T bet

PBL subsets were determined as described previously [2 (link), 5 (link)]. For analysis of determinants on the cell surface, PBL were incubated with fluorochrome-labelled monoclonal antibodies against CD3, CD4, CD25, CD28, CD62L, CD95, CD119, CD127, CD152, CD154, CD178, CD252, HLA-DR, and CD183 (CXCR3) (all from BD Biosciences). Intracellular determinants were stained with fluorochrome-labelled monoclonal antibodies against Foxp3, IFNγ (clone B27), IL4, IL10, Granzyme B, Perforin, T-bet (all from BD Biosciences), Helios (ebioscience, Frankfurt, Germany) and TGFß₁ (R&D systems, Wiesbaden). Briefly, PBL were incubated with combinations of monoclonal antibodies for 30 min as described and eight-color fluorescence was analyzed using a FACSCanto II triple-laser flow cytometer (BD Biosciences) [2 (link), 5 (link)]. When, in addition, intracellular proteins were studied, cell membranes were permeabilized using BD Perm/Wash buffer (BD Biosciences). At least 100,000 events were analyzed in the initial FSC/SSC dot plot. IFNγ monoclonal antibody used for cell separation (BD clone 4S.B3) and IFNγ monoclonal antibody used for cell staining (BD clone B27) were not competitive (data not shown).

Single-cell suspensions of brain and spinal cord tissue were prepared according to standard protocols. For cytofluorometry, following antibodies were used: CD4, CD8, CD45, CCR6, CXCR3, TCRβ, CD11c, CD11b, CD49b, CCR2, CD86, T-bet, RORγt, IL-17, IFN-γ, TNF-α, and IL-12 with conjugated fluorochromes (BD Biosciences). Antibodies were incubated with cells in PBS with 2% FBS for 30 min at 4°C, and then cells were analyzed. For intracellular staining of cytokines, cells were stimulated for 4 h in RPMI 1640 containing 10% FBS (Gibco), 10 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich), and 500 ng/mL ionomycin (Sigma-Aldrich) with addition of Brefeldin A (BD Biosciences). Antibodies for the cell surface markers were added to the cells in PBS with 2% FBS for 30 min on ice. After wash, cells were resuspended in Cytofix/Cytoperm buffer (BD Biosciences) for 20 min on ice, washed twice, and incubated with Abs for intracellular antigens (cytokines) in Perm buffer (30 min, on ice). For staining of transcriptional factors, unstimulated cells were used. Data were acquired using FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Splenic cells and isolated cells from siLP were stained with specific antibodies against mouse as follows: activated T-cells: CD4 (BD), CD44 (BD), CD62L (BD); regulatory T-cells: CD4 (BD), CD25 (BD), Foxp3 (ebioscience); Th17: CD3 (BD), RORγt (BD), IL-17 (BD) and Th1: CD3 (BD), T-bet (BD) and IFN-γ (BD). Surface and intracellular staining were performed as previously described^{26 (link)}.

Isolated cells from spleens and siLP were stained as follows: Regulatory T-cells: CD4 (BD), CD25 (BD), Foxp3 (ebioscience); Th17: CD3 (BD), RORγt (BD), IL-17 (BD) and Th1 (CD3 (BD), T-bet (BD) and IFN-γ (BD). The staining protocol was performed as previously described [23 (link)]. MACSQuant® Analyzers (Miltenyi Biotec) and VenturiOne® (AppliedCytometry) software were respectively used for data collection and analysis.

Cultured CD4⁺ T cells from healthy control subjects were stimulated with Sema7A for 3 h. Unstimulated cells or T cells treated with DSema7A were used as controls. The cells were washed twice with ice-cold PBS and lysed in lysis buffer consisting of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and cOmplete Mini protease inhibitor cocktail (Roche). For phosphorylation-specific immunoblot analysis, phosphatase inhibitors (10 mM NaF, 12.5 mM β-glycerophosphate, and 1 mM Na₃VO₄) were added to the lysis buffer. Whole-cell lysates were subjected to SDS-PAGE under reducing conditions. Immunoblotting was performed using antibodies specific to retinoic acid receptor-related orphan nuclear receptor γt (RORγt; BioLegend) and T-bet (BD Biosciences).
The CD14⁺ cells from patients with RA were stimulated with 10 ng/ml of recombinant Sema7A or DSema7A for 15 minutes. CD14⁺ cells stimulated with DSema7A were used as negative controls. The activation of FAK was evaluated by immunoblotting using antibodies specific to FAK (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-FAK (Tyr397) (BD Biosciences), and glyceraldehyde 3-phosphate dehydrogenase (Abcam, Cambridge, UK).