Crosstalk of regulatory T cells and tolerogenic dendritic cells prevents contact allergy in subjects with low zone tolerance  Ulrike Luckey, PhD, Talkea Schmidt, MSc, Nikolai Pfender, Maike Romer, Nadine Lorenz, MSc, Stefan F. Martin, PhD, Tobias Bopp, PhD, Edgar Schmitt, PhD, Alexey Nikolaev, Nir Yogev, PhD, Ari Waisman, PhD, Thilo Jakob, MD, Kerstin Steinbrink, MD  Journal of Allergy and Clinical Immunology  Volume 130, Issue 3, Pages 781-797.e11 (September 2012) DOI: 10.1016/j.jaci.2012.06.022 Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Pivotal role of CD4+CD25+FOXP3+ Treg cells for the development of LZT. A-C, Mice were treated with the anti-CD25 mAb (PC61) or a control antibody. Solvent (CHS) or TNCB (LZT) was applied epicutaneously 10 times. After recovery of CD4+CD25+FOXP3+ Treg cells (see Fig E1), animals were sensitized and challenged as described. D-F, DEREG mice were treated with DT for Treg cell depletion. Solvent (CHS) or tolerizing doses (LZT) of the hapten TNCB were applied epicutaneously, and subsequently, mice were sensitized and challenged (see Fig E2). Fig 1, A and D, The ear-swelling response was assessed after challenge (mean ± SD). Fig 1, B, C, E, and F, Skin-draining LNs were obtained for T-cell analysis. Fig 1, B and E, T-cell proliferation was depicted (mean value of triplicates in counts per minute) after restimulation. Data represent one of 4 (Fig 1, A-C) and one of 3 (Fig 1, D-E) independent experiments with similar results. Pooled data of release of IFN-γ and IL-2 by T cells of 4 (Fig 1, C) or 3 (Fig 1, F) independent experiments are shown (means ± SDs). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Pivotal role of CD4+CD25+FOXP3+ Treg cells for the development of LZT. A-C, Mice were treated with the anti-CD25 mAb (PC61) or a control antibody. Solvent (CHS) or TNCB (LZT) was applied epicutaneously 10 times. After recovery of CD4+CD25+FOXP3+ Treg cells (see Fig E1), animals were sensitized and challenged as described. D-F, DEREG mice were treated with DT for Treg cell depletion. Solvent (CHS) or tolerizing doses (LZT) of the hapten TNCB were applied epicutaneously, and subsequently, mice were sensitized and challenged (see Fig E2). Fig 1, A and D, The ear-swelling response was assessed after challenge (mean ± SD). Fig 1, B, C, E, and F, Skin-draining LNs were obtained for T-cell analysis. Fig 1, B and E, T-cell proliferation was depicted (mean value of triplicates in counts per minute) after restimulation. Data represent one of 4 (Fig 1, A-C) and one of 3 (Fig 1, D-E) independent experiments with similar results. Pooled data of release of IFN-γ and IL-2 by T cells of 4 (Fig 1, C) or 3 (Fig 1, F) independent experiments are shown (means ± SDs). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 CD4+CD25+ Treg cells activated by low amounts of contact allergen operate in an allergen-nonspecific manner in contrast to regulatory CD8+ T cells. A-C, After tolerization with TNCB, CD4+CD25+ T cells were purified from LN cells and adoptively transferred (AT) into naive WT mice. Tolerized mice (LZT) and mice with CHS without cell transfer served as control animals. Subsequently, recipients and control groups were sensitized and challenged with either TNCB or DNFB, respectively. Fig 2, A, The ear-swelling response was assessed after challenge (mean ± SD). Fig 2, B, T-cell proliferation is detected after restimulation (mean value of triplicates in counts per minute). One representative of 4 experiments is shown in Fig 2, A and B. Fig 2, C, Release of IFN-γ and IL-2 by T cells was assessed (means ± SD). Pooled data of 4 experiments are depicted. D and E, CD4+CD25+ T cells were purified from LN cells from solvent-treated (naive) or tolerized (activated) mice and adoptively transferred into naive animals. Tolerized (LZT) and solvent-treated (CHS) mice without cell transfer served as control animals. Recipients and control groups were sensitized and challenged. Fig 2, D, Ear thickness (mean ± SD) was assessed. E, T-cell proliferation is depicted as the mean counts per minute of triplicates after hapten-specific restimulation. One representative experiment of 3 is demonstrated in Fig 3, D and E. F, After tolerization with TNCB, CD8+ T cells were purified from LN cells and adoptively transferred (AT) into naive WT mice. Tolerized (LZT) mice and mice with CHS without cell transfer served as control animals. Subsequently, recipients and control groups were sensitized and challenged with either TNCB or DNFB, respectively. Increase in ear thickness (mean ± SD) is detected. One representative of 3 experiments is shown. *P < .05, **P < .01, and ***P < .001. n.s., Not significant. tol, Tolerized; sens/chall, sensitized/challenged. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Induction of an activated phenotype of CD4+CD25+FOXP3+ Treg cells during LZT. Mice were tolerized or solvent treated, and thereafter, some groups were sensitized. Flow cytometric analysis of CD4+CD25+FOXP3+ Treg cells of LN cells was performed. Percentages ± SD (left panel) and mean fluorescence intensity (MFI ± SD, right panel) of CCR7+ (A) or CD103+ (B) Treg cells (gated on CD4+CD25+FOXP3+ T cells) were analyzed after induction of tolerance or solvent treatment (24 hours after the last application of last tolerizing dose) or after sensitization (day 5 after sensitization). Pooled data of 4 experiments and 1 representative experiment after tolerization are shown. C, After tolerization (LZT) or solvent treatment (CHS), CD4+CD25+ T cells were purified from LN cells of CCR7−/− mice and adoptively transferred (AT) into naive WT mice. Tolerized (LZT) mice and mice with CHS without cell transfer served as control animals. Subsequently, recipients and control groups were sensitized and challenged. The ear-swelling response was assessed after challenge (mean ± SD). *P < .05 and **P < .01. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Induction of an activated phenotype of CD4+CD25+FOXP3+ Treg cells during LZT. Mice were tolerized or solvent treated, and thereafter, some groups were sensitized. Flow cytometric analysis of CD4+CD25+FOXP3+ Treg cells of LN cells was performed. Percentages ± SD (left panel) and mean fluorescence intensity (MFI ± SD, right panel) of CCR7+ (A) or CD103+ (B) Treg cells (gated on CD4+CD25+FOXP3+ T cells) were analyzed after induction of tolerance or solvent treatment (24 hours after the last application of last tolerizing dose) or after sensitization (day 5 after sensitization). Pooled data of 4 experiments and 1 representative experiment after tolerization are shown. C, After tolerization (LZT) or solvent treatment (CHS), CD4+CD25+ T cells were purified from LN cells of CCR7−/− mice and adoptively transferred (AT) into naive WT mice. Tolerized (LZT) mice and mice with CHS without cell transfer served as control animals. Subsequently, recipients and control groups were sensitized and challenged. The ear-swelling response was assessed after challenge (mean ± SD). *P < .05 and **P < .01. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 CD4+CD25+ Treg cells are required for generation of CD8+ Treg cells of mice with LZT. Depletion of CD4+CD25+FOXP3+ Treg cells was induced by injection of PC61 (anti-CD25 mAb; right panels, PC61). Control mice received an IgG control antibody (left panels, control). After tolerization, CD8+ T cells were obtained and adoptively transferred into naive WT mice (gray bars, squares). Mice without cell transfer served as control animals for CHS (black bars, squares) or LZT (white bars, squares). Recipients and control mice were sensitized and challenged. A and B, The allergic inflammatory response (Fig 4, A) and proliferation of T cells (Fig 4, B) were assessed as described in the legend for Fig 1. Fig 4, A and B, Results of one representative experiment of 3 independent experiments are shown. C, Release of IFN-γ and IL-2 by T cells was assessed (mean ± SD). Pooled data of 3 independent experiments are demonstrated. *P < .05, **P < .01, and ***P < .001. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Tolerogenic CD11c+ DCs are required for tolerance induction. Depletion of CD11c+ DCs was induced by injection of DT into CD11c-DTR mice. PBS administration served as a control. Subsequently, mice were tolerized (LZT) or treated with solvent (CHS), and after recovery of CD11c+ DCs, sensitization and challenge were conducted. In some experiments mice were reconstituted with CD11c+ DCs after depletion of DCs and before tolerance induction. A, The ear-swelling response was assessed after challenge (mean ± SD). B, T-cell proliferation after hapten-specific restimulation was detected (mean value of triplicates in counts per minute). One representative result of 3 experiments is shown. C, Amounts of IFN-γ and IL-2 by T cells was detected (mean ± SD). Pooled data of 3 independent experiments are depicted. *P < .05 and **P < .01. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 LZT is transferable by CD11c+ DCs and abrogated in the absence of CD8+CD11c+ DCs. A and B, Animals were tolerized or solvent treated. Thereafter, CD11c+ DCs were isolated from peripheral LNs and adoptively transferred to naive mice. Recipients and control animals (CHS and LZT without cell transfer) were sensitized and challenged. Skin inflammation (Fig 6, A) and T-cell proliferation (Fig 6, B) was assessed as described in the legend for Fig 1. One of 3 experiments with similar results is depicted. C-E, Solvent (CHS) or TNCB (LZT) was applied epicutaneously 10 times onto Batf3 mice lacking CD8+CD11c+ DCs. WT mice served as control animals. Subsequently, animals were sensitized and challenged, as previously described. Fig 6, C, The ear-swelling response was assessed after challenge (mean ± SD). Fig 6, D, T-cell proliferation was depicted (mean value of triplicates in counts per minute) after restimulation. Fig 6, C and D, Data represent one of 3 independent experiments with similar results. Fig 6, E, Pooled data of release of IFN-γ and IL-2 by T cells of 3 independent experiments are shown (means ± SD). *P < .05 and **P < .01. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 CD4+CD25+ Treg cells are required for activation of tolerogenic CD11c+ DCs followed by induction of hapten-specific regulatory CD8+ T cells of LZT. A and B, Mice were treated with anti-CD25 mAb to deplete Treg cells or with IgG control antibody. Subsequently, after tolerance induction (AT CD11c+ LZT) or solvent treatment (AT CD11c+ Ctrl), CD11c+ DC were transferred. Recipients and control animals (CHS and LZT without cell transfer) were sensitized and challenged. Fig 7, A, Ear thickness was assessed in mean ± SD after challenge. One representative experiment of 3 is shown. Fig 7, B, T-cell proliferation (mean values of triplicates in counts per minute) was detected. Results of one of 3 independent experiments are depicted. C and D, Injections of DT into CD11c-DTR mice were performed to deplete CD11c+ DCs. After tolerization (AT of CD8+ LZT) or solvent treatment (AT of CD8+ Ctrl), CD8+ T cells were obtained and injected into naive recipients. Recipients and control animals (CHS and LZT without cell transfer) were sensitized and challenged. Fig 7, C, Absolute increase in ear thickness (± SD) was assessed after challenge. Fig 7, D, Proliferation (mean values of triplicates in counts per minute) of T cells is shown. One representative result of 3 experiments is demonstrated. E, CD4+CD25+ Treg cells (Ly5.2) were obtained after tolerization (LZT) or solvent treatment (CHS) and labeled with calcein. Thereafter, CD4+CD25+ Treg cells were transferred into naive Ly5.1 WT recipients that were subsequently sensitized. Skin-draining LN cells were harvested, and calcein-positive Ly5.1+ CD11c+ DCs were detected by using flow cytometry. One of 3 representative experiments is shown. F, After transfer of tolerogenic or control CD45.2 CD11c+ DCs into the recipients (CD45.1 mice), tissue sections of skin-draining LNs were stained for FOXP3 (red nuclear staining) and CD45.2 DCs (depicted in green). Interactions of FOXP3+ Treg cells and CD45.2 DCs in mice with LZT or with CHS of representative tissue sections (left panels) and expressed as the mean number of Treg cells per DC in 20 high-power fields (right panel) are demonstrated. Bar scale = 50 μm. *P < .05, **P < .01, and ***P < .001 or as indicated. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 CD4+CD25+ Treg cells are required for activation of tolerogenic CD11c+ DCs followed by induction of hapten-specific regulatory CD8+ T cells of LZT. A and B, Mice were treated with anti-CD25 mAb to deplete Treg cells or with IgG control antibody. Subsequently, after tolerance induction (AT CD11c+ LZT) or solvent treatment (AT CD11c+ Ctrl), CD11c+ DC were transferred. Recipients and control animals (CHS and LZT without cell transfer) were sensitized and challenged. Fig 7, A, Ear thickness was assessed in mean ± SD after challenge. One representative experiment of 3 is shown. Fig 7, B, T-cell proliferation (mean values of triplicates in counts per minute) was detected. Results of one of 3 independent experiments are depicted. C and D, Injections of DT into CD11c-DTR mice were performed to deplete CD11c+ DCs. After tolerization (AT of CD8+ LZT) or solvent treatment (AT of CD8+ Ctrl), CD8+ T cells were obtained and injected into naive recipients. Recipients and control animals (CHS and LZT without cell transfer) were sensitized and challenged. Fig 7, C, Absolute increase in ear thickness (± SD) was assessed after challenge. Fig 7, D, Proliferation (mean values of triplicates in counts per minute) of T cells is shown. One representative result of 3 experiments is demonstrated. E, CD4+CD25+ Treg cells (Ly5.2) were obtained after tolerization (LZT) or solvent treatment (CHS) and labeled with calcein. Thereafter, CD4+CD25+ Treg cells were transferred into naive Ly5.1 WT recipients that were subsequently sensitized. Skin-draining LN cells were harvested, and calcein-positive Ly5.1+ CD11c+ DCs were detected by using flow cytometry. One of 3 representative experiments is shown. F, After transfer of tolerogenic or control CD45.2 CD11c+ DCs into the recipients (CD45.1 mice), tissue sections of skin-draining LNs were stained for FOXP3 (red nuclear staining) and CD45.2 DCs (depicted in green). Interactions of FOXP3+ Treg cells and CD45.2 DCs in mice with LZT or with CHS of representative tissue sections (left panels) and expressed as the mean number of Treg cells per DC in 20 high-power fields (right panel) are demonstrated. Bar scale = 50 μm. *P < .05, **P < .01, and ***P < .001 or as indicated. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 CD4+CD25+ Treg cell–derived IL-10 is critical during the induction phase of LZT. A, IL-10 release from CD4+, CD4+CD25+, and CD8+ T cells was assessed by means of ELISpot after hapten-specific restimulation. The results of one of 2 representative experiment are demonstrated, and the pooled data of 5 animals are depicted. B, In vivo IL-10 transcription of CD4+, CD4+CD25+, and CD8+ T cells from skin-draining LNs was analyzed by using IL-10 transcriptional reporter mice (Vert-X). Pooled data (n = 19 mice per group) from 4 independent experiments are depicted (percentage of positive cells ± SEM). C, CD4+ and CD8+ or CD4+ T cells depleted of CD25+ Treg cells, respectively, were obtained from untreated WT or IL-10−/− mice for reconstitution of T cell–deficient Rag1−/− mice. Subsequently, the mice were repeatedly painted with tolerizing doses of TNCB (LZT) or solvent treated (CHS), followed by sensitization and challenge. The allergic skin inflammation was assessed based on the increase in ear swelling (mean ± SD). D, Animals were treated with an IL-10 receptor blocking antibody (aIL-10R) or isotype control. Ear thickness in absolute values ± SDs was assessed 24 hours later. One of 2 representative experiments is demonstrated. *P < .05, **P < .01, and ***P < .001. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 CD4+CD25+ Treg cell–derived IL-10 is critical during the induction phase of LZT. A, IL-10 release from CD4+, CD4+CD25+, and CD8+ T cells was assessed by means of ELISpot after hapten-specific restimulation. The results of one of 2 representative experiment are demonstrated, and the pooled data of 5 animals are depicted. B, In vivo IL-10 transcription of CD4+, CD4+CD25+, and CD8+ T cells from skin-draining LNs was analyzed by using IL-10 transcriptional reporter mice (Vert-X). Pooled data (n = 19 mice per group) from 4 independent experiments are depicted (percentage of positive cells ± SEM). C, CD4+ and CD8+ or CD4+ T cells depleted of CD25+ Treg cells, respectively, were obtained from untreated WT or IL-10−/− mice for reconstitution of T cell–deficient Rag1−/− mice. Subsequently, the mice were repeatedly painted with tolerizing doses of TNCB (LZT) or solvent treated (CHS), followed by sensitization and challenge. The allergic skin inflammation was assessed based on the increase in ear swelling (mean ± SD). D, Animals were treated with an IL-10 receptor blocking antibody (aIL-10R) or isotype control. Ear thickness in absolute values ± SDs was assessed 24 hours later. One of 2 representative experiments is demonstrated. *P < .05, **P < .01, and ***P < .001. n.s., Not significant. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 9 Immune mechanisms during the induction phase of LZT. Repeated applications of low subimmunogenic doses of contact allergens induce a non–hapten-specific activation of IL-10–producing CD4+CD25+FOXP3+ Treg cells that interact with LN-related CD8+CD11+ DCs. This crosstalk results in a tolerogenic phenotype of DCs that induce the generation of CD8+ Treg cells of mice with LZT. These CD8+ Treg cells of mice with LZT efficiently inhibit the development of TC1-mediated allergic skin immune responses in a hapten-specific manner. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Evaluation of Treg cell depletion after application of an anti-CD25 mAb (PC61) during tolerization. A, Treatment of mice was performed as depicted. B, Flow cytometric analysis was conducted to analyze percentages of CD4+FOXP3+ (upper panel) and FOXP3+CD25+ (gated on CD4+ T cells, lower panel) Treg cells at day 0 (before treatment), days 3 and 12 (phase of tolerization), and day 27 (before sensitization) after application of the anti-CD25 mAb (PC61). Representative data of 6 individual experiments are depicted. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Efficacy of Treg cell depletion in DEREG mice. A, Mice were injected with DT before tolerization. B and C, Flow cytometric analysis of skin-draining LN cells (Fig E2, B) and peripheral blood (Fig E2, C) was performed. Percentages of CD4+FOXP3+ Treg cells (lymphocyte gate, upper panels) and FOXP3+CD25+ Treg cells (gated on CD4+ T cells, lower panels) were analyzed before treatment at days 0, 1, and 3 (phase of tolerization) and days 5 and day 7 (before sensitization) after application of DT. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Percentages of CD4+CD25+FOXP3+ Treg cells are equivalent in tolerized versus control mice. Animals were painted with either TNCB or solvent during tolerization and subsequently sensitized and challenged. LNs were obtained after tolerization, sensitization, and challenge. Analyses were performed for CD25+ (A-C) and CD25high (D-F) T cells. Expression of CD4 and CD25 (Fig E3, A and D) and FOXP3 (gated on CD4+CD25+ T cells; Fig E3, B and E) was analyzed by means of flow cytometry. One representative experiment (Fig E3, A, B, D, and E) and pooled data of 4 experiments (Fig E3, C and F) are demonstrated. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Percentages of CD4+CD25+FOXP3+ Treg cells are equivalent in tolerized versus control mice. Animals were painted with either TNCB or solvent during tolerization and subsequently sensitized and challenged. LNs were obtained after tolerization, sensitization, and challenge. Analyses were performed for CD25+ (A-C) and CD25high (D-F) T cells. Expression of CD4 and CD25 (Fig E3, A and D) and FOXP3 (gated on CD4+CD25+ T cells; Fig E3, B and E) was analyzed by means of flow cytometry. One representative experiment (Fig E3, A, B, D, and E) and pooled data of 4 experiments (Fig E3, C and F) are demonstrated. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Evaluation of CD11c+ DC depletion. A, CD11c-DTR mice were injected with DT at days −1 and 0. Subsequently, mice were tolerized by means of 4 tolerizing doses every 12 hours. B, Numbers of CD11c+ DCs in skin-draining LNs were assessed by using flow cytometry at days 0, 1, 2, and 7 after treatment with DT. For the experiments, mice were tolerized by application of low amounts of TNCB or treated with solvent in the absence of CD11c+ DCs. After recovery of CD11c+ DCs, sensitization and challenge were performed. C, Percentages of CD4+CD25+FOXP3+ Treg cells in draining LNs after DT treatment are demonstrated. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Increased CD80 expression on LN DCs after depletion of Treg cells. Depletion of CD4+CD25+FOXP3+ Treg cells was induced by means of injection of PC61 (anti-CD25 mAb). Mice were tolerized (LZT) or treated with solvent (controls). The phenotype of LN DCs was assessed by means of flow cytometry after tolerance induction. A and B, Percentages of CD11c+ MHC class IIhigh DCs in LN cells were demonstrated as mean ± SD (Fig E5, A), and mean fluorescence intensity (MFI) of CD80 expression of CD11c+ MHC class IIhigh DCs was depicted as percentages ± SDs of CHS controls (= 100%; Fig E5, B). C, Gating strategy for CD11c+ MHC class IIhigh DCs. FSC, Forward scatter; SSC, side scatter. *P < .05. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Identical percentages of calcein-positive CD4+ and CD8+ T cells and B cells in mice with LZT and mice with CHS after transfer of calcein-labeled CD4+CD25+ Treg cells. CD4+CD25+ Treg cells were obtained from skin-draining LN cells of Ly5.2 C57BL/6 WT mice after application of solvent (CHS) or tolerizing doses (LZT) of the hapten TNCB, labeled with calcein, and injected intravenously into Ly5.1 WT animals. The recipients were sensitized after 24 hours, and 72 hours after sensitization, skin-draining LNs were harvested and calcein-positive Ly5.2. Treg cells (A) and the calcein transfer in Ly5.1 CD4+ T cells (B), CD8+ T cells (C), and B220+ B cells (D) were detected by using flow cytometry. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 LZT is also efficiently induced after sensitization. A, Mice were sensitized and subsequently treated with solvent (CHS) or tolerizing doses (0.45 μg) of TNCB (LZT after sensitization). B and C, After challenge, the ear-swelling response was assessed (mean of absolute values ± SDs; Fig E7, B) and T-cell proliferation was detected (mean values of triplicates in counts per minute; Fig E7, C) after restimulation. D, Release of IFN-γ and IL-2 by T cells is shown. Data represent one of 3 independent experiments with similar results. **P < .01. Journal of Allergy and Clinical Immunology 2012 130, 781-797.e11DOI: (10.1016/j.jaci.2012.06.022) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions