Mice transgenic for LYP-W620 (TgLYPW) show overexpression of LYP in DP thymocytes.

<p>A, LYP protein expression in TgLYPW thymocytes. Total thymocytes from TgLYPW (lane 2) or a non-Tg littermate (lane 1) and TgLYPW^C227S (lane 4) or a non-Tg littermate (lane 3) were lysed and subjected to immunoprecipitation (IP) using an anti-HA antibody (Ab). Panel shows Western blotting using an anti-LYP Ab. Data are representative of 3 independent experiments with similar results. B, LYP phosphatase activity in TgLYPW thymocytes. Anti-HA IPs were performed from lysates of total thymocytes from TgLYPW and TgLYPW^C227S mice. Graphs show the phosphatase activity of LYP as assessed by dephosphorylation of the fluorescent substrate DiFMUP. Data are representative of 2 independent experiments with similar results. C–D, LYP-W620 transgene expression in thymocyte subpopulations. Expression of LYPW (C) or LYPW^C227S (D) was assessed by intracellular staining using a fluorophore-conjugated anti-HA Ab in DN (upper left panel), DP (upper right panel) CD4SP (lower left panel) and CD8SP (lower right panel) thymocytes from Tg mice (black graphs) and control non-Tg littermates (grey filled graphs). E, Quantification of overexpression of LYPW relative to endogenous Pep in DP thymocytes of TgLYPW mice. mRNA encoding LYP and Pep was quantified by qPCR from sorted DP thymocytes from control BALB/c (white bar) and TgLYPW (striped bar) mice, using a primer pair that amplifies both human <i>PTPN22</i> and mouse <i>Ptpn22</i> mRNAs. Graph shows relative expression levels of total <i>PTPN22</i> after normalization to the mouse housekeeping gene <i>Polr2a</i>. Data are average and SE of 3 biological replicates.</p

Overexpression of LYPW inhibits TCR signaling in DP thymocytes.

<p>A–B, Overexpression of LYPW causes reduced activation of Erk in thymocytes. Total thymocytes from TgLYPW or TgLYPW^C227S (striped bars) or their respective non-Tg littermates (white bars) were stimulated with 20 µg/ml anti-CD3 and 10 µg/ml anti-Armenian Hamster IgG1 crosslinker for 2.5 minutes. A, Graph shows phosphorylation of Erk in total thymocyte lysates assessed using the PathScan® phospho-p44 MAPK (Thr202/Tyr204) sandwich ELISA kit. Histogram shows mean and range of fold induction of at least 3 biological replicates. B, Phosphorylation of Erk in DP thymocytes was assessed by phosphoflow analysis after intracellular staining with an anti-pErk Ab. Fold induction of Erk phosphorylation was normalized within each experiment relative to the sample with the highest induction. Histogram shows mean and range of at least 3 biological replicates. C, Overexpression of LYPW causes reduced T cell activation in DP thymocytes. Thymocytes from TgLYPW or TgLYPW^C227S (grey graphs) and their respective non-Tg littermates (black graphs) were cultured in the presence of 10 µg/ml (long dashed graphs) or 25 µg/ml (solid graphs) anti-CD3, or media alone (dotted graphs), for 18 hours. Graphs shows expression of CD69 in DP thymocytes as assessed by flow cytometry analysis after staining with an anti-CD69 Ab. Median fluorescence intensity (MFI) values are indicated on each graph. Graphs are representative of at least 3 biological replicates with identical results.</p

OT-1 thymocytes overexpressing LYP-W620 show similar antigen sensitivity as non-Tg thymocytes.

<p>Freshly isolated OT-1 transgenic thymocytes from TgLYP (black circles), TgLYP^C227S (black triangles) or control non-Tg mice (white diamonds) were co-cultured overnight together with RMA cells and the indicated doses of SIINFKL peptide (N4, left panel), of the low and very low affinity altered peptide ligands SIITFEKL (T4, middle panel) and SIIVFEKL (V4, right panel). Graphs show normalized dose-response values of peptide concentration vs the fraction of maximum numbers of residual DP (CD4 and CD8 bright) thymocytes.</p

LYPW overexpression does not alter thymic repertoire and autoimmune phenotype of Skg mice.

<p>A–B, Overexpression of LYPW does not alter Vβ repertoire or numbers of CD4⁺Foxp3⁺ thymocytes in Skg/WT or Skg/Skg mice. Left panel shows average and SE % Vβ positive CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ thymocytes from Skg/WT (A) or Skg/Skg (B) TgLYPW (striped bars, n = 3 for Skg/WT and n = 6 for Skg/Skg) and control non-Tg littermates (white bars, n = 3 for Skg/WT and n = 5 for Skg/Skg) as assessed by flow cytometry analysis after staining with anti-Vβ3, -Vβ5 and -Vβ8 antibodies. Right panel shows mean and range of CD4⁺Foxp3⁺ (first and second bar) and total (third and fourth bars) thymocytes from the same Skg/WT (A) or Skg/Skg (B) TgLYPW mice (striped bars) or non-Tg littermates (white bars). C, Overexpression of LYPW does not alter the course of mannan-induced arthritis and the frequency of Th17 cells in peripheral lymph node (LN) of arthritic Skg mice. Left panel shows arthritis score (measured as ankle swelling in mm) of Skg/Skg TgLYPW mice (black circles, n = 6) and littermates non-Tg Skg/Skg mice (white circles, n = 5) followed-up for 40 days after a single i.p. injection of 20 mg mannan dissolved in 200 µl PBS. One month following mannan-injection, LN cells from Skg/Skg TgLYPW mice (black circles, n = 15) or non-Tg Skg/Skg littermates (white circles, n = 16) were stimulated with 20 ng/ml PMA and 2 mM ionomycin for 5 hours. Right panel shows % Th17⁺ cells of the CD4⁺ T cell population as assessed by flow cytometry analysis after intracellular staining with an anti-IL17 antibody.</p

Cell distribution in thymus of TgLYPW and TgLYPW^C227S.

<p>Table shows the basic statistics (average±SD) of cell counts in transgenic animals and non transgenic littermates.</p

Polyclonal thymocytes undergo similar levels of negative selection in the presence of absence of transgenic LYP-W620.

<p>A, Lethally irradiated Rip-mOva mice (continuous graphs and black symbols) or C57BL/6 mice (dotted graphs and crossed symbols) were reconstituted with bone marrow harvested from Vβ5xLYPW (circles) or Vβ5 control (diamonds) mice. 10 weeks after the reconstitution the mice were infected with a strain of <i>Listeria monocytogenes</i> expressing Ovalbumin (Lm-Ova). Splenocytes were harvested at 8 days after the infection and briefly <i>in vitro</i> re-stimulated with titrated doses of SIINFEKL peptide. Afterwards, the cells were intracellularly stained for IFNγ. Peptide-dose response curves showing the frequency of IFNγ producing CD8⁺ T cells as fraction of maximum response are presented. B, TgLYPW, TgLYP^C227S and control non-Tg mice contain similar numbers of low avidity auto-reactive T cells. RipxTgLYPW (left panel, black circles), RipxTgLYPW^C227S (right panel, black triangles) and control Rip mice (left and right panels, black diamonds) were infected with Lm-Ova and 4 weeks later challenged by a strain of <i>Vesicular stomatitis virus</i> expressing Ova (VSV-Ova). On day 6 after the primary (left side of each panel) or the secondary infection (right side of each panel) blood was drawn from the mice and PBMC were briefly re-stimulated with SIINFEKL peptide. The number of IFNγ producing CD8⁺ T cells was determined by intracellular cytokine staining. Panels show the frequency of Ova-specific T cells.</p