IL-6 overexpression and immune activation in the spleen of Il12rb2 KO mice older than 12 months. (A) Expression of the CD69 activation marker on splenocytes from an 18-month-old Il12rb2 KO mouse and an age-matched WT mouse (insets). Cells were stained with CD69 mAb in combination with anti-B220, CD3, CD4, or CD8 mAb and gated on the latter markers. Dot-plots are shown. (Bi-ii) Immunohistochemical staining of spleen tissue sections from an 18-month-old WT mouse (Bi) and an age-matched Il12rb2 KO mouse (Bii) with anti–IL-6 mAb (magnification, × 400). (Biii) Flow cytometric analysis of intracellular IL-6 in splenocytes from an 18-month-old Il12rb2 KO mouse (KO, right profile) and an age-matched WT mouse (WT, middle profile). Isotypic control staining (isotype), shown in the left profile, was superimposable for both animals. Cells were cultured without stimuli for 16 hours in the presence of brefeldin A to allow intracellular accumulation of synthesized IL-6, permeabilized, and analyzed. (C) Intracellular expression of IL-6 was investigated by flow cytometry following double staining of splenocytes from an 18-month-old Il12rb2 KO mouse and an age-matched WT mouse (insets) with anti-B220, CD3, CD4, CD8, or CD11c, and anti–IL-6 mAbs. Cells were cultured as indicated in panel B, permeabilized, and analyzed setting the gate on B220⁺, CD3⁺, CD4⁺, CD8⁺, or CD11c⁺ splenocytes. Dot-plots are shown. (D) Tricolor staining of splenocytes from an 18-month-old Il12rb2 KO mouse with B220 or CD3 mAbs in combination with CD69 and anti–IL-6 mAbs. Gate was set on B220⁺, IL-6⁺ or CD3⁺, IL-6⁺ cells. Dot-plots are shown.