Dissecting the maturation of activated B cells using single-cell RNA-sequencing
Raghav Chawla1,2,3, Michelle Wray-Dutra2, David Rawlings2, Cole Trapnell1*, Richard James2*
1University of Washington, 2Seattle Children’s Hospital, 3Fred Hutchinson Cancer Research Center
B cells constitute a principal component of humoral immunity and their dysregulation leads to numerous human diseases. Following activation, B cells undergo terminal differentiation within secondary lymphoid organs, giving rise to antibody-secreting plasma cells.
We set out to dissect the components of the murine spleen at a single-cell level and establish a precise differentiation trajectory as activated B cells undergo final maturation. To this end, we immunized wildtype mice with degenerating sheep red blood cells and prepared single-cell suspensions from spleens harvested at different time points post-immunization. Single-cell cDNA libraries were generated using the 10X Genomics Chromium platform and sequenced on an Illumina NextSeq 500. The sequencing data was analyzed using Cell Ranger and Monocle 2.
We obtained single-cell expression profiles from 11,000 cells across 8 samples. Using unsupervised clustering, we identified cell types and defined their expression signatures in a completely unbiased manner. Besides validating the known splenic B-cell subsets, we found a previously uncharacterized naïve B-cell population with a strong interferon response signature. Differential gene expression analysis also revealed that naïve B cells exhibit a much lower total mRNA abundance than activated B or plasma cells and uncovered novel insights into the patterns of immunoglobulin expression. Finally, we generated branched trajectories which suggest that early B-cell maturation into plasma cells occurs primarily via an extrafollicular pathway rather than via the germinal center.
In conclusion, we used single-cell RNA-sequencing to a) dissect the cellular components of the murine spleen, thereby defining the transcriptional signatures of multiple B-cell subtypes and maturational stages, and b) gain novel insights into the kinetics of B-cell maturation.