Anti icos

Kidneys were fixed with 4% formalin, embedded in paraffin, and cut into 4 um serial slices. After deparaffinization and rehydration, Citrate buffer (pH 6.0) or Ethylene Diamine Tetraacetic Acid (EDTA, pH 9.0) was used for antigen retrieval. For immunohistochemistry staining, after being blocked with 10% H₂O₂ for 15 min and 5% serum for 30 min at room temperature, the paraffin sections were incubated at 4 °C overnight with primary antibodies. The primary antibodies included anti-IL-21 (ABclonal, Wuhan, China), anti-CXCR5 (Bioss, Beijing, China), anti-ICOS (eBioscience, San Diego, CA, USA), anti-PD1 antibodies (CST, Framingham, MA, USA). Slices were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Then, 3,3′-diaminobenzidine (DAB) was visualized. For immunofluorescence (IF) staining, paraffin sections were blocked with 5% serum at room temperature for 30 min, and then incubated with primary antibodies at 4 ℃ overnight. The primary antibodies included anti-CD4 (Biolegend, San Diego, CA, USA), anti-PD1 (CST, Framingham, MA, USA), anti-CXCR5 (Bioss, Beijing, China), anti-ICOS (eBioscience). Slices were incubated with fluorescence-labeled secondary antibodies and developed with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using an Olympus microscope.

To study ICOS signaling, we activated and restimulated cells with anti-ICOS similarly to previously described ICOS restimulation conditions (Rolf et al., 2010a (link)). Briefly, WT or FOXO1-GFP naive CD4 (CD69⁻CD25⁻CD4⁺) T cells were purified by negative depletion and activated with anti-CD3 (2C11), 1 μg/ml anti-CD28 plus or minus 10 μg/ml anti-IFN-γ, 10 μg/ml anti-IL-4, 50 ng/ml IL-6, and 10 ng/ml IL-21 (iTfh conditions) in RP10 for 48 hr. After 48 hr, the cells were rested in RP10 for 24 hr. Following the rest, the cells were restimulated with soluble anti-CD3 0.5 μg/ml, goat anti-hamster 20 μg/ml (Vector Labs, Burlingame, CA) with or without stimulatory 2 μg/ml anti-ICOS (Clone: C398.4A, eBioscience). To determine whether ICOS signaling inactivated, we collected FOXO1 cells at 30 m or 24 hr post restimulation and analyzed FOXO1-GFP compared to DRAQ5 staining using AMNIS ImageStream and BD LSR Fortessa analysis. To determine whether ICOS signaling through FOXO1 might be involved in ICOS upregulation of BCL6, we left WT or Foxo1^KO CD4 T cells in culture for 24 hr post restimulation and analyzed expression of Tfh markers by flow cytometry.

For surface staining, single-cell suspensions were stained with anti-PDCA1, anti-CXCR5 (BD Biosciences), anti-CD11c, anti-CD86, anti-CD80, anti-MHC-II, anti-CD4, anti-CD3, anti-CD8, anti-CD25, anti-CD19, anti-B220, anti-GL-7, and anti-ICOS antibodies (all from eBioscience, Germany). To analyze Foxp3, cells were fixed and permeabilized with FOXP3 staining buffer set (eBioscience, Germany) following the manufacturer's instructions and stained with anti-Foxp3 antibodies (eBioscience, Germany). Flow cytometry was carried out using FACSCanto II device (BD Biosciences, Germany). Data analysis was performed using FCS Express Software.