The toll-like receptor 4 ligands Mrp8 and Mrp14 play a critical role in the development of autoreactive CD8 + T cells

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1 1 SUPPLEMENTARY INFORMATION The toll-like receptor 4 ligands Mrp8 and Mrp14 play a critical role in the development of autoreactive CD8 + T cells Karin Loser 1,2,6, Thomas Vogl 2,3, Maik Voskort 1, Aloys Lüken 3, Verena Kupas 1, Wolfgang Nacken 4, Lars Klenner 1, Annegret Kuhn 1, Dirk Föll 3, Lydia Sorokin 5, Thomas A. Luger 1,2, Johannes Roth 2,3 & Stefan Beissert 1,2 1 Department of Dermatology, University of Münster, Münster, Germany; 2 Interdisciplinary Center of Clinical Research (IZKF), University of Münster, Münster, Germany; 3 Institute of Immunology, University of Münster, Münster, Germany; 4 Institute of Molecular Virology, University of Münster, Münster, Germany; 5 Institute of Pathobiochemistry, University of Münster, Münster, Germany Correspondence should be addressed to K.L. (loserk@uni-muenster.de) SUPPLEMENTARY DATA It has been demonstrated that at least in human autoimmune disorders, such as systemic lupus erythematosus (LE), CD3 + CD4 CD8 T cells () are expanded and produce substantial amounts of IL Therefore, we quantified the IL-17 secretion in DN wild-type, -transgenic and x mice. In contrast to cells isolated from subjects with LE, from autoimmune-prone -transgenic mice expressed only low levels of IL-17 and, more importantly, the IL-17 production of this cell population was not altered by Mrp8 and Mrp14 exposure (Supplementary Fig. 2a,b). Of note, we also did not detect increased IL-17 levels in from transgenic mice with overt disease compared to controls (Supplementary Fig. 2a,b).

2 2 Moreover, in mouse models of lupus as well as in subjects with LE it has been shown that are critically involved in the pathogenesis 1,2. Therefore, we analyzed whether Mrp8 and Mrp14 might also modulate the pathogenicity of in CD4L-induced systemic autoimmunity. As demonstrated in Supplementary Fig. 2c,d wild-type mice that received from autoimmune-prone -transgenic donors did not develop dermatitis and we did not detect autoantibodies in the serum or nephritis (Supplementary Fig. 2c,d). Furthermore, adoptively transferred from x mice did not induce autoimmunity in the recipients indicating that in CD4L-induced systemic autoimmunity might play a minor role in disease development and Mrp8 as well as Mrp14 did not influence the pathogenicity of DN T cells. Of note, also from either -transgenic or x mice failed to induce autoimmunity in wild-type recipients (Supplementary Fig. 2c,d). Next we analyzed whether Mrp8 and Mrp14 proteins up-regulated the IL-17 expression in or upon in vitro stimulation. As shown in Supplementary Figure 3a Mrp8 and Mrp14 induced only feeble levels of IL-17 in from wild-type, x or -transgenic mice although the T cells were co-cultured with epidermal LC from autoimmune-prone -transgenic mice (Supplementary Fig. 3a). Of note, Mrp8 and Mrp14 did not up-regulate IL-17 in (Supplementary Fig. 3b). Worth mentioning, that neither Mrp8 CD4 + nor were able to induce systemic autoimmunity in wild-type recipients upon adoptive cell transfer (Supplementary Fig. 3c e).

3 3 SUPPLEMENTARY METHODS Immunohistochemistry and immunofluorescence staining. Staining was performed on cryostat sections (3 5 µm) of mouse or human skin according to standard methods. We incubated slides in the appropriate dilutions of primary antibodies (antibodies to human and mouse Mrp8 or Mrp14 were provided by T.V.) and subsequently stained them with Alexa Fluor 488-, Alexa Fluor 568- or horseradish peroxidase (HRP)-coupled secondary antibodies (Invitrogen). We visualized peroxidase activity using 3 amino 9 ethyl carbazol (AEC) and counterstained tissues with MAYER'S hemalaun solution (Merck). In some experiments nuclei were stained with 4',6 diamidino 2 phenylindole (DAPI). qpcr. We extracted RNA from human or mouse CD8 + T cells, mouse or mouse using RNeasy columns (Qiagen). Subsequently, cdna was synthesized from 1 µg of total RNA using the Reverse Transcription System (Fermentas). We performed real-time quantitative PCR in an ABI PRISM 76 cycler (Applied Biosystems) using mouse as well as human TaqMan gene expression assays (Applied Biosystems). All mrna levels are normalized to beta-actin. Cytokine quantification. We assayed the cytokine activity in culture supernatants using FlowCytomix kits (BenderMed Systems). In some experiments T cells were by a combination of antibodies to CD3 (clone 145-2C11) and CD28 (clone 37.51, 1 µg ml 1 each antibody) prior to cytokine quantification.

4 4 SUPPLEMENTARY REFERENCES 1. Crispìn, J.C. et al. Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys. J. Immunol. 181, (28). 2. Kang, H.K., Liu, M. & Datta S. K. Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigenspecific regulatory T cells and contraction of inflammatory Th17 cells. J. Immunol. 178, (27).

5 5 SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1 Neither nor from autoimmune-prone -transgenic mice showed increased IL-17 secretion or induced autoimmunity in wild-type recipients (a) IL-17 mrna expression in CD8 + T cells, or DN T cells from wild-type (WT), -transgenic () or x mice (n = 3 mice each group) was analyzed by qpcr, * P <.5 versus -transgenic mice. (b) Flow cytometry of cervical lymph nodes from, x, and WT mice (n = 3 mice each group). Cells were gated for or and IL-17 expression is shown in a representative histogram overlay. FACS staining was performed after cell permeabilization. (c) Typical skin pathology of WT mice 4 weeks after adoptive transfer of or from either WT, or x mice. (d) Upper part: detection of autoantibodies in the serum from WT recipients of or DN T cells from WT, or x donors (scale bar 25 = µm). Lower part: detection of immunoglobulin depositions in the kidneys of WT mice after adoptive transfer of or from WT, or x donors by immunofluorescence staining of renal tissue using an antibody directed against IgG (scale bar = 25 µm). Supplementary Figure 2 Mrp8 or Mrp14 did not modulate the IL-17 secretion or the pathogenicity of CD4 + and. (a,b) IL-17 secretion in (a) or DN T cells (b) from wild-type (WT) mice, -transgenic mice () before and after onset of systemic autoimmunity, and x mice (n = 3 mice each group) was quantified. and were co-cultured with LC from autoimmune-prone -transgenic mice and with Mrp8 or Mrp14 proteins. (c) Typical skin phenotype of WT mice 4 weeks after adoptive transfer of or that were co-cultured with LC from autoimmune-prone -transgenic mice and

6 6 with Mrp8 prior to injection into recipient mice. (d) Immunoglobulin depositions in the kidneys from WT recipients of Mrp8 CD4 + or were visualized by immunofluorescence staining using an antibody directed against IgG. Representative images are shown (scale bar = 25 µm). (e) Mrp8 and Mrp14 concentration in the serum of WT mice 4 weeks after injection of Mrp8 CD4 + or were analyzed by ELISA (n = 3 mice each group).

7 a WT x 12 CD8 + T cells IL-17 expressio on (n-fold, relative to b-actin) Y Data * IL-17 expressio on (n-fold, relative to b-actin) Y Data IL-17 expressio on (n-fold, relative to b-actin) Y Data IFN-g b Isotype control WT x c WT + CD4 + T cells from WT WT + CD4 + WT + CD4 + x Gated for Gated for Counts IL IL-17 WT + DN T cells from WT WT + DN WT + DN x d WT + CD4 + WT + CD4 + WT + CD4 + WT + DN WT + DN WT + DN T cells T cells from WT from WT x x Loser K. et al. Supplementary Fig. 1

8 a Stimulated with PBS Stimulated with Mrp8 Stimulated with Mrp14 c WT + PBS WT + Mrp8 6 IL-17 (pg ml 1 ) wt x before onset of with overt disease disease LC from WT + PBS WT + Mrp8 b Stimulated with PBS Stimulated with Mrp8 Stimulated with Mrp14 d WT + PBS WT + Mrp8 6 5 IL L-17 (pg ml 1 ) wt x before onset of disease with overt disease LC from WT + PBS WT + Mrp8 e Mrp8/Mrp1 14 (ng ml 1 ) 1,2 1, WT + PBS stim. WT + Mrp8 stim. WT + PBS stim. WT + Mrp8 stim. Loser K. et al. Supplementary Fig. 2