Molecular and immunophenotypic characterization of lymphoid infiltrates in the kidney and the liver. (Ai) Flow cytometric detection of intracellular IL-6 in liver-cell suspension from a 20-month-old Il12rb2 KO mouse. Open profile is anti–IL-6 mAb staining; dark profile is isotypic control staining. 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, gating on lymphocytes according to physical parameters. Staining with anti–IL-6 mAb of liver cells from an age-matched WT mouse yielded completely negative results (not shown). (Aii-iv) Dot-plots showing the results of double staining with anti-B220, CD4 or CD8, and anti–IL-6 mAbs. Gate was set on B220⁺, CD4⁺, or CD8⁺ splenocytes. (B) Ig gene rearrangement studies by Ig CDR3 spectratyping. DNA was extracted from the spleen of a 19-month-old WT (Bi) and an age-matched Il12rb2 KO mice (Bii), as well from the liver (Biii) and the kidney (Biv) of the latter mouse. PCR amplification was performed by using a set of forward primers designed on an IgV_H chain sequence available on the IMGT database (http://imgt.cines.fr) that detects approximately 55% of IgV_H regions (see “Materials and methods” for further details). Products were run on an automated sequenator and analyzed with Genescan software (Applied Biosystems).