Specific pre-plasma cell states and local proliferation at the dark zone – medulla interface characterize germinal center-derived plasma cell differentiation in lymph node (Figure 6)

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. We collected draining lymph node from mice that were immunized since 14 days and gavaged since 3 days. Freshly dissected draining lymph nodes were dryed on absorbent paper, embedded in OCT, snap frozen in isopentane over dry ice, and stored at -80°C until processing. On the day of the experiment, 10 µm-thick cryosections were prepared from the region of interest using a cryostat. Four sections separated by 50µm in depth were placed on a Visium slide within the designated capture areas. Hematoxylin and eosin (H&E) staining was performed according to the manufacturer's guidelines with the following modifications: hematoxylin incubation for 30 seconds, bluing buffer for 5 seconds, and eosin incubation for 1 minute 30 seconds, all at room temperature. Following washing, brightfield images of the stained sections were acquired before proceeding to subsequent steps of the 10x Genomics Visium Spatial Gene Expression protocol. The tissue was embedded in OCT medium and a 10 µm section was cut on a cryostat and deposited on the capture area of the Visium slide following the guidelines of the 10x Genomics Visium Spatial Gene Expression protocol. Briefly, we performed permeabilization (18 min), reverse transcription, second strand synthesis, denaturation, cDNA amplification (16 cycles of PCR), and library construction according to the manufacturer’s instructions. The resulting libraries were sequenced on an Illumina NextSeq2000, generating an average of 120 million reads per library (Read 1: 28 cycles, Read i7: 10 cycles, Read i5: 10 cycles, Read 2: 79 cycles).