α cd86

Tannerella forsythia wild-type and Gtf-deficient mutants were irradiated three times with UV light using maximum UV energy settings for 1 min, each, (Ultraviolet Crosslinker CL-1000; UVP, Upland, CA, United States) for inactivation. Cell integrity after treatment was verified by scanning electron microscopy using an Inspect S50 scanning electron microscope (FEI, Eindhoven, Netherlands) (Tomek et al., 2014 (link)).
Murine and human iDCs were matured in a 48-well plate with inactivated T. forsythia wild-type, ΔTanf_01245 (ΔgtfS), ΔTanf_01290 (ΔgtfI), or ΔTanf_01305 (ΔgtfE) cells, at concentrations of 10⁶, 10⁷ and 10⁸ cfu/ml. iDCs stimulated with 100 ng/ml LPS (E. coli strain O111:B4, Calbiochem, EMD Chemicals, San Diego, CA, United States) served as a positive control. Stimulated DCs were incubated at 37°C for 24 h. Supernatants were frozen for later use, DCs were harvested. DC maturation was monitored by visual and flow cytometric evaluation of typical DC morphology (LSR II flow cytometer, BD Biosciences), and expression of cell-surface markers (α-CD80, α-CD86, α-MHC-II; eBioscience) after 6 h and 24 h, respectively.

Poly (D, L-lactic-co-glycolic acid) (75:25 PLGA, Resomer® RG 752H, M_W 4A) was purchased from Lakeshore Biomaterials (Birmingham, AL, USA). Ovalbumin (OVA_257-264) and dimethyl-dioctadecyl-ammonium bromide (DDAB) were obtained from Sigma (St, Louis, MO, USA). Methylene chloride (AR grade) and absolute ethyl alcohol (AR grade) were purchased from Beijing Chemical Reagent Company (Beijing, China). Concanavalin A, Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco's modified Eagle medium (DMEM) medium, and fetal bovine serum (FBS) were supplied by Gibco (Grand Island, NY, USA). Alexa 635-phalloidin and Lysol-Tracker probes were purchased from Invitrogen (Grand Island, NY, USA). FITC was obtained from Sigma-Aldrich. Fluorochrome-labeled α-MHCI, α-MHC II, α-CD86, α-CD80, α-CD11c, α-CD69, α-CD44, α-CD62L, α-CD4, α-CD8 and α-CD19 antibodies were supplied by eBioscience (San Diego, CA, USA). Recombinant mouse GM-CSF and IL-4 were obtained from Peprotech (Rocky Hill, NJ, USA). Mouse cytokine ELISA kits were purchased from eBioscience. OVA derived (H-2 Kb, SIINFEKL) specific MHC I pentamers was purchased from ProImmune (Oxford, UK). p38 MAPK inhibitor SB203580 was purchased from Cell Signaling Technology (Danvers, MA, USA).