Jes5 2a5

For macrophage–T cell co-cultures, 2 × 10⁴ BMDMs differentiated in the above conditions were seeded together with 5 × 10⁴ sorted CD8⁺ T cells and stimulated for 3 d with anti-CD3 (1 µg ml^–1; 145-2C11, BD Biosciences) and anti-CD28 (0.5 µg ml^–1; 37.51, BD Biosciences), in the presence or absence of anti-IL-10 blocking antibodies (4 or 10 μg ml^–1; JES5-2A5, eBioscience).
For macrophage conditioning with B cells followed by T cell co-cultures, purified LN B cells were stimulated with anti-IgM (8 µg ml^–1) plus anti-CD40 (10 µg ml^–1; BD Biosciences) for 3 d. Bone marrow cells were differentiated with M-CSF for 1 d, and adherent cells were then cultured with or without activated B cells (1 × 10⁵ cells) for five more days in the presence of M-CSF and further polarized for 6 h with IL-10 (10 ng ml^–1). Macrophage–T cell co-cultures were performed as described above. After 3 d of co-culture, CD8⁺ T cells were analysed by flow cytometry, and the concentration of IFNγ and TNF in the supernatant was quantified using the Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences).

Antibodies for immunofluorescence were purchased from the following sources: F4/80 (0.2 μg/ml, BM8, OriGene, Rockville, MD, USA), TNF-α (2.5 μg/ml, MP6-XT3, eBioscience, San Diego, CA, USA), IL-10 (2.5 μg/ml, JES5-2A5, eBioscience), fibronectin (0.7 μg/ml, Polyclonal, invitrogen, Carlsbad, CA, USA), and collagen IV (2.5 μg/ml, Polyclonal, Invitrogen). Secondary antibody were goat anti-rat IgG Alexa Fluor® 488 (2 μg/ml, BioLegend, San Diego, CA, USA), streptavidin Alexa Fluor® 594 (0.5 μg/ml, BioLegend), goat anti-rabbit IgG Alexa Fluor® 488 (2 μg/ml, Invitrogen).
The following antibodies (BioLegend) were used for flow cytometry: APC anti-mouse F4/80 (2.5 μg/ml, BM8), PE/Cy7 anti-mouse CD86 (1.25 μg/ml, GL-1), PE anti-mouse CD206 (1.25 μg/ml, C068C2), PE/Cy7 anti-mouse TNF-α (5 μg/ml, MP6-XT2), and Alexa Fluor® 488 anti-mouse IL-10 (12.5 μg/ml, JES5-16E3).