Anti il 17

Cell suspensions from the spleen and lymph nodes were stained with fluorescence-conjugated anti-CD4, anti-IFN-gamma, and anti-IL-17 (BD Biosciences, USA) for analysis of CD4⁺ T, Th1, and Th17 cells.

The expression markers on T cells were determined by FACS analyses after surface or intracellular staining with anti-mouse specific antibodies conjugated with Alexa Fluor 488, PE, FITC, allophycocyanin (APC), or PerCP.cy5.5. These mouse antibodies included: anti-CD3, anti-CD4, anti-CD8, anti-IFN-γ, anti-IL-4, anti-IL-17, and anti-FoxP3, which were purchased from BD Biosciences. For intracellular staining, T cells were stimulated with PMA (1 μg/ml) and ionomycin (2 μg/ml) (Sigma-Aldrich) for 5 hours in the presence of GolgiStop (BD Biosciences) before the intracellular staining with antibodies. All stained cells were analyzed on a LSR II cytometer (BD Bioscience) and data analyzed with FlowJo software (Tree Star).

T cell differentiation assays were, respectively performed as previously described [15 (link)]. In brief, irradiated EG7 tumor cells (1 x 10⁶) were loaded to Atg5^flox/flox or LysM-Atg5^-/- BMDM (1 x 10⁵) to facilitate phagocytosis and antigen processing. Two hours after the EG7 cells were loaded, they were extensively washed, and then cultured with CD4⁺ or CD8⁺ T cells from wild-type mice for 96 h. Cells were stained with anti-CD3, -CD4, or -CD8 mAbs, fixed and permeabilized with Cytofix / Cytoperm (BD Biosciences). Cells were then further stained with anti-IFN-γ, anti-IL-4, anti-IL-17, anti-Foxp3, anti-granzyme-B and isotype control mAb (BD Biosciences), and analyzed by flow cytometry.

Mouse splenocytes were harvested 4 weeks after booster immunizations, followed by stimulation with 6 μg/mL OMPs isolated from H. pylori 7.13 for 24 h, according to our previous report (Liu et al., 2008 (link)). Subsequently, supernatants of splenocytes were collected, and splenic cytokines of the stimulated cells were measured by corresponding ELISA kits. Briefly, monoclonal anti–interferon-γ (anti- IFN-γ), anti-IL-4, anti-IL-13, anti-IL-17, and anti-IL-12 (p40) antibodies (BD Biosciences, Mountain View, California, United States) were coated onto 96-well plates. Next, the samples were blocked with PBS containing 1% BSA, added to triplicate wells, and incubated overnight at 4°C. Then, the wells were washed and incubated with biotinylated monoclonal anti–IFN-γ, anti-IL-4, anti-IL-13, anti-IL-17, and anti–IL-12 (p40) antibodies (BD Biosciences, Billerica, Massachusetts, United States). Horseradish peroxidase (HRP)–labeled anti-biotin antibody (Vector Laboratories, Burlingame, California, United States) was then added. To develop the reaction, 3,3′,5,5′-tetramethyl-benzidine (Moss Inc., Pasadena, California, United States) was added, which was stopped with 0.5 N HCl. Finally, a standard curve was generated based on mouse recombinant (r) IFN-γ, IL-4, IL-13, IL-17, and IL-12 (p40).

Splenocytes and lymph node cells were isolated from CIA mice, as described previously [14 (link)]. For intracellular cytokine detection, cells were stimulated with PMA (50 ng/ml), ionomycin (1 μg/ml) and brefeldin A (10 μg/ml) for 4 h. Specific antibodies against mouse CD4 and CD25 were then added to the cells for 30 min before fixation and permeabilization with Perm/Fix solution (eBioscience, Paris). Cells were finally labelled with anti-IL-10, anti-IL-17, and anti-IFN-γ antibodies (BD Biosciences, Le Pont-de-Claix). Samples were analysed on a FACS Canto II and the analysis performed using the BD FACSdiva software (BD Biosciences).

For cytokine detection, cells were stimulated with phorbol myristate acetate (50 ng/mL; Sigma-Aldrich), ionomycin (1 μM; Sigma-Aldrich) for 4 hours with protein transport inhibitor (GolgiPlug 1 μl/ml, GolgiStop 2/3 μl/ml; BD Bioscience) in complete medium (CM) before staining. Cells were first stained extracellularly with specific antibodies against human CD3, CD4, CD8, CD45RA, CD45RO (BD Bioscience), then were fixed and permeabilized with Fixation/Permeabilization solution (eBioscience), and finally were stained intracellularly with anti-IL-2, anti-IL-17, anti-tumor necrosis factor-α (TNF-α), anti-interferon-γ (IFN-γ), anti-Granzyme B, anti-Ki67 and anti-EZH2 (BD Biosciences). Samples were acquired on flow cytometry (LSR II; Becton Dickinson) and data were analyzed with DIVA software (BD Biosciences) [35 (link)]. The gating strategy was shown in Supplementary Figure S2. KLRG1⁺ and KLRG1⁻ T cells were purified and sorted with FACSAria cell sorter (Becton Dickinson).