suppressor T cells.J Exp Med. 2001; 193: F41-F46Crossref PubMed Google Scholar; Taylor et al., 2001Taylor P.A. Noelle R.J. Blazar B.R. CD4+CD25+ immune regulatory T cells are required for induction of tolerance to alloantigen via costimulatory blockade.J Exp Med. 2001; 193: 1311-1318Crossref PubMed Scopus (477) Google Scholar; Thorstenson and Khoruts, 2001Thorstenson K.M. Khoruts A. Generation of anergic and potentially immunoregulatory CD25+CD4T cells in vivo after induction of peripheral tolerance with intravenous or oral antigen.J Immunol. 2001; 167: 188-195Crossref PubMed Scopus (374) Google Scholar; Zhang et al., 2001Zhang X. Izikson L. Liu L. Weiner H.L. Activation of CD4+CD25+ regulatory T cells by oral antigen administration.J Immunol. 2001; 167: 4245-4253Crossref PubMed Scopus (398) Google Scholar). There is evidence that expansion of Treg by iDC is associated with tolerance induction (Roncarolo et al., 2001Roncarolo M.G. Levings M.K. Traversari C. Differentiation of T regulatory cells by immature dendritic cells.J Exp Med. 2001; 193: F5-F9Crossref PubMed Google Scholar). NF-κB has emerged as a key transducer of inflammatory signals for the DC maturation program (Li and Verma, 2002Li Q. Verma I.M. NF-κB regulation in the immune system.Nat Rev Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3088) Google Scholar). NF-κB complexes comprise homodimers or heterodimers of structurally related proteins, including p50, p52, relA (p65), c-Rel, and RelB, and are present in an inactive form in the cytosol bound to the inhibitor protein, IκB. Inflammatory cytokines such as IL-1 and tumor necrosis factor-α and by-products of bacterial and viral infections promote nuclear translocation of NF-κB by inducing phosphorylation- and ubiquitin-dependent degradation of IκB. Activated NF-κB then regulates various immune responses, including upregulation of co-stimulatory molecules and immunostimulatory cytokines as well as cell survival. Recently, oligodeoxynucleotides (ODNs) bearing consensus binding sequences of specific transcription factors have been explored as tools for manipulating gene expression on living cells (Mann and Dzau, 2000Mann M.J. Dzau V.J. Therapeutic applications of transcription factor decoy oligonucleotides.J Clin Invest. 2000; 106: 1071-1075Crossref PubMed Scopus (202) Google Scholar). This strategy involves the intracellular delivery of a “decoy” ODN, which occupies a DNA-binding site and blocks subsequent binding of proteins to the promoter regions of target genes. Based on the immunoregulatory effects, NF-κB decoy ODN has been investigated in several studies for prevention of myocardial infarction, anti-inflammatory gene therapy for cystic fibrosis patients, prevention of rheumatoid arthritis, and prolongation of cardiac allograft survival (Morishita et al., 1997Morishita R. Sugimoto T. Aoki M. Kida I. Tomita N. Moriguchi A. et al.In vivo transfection of cis element “decoy” against nuclear factor-κB binding site prevents myocardial infarction.Nat Med. 1997; 3: 894-899Crossref PubMed Scopus (594) Google Scholar; Tomita et al., 1999Tomita T. Takeuchi E. Tomita N. Morishita R. Kaneko M. Yamamoto K. et al.Suppressed severity of collagen-induced arthritis by in vivo transfection of nuclear factor κB decoy oligodeoxynucleotides as a gene therapy.Arthritis Rheum. 1999; 42: 2532-2542Crossref PubMed Scopus (218) Google Scholar; Giannoukakis et al., 2000Giannoukakis N. Bonham C.A. Qian S. et al.Prolongation of cardiac allograft survival using dendritic cells treated with NF-κB decoy oligodeoxyribonucleotides.Mol Ther. 2000; 1: 430-437Crossref PubMed Scopus (134) Google Scholar; Griesenbach et al., 2000Griesenbach U. Scheid P. Hillery E. de Martin R. Huang L. Geddes D.M. et al.Anti-inflammatory gene therapy directed at the airway epithelium.Gene Therapy. 2000; 7: 306-313Crossref PubMed Scopus (59) Google Scholar; Bonham et al., 2002Bonham C.A. Peng L. Liang X. Chen Z. Wang L. Ma L. et al.Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-κB oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig.J Immunol. 2002; 169: 3382-3391Crossref PubMed Scopus (124) Google Scholar). In these studies, the purpose of employing NF-κB decoy ODN was to induce immunosuppressive effects in target organs, although it remained unclear whether tolerance was induced in an antigen-specific manner. It is therefore of great interest to determine whether NF-κB decoy ODN can induce tolerance to DTH. In the present study, we therefore investigated whether topical application of NF-κB decoy ODN to the skin of BALB/c mice would induce tolerance to DTH caused by responsible exogenous antigens. When NF-κB decoy ODN ointment (2%) was applied to the shaved abdominal skin of BALB/c mice 24 hours before sensitization with ovalbumin (OVA) at the same site, DTH responses were significantly suppressed (Figure 1a). Mice treated with scramble decoy ointment, in contrast, did not show any suppressive effect. To analyze tolerance induction in recall responses of DTH, all mice were first sensitized with OVA on day 0 and then NF-κB decoy ODN was applied on day 6, 24 hours before a second sensitization on day 7. As in the first experiment, DTH responses were again suppressed (Figure 1b), suggesting that NF-κB decoy ODN might induce peripheral tolerance to OVA. To investigate whether the effects of NF-κB decoy ODN were local or systemic, mice were sensitized with OVA on the back after topical application of NF-κB decoy ODN to the abdominal skin. As DTH was suppressed only when OVA and NF-κB ODN were applied to the same area (Figure 1c), the actions appear to be mediated by local immunosuppression. We next performed adoptive transfer experiments. Single-cell suspensions were prepared from regional lymph nodes (LNs) from sensitized and tolerant mice as shown in Figure 1a and transferred to naïve mice via the tail vein before sensitization and DTH elicitation. As shown in Figure 1d, DTH was suppressed in mice receiving T cells from tolerant mice, but not from sensitized mice. Thus, the tolerance induced by NF-κB decoy ODN treatment is mediated by regulatory cells. We next evaluated antigen specificity of the tolerance induced by NF-κB decoy ODN in vivo by antigen-crossing assays. After the adoptive transfer of 5 × 106 LN cells from mice tolerant to OVA or hen egg lysozyme (HEL), mice were sensitized and elicited by these antigens in a crisscross manner. Tolerance was observed only when the mice receiving LN cells from tolerant mice were stimulated with the same antigen used for tolerance induction (Figure 2). These findings indicate that the topical NF-κB decoy ODN induces antigen-specific tolerance in vivo. To determine whether CD4+CD25+ Treg population is responsible for tolerance induction in our system, we first employed in vivo depletion experiments using anti-CD25 mAb (PC61). Inhibition of DTH by NF-κB decoy ODN was almost completely abrogated by PC61 treatment, indicating the involvement of CD25+ T cells in the suppression (Figure 3a). PC61 treatment in sensitized and naïve mice had neither stimulatory nor suppressive effects on DTH (Figure 3a and data not shown). To investigate the percentage of CD4+CD25+ T cells in each group, LN cells from naïve, sensitized, and tolerant mice were analyzed by FACS (Figure 3b). The value was largest in tolerant mice but the number in sensitized mice was also increased. Mean fluorescence intensity of CD25 expression on CD4+CD25+ T cells, however, was significantly increased in tolerant mice but not in sensitized mice (Figure 3f), suggesting a qualitative difference between the two CD4+CD25+ populations. To confirm that CD4+CD25+ T cells were responsible for the tolerance, CD4+CD25+ and CD4+CD25− T cell populations were prepared using magnetic beads and transferred to OVA-sensitized mice. The results showed the DTH response to be suppressed in mice that had received with CD4+CD25+ T cells from sensitized and tolerant mice, but not naïve mice (Figure 4). No suppressive effects were observed in mice injected with CD4+CD25− T cells. Together with the results shown in Figure 3, these findings imply that the topical application of NF-κB decoy ODN in combination with OVA sensitization could induce OVA-specific tolerance through efficient activation of the pre-existing OVA-specific Treg population. The unexpected suppressive effects against DTH observed in the mice receiving CD4+CD25+ T cells from sensitized mice may reflect CD4+CD25+ enrichment effects, as adoptive transfer of unfractionated LN cells from sensitized mice did not suppress DTH responses, suggesting the frequency of Treg in sensitized mice to be substantially lower than that of tolerant mice as shown in Figure 1d. In addition to enrichment effects, a certain CD4+CD25+ Treg population might be activated even in the absence of NF-κB decoy ODN, as only a weak suppressive effect against DTH was observed with CD4+CD25+ T cells from naïve mice (Figure 4). Alternatively, other regulatory CD4+ T cell populations (Groux, 2003Groux H. Type 1 T-regulatory cells: their role in the control of immune responses.Transplantation. 2003; 75: 8S-12SCrossref PubMed Google Scholar) expressing CD25 as an activation marker might be activated. In general, activation and polarization of T cells are orchestrated by antigen-presenting cells such as DC (Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (11820) Google Scholar). Langerhans cells play the roles in capturing, processing, and presenting antigens to T cells in draining LNs. Recently, several reports provided evidence that iDC lacking expression of co-stimulatory molecules and immunostimulatory cytokines can induce tolerance (Lutz and Schuler, 2002Lutz M.B. Schuler G. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?.Trends Immunol. 2002; 23: 445-449Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). We postulated that topical application of NF-κB decoy ODN affects maturation of DC. Therefore, DC migration from the skin to LNs was first investigated by topical application with FITC, because FITC has been used not only as a hapten but also for cell tracking (Macatonia et al., 1987Macatonia S.E. Knight S.C. Edwards A.J. Griffiths S. Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothicyanate. Functional and morphological studies.J Exp Med. 1987; 166: 1654-1667Crossref PubMed Scopus (510) Google Scholar). At 24 hours after NF-κB decoy, ODN was applied to shaved abdominal skin, 0.5% FITC was painted on the same area and draining LN cells were prepared after a further 24 hours as described previously and stained with phycoerythrin (PE)-conjugated CD11c. As shown in Figure 5a, migration of CD11c+FITC+ cells was decreased by topical NF-κB decoy ODN application. When epidermal sheets were stained with major histocompatibility complex II (MHC II) after OVA sensitization following topical application of NF-κB decoy ODN, a larger number of resident Langerhans cells was observed than in the case with OVA sensitization alone (data not shown). Interestingly, resident Langerhans cells had an activated phenotype with longer dendrites and enlarged cell size in NF-κB decoy ODN-treated mice (data not shown). Further FACS analysis revealed that topical NF-κB decoy ODN substantially inhibits upregulation of CD80, CD86, and MHC II on FITC-positive cells (Figure 5b). In order to assess the functions of DC from NF-κB decoy ODN-treated mice, their stimulatory capacity for antigen-specific T cells was examined. DC were isolated from LNs by MACS (Miltenyi Biotec, Gladbach, Germany) using anti-CD11c microbeads 24 hours after sensitization by OVA with or without NF-κB decoy ODN and were co-incubated with DO.11.10 CD4+ T cells expressing T cell receptors for MHC II (I-Ad) plus OVA peptide (323–339) for 3 days. DC from tolerant mice showed diminished secretion of IFN-γ (Figure 5c). Scramble decoy ODN had no suppressive effect. The results indicate that DC from NF-κB decoy ODN-treated mice are at least functionally immature, although they do not show the typical surface phenotype of iDC. Regulation of DC maturation and Treg induction is a key to tolerance induction. Here we showed that topical NF-κB decoy ODN application induces Treg-mediated tolerance against antigen-specific DTH by suppressing migration and maturation of DC. Recent studies have revealed that peripheral tolerance is induced by iDC lacking expression of MHC-class II, co-stimulatory molecules, and immunostimulatory cytokines (Lutz and Schuler, 2002Lutz M.B. Schuler G. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?.Trends Immunol. 2002; 23: 445-449Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). DC in peripheral tissues, such as epidermal Langerhans cells, remain immature in the steady state, expressing only a small quantity of MHC II and virtually no co-stimulatory molecules. Once iDC capture antigens by endocytosis, they migrate into T cell areas of regional LNs and present processed antigenic peptides to T cells in the context of MHC molecules (Inaba et al., 1997Inaba K. Pack M. Inaba M. Sakuta H. Isdell F. Steinman R.M. High levels of a major histocompatibility complex II-self peptide complex on dendritic cells from the T cell areas of lymph nodes.J Exp Med. 1997; 186: 665-672Crossref PubMed Scopus (232) Google Scholar; Huang et al., 2000Huang F.P. Platt N. Wykes M. Major J.R. Powell T.J. Jenkins C.D. et al.A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes.J Exp Med. 2000; 191: 435-444Crossref PubMed Scopus (763) Google Scholar; Lutz and Schuler, 2002Lutz M.B. Schuler G. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?.Trends Immunol. 2002; 23: 445-449Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). DC maturation simultaneously occurs during such processes, and they express high amount of MHC II, co-stimulatory molecules and immunostimulatory cytokines. It is an established fact that mature DC can activate T cells by delivering signals through T cell receptor and co-stimulatory molecules (Viola and Lanzavecchia, 1996Viola A. Lanzavecchia A. T cell activation determined by T cell receptor number and tunable thresholds.Science. 1996; 273: 104-106Crossref PubMed Scopus (860) Google Scholar). Recent studies have, however, also revealed that peripheral tolerance is induced by iDC lacking expression of MHC II, co-stimulatory molecules, and immunostimulatory cytokines (Steinman et al., 2000Steinman R.M. Turley S. Mellman I. Inaba K. The induction of tolerance by dendritic cells that have captured apoptotic cells.J Exp Med. 2000; 191: 411-416Crossref PubMed Scopus (994) Google Scholar; Lutz and Schuler, 2002Lutz M.B. Schuler G. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?.Trends Immunol. 2002; 23: 445-449Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar; Nouri-Shirazi and Guinet, 2002Nouri-Shirazi M. Guinet E. Direct and indirect cross-tolerance of alloreactive T cells by dendritic cells retained in the immature stage.Transplantation. 2002; 74: 1035-1044Crossref PubMed Scopus (65) Google Scholar). In addition, tolerance can be induced by partially mature DC, which express MHC II, CD80, and CD86 but lack secretion of IL-12, IL-6, and tumor necrosis factor-α (Groux, 2003Groux H. Type 1 T-regulatory cells: their role in the control of immune responses.Transplantation. 2003; 75: 8S-12SCrossref PubMed Google Scholar). Other groups have reported that murine acute graft-versus-host disease can be prevented by DC modified by 1α,25-dihydroxyvitamin D3 (Sato et al., 2003Sato K. Yamashita N. Yamashita N. Baba M. Matsuyama T. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse.Immunity. 2003; 18: 367-379Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) and that tolerance can be induced by the generation of Treg by UV-damaged Langerhans cells in the contact hypersensitivity model (Schwarz et al., 2004Schwarz A. Maeda A. Wild M.K. Kernebeck K. Gross N. Aragane Y. et al.Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity.J Immunol. 2004; 172: 1036-1043Crossref PubMed Scopus (169) Google Scholar; Schwarz et al., 2005Schwarz A. Maeda A. Kernebeck K. van Steeg H. Beissert S. Schwarz T. Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair.J Exp Med. 2005; 201: 173-179Crossref PubMed Scopus (166) Google Scholar). In our study, topical NF-κB decoy ODN generated DC expressing a high level of MHC II, but relatively low levels of co-stimulatory molecules, similar to the case with DC treated with vitamin D3 (Sato et al., 2003Sato K. Yamashita N. Yamashita N. Baba M. Matsuyama T. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse.Immunity. 2003; 18: 367-379Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Discordant upregulation between expression of MHC II and co-stimulatory molecules in the sensitization phase may thus be critical for induction of tolerance. It is well known that individual surface molecules and cytokines have significant roles in the induction of peripheral tolerance. However, this may vary according to the peripheral environment such as the severity of inflammation. Several groups have investigated the links between DC maturation and NF-κB activity using adenoviral transfection of IκB or suppression of IκB kinase, and showed that activated NF-κB plays significant roles in DC maturation including the induction of co-stimulatory molecules and cytokines such as IL-6 and IL-12 (Rescigno et al., 1998Rescigno M. Martino M. Sutherland C.L. Gold M.R. Ricciardi-Castagnoli P. Dendritic cell survival and maturation are regulated by different signaling pathways.J Exp Med. 1998; 188: 2175-2180Crossref PubMed Scopus (586) Google Scholar; Yoshimura et al., 2001bYoshimura S. Bondeson J. Foxwell B.M.J. Brennan F.M. Feldmann M. Effective antigen presentation by dendritic cells is NF-κB dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines.Int Immunol. 2001; 13: 675-683Crossref PubMed Scopus (192) Google Scholar). When NF-κB activity is artificially suppressed using proteasome inhibitors, adenoviral transfection of IκB, or NF-κB decoy ODN, the antigen-presenting functions of DC are also impaired, showing insufficient expression of MHC II, co-stimulatory molecules (CD80, CD86, and CD40), or immunostimulatory cytokines (IL-12 and tumor necrosis factor-α) (Yoshimura et al., 2001aYoshimura S. Bondeson J. Brennan F.M. Foxwell B.M.J. Feldmann M. Role of NFκB in antigen presentation and development of regulatory T cells elucidated by treatment of dendritic cells with the proteasome inhibitor PSI.Eur J Immunol. 2001; 31: 1883-1893Crossref PubMed Scopus (100) Google Scholar; O'Sullivan and Thomas, 2002O'Sullivan B.J. Thomas R. CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-κB.J Immunol. 2002; 168: 5491-5498Crossref PubMed Scopus (130) Google Scholar). Another report documented evidence that expression of a new co-stimulatory molecule homologue B7-H also requires NF-κB (Li and Verma, 2002Li Q. Verma I.M. NF-κB regulation in the immune system.Nat Rev Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3088) Google Scholar). Recent data suggest that regulatory T cells play a key role in peripheral tolerance (Takahashi et al., 1998Takahashi T. Kuniyasu Y. Toda M. Sakaguchi N. Itoh M. Iwata M. et al.Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state.Int Immunol. 1998; 10: 1969-1980Crossref PubMed Scopus (1276) Google Scholar; Shevach, 2001Shevach E.M. Certified professionals: CD4+CD25+ suppressor T cells.J Exp Med. 2001; 193: F41-F46Crossref PubMed Google Scholar; Taylor et al., 2001Taylor P.A. Noelle R.J. Blazar B.R. CD4+CD25+ immune regulatory T cells are required for induction of tolerance to alloantigen via costimulatory blockade.J Exp Med. 2001; 193: 1311-1318Crossref PubMed Scopus (477) Google Scholar; Thorstenson and Khoruts, 2001Thorstenson K.M. Khoruts A. Generation of anergic and potentially immunoregulatory CD25+CD4T cells in vivo after induction of peripheral tolerance with intravenous or oral antigen.J Immunol. 2001; 167: 188-195Crossref PubMed Scopus (374) Google Scholar; Zhang et al., 2001Zhang X. Izikson L. Liu L. Weiner H.L. Activation of CD4+CD25+ regulatory T cells by oral antigen administration.J Immunol. 2001; 167: 4245-4253Crossref PubMed Scopus (398) Google Scholar) under the influence of iDC or maturing DC (Min et al., 2003Min W.P. Zhou D. Ichim T.E. Strejan G.H. Xia X. Yang J. et al.Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance.J Immunol. 2003; 170: 1304-1312Crossref PubMed Scopus (220) Google Scholar; Roelofs-Haarhuis et al., 2003Roelofs-Haarhuis K. Wu X. Nowak M. Fang M. Artik S. Gleichmann E. Infectious nickel tolerance: a reciprocal interplay of tolerogenic APCs and T suppressor cells that is driven by immunization.J Immunol. 2003; 171: 2863-2872Crossref PubMed Scopus (35) Google Scholar). At present, at least four regulatory T cells can be identified based on expression of cell surface markers, secretion of cytokines, and suppression mechanisms (Groux, 2003Groux H. Type 1 T-regulatory cells: their role in the control of immune responses.Transplantation. 2003; 75: 8S-12SCrossref PubMed Google Scholar). Here, we demonstrated that the pre-existing OVA-specific CD4+CD25+ Treg are dominantly activated by NF-κB decoy ODN plus OVA and induce antigen-specific tolerance to DTH responses in vivo. These observations are in line with the recent reports that immature and maturing DC can induce proliferation of CD4+CD25+ T cells in an antigen-specific manner both in vivo and in vitro (Walker et al., 2003Walker L.S.K. Chodos A. Eggena M. Dooms H. Abbas A.K. Antigen-dependent proliferation of CD4+CD25+ regulatory T cells in vivo.J Exp Med. 2003; 198: 249-258Crossref PubMed Scopus (506) Google Scholar; Yamazaki et al., 2003Yamazaki S. Iyoda T. Tarbell K. Olson K. Velinzon K. Inaba K. Steinman R.M. Direct expansion of functional CD25+CD4+ regulatory T cells by antigen-processing dendritic cells.J Exp Med. 2003; 198: 235-247Crossref PubMed Scopus (751) Google Scholar). CD4+CD25+ Treg suppress bystander T cells in an antigen nonspecific manner by direct cell-to-cell interaction in vitro once they are activated (Thornton and Shevach, 1998Thornton A.M. Shevach E.M. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production.J Exp Med. 1998; 188: 287-296Crossref PubMed Scopus (2100) Google Scholar; Thornton and Shevach, 2000Thornton A.M. Shevach E.M. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific.J Immunol. 2000; 164: 183-190Crossref PubMed Scopus (1037) Google Scholar). However, antigen-specific stimulation via T cell receptor is required for the expansion and induction of suppressive properties of Treg (Shevach, 2001Shevach E.M. Certified professionals: CD4+CD25+ suppressor T cells.J Exp Med. 2001; 193: F41-F46Crossref PubMed Google Scholar). The suppression mode, whether antigen specific or nonspecific, remains controversial (Shevach, 2001Shevach E.M. Certified professionals: CD4+CD25+ suppressor T cells.J Exp Med. 2001; 193: F41-F46Crossref PubMed Google Scholar; Sakaguchi, 2004Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2795) Google Scholar). In this regard, the strategy employed in this study provides a new path for antigen-specific tolerance induced by CD4+CD25+ Treg. Our data indicate that topical application of NF-κB decoy ODN can suppress DC migration and maturation leading to regulation of the immunostimulatory capacity. After NF-κB decoy ODN treatment, a sizable DC population still remains in the peripheral tissue after capturing exogenous antigen. Even if these cells migrate into draining LNs, their presentation of antigens to T cells via MHC II is limited by insufficient co-stimulatory signaling. Inadequate antigen presentation by modified DC may lead to the induction of Treg. Here we induced Treg through a combination of topical NF-κB decoy ODN treatment and antigen stimulation. At present, we consider that antigen stimulation of iDC without NF-κB signaling is critical for the induction of tolerogenic DC that can activate Treg efficiently. In UV-induced tolerance to contact hypersensitivity, intravenous transfer of Treg could not suppress contact hypersensitivity challenge in sensitized mice (Schwarz et al., 2004Schwarz A. Maeda A. Wild M.K. Kernebeck K. Gross N. Aragane Y. et al.Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity.J Immunol. 2004; 172: 1036-1043Crossref PubMed Scopus (169) Google Scholar), which is distinct from our data. However, in our study, secondary antigen sensitizations were performed after adoptive transfer of Treg, while mice were elicited immediately after transfer into UV-induced contact hypersensitivity tolerance study. For tolerance induction, therefore, antigen presentation by iDC or modified DC in the presence of enriched Treg may be critical to prevent expansion of effector cells. A number of studies have proven that adoptive transfer of allogeneic DC cultured with NF-κB decoy ODN prolongs survival of cardiac allografts (Giannoukakis et al., 2000Giannoukakis N. Bonham C.A. Qian S. et al.Prolongation of cardiac allograft survival using dendritic cells treated with NF-κB decoy oligodeoxyribonucleotides.Mol Ther. 2000; 1: 430-437Crossref PubMed Scopus (134) Google Scholar; Bonham et al., 2002Bonham C.A. Peng L. Liang X. Chen Z. Wang L. Ma L. et al.Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-κB oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig.J Immunol. 2002; 169: 3382-3391Crossref PubMed Scopus (124) Google Scholar). However, this approach involves considerable cost and time because DC must be isolated from the peripheral blood, incubated with NF-κB decoy ODN in vitro and then injected intravenously. In addition, this method is only effective for immune tolerance to allogeneic antigens. Here we demonstrated peripheral immune tolerance to two distinct antigens by a combination of antigen stimulation and topical application of NF-κB decoy ODN. This strategy is very simple and less expensive. Therefore, application of this strategy might be able to regulate immune responses against antigens that cause allergic or autoimmune diseases if the responsible antigens can be identified. The immune suppression induced by NF-κB decoy ODN in the present study was restricted to the applied skin area. Intravenous injection of tolerogenic DC may induce systemic immune suppression effectively, but one distinct advantage of tolerance induction by topical NF-κB decoy ODN treatment may be the prevention of adverse effects associated with systemic administration. Systemic suppression of NF-κB is likely to be harmful, given that knockout mice for various NF-κB signaling components suffer from immune deficiency or lack lymphocyte activation (Morishita et al., 1997Morishita R. Sugimoto T. Aoki M. Kida I. Tomita N. Moriguchi A. et al.In vivo transfection of cis element “decoy” against nuclear factor-κB binding site prevents myocardial infarction.Nat Med. 1997; 3: 894-899Crossref PubMed Scopus (594) Google Scholar). In support of this concept, a topical NF-κB decoy ODN treatment for atopic dermatitis in mice was found to be highly effective (Nakamura et al., 2002Nakamura H. Aoki M. Tamai K. Oishi M. Ogihara T. Kaneda Y. et al.Prevention and regression of atopic dermatitis by ointment containing NF-κB decoy oligodeoxynucleotides in NC/Nga atopic mouse model.Gene Therapy. 2002; 9: 1221-1229Crossref PubMed Scopus (102) Google Scholar). In addition, it has been revealed that cutaneous immunization with autoantigenic peptides prevents experimental allergic encephalomyelitis (Bynoe et al., 2003Bynoe M.S. Evans J.T. Viret C. Janeway Jr, C.A. Epicutaneous immunization with autoantigenic peptides induces T suppressor cells that prevent experimental allergic encephalomyelitis.Immunity. 2003; 19: 317-328Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In conclusion, a new strategy to induce antigen-specific peripheral tolerance by topical application of NF-κB decoy ODN has great potential for the control of allergic diseases and autoimmune disorders. Further studies of this approach are clearly warranted. Female 6- to 12-week-old BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). Mice transgenic for the OVA323–339-specific and I-Ad-restricted DO11.10 T cell receptorαβ on a BALB/c genetic background (Murphy et al., 1990Murphy K.M. Heimberger A.B. Loh D.Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+ TCRlo thymocytes in vivo.Science. 1990; 250: 1720-1723Crossref PubMed Scopus (1627) Google Scholar) were kindly provided by Dr K. Murphy (University of Washington, St, Louis, MO) via Dr Y. Koide (Hamamatsu University School of Medicine, Hamamatsu, Japan). All procedures with animals were carried out in accordance with institutionally approved protocols, following the principles of laboratory animal care formulated by the National Society for Medical Research. Five mice were used in each group in all experiments. At least three sets of experiments were performed independently to obtain results. FITC-conjugated anti-CD11c (HL3), FITC- or PE-conjugated anti-I-Ad (2G9), and biotin-conjugated anti-CD25 (7D4) were purchased from BD Biosciences Pharmingen (San Diego, CA), PE-conjugated anti-CD80 (16-10A1), anti-CD86 (GL1), and anti-CD4 (GK1.5) mAb were from e-Bioscience (San Diego, CA), FITC-conjugated streptavidin was from EMD Biosciences, Inc. (San Diego, CA), and an anti-CD25 mAb (PC61) was kindly provided by Dr O. Taguchi (Aichi Cancer Center Research Institute, Nagoya, Japan). The IgG fraction of anti-CD25 (PC61) was obtained by protein G-Sepharose (Amersham Biosciences, Piscataway, NJ). OVA and HEL (Sigma-Aldrich, St Louis, MO) were used for sensitization and elicitation of DTH. FITC isomer I was purchased from Sigma-Aldrich. NF-κB decoy ODN ointment (2%) was prepared as described below. NF-κB decoy or scramble ODN were synthesized by QIAGEN GmbH (Hilden, Germany), and mixed with 5% stearyl alcohol and white petroleum. Their sequences were as follows (NF-κB binding sequences underlined): NF-κB decoy ODN5′-CCTTGAAGGGATTTCCCTCC-3′3′-GGAACTTCCCTAAAGGGAGG-5′ Scrambled ODN5′-TTGCCGTACCTGACTTAGCC-3′3′-AACGGCATGGACTGAATCGG-5′ The sequences of this NF-κB decoy ODN have been shown to bind NF-κB (Morishita et al., 1997Morishita R. Sugimoto T. Aoki M. Kida I. Tomita N. Moriguchi A. et al.In vivo transfection of cis element “decoy” against nuclear factor-κB binding site prevents myocardial infarction.Nat Med. 1997; 3: 894-899Crossref PubMed Scopus (594) Google Scholar). Time schedules for individual experiments are detailed in the corresponding figures. Twenty milligrams of NF-κB ODN ointment was applied on the shaved abdominal skin of BALB/c mice in each case. Mice were sensitized by 1 mg OVA or 500 μg HEL in an emulsion of 100 μl phosphate buffered saline (PBS) (Sigma-Aldrich) and 100 μl incomplete Freund's adjuvant (Sigma-Aldrich), and DTH was elicited with 100 μg OVA (2 mg/ml) or 50 μg HEL (4 mg/ml) in PBS. Elicitation was performed by subcutaneous injection of antigens into left footpads 7 days after final sensitization, if not otherwise stated. Fifty microliters of PBS were injected into right footpads as a negative control. Footpad swelling was assayed by the thickness of the left footpad minus that of the right footpad at 24 or 48 hours after elicitation. Footpad thickness was measured with a digimatic gauge (Mitsutoyo Corporation, Kanagawa, Japan). For adoptive transfer experiments, inguinal and popliteal LNs were harvested from mice 3 days after elicitation, mashed with slide glasses, and passed through nylon mesh before centrifugation at 1,500 r.p.m. for 10 minutes. After two washes, LN cells were suspended in PBS and injected into tail veins (each mouse received 5 × 106 LN cells in 300 μl of PBS from one mouse). Mice were injected with 0.2 mg PC61 mAb in 500 μl PBS intraperitoneally as described previously (Onizuka et al., 1999Onizuka S. Tawara I. Shimizu J. Sakaguchi S. Fujita T. Nakayama E. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor α) monoclonal antibody.Cancer Res. 1999; 59: 3128-3133PubMed Google Scholar; Sakaguchi, 2004Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2795) Google Scholar). LN cells were prepared from sensitized or tolerant mice as described above, and CD4+CD25+ T cells were positively separated from CD4+CD25− T cells with a MACS CD4+CD25+ Regulatory T cell isolation kit (Miltenyi Biotec, Gladbach, Germany). The percentages of separated CD4+CD25− and CD4+CD25− T cells in the LN cells were approximately 30 and 2%, respectively. Mice were sensitized with OVA on day 0 and injected intraveneously with 2 × 105 CD4+CD25+ or CD4+CD25− T cells in 300 μl PBS on day 6. Elicitation followed on day 7. Mice were painted with 0.5% FITC in 1:1 acetone/dibutyl phthalate (400 μl) (Sigma-Aldrich) 24 hours after topical application of NF-κB decoy ODN. LN cells were prepared from axillary, inguinal, and popliteal LNs and stained with PE-conjugated CD11c mAb. A possible population containing DC was gated with forward and side scatters and the percentage of CD11c+FITC+ cells was evaluated by FACS, along with the expression of MHC II, CD80, and CD86 on FITC+ cells. After the application of NF-κB decoy ODN or scramble decoy and the sensitization with OVA, CD11c+ DC were isolated from cell suspensions of LNs with anti-CD11c microbeads (Miltenyi biotec). CD4+ T cells were isolated from splenocytes of DO11.10 transgenic mice using the CD4+ T cell isolation kit (Miltenyi Biotec). CD4+ T cells (2 × 105/well) were incubated with DC (4 × 104/well) in 96-well microplates (stimulator:responder ratio=1:5). After 72 hours incubation, release of IFN-γ was assayed by ELISA (R&D systems, Minneapolis, MN). Single-cell suspensions were prepared as described above and blocked with rat or mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) on ice for 10 minutes before antibody staining. Cells were then washed in Hank's balanced salt solution (Sigma-Aldrich) with 2% fetal bovide serum (Sigma-Aldrich) twice, and stained with mAb on ice for 30 minutes. They were washed twice and suspended in Hank's balanced salt solution and analyzed by FACScan (BD Biosciences Immunocytochemistry Systems, San Diego, CA). In some experiments, cells were stained with FITC-conjugated streptavidin (Jackson ImmunoResearch Laboratories) after reaction with biotin-conjugated mAb. All data were statistically analyzed using Student's t-test. Bar graphs are presented as mean±standard error of the mean value. The author states no conflict of interest. This work was in part supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank K Tamai for advice on construction of NF-κB decoy ODN and preparation of the ointment containing NF-κB decoy ODN, as well as J. Krutmann for critically reading the manuscript." @default.
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• W1965575960 title "Antigen-Specific Peripheral Tolerance Induced by Topical Application of NF-κB Decoy Oligodeoxynucleotide" @default.
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