Meca79

Lymph nodes were fixed in 10% neutral-buffered formalin and embedded in paraffin. Tissue sections were typically 4-μm thick and were examined after staining with hematoxylin and eosin (H&E). For immunohistochemistry, the sections were first boiled in a citrate-based solution to retrieve antigens and subsequently quenched in 3% hydrogen peroxide. The antibodies used included anti-FOXP3 (Cat. MAB8214, R&D systems, Minneapolis, MN), anti-PD-1 (Cat. AF1021, R&D systems), anti-CD3 (Cat. ab5690, Abcam, Cambridge, MA), anti-PAX5 (Cat. sc-1974, Santa Cruz Biotechnology, Dallas, TX), anti-CD4 (Cat. 14–0042, eBioscience, San Diego, CA), anti-CD8 (Cat. 14–0081, eBioscience), anti-CD21 (Cat. Ab75985, Abcam), MECA79 (Cat. 53–6036-80, eBioscience) and anti-HA (Cat. 3724, Cell Signaling Technology) antibodies. After biotinylated secondary antibody treatment, visualization was carried out with Vectastain ABC-HRP kit (Vector Laboratories, Burlingame, CA) or ABC-AP kit (Vector Laboratories) in combination with DAB or with VectorRed (Vector Laboratories) respectively.

Lymph nodes were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were typically four μm thick and stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were boiled in citrate-based solution to retrieve antigens and subsequently quenched in 3% hydrogen peroxide. Antibodies used included anti-CD21 (Cat. ab75985, Abcam, Cambridge, MA, USA), MECA79 (Cat. 53-6036-80, eBioscience, San Diego, CA, USA), anti-PD-1 (Cat. AF1021, R&D systems, Minneapolis, MN, USA), anti-CD3 (Cat. ab5690, Abcam), anti-PAX5 (Cat. sc-1974, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD4 (Cat. 14–0042, eBioscience), anti-CD8 (Cat. 14–0081, eBioscience), anti-CD138 (Cat. AF3190, R&D Systems) and anti-HA (Cat. 3724, Cell Signaling Technology). Horseradish peroxidase-coupled secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were used in combination with DAB for visualization. For immunofluorescence staining, sections were stained with Alexa Fluor 488 conjugated anti-GL7 (Cat. 144611, BioLegend, San Diego, CA, USA) and anti-CD4 (Cat. 14–0042, eBioscience). Secondary antibody for anti-CD4 was goat anti-Rat IgG (H + L) Alexa Fluor 594 (Cat. A11007, Invitrogen, Carlsbad, CA, USA).