Visualizing germinal center B cell dynamics at single cell transcriptome resolution. Mus musculus

During a germinal center (GC) response, B cells diversify their immunoglobulin (Ig) genes by somatic hyper-mutation (SHM) and undergo clonal expansion and positive selection thereby enabling the generation of higher affinity antibodies. We have analyzed the genomic states underlying GC B cell dynamics by single cell RNA-Seq. Profiling of antigen specific GC B cells during the peak of the response, revealed four distinctive genomic states characterized by antigen presentation, apoptotic, mitochondrial and mitotic gene expression modules. Intersection of genomic states and Ig heavy-chain (Igh) class-switch trajectory suggested that mitochondrial machinery is utilized to support class-switch recombination (CSR). Furthermore, by analyzing the transcriptomes of B cells with varying affinity BCR sequences that assembled from single-cell RNA-seq data through a novel algorithm, we show that high affinity GC B cells manifest enhanced mitotic and BCR signaling transduction, but compromised antigen processing and presentation gene expression modules. Thus, we are developing a comprehensive framework of the genomic states and molecular pathways underlying GC B cell dynamics. Overall design: C56BL/6J (Jax 664) mice were immunized intraperitoneally with 100 μg NP(23)-KLH (Biosearch Technologies) mixed with 50% (v/v) Alum (Thermo Scientific) and 1 μg LPS (Sigma). Splenocyte suspensions were prepared in MACS buffer (pH 7.4 PBS plus 1% FBS and 5 mM EDTA) on day 13 post immunization and blocked with 25 μg/ml 2.4G2 (BD) for 15 min on ice. Then cells were labeled with 2 μg/ml biotin anti-CD3, 1 μg/ml biotin anti-CD4, 1 μg/ml biotin anti-CD8, 2.5 μg/ml biotin anti-CD11C and 2.5 μg/ml biotin anti-IgD for 25 min at 4°C. After washing four times, cells were further labeled with anti-biotin beads (Miltenyi Biotec) for 20 min at 4°C. The magnetic columns were used to deplete non-GC B cells according to standard protocols (Miltenyi Biotec). The effluent cells were further labeled with NP-PE, B220, Fas and GL7 antibodies (Supplementary Table 1) for 30 min at 4 °C. GC B cells were sorted as 7AAD–B220+Fas+GL7hi with FACSAria II (BD) with 70 μm nozzle. Single GC B cells were prepared using the C1TM Single-Cell Auto Prep System (Fluidigm, San Fransisco, CA), according to the manufacturer’s instructions. 182 scRNA Seq libraries were sequenced per HiSeq 2500 gel with 75bp paired-end sequencing.