Figure 2. TMPD lipogranulomas contain class switched B cells.

A, Lipogranulomas express AID. Left, cDNA from TMPD or mineral oil lipogranulomas, spleen, or peritoneal cells was amplified using primers specific for AID or β-actin and analyzed by agarose gel electrophoresis. Right, AID mRNA was quantified by real-time PCR normalized to 18S RNA. B, μ and γ H-chain transcripts. Lipogranuloma and spleen cDNA from mineral oil or TMPD treated mice was tested for expression of IgM and IgG1 by PCR using VHF1-8 and Cμ or Cγ1 reverse primers, respectively. Left, agarose gel of amplified PCR products from lipogranulomas or spleen. γ1 transcripts are seen strongly in two TMPD lipogranulomas (TMPD, lanes 1 and 4) and weakly in another (TMPD, lane 3). Right, frequencies of IgM and IgG1 production in TMPD vs. mineral oil lipogranulomas (4 individual lipogranulomas/mouse, 3 mice/group). C, Circle transcripts. Presence of isotype specific I promoter-Cγ2a transcripts in lipogranulomas from TMPD-treated but not in mineral oil treated mice. Circle transcripts were detected using Iγ2aF forward primer and CμR reverse primer. PCR product sizes closely approximated the expected sizes of 458 and 318 bp (14) (arrows). PCR using B-actin primers was used as a loading control. D, Direct immunofluorescence for IgM (top) and IgG (bottom) producing cells in lipogranulomas from mineral oil or TMPD-treated mice (green). Nuclei were visualized by DAPI staining (blue). Both IgM and IgG producing cells were detected in ectopic lymphoid tissue from TMPD-treated mice, but only IgM producing cells in tissue from mineral oil-treated mice.