Additional file 1 of Tissue plasminogen activator worsens experimental autoimmune encephalomyelitis by complementary actions on lymphoid and myeloid cell responses
Creators: name: Hélie, Pauline
name: Camacho-Toledano, Celia
name: Lesec, Léonie
name: Seillier, Célia
name: Miralles, Antonio J.
name: Ortega, Maria Cristina
name: Guérit, Sylvaine
name: Lebas, Héloïse
name: Bardou, Isabelle
name: Sol, Virginia Vila-Del
name: Vivien, Denis
name: Mauff, Brigitte Le
name: Clemente, Diego
name: Docagne, Fabian
name: Toutirais, Olivier
Publisher: name: figshare
Year: 2021
Subjects: subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine

description: Additional file 1: Suppl Figure 1: Gating strategy and lymphoid cell response after EAE in WT and tPA-/- mice. (A) Representative gating strategy for absolute cell count analysis of leukocyte subsets in spinal cord homogenates (WT mouse, plateau phase of EAE). (B) CD3+ leukocytes were gated for subpopulation analysis after using scattergram gates for viable leukocytes (in A). (C) representative flow cytometry plots for the assessment of intracellular cytokines IFNγ and IL-17 in CD4+ cells after EAE in WT and tPA-/-. Suppl Figure 2: T cell infiltration in the spinal cord of WT and tPA-/- EAE mice. Photomicrographs show representative images (from N=3) of indicated markers in spinal cord tissue samples of WT and tPA-/- mice at the EAE plateau phase (d20±3) from (A) cervical, (B) upper thoracic, and (C) lumbar sacral. Suppl Figure 3: Glunomab does not prevent the effect of tPA on T cells. Splenocytes activated with both anti-CD3ε and anti-CD28 antibodies were treated or not with tPA at 10 μg/mL in presence of Glunomab or the isotypic antibody for 4 days. Proliferation index (%) of (A) CD4+ and (B) CD8+ T cells in the indicated conditions. Results are expressed as mean + SEM (N=3). *P&lt;0.05 vs control #P&lt; 0.05 vs indicated experimental group. Suppl Figure 4: Myeloid response of WT and tPA-/- mice at the plateau phase of EAE. Representative gating strategy for absolute cell count analysis of leukocyte subsets in spinal cord homogenates of WT and tPA-/- mice. CD3- leukocytes were gated for subpopulation analysis after using scattergram gates for viable leukocytes (see Suppl.fig 1A). Suppl Figure 5: Myeloid response to tPA treatment. Representative flow cytometry plots for (A) dendritic cells and (B) macrophages after tPA treatment (0-20 μg/mL). Suppl Figure 6: Dynamic modification of immunogenic and tolerogenic DC phenotype along the EAE clinical course. CD40 is highly present at the moment of maximum affectation and decreased when symptoms partially recover. PD-L1 is increased after the recovery of the clinical symptoms. Asymptomatic immunized mice showed a high presence of PD-L1+ tolerogenic DCs. C.S= clinical score and d.p.i= day post-immunization. Suppl Figure 7: Effect of tPA on APC maturation is not mediated by its ability to interact with NMDA receptor. Splenocytes extracted from EAE mice at the peak of the clinical course were treated with 2 μg/mL tPA in presence of Glunomab or an isotypic antibody (A-H). The percentages of MHC-II+CD80+CD86+ immunogenic and MHC-II+CD80-CD86- tolerogenic APCs were determined by flow cytometry. Results are expressed as mean + SEM (N=5); *P&lt; 0.05, **P&lt;0.01 and ***P&lt;0.001.