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) 1

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. Mice were primed with OVA in Alum, for some of them also boosted with OVA in Alum, and we investigated cells 4 days after tamoxifen gavage, at day 10 and day 20 of either the primary response (D10P, D20P), or the secondary response after boosting (D10S, D20S), using the FACS-based 5-prime-end sequencing (FB5P-seq) method for integrative scRNA-seq analysis described by Attaf et al. (DOI : 10.3389/fimmu.2020.00216). Individual cells were sorted into a 96-well PCR plate, with each well containing 2 µL of lysis buffer. Index sort mode was activated to record the fluorescence intensities of all markers for each individual cell. Flow cytometry standard (FCS) files from the index sort were analyzed using FlowJo software, and compensation parameters were exported as CSV tables for subsequent bioinformatic analysis. Immediately after sorting, plates containing individual cells were stored at -80°C until further processing. Following thawing, reverse transcription was performed, and the resulting cDNA was preamplified for 22 cycles. Libraries were then prepared according to the FB5P-seq protocol. The FB5P-seq data were processed to generate both a single-cell gene count matrix and single-cell B cell receptor (BCR) repertoire sequences for B cell analysis. Two separate bioinformatic pipelines were employed for gene expression and repertoire analysis, as detailed in Attaf et al. (DOI : 10.3389/fimmu.2020.00216).