Cell Type-Specific Expression and Functional Specialization of CXCL12 Isoforms

We analysed somatic hypermutation of Ig genes in mice carrying mutations in the CXCL12 gene that prevent anchoring of this chemokine to the Extra Cellular Matrix (ECM). Two types of mutant strains were analysed: CXCL12gagtm mice, which carry mutations that abrogate CXCL12/ECM interactions; and CXCL12gammaKO, where expression of the gamma isoform of CXCL12 was specifically abrogated.A total of 10 collections of sequences were generated by PCR and processed for Single Molecule Real-Time (SMRT) sequencing on a PacBio Sequel II system (Biomics Platform, Institut Pasteur):1) NAIVE B CELLS (IgDhi YFP-) from unimmunised AID-Cre-YFP mice (Crouch et al. 2007. J Exp Med 204:1145)2) MEMORY B CELLS (IgD-YFP+) from reconstituted control mice3) MEMORY B CELLS (IgD-YFP+) from reconstituted CXCL12gagtm mice4) MEMORY B CELLS (IgD-YFP+) from reconstituted control mice5) MEMORY B CELLS (IgD-YFP+) from reconstituted CXCL12gammaKO miceSequences 2-4 were generated by amplifying a 624bp fragment containing the V-D-JH4 recombination region of most VHJ558 family members, 3-4 months after reconstitution with AID-Cre-YFP BM6) NAIVE B CELLS (IgDhiYFP-) from CXCL12gagtm mice7) GERMINAL CENTER B CELLS (IgD-CD95+CD38-) from control mice8) GERMINAL CENTER B CELLS (IgD-CD95+CD38-) from CXCL12gagtm mice9) GERMINAL CENTER B CELLS (IgD-CD95+CD38-) from control mice10) GERMINAL CENTER B CELLS (IgD-CD95+CD38-) from CXCL12gammaKO miceSequences 6-10 were generated by amplifying a 500bp fragment containing VH186.2 rearranged to JH2, 13 days after immunisation with NP16-CGG