Specific pre-plasma cell states and local proliferation at the dark zone – medulla interface characterize germinal center-derived plasma cell differentiation in lymph node (Figure2) II

High affinity antibody-producing plasma cell (PC) production in germinal centers (GC) is crucial for antibody-mediated immune protection after vaccination or infection. The selection of high affinity B cells in the GC light zone instructs PC differentiation in a subset of cells, but the phenotype, differentiation trajectory and spatial localization of those prePC intermediates remain to be characterized. Here, we have used a mouse model to track GC-derived B cells with integrative single-cell and spatial analyses in draining lymph node after immunization. We first identified putative prePC in scRNA-seq datasets, then enriched those cells through their specific surface phenotype for further analysis of their gene expression trajectories and BCR repertoire. We found a continuum of actively proliferating transitional states bridging selected LZ GC B cells and recently exported PCs, with gradually increasing levels of endoplasmic reticulum stress-associated genes and immunoglobulin transcripts. Spatial analyses revealed that recently differentiated PC continued their maturation and proliferation at the interface between the DZ and extensions of the lymph node medulla. Our results provide insights into the intermediate stages and microenvironmental factors involved in the differentiation of GC B cells into PC, with implications for vaccine development and understanding antibody responses. Overall design: Mice were primed with NP-KLH in Sigma Adjuvant System, and we investigated cells 5 days after tamoxifen gavage, at day 16 of the primary response, using droplet-based 5'-end scRNA-seq. For experiments described in Figure 2a, cells from draining lymph nodes were washed and resuspended in FACS buffer (PBS containing 5% FCS and 2 mM EDTA) at a concentration of 10E8 cells/ml. Samples from different mice and different time points were processed separately using cell hashing as described (DOI : 10.1038/s41592-019-0392-0). Cells were individually stained with distinct barcoded anti-mouse CD45 antibodies (in-house conjugated) in FACS buffer for 30 minutes on ice. Subsequently, cells were washed and stained with a mix of primary antibodies, then Live/Dead Fixable Aqua Dead Cell Stain (Thermofisher). Live cells of interest (IgDneg eYFP+ PC and B cells) from each sample were bulk-sorted using a BD Influx cell sorter. PC and non-PC were captured in distinct wells for droplet-based scRNA-seq to maximize the recovery of BCR sequence information from non-PC. Within each fraction, cells from different samples were pooled and loaded for the 10x Genomics Single Cell 5' v2 workflow. Libraries were prepared according to the manufacturer's instructions with modifications for generating B cell receptor (BCR) sequencing libraries. Following cDNA amplification, SPRI select beads were used to separate the large cDNA fraction derived from cellular mRNAs (retained on beads) from the hashtag oligonucleotide (HTO)-containing fraction (in supernatant). For the mRNA-derived cDNA fraction, 50 ng were used to generate the transcriptome library, and 10-20 ng were used for BCR library construction. Gene expression libraries were prepared according to the manufacturer's instructions. For BCR libraries, heavy and light chain cDNAs were amplified by two rounds of PCR (6 cycles + 8 cycles) using external 12 primers recommended by 10x Genomics. Approximately 800 pg of purified amplified cDNA was tagmented using the Nextera XT DNA Sample Preparation kit (Illumina) and amplified for 12 cycles using the SI-PCR forward primer (10x Genomics) and a Nextera i7 reverse primer (Illumina). For the HTO-containing fraction, 5 ng were used to generate the HTO library. The resulting libraries were pooled and sequenced on an Illumina NextSeq2000 platform with single-indexed paired-end kits following the manufacturer's guidelines.