A progressive increase in the breadth and specificity of autoantibodies over time, termed epitope spreading, drives pathogenic targeting of an ever-widening repertoire of self-components in many autoimmune diseases. Ostensibly, this progressive inclusion of additional B cell clones into an ongoing autoreactive response can occur through linked recognition, whereby proto-autoreactive B cells recognize distinct antigenic epitopes, which carry shared T cell epitopes. In a murine model displaying epitope spreading resembling that observed in systemic lupus erythematosus, we find that the epitope spreading process is compartmentalized by MHC. Antigen presentation by B cells carrying two MHC haplotypes can bridge the MHC barrier between two compartments of B cells that do not share MHC haplotypes, by communicating with two separate pools of MHC-restricted T cells. This leads to inclusion of distinct and diverse B cell reactivities in germinal centers. Our findings demonstrate a formidable capacity of B cells to drive the autoreactive response.
To better understand the immune landscape of the no-bridge and bridge chimeras, we leveraged the BD Rhapsody platform to perform linked single-cell Abseq, immune profiling and BCR/TCR analyses. We analyzed total splenocytes from two no-bridge chimeras, and total splenocytes enriched for GC B cells from a bridge chimera.
For single-cell mRNA and repertoire sequencing, total splenocytes were purified from spleens of two no-bridge chimeras as indicated under Tissue Harvest and Preparation, followed by negative selection with biotinylated Ter-119, anti-biotin microbeads and magnetic separation. For one bridge chimera, the spleen was split in half, and one half was subjected to the same total splenocyte purification whereas the other half was used for purification of GC B cells using a PNA microbead kit (130-110-479, Miltenyi Biotec) according to the manufacturer's instructions, and the two fractions were mixed to generate a GC B cell enriched splenocyte sample.
From each sample, 1x106 cells were added 100 ul of AbSeq staining mix containing a cocktail of 20 Abseq antibodies, and incubated for 60 minutes. Cells were washed three times with 2 ml BD staining buffer (250 g for 8 minutes centrifugation) and for each sample, 20,000 cells were loaded onto a BD Rhapsody cartridge and processed on the Rhapsody Express module following the manufacturer's instructions. Each sample was subsequently split into two (A and B) to be able to verify technical reproducibility, and each subsample was used for generation of 4 libraries: (1) single-cell targeted library using the Mouse Immune Response Panel (Catalog No. 633753, BD) containing primer pairs targeting 397 genes commonly found in mouse immune cells and a BD Rhapsody Panel supplement (Part number 633742, BD) containing an additional 45 target genes, for a total of 442 targets; (2) Abseq library; (3) BCR V(D)J library; and (4) TCR V(D)J library.
Cell information / gene expression violin plot / box plot
In this tab, users can visualise the gene expression or continuous cell information
(e.g. Number of UMIs / module score) across groups of cells (e.g. libary / clusters).
Proportion / cell numbers across different cell information
In this tab, users can visualise the composition of single cells based on one discrete
cell information across another discrete cell information.
Usage examples include the library or cellcycle composition across clusters.
In this tab, users can visualise the gene expression patterns of
multiple genes grouped by categorical cell information (e.g. library / cluster).
The normalised expression are averaged, log-transformed and then plotted.