α-MSH-Stimulated Tolerogenic Dendritic Cells Induce Functional Regulatory T Cells and Ameliorate Ongoing Skin Inflammation  Matteo Auriemma, Thomas Brzoska, Lars Klenner, Verena Kupas, Tobias Goerge, Maik Voskort, Zuotao Zhao, Tim Sparwasser, Thomas A. Luger, Karin Loser  Journal of Investigative Dermatology  Volume 132, Issue 7, Pages 1814-1824 (July 2012) DOI: 10.1038/jid.2012.59 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 α-Melanocyte-stimulating hormone (α-MSH) induces tolerogenic dendritic cells (DCs). (a) Animals were injected with α-MSH or phosphate-buffered saline (PBS), and 48hours after elicitation of contact hypersensitivity (CHS) ear swelling was assessed. Data are shown as mean ear swelling±SD and are representative of 12 mice in each group (*P<0.05 vs. PBS-treated mice). (b) Flow cytometry of T cells from mice injected with PBS or α-MSH (n=8 mice in each group) 48hours after elicitation of CHS. (Left) Representative dot blots and (right) statistical analyses of Foxp3+ cells in regional lymph nodes are shown; cells are gated for CD4. Foxp3 staining was performed after cell permeabilization (*P<0.05 vs. PBS-treated mice). (c, d) Bone marrow–derived DCs (bmDCs) were stimulated with α-MSH or PBS, and the expression of (c) CD80, CD86, PD-L1, PD-L2, and (d) CD205 was quantified by flow cytometry or real-time PCR. Histogram overlays are gated for CD11c, and one representative out of three independent experiments is shown. (e) The IL-10 concentration in culture supernatants from α-MSH- or PBS-stimulated DCs was quantified using FlowCytomix kits, and IL-10 mRNA levels were analyzed by real-time quantitative PCR (*P<0.05 vs. PBS-stimulated DCs). (f) DCs from C57BL/6Je/e mice were stimulated with α-MSH, and the expression of CD80, CD86, and CD205 was analyzed by flow cytometry. Histogram overlays are gated for CD11c, and one representative experiment is shown. Journal of Investigative Dermatology 2012 132, 1814-1824DOI: (10.1038/jid.2012.59) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 α-Melanocyte-stimulating hormone (α-MSH)-stimulated DCs expand functional regulatory T cells (Tregs). (a) CD4+ T cells from cocultures with α-MSH-stimulated or control DCs were analyzed for the expression of Foxp3, and (left) one representative dot-plot and (right) statistical analyses of four independent experiments are shown (*P<0.05 vs. CD4+ T cells from cocultures with phosphate-buffered saline (PBS)-stimulated DCs). Foxp3 staining was performed after cell permeabilization and cells are gated for CD4. (b) CD4+ T cells from cocultures with α-MSH-stimulated or control DCs were analyzed for the expression of Foxp3, Nrp-1, and CTLA-4 by flow cytometry. CTLA-4 and Foxp3 staining was performed after cell permeabilization, and (top) representative histogram overlays and (bottom) statistical analyses of four independent experiments are shown (*P<0.05 vs. CD4+ T cells from cocultures with PBS-stimulated DCs). Cells are gated for CD4+Foxp3+. (c) The relative mRNA levels of Foxp3, IL-10, Nrp-1, CTLA-4, and TGF-β were quantified in CD4+ T cells from cocultures with α-MSH- or PBS-stimulated DCs. Data are shown as mean±SD (*P<0.05 vs. CD4+ T cells from cocultures with PBS-stimulated DCs). (d) CD4+ T cells were purified from cocultures with α-MSH- or PBS-treated DCs, and proliferation assays were performed by stimulating freshly isolated CD4+CD25- T cells with anti-CD3 and anti-CD28 in the absence or presence of CD4+ T cells from cocultures. Mean values of 3H-thymidine incorporation±SD are shown (*P<0.05 vs. effector T cells+CD4+ T cells from cocultures with PBS-stimulated DCs). (e) Wild-type (wt) mice were injected with CD4+ T cells from cocultures with α-MSH- or PBS-stimulated DCs 24hours before induction of contact allergy. Data are shown as mean ear swelling assessed 48hours after elicitation of contact hypersensitivity (CHS) and are representative of 15 mice in each group (*P<0.05 vs. transfer of CD4+ T cells from cocultures with PBS-stimulated DCs). (f) CD4+ T cells were cocultured with α-MSH- or PBS-stimulated DCs. Subsequently, the numbers of Foxp3+Helios+ and Foxp3+Helios- Tregs were quantified by flow cytometry. Cells are gated for CD4; Foxp3 and Helios staining was performed after cell permeabilization; and one representative experiment is shown. c.p.m., counts per minute. Journal of Investigative Dermatology 2012 132, 1814-1824DOI: (10.1038/jid.2012.59) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 α-Melanocyte-stimulating hormone (α-MSH) ameliorates ongoing psoriasis by inducing regulatory T cells (Tregs). (a) Typical skin pathologies of BALB/c mice topically treated with imiquimod for 8 days and injected intravenously (i.v.) with α-MSH or anti-tumor necrosis factor-α (TNF-α) (n=8 mice in each group). Hematoxylin and eosin (H&E) staining of skin tissue: original magnification × 200, bar=25μm. (b) DCs from regional lymph nodes of mice injected with α-MSH expressed elevated levels of IL-10. Cells are gated for major histocompatibility complex (MHC-II+), and IL-10 staining was performed after cell permeabilization. One representative histogram overlay is depicted. (c, left) Increased numbers of Tregs and (right) upregulated IL-10 mRNA expression in CD4+ T cells from lymph nodes draining cutaneous lesions of α-MSH-treated mice (*P<0.05 vs. injection of phosphate-buffered saline (PBS)). (d) Reduced Th17 numbers in lymph nodes from α-MSH-injected mice. Cells are gated for CD4, and IL-17 staining was performed after cell permeabilization. (Left) One representative dot-plot for each group and (right) statistical analyses of % IL-17-expressing CD4+ T cells from regional lymph nodes are shown (*P<0.05 vs. injection of PBS). (e) α-MSH decreased the expression of Th17-associated genes in CD4+ T cells from lymph nodes draining cutaneous lesions. Relative mRNA levels of RORγt, IL-17, and IL-22 are shown from n=6 mice in each group (*P<0.05 vs. injection of PBS). Journal of Investigative Dermatology 2012 132, 1814-1824DOI: (10.1038/jid.2012.59) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 α-Melanocyte-stimulating hormone (α-MSH)-stimulated DCs ameliorate imiquimod-induced psoriasis. (a) Typical skin pathologies of BALB/c mice treated with imiquimod and injected with Thy1.1+ phosphate-buffered saline (PBS)- or α-MSH-stimulated DCs (n=8 mice in each group). Hematoxylin and eosin (H&E) staining of skin tissue: original magnification × 200, bar=25μm. (b) Immunofluorescence staining of regional lymph nodes and lesional skin from mice treated with imiquimod and injected with PBS- or α-MSH-stimulated DCs using antibodies against CD4, Foxp3, and IL-17. One representative image is shown, and CD4+Foxp3+ regulatory T cells (Tregs) and CD4+IL-17+ Th17 cells are marked by arrows. Original magnification × 400, bar=25μm. DAPI, 4′,6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2012 132, 1814-1824DOI: (10.1038/jid.2012.59) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 α-Melanocyte-stimulating hormone (α-MSH) induces tolerogenic human dendritic cells (DCs) capable of generating functional regulatory T cells (Tregs). (a) DCs from peripheral blood were stimulated with α-MSH or phosphate-buffered saline (PBS), and the expression of CD80, CD86, and IL-10 was quantified by flow cytometry. Histogram overlays are gated for HLA-DR, and one representative experiment is shown. IL-10 staining was performed after cell permeabilization. (b) CD4+ T cells from cocultures with α-MSH-stimulated DCs were analyzed for the expression of Foxp3 and CD25 by flow cytometry. Cells are gated for CD4; Foxp3 staining was performed after cell permeabilization; and (left) one representative experiment and (right) statistical analyses of n=7 donors are shown (*P<0.05 vs. CD4+ T cells from cocultures with PBS-stimulated DCs). (c) Freshly isolated CD4+CD25- effector T cells were activated with anti-CD3 and anti-CD28 in the absence or presence of CD4+ T cells from cocultures with PBS- or α-MSH-stimulated DCs. Mean values of 3H-thymidine incorporation±SD are shown from one out of three independent experiments (*P<0.05 vs. effector T cells+CD4+ T cells from cocultures with PBS-stimulated DCs). (d) Signaling via MC-1R is critical for the α-MSH-mediated induction of tolerogenic DCs. HLA-DR+ DCs were purified from peripheral blood of individuals with loss-of-function variants in the MC-1R gene, stimulated with α-MSH, and the expression of CD86 was analyzed by flow cytometry. Cells are gated for HLA-DR, and one representative histogram overlay is shown. c.p.m., counts per minute. Journal of Investigative Dermatology 2012 132, 1814-1824DOI: (10.1038/jid.2012.59) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 α-Melanocyte-stimulating hormone (α-MSH)-stimulated DCs induce functional regulatory T cells (Tregs) capable of inhibiting the activity of Th17 cells from individuals with psoriasis. (a) CD4+ T cells were purified from peripheral blood of healthy donors or individuals with psoriasis and cocultured with α-MSH-stimulated DCs. The numbers of Foxp3+ Tregs were quantified by flow cytometry from n=8 individuals in each group, and Foxp3 staining was performed after cell permeabilization. (b) Coculture with α-MSH-stimulated DCs restored the suppressor function in CD4+ T cells from individuals with psoriasis. Proliferation assays were performed by mixing CD4+ T cells from cocultures with phosphate-buffered saline (PBS)- or α-MSH-stimulated DCs and freshly isolated, anti-CD3/anti-CD28-activated CD4+CD25- effector T cells from the same individuals. Mean values of 3H-thymidine incorporation±SD are shown from one out of three independent experiments (*P<0.05 vs. proliferation of effector T cells alone). (c) Reduced IL-17 expression in CD4+ T cells from individuals with psoriasis after coculture with α-MSH-stimulated DCs. CD4+ T cells were purified from cocultures, and the IL-17 expression was quantified by flow cytometry in n=7 individuals in each group. IL-17 staining was performed after cell permeabilization (*P<0.05 vs. coculture with PBS-stimulated DCs). (d) CD4+ T cells from individuals with psoriasis that have been cocultured with α-MSH-stimulated DCs show decreased relative mRNA levels of IL-17, IL-22, and RORc (n=9 individuals in each group; *P<0.05 vs. coculture with PBS-stimulated DCs). (e) CD4+ T cells from cocultures with α-MSH-stimulated DCs suppress the proliferation of Th17 cells. CD4+ T cells were purified from cocultures with α-MSH-stimulated or control DCs and mixed with freshly isolated CD4+ T cells from individuals with psoriasis. Subsequently, the numbers of IL-17-expressing cells were quantified by flow cytometry in n=5 individuals in each group (*P<0.05 vs. coculture with PBS-stimulated DCs). c.p.m., counts per minute. Journal of Investigative Dermatology 2012 132, 1814-1824DOI: (10.1038/jid.2012.59) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions