<i>Cre</i> EYFP reporter expression in the skin of OX40^Cre strains.

<p>(A) Fluorescent stereomicroscope images of epidermal skin layer taken from the underside (×10 magnification). (B) Confocal images of skins sections from 12–16 week old mice of the indicated mice showing expression of EYFP and counter stained with DAPI. (C) EYFP expression in hair follicle of healthy <i>OX40^Cre Ikbk2^fx/wt</i> mice showing DAPI and EYFP separately and together. White scale bar indicates 10 µm size. Data are representative of five or more mice from three independent experiments.</p

Skin pathology in <i>OX40^Cre Ikbk2^fx/fx</i> mice is associated with TNF production and increased apoptosis.

<p>(A) Confocal images of skins sections from 12–16 week old mice of the indicated strain were analysed for EYFP expression (green), stained with DAPI (blue) and for expression of TNF (red). White scale bar indicates 10 µm size. (B) Apoptosis was assessed in skin sections from <i>OX40^Cre Ikbk2^fx/fx and OX40^Cre Ikbk2^fx/wt</i> mice by TUNNEL assay and counterstained with eosin. Light microscopy was performed at 10× magnification.</p

Skin pathology in <i>OX40^Cre Ikbk2^fx/fx</i> mice is not T cell dependent.

<p>(A) Photograph shows skin pathology in a representative <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> mouse (fx/fx) compared with a littermate <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/wt</i> control (fx/wt). White dotted line indicates the border between dermis and epidermis. (B) Images show skin sections from 12–16 week old mice of the indicated strain stained with H&E and taken at 10× magnification. (C) Confocal images of skins sections from 12–16 week old mice of the indicated mice stained for expression of CD45, cytokeratin 5 or cytokeratin 6 (red) and counter stained with DAPI. White scale bar indicates 10 µm size. (D) Graph shows progress of disease development in <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> mice (blue, n = 18) as compared with Rag1 sufficient <i>OX40^Cre Ikbk2^fx/fx</i> mice (black lines, n = 9). (E) Bar chart shows mean time of disease progression to score 2 in <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> mice as compared with Rag1 sufficient <i>OX40^Cre Ikbk2^fx/fx</i> mice. Data are representative (A–C) of three independent experiments or are pooled (D–E) from three independent experiments.</p

Activation of WT T cells in <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> mice.

<p>Eight week old <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> and <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/wt</i> controls were reconstituted with 5×10⁷ lymph node T cells from WT C57Bl6^NIMR mice. Two weeks later, number and phenotype of transferred T cells in different recipients was determined. (A) Contour plots are of CD25 vs CD44 expression by CD4⁺ TCR^hi T cells (top row) and side scatter (SSC) vs CD44 by CD8⁺ TCR^hi T cells (bottom row) from dLN of <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> (<i>Ikbk2</i> fx/fx) and <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/wt</i> (<i>Ikbk2</i> fx/wt) hosts. Numbers indicate % of cells in the adjacent gate. Data are representative of at least five mice per strain. (B) Scatter charts show absolute numbers of CD4⁺ TCR^hi or CD8⁺ TCR^hi T cells in skin draining lymph nodes (dLN), mesenteric lymph nodes (mLN) or spleen (SPN) in host <i>Rag1^−/− OX40^Cre Ikbk2^fx/fx</i> mice (fx/fx) or control <i>Rag1^−/− OX40^Cre Ikbk2^fx/wt</i> mice (fx/wt). Data are pool of two independent experiments.</p

<i>OX40^Cre Ikbk2^fx/fx</i> mice exhibit skin epidermis hyperplasia.

<p>(A) Images show skin sections from 12–16 wk old mice of the indicated strain stained with H&E and taken at 10× magnification. White dotted line indicates the border between dermis and epidermis. (B) Confocal images of skins sections from 12–16 week old mice of the indicated mice stained for expression of CD45, cytokeratin 5 or cytokeratin 6 (red) and counter stained with DAPI. White scale bar indicates 10 µm size. Data are representative of 3 independent experiments and at least two mice per strain experiment.</p

Development of skin pathology in <i>OX40^Cre Ikbk2^fx/fx</i> mice.

<p>(A) Photographs show dorsal aspect of <i>OX40^Cre Ikbk2^fx/fx</i> mice with varying degrees of skin pathology. Scoring was established according to extent of involvement of the dorsal surface: 0 - no pathology, 1 - <10% surface, 2 - 10–25% surface, 3 - >25% surface. Mice with >50% dorsal skin involvement and/or weight loss >20% were culled. (B) Graph shows average mouse score for <i>OX40^Cre Ikbk2^fx/fx</i> mice over time (n = 9).</p

Lymphoid hyperplasia and T cell activation in <i>OX40^Cre Ikbk2^fx/fx</i> mice.

<p>(A) Images show axillary and brachial lymph nodes (top four nodes) as compared with mesenteric chain and spleen from the indicated mouse strains, taken between 12–16 weeks of age. (B) Scatter charts show absolute numbers of total lymphocytes, CD4⁺ TCR^hi or CD8⁺ TCR^hi T cells in skin draining lymph nodes (dLN), mesenteric lymph nodes (mLN) or spleen (SPN) in <i>OX40^Cre Ikbk2^fx/fx</i> mice (fx/fx, n = 6) or control <i>OX40^Cre Ikbk2^fx/wt</i> mice (fx/wt, n = 4). (C) Density plots are of CD25 vs CD44 expression by CD4⁺ TCR^hi T cells (top row) and side scatter (SSC) vs CD44 by CD8⁺ TCR^hi T cells (bottom row) from dLN of the indicated mouse strains. Data are representative of six independent experiments.</p

Ptpn22 variants do not alter BMDC receptor mediated phagocytosis.

<p><b>(A-B)</b> Wild type (WT) and <i>Ptpn22</i>^-/- derived bone marrow derived dendritic cells (BMDC) were harvested and cell surface CD206 was determined by flow cytometry. <b>(A)</b> Representative CD206 expression profiles on WT (black solid line) and <i>Ptpn22</i>^<i>-/-</i> (black dashed line) BMDC. <b>(B)</b> CD206 Geometric Mean Fluorescent Intensity (GMFI). N = 11 independent experiments; bars represent mean ± s.e.m. <b>(C)</b> WT and <i>Ptpn22</i>^<i>-/-</i> BMDC were incubated with ovalbumin-AF488 for 0–60 minutes at 37°C prior to CD11c staining. N = 4 independent experiments; line represents the mean ± s.d. <b>(D)</b> WT and <i>Ptpn22</i>^<i>R619W</i> BMDC were incubated with ovalbumin-AF488 for 0–60 minutes at 37°C prior to CD11c staining. N = 3 independent experiments; line represents the mean ± s.d.</p

Ptpn22 is dispensable for antigen degradation and presentation.

<p><b>(A-B)</b> Wild type (WT) and <i>Ptpn22</i>^<i>-/-</i> BMDC were incubated with ovalbumin-AF594 coated beads for 0–5 hours at 37°C. Non-internalised beads were excluded by staining with rabbit anti-ovalbumin followed by F(ab’)₂ anti-rabbit-AF647. Cells were lysed and the fluorescent intensity of AF594⁺ AF647^- beads determined by flow cytometry. <b>(A)</b> Ovalbumin-AF594 Geometric Mean Fluorescent Intensity (GMFI). N = 2 independent experiments ± s.d. <b>(B)</b> Proportion of internalised beads from WT and <i>Ptpn22</i>^<i>-/-</i> BMDC that have reduced ovalbumin-AF594 fluorescence. N = 2 independent experiments ± s.d. <b>(C)</b> WT and <i>Ptpn22</i>^<i>-/-</i> BMDC were incubated with GFP or GFP-Eα for 18 hours followed by staining for Eα_52–68 in I-A^b (YAe). The percentage of YAe⁺ live, singlet, CD11c⁺ BMDC was determined by flow cytometry. N = 3 independent experiments; bars represent mean ± s.d. <b>(D-E)</b> CellTrace Violet (CTV) labelled WT CD4⁺ OT.II T-cells were incubated with CellTrace Far Red (CFTR) stained OVA_323-339 peptide pulsed LPS matured WT or <i>Ptpn22</i>^<i>-/-</i> BMDC for 0–120 minutes at 37°C, and the proportion of CTV⁺ CTFR⁺ conjugates within the CTV⁺ T-cell population determined by flow cytometry. <b>(D)</b> Representative flow cytometry plots showing conjugates (top, right hand quadrant, CTV⁺ CTFR⁺). <b>(E)</b> Proportion of DC:T-cell conjugates within CTV⁺ gate. N = 4 independent experiments; line represents mean ± s.d. Data <b>(C)</b> determined non-significant by unpaired T-test. Differences between genotypes in <b>(E)</b> were deemed non-significant by two-way ANOVA with Sidak’s Multiple comparison test.</p

Ptpn22 variants do not modulate BMDC dependent OT.II T-cell activation.

<p><b>(A-E)</b> Wild type (WT), <i>Ptpn22</i>^<i>-/-</i> and <i>Ptpn22</i>^<i>R619W</i> BMDC were stimulated overnight in the presence or absence of OVA_323-339 (0.01–1 μM) or ovalbumin (0.01–1 μM). BMDC were harvested and co-cultured with CellTrace Violet (CTV) labelled CD4⁺ OT.II T-cells at a 1:2 BMDC:T-cell ratio. <b>(A-B)</b> 24 hour Geometric Mean Fluorescent Intensity (GMFI) surface expression of CD69 determined on live, singlet, CD4⁺ T-cells. <b>(A)</b> N = 3 independent experiments and <b>(B)</b> N = 4 independent experiments; bars represent mean ± s.d. <b>(C-D)</b> Co-culture supernatants were assessed for <b>(C)</b> IL-2 after 24 hours and <b>(D)</b> IFNγ after 144 hours. N = 3 independent experiments; bars represent mean ± s.d. <b>(E)</b> At day 6 the proportion of CD4⁺ T-cells within each CTV generation was determined by flow cytometry. N = 3 independent experiments, lines represent mean ± s.d. <b>(F-H)</b> CTV labelled CD45.1⁺ CD4⁺ TCR Vα2⁺Vβ5⁺ OT.II T-cells were adoptively transferred i.v. into CD45.2⁺ WT or <i>Ptpn22</i>^<i>-/-</i> recipient mice followed by i.p. immunisation of PBS or ovalbumin (100 μg/mouse). Spleens were assessed after 48h <b>(G)</b> or 96h <b>(F, H)</b> for CTV dilution within the CD45.1⁺ CD4⁺ TCR Vα2⁺Vβ5⁺ population by flow cytometry. <b>(F)</b> Representative flow cytometry plot showing CTV dilution following i.p. of PBS (black dashed line) or 10 μg ovalbumin (black line, grey fill). <b>(G-H)</b> Lines represent mean ± s.d., N = 3 <b>(G)</b> and N = 5 <b>(H)</b>. Differences between genotypes <b>(A-E, G, H)</b> were deemed non-significant by two-way ANOVA with Sidak’s Multiple comparison test.</p