Anti cd138

Anti-CD4, anti-CD8, anti-CD11c, anti-CD11b, anti-MHC-II, anti-CD80, anti-CD86, anti-CD335, anti-CD3, anti-IFN-γ (all BD Biosciences, Heidelberg, Germany), anti-CD62L, anti-TNF-α, anti-Foxp3 (all eBioscience, Frankfurt, Germany), anti-CD160, anti-CD138, and anti-IL-10 (Biolegend, London, UK) were used as fluorescein isothiocyanate, pacific blue, phycoerythrin (PE), BD Horizon V450, allophycocyanin, AlexaFlour647, PE-cyanin 7, or peridinin-chlorophyll protein conjugates. Dead cells were identified by staining with the fixable viability dye eFlour 780 (eBioscience, Frankfurt, Germany). Intracelluar staining for Foxp3 was performed with the Foxp3 staining kit (eBioscience, Frankfurt, Germany) according to the manufacturer’s recommendations. Cytokine production from freshly isolated splenocytes was measured by stimulating cells with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, München, Germany) and 100 µg/ml ionomycin (Sigma-Aldrich, München, Germany) for 4 or 6 h (IL-10), respectively, in the presence of 5 µg/ml Brefeldin A (for IFN-γ, TNF-α staining) and 5 µg/ml Monensin (for IL-10 staining), treating with 2% paraformaldehyde and 0.1% NP40, and staining with the respective antibody cocktail. Flow cytometric expression analyses were performed with an LSR II instrument using DIVA software (BD Biosciences, Heidelberg, Germany).

The cells were resuspended in ice-cold PBS containing 1% FBS and were stained with anti-CD3, anti-CD4, anti-CD8, anti-B220, anti-CD38, anti-CD138, anti-PD-1, anti-CXCR5, anti-CD62L, anti-OX40, anti-IgD, anti-CD95, anti-GL7, anti-CD11c, anti-CD80, anti-CD86, anti-MHC II, anti-CD40, anti-CD69, anti-CD11b, anti-Gr-1, and anti-F4/80 antibodies (Biolegend). Intracellular staining of IFN-γ, TNF-α, IL-17a, Helios, and Foxp3 was performed after 4 h of stimulation with PMA and ionomycin following a standard protocol (BD Biosciences). The results were obtained on a BD FACS Fortessa flow cytometer and were analyzed using FlowJo software.

iGB cells were generated as above. On day 4, fibroblasts and in vitro-differentiated plasmablasts (generally less than 10% frequency) were removed using biotinylated anti-H-2Kd (catalog no. 116604, BioLegend) and anti-CD138 (catalog no. 142511, BioLegend) followed by anti-biotin microbeads (Miltenyi Biotec); negative selection was performed using LS columns (Miltenyi Biotec). In some experiments, FACS was used to purify tdTomato⁺ iGB cells. For competitive experiments, purified WT iGBs (CD45.1/2⁺) were mixed 1:1 (ratio confirmed by flow cytometry before transfer) with CD45.2⁺ tdTomato⁺ Tfam^−/− iGBs and injected intravenously (6 × 10⁶ total cells in competitive setting or 3 × 10⁶ cells in noncompetitive setting) into CD45.1⁺ or CD45.2⁺ congenic hosts that were immunized with SRBC according to the enhanced protocol to maximize the recruitment of transferred iGB cells into the GC. On day 6 after iGB adoptive transfer, spleens were collected and analyzed by flow cytometry and confocal imaging to assess GC entry.