Anti igd

Cells were harvested and washed with FACS buffer. For surface staining, the 1 × 10⁶ cells in 100-μL single-cell suspension were stained with antibodies for 30 minutes in the dark at 4 °C. The following antibodies were used: live and dead cell stain FVS (BD Biosciences), anti-CD3, anti-CD4, anti-CD8, anti-CD103/ITGAE, anti-CD19, anti-CD27, anti-IgD, anti-CD38, and anti-CD138 (BD Biosciences, San Jose, CA); and anti-CD69, anti-CD18/ITGB2, anti-KLRG1 (BioLegend, San Diego, CA). For intracellular cytokine staining, immune cells were incubated in complete RPIM1640 containing 10% FBS and leukocytes activation cocktail with GolgiPlug (BD Biosciences) at 37 °C for 5 hours, and then surface-stained cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) for 20 minutes at 4 °C. Subsequently, cells were stained with anti-GZMB, anti-GZMK, and anti-IgA (BD Biosciences) for 30 minutes at 4 °C. For staining TFs, cells were fixed and permeabilized with Transcription Factor Buffer Set (BD Biosciences). Briefly, after surface staining, cells were fixed and permeabilized for 50 minutes at 4 °C, then washed and stained with ATF5 (Abcam, Cambridge, UK) for 50 minutes at 4 °C. FCM was carried out using Celesta (BD Biosciences) and analyzed using FlowJo software (version 10.6.2, Tree Star).

Spleens sections were harvested, embedded in Tissue-Tek and ‘snap-frozen’ in dry ice and stored at −80 °C. Cryostat sections (8 μm in thickness) were prepared, air-dried and fixed in ice-cold acetone for 15 min. Sections were blocked with 5% rat serum and stained with the following antibodies: anti-IgG1, anti-IgD, anti-CD3 or anti-VCAM-1 antibodies (BD Biosciences PharMingen). Antibodies were diluted (1:50) in PBS containing 5% rat serum. Sections were further mounted with CYTOSEAL 60 (Electron Microscopy Sciences) and analysed with an Olympus FV1000 microscope (Olympus) using a × 20 objective, and the images were acquired with Olympus Fluoview Version 2.1 software.