Longitudinal scRNAseq profiling of memory B cells in mRNA vaccinated individuals post BA.1-breakthrough infection. Longitudinal scRNAseq profiling of memory B cells in mRNA vaccinated individuals post BA.1-breakthrough infection

Memory B cells play a fundamental role in host defenses against viruses. This dataset aimed at understanding the recruitment and remodeling of the memory B cell repertoire in the context of BA.1-breakthrough infection in BNT162b2 mRNA vaccinated individuals. All four donors enrolled in the study had received a booster (3rd dose) of BNT162b2 mRNA vaccine and had no history of prior SARS-CoV-2 infection. All four experienced a documented breakthrough infection between 12/24/2021 and 01/30/2022 when BA.1 was responsible for > 85% of SARS-CoV-2 infections in France. All donors were sampled at Henri Mondor University Hospital (AP-HP, Paris France), and samples used for scRNA-seq were collected shortly after breakthrough infection (PO_M0 samples; between day 7 and day 18) and 5 to 6 months after infection (PO_M6 samples; between day 152 and day 173). Clinical and biological characteristics of these patients are summarized in the Patient_information.csv file. For each sample, an initial pool of 50.000 total peripheral CD3-CD14-CD56- CD19+ IgD- cells was always sorted and afterward, to enrich for cells of interest, only CD19+CD38low antibody secreting cells (ASCs), CD19hi IgD+ cells and SARS-CoV-2 Spike/RBD PE/tetramer positive B cells were sorted, leading to approximately 55000-60000 total sorted cells per sample. Sorted cells were then counted and up to 20 000 cells were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries for gene expression (mRNA), ADT and VDJ BCR libraries were generated using Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. PBMCs were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and approximately 15x106 cells were then resuspended in 100µL PBS 2%FBS and incubated for 40 minutes at 4°C with a decoy tetramer (biotinylated Bovine Serum albumin coupled with BV785 streptavidin) and Hu-1 Spike, BA.1 Spike, Hu-1 RBD and BA.1 RBD tetramers (constructed using PE-labelled TotalSeqC® streptavidin with different barcodes for each individual antigens (see feature_reference.csv.gz files).). Cells were washed, resuspended in 100µL PBS 2%FBS and stained with a cocktail of fluorochrome conjugated (CD3, CD14 both APC-H7 at 1:100 each; CD15 and CD56 BV785 at 1:100 each, CD19 PECF594 at 1:100, IgD FITC at 1:100, CD38 PercP-Cy5.5 at 1:100) and TotalSeqC® (CD38, CD27, CD71, CD21, CD11c, CD39, FCRL5, CD95 all at 1:40 (all obtained from BioLegend)) antibodies for 40 minutes on ice. Viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, 1:200) incubated with conjugated antibodies. Two distinct sorts were performed for each donor: one at the early time-point (PO_M0) and one at the 6 months’ time-point (PO_M6).

记忆B细胞（Memory B cells）在宿主抗病毒防御中发挥核心作用。本数据集旨在解析接种BNT162b2 mRNA疫苗的个体在感染BA.1毒株突破感染后，记忆B细胞库的招募与重塑模式。
本研究纳入的4名受试者均已完成BNT162b2 mRNA疫苗加强针（第3剂）接种，且无既往SARS-CoV-2感染史。4名受试者均在2021年12月24日至2022年1月30日期间经确诊发生突破感染，彼时BA.1毒株占法国SARS-CoV-2感染病例的85%以上。
所有受试者均在法国巴黎亨利·蒙多大学医院（AP-HP）完成采样，样本采集分为两个时间节点：突破感染后短期采集的PO_M0样本（第7至18天），以及感染后5至6个月采集的PO_M6样本（第152至173天）。受试者的临床与生物学特征汇总于"Patient_information.csv"文件中。
针对每份样本，首先均分选得到初始总量为50000个的外周血CD3⁻CD14⁻CD56⁻CD19⁺IgD⁻细胞；后续为富集目标细胞群，进一步分选得到CD19⁺CD38low抗体分泌细胞（antibody secreting cells, ASCs）、CD19hiIgD⁺细胞以及SARS-CoV-2刺突蛋白/RBD PE/四聚体阳性B细胞，最终每份样本分选得到的总细胞数约为55000~60000个。
分选完成的细胞经计数后，最多取20000个细胞上样至10x Chromium Controller中，制备乳液单细胞凝胶微珠。基于10x Genomics的Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1并配合特征条形码技术，按照厂商操作手册完成基因表达（mRNA）、ADT及VDJ BCR文库的单细胞RNA测序（scRNA-seq）建库。
外周血单个核细胞（PBMCs）最初通过标准密度梯度离心从静脉血样本中分离，并在-150℃冻存后使用。解冻时采用含10%胎牛血清的RPMI-1640（Gibco）培养基重悬，洗涤两次后，取约15×10⁶个细胞重悬于100μL含2%胎牛血清的PBS中，于4℃孵育40分钟，所用试剂包括诱饵四聚体（生物素化牛血清白蛋白偶联BV785链霉亲和素），以及Hu-1刺突蛋白、BA.1刺突蛋白、Hu-1 RBD及BA.1 RBD四聚体（采用PE标记的TotalSeqC®链霉亲和素构建，每种抗原对应不同条形码，详见"feature_reference.csv.gz"文件）。
细胞洗涤后重悬于100μL含2%胎牛血清的PBS中，加入荧光素偶联抗体组合（CD3、CD14均采用APC-H7标记，稀释比例1:100；CD15、CD56均采用BV785标记，稀释比例1:100；CD19采用PECF594标记，稀释比例1:100；IgD采用FITC标记，稀释比例1:100；CD38采用PercP-Cy5.5标记，稀释比例1:100）及TotalSeqC®抗体（CD38、CD27、CD71、CD21、CD11c、CD39、FCRL5、CD95，稀释比例1:40，均购自BioLegend），于冰上孵育40分钟。活细胞通过LIVE/DEAD Fixable Aqua Dead Cell Stain Kit（Thermo Fisher Scientific，稀释比例1:200）进行鉴定，该试剂与偶联抗体同步孵育。
每名受试者均完成两次独立分选：一次为早期时间点（PO_M0），另一次为6个月时间点（PO_M6）。