Isolation of Lung and Lymph Node Immune Cells

Blood was collected by cardiac puncture, and serum was stored at −80 °C. Immune cells were isolated from the mediastinal lymph nodes (MLN) and lung as previously described^{56 (link),83 (link)}. After hypotonic lysis to remove erythrocytes, single cell suspensions were prepared. CD45^negative (non-hematopoietic cells) were isolated from the lung using protease digestion^{84 (link)}. Briefly, 1% low melting point agarose was inserted into the lung via the trachea, and the tissue digested with dispase (Stem Cell Technologies, Cambridge, MA). CD45^negative cells were isolated from the resulting cell suspension using magnetic beads conjugated to an anti-mouse CD45 antibody (Miltenyi Biotec, Auburn, CA).