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. Author manuscript; available in PMC: 2016 Aug 1.
Published in final edited form as: J Immunol. 2015 Jun 24;195(3):853–864. doi: 10.4049/jimmunol.1403114

Figure 7. Anti-insulin L chains are selected from the pre-immune repertoire to enter germinal center reactions.

Figure 7

Spleens were harvested from unimmunized VH125SD B6 mice, or from mice 4 d following immunization with insulin-BRT. Flow cytometry sorting was using to purify insulin-binding B cells (B220+ live lymphocytes), that were further gated in immunized mice as IgM+ non-GC (GL7 Fas), IgM+ GC (GL7+ Fas+), or IgM GC populations. Isolated RNA was transcribed to cDNA, and Igκ genes were amplified by PCR. Sequences were analyzed using the Ig BLAST database (see Methods). (A-D) The number of clones identified by the indicated Vκ is divided by the total number of clones analyzed for each immunization group. Vκ4–74 (black), Vκ4–57–1 (gray), all other Vκ (white). (E-H) Igκ clone sequences were compared to germline sequences, excluding the 5’ region that correlated with degenerate primers used for amplification. The number of sequences that possess the indicated number of nucleotide changes, 0 (white), 1 (striped), 2 (gray), or > 3 (black), is shown for each immunization group.