Evolution du profil cutané après traitement orthodontique précoce

LYON1-BU Santé Odontologie (693882213) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Specificity of OX110, OX131 and OX132 mAb.

<p>Streptavidin coupled magnetic beads were coated with biotinylated chimeric proteins containing extracellular domains of members of the mouse CD200R family and rCD4d3+4 and a biotinylation site (as indicated on top of each column). The binding of the mAb indicated in each row was analyzed by flow cytometry. (A) Protein coating levels for each group of magnetic beads were tested by staining with OX68 (CD4 mAb) (tinted solid line) or OX21 (control mAb) (thin line). Flow cytometry plot named rCD4 d3+4 indicates coating level for biotinylated rCD4d3+4 only. (B–D) OX110 (B), OX131 (C) and OX132 (D) mAb were used to stain magnetic beads coated with the chimeric proteins indicated above each column (tinted solid line), or control beads coated with biotinylated rCD4d3+4 only (dashed line). Data are representative of three experiments.</p

CD200R and CD200RLc expression on mouse leukocytes.

<p>(A) <b>Left panel:</b> Gating strategy for macrophage and neutrophils in peritoneal aspirates of naive (top) and zymozan stimulated (bottom) mice. For each mouse CD11b+ cells were gated in CD11b-forward scatter plot (left) then in these populations Gr-1(−)F4/80(+) population was gated as resident macrophages (top right), Gr-1(+)F4/80(−) population was gated as neutrophils (bottom right), Gr1(+)F4/80(+) population was gated as inflammatory macrophages (bottom right). <b>Right panel:</b> expression of CD200R (top row) and CD200RLc (bottom row) in macrophage and neutrophil populations (tinted solid lines) compared to control mAb (dashed lines). (B) <b>Left Panel:</b> Gating strategy for different <i>in vitro</i> cultures of mouse bone marrow cells. Cells grown for mast and basophil differentiation were first gated for FcεRI expression in FcεRI- forward scatter plot (top left). FcεRI(+) cells were further gated as C-kit(+)CD49b(−) mast cells (top right) and C-kit(−) CD49b(+) basophils (top right) in C-kit-CD49b plot. Cells cultured in IL-5 supplemented media were gated as CD11b(+)Siglec F(+) eosinophils in CD11b-Siglec F plot (bottom left). Cells cultured in GMCSF supplemented media were gated as CD11c(+)MHC II(+) dendritic cells in CD11c-MHC II plot (top right). <b>Right panel:</b> Expression of CD200R (top row) and CD200RLc (bottom row) in <i>in vitro</i> cultures of mouse bone marrow cells (tinted solid lines) (C) <b>Left panel:</b> Gating strategy for lymphocytes derived from mouse spleen. B cells were gated for B220 expression in B220-forward scatter plot (top left). NK cells were gated as double positives in NK1.1-CD49b plot (top right). T cells were first gated as CD3(+) population in CD3-forward scatter plot (bottom left), then this population was further gated into CD4(+) and CD8(+) cells in CD4/CD8 plot (bottom right). <b>Right panel:</b> Expression of CD200R in unstimulated splenocytes (top row), expression of CD200RLc in unstimulated splenocytes (middle row) and expression of CD200R in <i>in vitro</i> stimulated splenocytes (LPS for B cells, CD3 mAb for T cells and IL-2 for NK cells) (bottom row) are shown (tinted solid lines) compared to control mAb (dashed lines).</p

The CD200/CD200R interaction can be blocked by OX131 but not OX110 mAb.

<p>A) Biotinylated rCD4d3+4 (dashed line), CD200R(1) rCD4d3+d4 (solid black line) and CD200R(2) rCD4d3+4 (solid grey line) proteins were immobilized onto streptavidin coated CM5 chips (681, 726, 704 response units respectively). The changes in response units (RU) upon sequential injection of different soluble proteins (boxed and indicated by vertical dots) are shown. (Both antibodies were injected three consecutive times to ensure saturation on the immobilized proteins.) (B) Table showing the increase in response units upon injection of soluble CD200 compared to the pre-injection states for each flow cell. The values for the control rCD4d3+4 indicate the signal due to the high protein content of the CD200 sample.</p

Repertoire of CD200R family genes and predicted reactivity of mAb.

<p>(A) The different genes present in common mice strains from genomic and biochemical analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063325#pone.0063325-Wright1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063325#pone.0063325-Akkaya2" target="_blank">[16]</a>. (B) The predicted gene products recognised by the three mAb from this study.</p

The effects of CD200R and CD200 mAb on T cell activation.

<p>(A) Schematic representation of the assay showing engagement of Moth Cytochrome C peptide (MCC), MHC-TCR and CD200/CD200R between the antigen presenting cell (APC) (CHO cells stably expressing IE^k and CD200) and 2B4 Reay T cells expressing CD200R(1) with IL-2 production as the readout. (B) <b>Left panel</b>: flow cytometry plots showing IE^k expression on CHO-IE^k CD200 cells (tinted solid line) compared to untransfected CHO cells (dashed line). <b>Right panel</b>: flow cytometry plots showing CD200 mAb staining of CHO IE^k CD200 cells (tinted solid line) compared to isotype control (dashed line). (C) Effect of different mAb on IL-2 release. Either CHO-IE^k cells <b>(left panel)</b> or CHO-IE^k CD200 cells <b>(right panel)</b> were used together with CD200R(1) expressing T cells and IL-2 concentrations were measured. Effects of mAb were statistically compared with no mAb applied condition (representative of more than seven experiments). (D) Changes in IL-2 secretion from CD200R(1) 2B4 Reay cells in response to increasing doses of mAb. CHO-IE^k CD200 cells as APC. IL-2 levels obtained with OX90 and OX131 mAb were compared (representative of three experiments). (E) 10 µg/ml of OX110, OX131, OX132 (control) or no mAb were immobilized onto plates together with 0.5 µg/ml of CD3 mAb. CD200R(1) expressing 2B4 Reay cells were incubated overnight and IL-2 release in each condition was assayed. IL-2 levels obtained by each mAb stimulated condition were statistically compared with no mAb stimulated control. (representative of three experiments). Statistically significant (p<0.05) results were shown with asterix. (*) indicates p<0.05, (**) indicates p<0.01 and (***) indicates p<0.001 (C–E).</p

CD200RLc and CD200RLe can generate activating signals when triggered by specific mAb.

<p>(A) Flow cytometry plots showing expression of CD200RLc (left panel) and CD200RLe (right panel) (solid tinted lines) compared to the control mAb (dashed lines) on RBL.2H3 cells stably transduced with either CD200RLc or CD200RLe together with mouse DAP12. (B) RBL.2H3 cells, transduced with CD200RLc and DAP12, CD200RLe and DAP12 or mock vectors, were plated and soluble OX110, OX131, OX132 or no mAb were tested by overnight stimulation. The level of degranulation was measured by assaying β-hexosaminidase in the cell lysate and supernatants. Antibody stimulated groups were compared with no antibody control group with statistically significant results indicated by *** (p<0.001; representative of three experiments).</p

CD200R(1) and CD200R(2) differ in binding with CD200R mAb but not in binding with CD200.

<p>(A–C) Flow cytometry plots showing 2B4 Reay cell line stably transduced with CD200R(1) (A), CD200R(2) (B) or mock vector (C) which were stained with OX110 (dashed line), OX131 (solid line) or control antibody (tinted). (D) Flow cytometry plot showing the binding of CD200 coated fluorescent beads to 2B4 Reay cells transduced with CD200R(1) (solid line), CD200R(2) (dashed line) but not vector control (dark grey filled). The double peak in panel D is due to some clumping of the beads.</p

The B subunit of Escherichia coli heat-labile enterotoxin inhibits Th1 but not Th17 cell responses in established experimental autoimmune uveoretinitis

PURPOSE. To investigate the efficacy of the B subunit of Escherichia coli heat-labile enterotoxin (EtxB) in the treatment of ocular autoimmune disease. Murine experimental autoimmune uveoretinitis (EAU) is an animal model of autoimmune posterior uveitis initiated by retinal antigen-specific Th1 and Th17 CD4 T cells, which activate myeloid cells, inducing retinal damage. EtxB is a potent immune modulator that ameliorates other Th1-mediated autoimmune diseases, enhancing regulatory T-cell activity. METHODS. EAU was induced in B10.RIII mice by immunization with peptide hIRBP. Disease severity was measured by clinical and histologic assessment, and functional responses of macrophages (Mφs) and T cells were assessed, both in vivo and in cocultures in vitro. EtxB was administered intranasally daily for 4 days, starting either 3 days before or 3 days after EAU induction. RESULTS. Preimmunization treatment with EtxB protected mice from EAU, limiting both the number and the activation status of retinal infiltrating immune cells. Treatment after EAU induction did not alter the disease course, despite suppression of IFN-γ. Although EtxB treatment of in vitro cocultures of T cells and Mφs increased IL-10 production, EtxB treatment in vivo increased the proportion and number of IL-17-producing CD4 cells infiltrating the eye. CONCLUSIONS. EtxB preimmunization protects mice from EAU induction by inhibiting Th1 responses, but the resultant reduction in IFN-γ responses by EtxB does not effect infiltration or structural damage in established EAU, where Th17 responses predominate. These data highlight the critical importance of the dynamics of T-cell phenotype and infiltration during EAU when considering immunomodulatory therapy