Coupled analysis of transcriptional states and somatic hypermutation in germinal center B cells reveals role for oxidative phosphorylation in positive selection

In this work we illustrate the power of coupling single cell analysis of transcriptional states with somatic hypermutation of germinal center B cells to reveal unanticipated roles of metabolic programs in the positive selection. We use massively parallel 5'-end scRNA-seq to simultaneously capture transcriptome states and mutation profiles of IgH variable genes in individual cells. Our computational analyses reveal that the OXPHOS module is enhanced in GC B cells undergoing positive selection in response to different antigens. Through deleting the Cox10 gene, which encodes a cytochrome oxidase assembly factor, specifically in GC B cells, we demonstrate that OXPHOS activity is required for cell division and positive selection. Furthermore, we show that chemical augmentation of OXPHOS activity facilitates the positive selection of GC B cells in vivo. Our findings therefore reveal that tuning of OXPHOS activity by increased affinity BCRs is critical for clonal expansion and positive selection. Six to eight weeks old C56BL/6J (Jax 000664) mice were immunized intraperitoneally with 100 μg NP(23)-KLH (Biosearch Technologies) or OVA (Sigma) mixed with 50% (v/v) Alum (ThermoFisher Scientific) and 1 μg LPS (Sigma). NP-specific GC B cells were sorted as 7AAD–B220+FashiGL-7hiNP+ on day 13 post immunization using MA900 (Sony) with 100 μm Chip at 4 °C. For the immunization with OVA, total GC B cells were sorted as 7AAD–B220+FashiGL-7hi for scRNA-seq. Single cells were captured via the GemCode Single Cell Platform using the GemCode Gel Bead, Chip and 5’-end Library Kits (10X Genomics), according to the manufacturer’s protocol. Libraries were sequenced on a NovaSeq (Illumina). Additionally, OVA-binding GC B cells were sorted for bulk BCR library generation and sequenced on Miseq (Illumina).