Recombinant mouse il 10

For B-T cell co-culture experiments B cells served as APCs and were stimulated for 24 h with commensals as described previously (“-primed B cells”). Prior to co-cultivation with naïve CFSE-labeled OT-II CD4⁺ T cells, B cells were incubated with 10 μg/mL Ova-Peptide (ISQAVHAAHAEINEAGR, EMC) for 2 h at 37°C. B cells were washed and supernatant was exchanged with fresh media (“-pulsed B cells”). Primed and pulsed B cells and naïve T cells were co-cultured at different ratios for 72 h at 37°C and 100 ng/mL purified anti-mouse IL-10 antibody (Clone: JES5-2A5, BioLegend) was added to certain samples.
For CD11c⁺ dendritic cells maturation assay, naïve B cells and differentiated immature BMDCs were simultaneously stimulated with bacteria at MOI 1 and co-cultured at a ratio of 5:1 (B cells/BMDCs). To show the effect of indirect cell-cell interaction, naïve B cells and BMDCs were additionally co-cultured in Transwells (pore-size 0.4 μm) and stimulated with bacteria at MOI 1. To mimic the influence of soluble IL-10 produced by B cells on the maturation of BMDCs, 10 μg/mL recombinant mouse IL-10 (BioLegend) were added to BMDC mono-cultures.

LPS (E. coil O111:B4) used for cell culture studies was purchased from Calbiochem (San Diego, CA, USA; cat# 437627), and for animal studies, it was purchased from Sigma-Aldrich (St. Louis, MO, USA; cat# L3012). IL-10, tumor necrosis factor alpha (TNFα), and interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kits and salmeterol (a long-acting selective β2-adrenergic receptor agonist) were obtained from R&D Systems (Minneapolis, MN, USA). Recombinant mouse IL-10, Ultra-LEAF™ Purified anti-mouse IL-10 antibody, and isotype IgG were from Biolegend (San Diego, CA, USA). The anti-mouse IL-10 antibody from Biolegend was used as the detecting/capture antibody for ELISA/ enzyme-linked immunospot (ELISPOT) assay and for neutralization of mouse IL-10 bioactivity in vivo and in vitro (https://www.biolegend.com/en-us/products/ultra-leaf-purified-anti-mouse-il-10-antibody-17764, accessed on 6 August 2021). The specificity and utility of this antibody were further validated in our laboratory. We found that enhanced IL-10 protein level was detected in the supernatant of LPS-stimulated mouse primary cell cultures by the R&D IL-10 ELISA kit, but no significant difference between vehicle and treatment with LPS plus anti-mouse IL-10 antibody was observed.

3-4x10⁵ CD4⁺ T cells were plated per well and immobilised by coating plates with 0.6μg Corning Cell Tak adhesive (354240, In Vitro Technologies Inc., QLD, Australia) or 1μg Poly-D-lysine (P6407, Sigma Aldrich, NSW, Australia) prior to running the Glycolysis stress test assay (SEA103020100, In Vitro Technologies Inc., QLD, Australia) as per manufacturer’s instructions. XF Base medium (SEA103335100, In Vitro Technologies Inc., QLD, Australia), supplemented with 1mM L-Glutamine (G7513, Sigma Aldrich, NSW, Australia) was prepared fresh on the day and pH adjusted to 7.4 at 37°C. For Fig 4G, 100ng/mL of recombinant mouse IL-10 (575806, BioLegend, California, USA) was added to port A, glucose to port B, oligomycin to port C and 2-DG to port D.