The RNA binding protein PTBP1 is necessary for B cell selection in germinal centres

Antibody affinity maturation occurs in germinal centres (GC) where B cells cycle between the light zone (LZ) and the dark zone. In the LZ GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from T helper cells that promotes their rapid proliferation. Here we show that the RNA binding protein PTBP1 is necessary for the progression of GC B cells through late S-phase of the cell cycle and for affinity maturation. PTBP1 is required for the expression of the c-MYC-dependent gene programme induced in GC B cells receiving T cell help and directly regulates the alternative splicing of transcripts increased during positive selection to promote anabolic metabolism and cell proliferation. mRNAseq libraries were generated from light zone and dark zone germinal centre B cells. Light zone and dark zone germinal centre B cells were FACS-sorted from mice immunised with alum precipitated NP-KLH (7 days before cell sorting). Erythrocytes were lysed and GC B cells were enriched before FACS sorting by depleting cells stained with biotinylated anti IgD, CD3e, Gr1 and Ter119 antibodies by MACS. Four biological replicates were carried out per condition. In each biological replicate, GC B cell-enriched splenocytes from 3 to 5 animals of the same genotype and sex were pooled together before FACS sorting. LZ and DZ GC B cells were sorted from the same GC B cell enriched samples. LZ and DZ GC B cells from control CD79a+/+Ptbp1fl/fl and CD79acre/+Ptbp1fl/fl cKO mice were sorted on the same day. Two biological replicates per condition were from females and two from males.