Anti ifn γ

Fixable Viability Dyes (eBioscience) were used at 1:1000 dilution to label dead cells. For surface staining, cells were stained with the following fluorescence-conjugated antibodies (eBioscience, Biolegend, Tonbo): anti-CD4 (RM4-5), anti-CD8 (53–6.7), anti-CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-Thy1.1 (OX-7), IL-1R (JAMA-147), IL-6Rα (D7715A7) were used at 1:400 dilution. For intracellular staining, surface stained cells were fixed and permeabilized with a Foxp3 staining kit (eBioscience) according to manufacturer’s instruction and were stained with the following antibodies: anti-Foxp3 (FJK-16s), anti-Rorγt (AFKJS-9), anti-Id2 (ab166708; Abcam), anti-IFN-γ (XMG1.2), anti-IL-17A (TC11-18H10.1), anti-IL-17F (eBio18F10), anti-IL-22(1H8PWSR), anti-IL10 (JES5⁻16E3) were used at 1:200 dilution. For intracellular cytokine staining of cytokines, cells were stimulated by phorbol myristate acetate (PMA) and ionomycin for 6 h in the presence of Golgi-Plug (555029, BD) or Golgi-Stop (554724, BD). Data from the stained cells were collected with LSR Fortessa flow cytometer analyzer equipped with 5 lasers with DIVA software (BD Biosciences) and were analyzed by FlowJo software (Treestar). Gating strategies for FACS sorting are described in Supplementary Fig. 11.

Cervical dislocation was performed to sacrifice the animals. The lumbar enlargement of the spinal cords were isolated from animals and instantly fixed in 4% paraformaldehyde for a minimum of 48 h. The spinal cords were frozen and cut into 6 μm sheets, stained with hematoxylin and eosin (H&E) as well as luxol fast blue (LFB). The sections were blocked using 5% goat serum for 30 min. Next, overnight incubations at 4 °C were done with anti-CD4 (CST, USA), anti-IFN-γ (Abcam, UK), anti-IL-2 (Abcam, UK), anti-IL-4 (Abcam, UK) and anti-IL-17A (Abcam, UK). Next, they were incubated with Alexa Fluor 488 secondary antibody (Abcam, UK). A fluorescence light microscope (Zeiss, Germany) was used for examination.

The proteins in the sample were harvested as described above. The experiment procedure was also following our previous publication [18 (link)]. The antibodies used were listed as below: anti-JAK1, anti-JAK2, anti-STAT1, anti-pSTAT1, anti-STAT3, anti-pSTAT3 (Cell Signaling, Beverly, MA, USA; catalog number:#3344, #3230, #9172, #7649, #9139, #9136); anti-SOCS3 (GeneTex, Irvine, CA, USA; catalog number:GTX23693); anti-IFN-γ (Abcam, Cambridge, UK; catalog number:ab9657); anti-TLR-4 (Proteintech, Rosemont, IL, USA; catalog number:19811-1-AP); anti-IL-6 (Bioworld Technology, Bloomington, MN, USA; catalog number:BS6419); at room temperature (RT) for 1 h. The protein bands on the membrane were detected with an ECL-Plus Western Blot Detection system (GE Healthcare UK LTD) according to the instructions of the manufacturer. All experiments were replicated at least thrice.

Immunohistochemical (IHC) analysis was performed using antibodies against Ki-67, VEGF, CD31 (Cell Signaling Technology, USA) respectively. For immunofluorescence (IF) staining, sections were incubated with anti-CD56, anti-IFNγ, anti-TNF-α antibodies (Abcam, Cambridge, UK). Next, slides were mounted with Vectashield mounting media containing DAPI and were analyzed under fluorescence microscope.

Anti-PPRV-N, anti-PPRV-H and anti-PPRV-V monoclonal antibody were provided by the China Animal Health and Epidemiology Center (Qingdao, China). The following primary antibodies were used: anti-CD63 (1:1500; Santa Cruz), anti-CD81 (1:1000; Santa Cruz), anti-SLAM (1:2000; Santa Cruz), anti-GAPDH (1:2000; Invitrogen), anti-IL10 (1:1500; ABclonal), anti-IFNa (1:1500; ABclonal), anti-IFNγ (1:1500; Abcam), or anti-TNFa (1:1000; Abcam). Secondary antibodies: HRP-conjugated mouse anti-rabbit IgG (1:15000; Cell Signaling Technology), HRP-conjugated goat anti-mouse IgG (1:20000; Sigma-Aldrich), HRP-conjugated goat anti-rabbit IgG (1:15000; Sigma-Aldrich), PE-conjugated goat anti-rabbit IgG (1:20000; TransGen Biotech), fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:15000; Sigma-Aldrich), tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG (1:15000; Sigma-Aldrich).