Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

Here we describe tissue-resident memory B lymphocytes of spleen and bone marrow. Single cell transcriptomes and B cell receptor repertoires identify several exclusive populations of isotype-switched memory B cells (Bsm) in murine spleen and bone marrow, and one interconnected population of 10-20%. A population of marginal zone-like Bsm is located exclusively in the spleen while a novel population of quiescent Bsm is located exclusively in the bone marrow. Cells of two further populations, present in both, spleen and bone marrow, differ in repertoire between the two organs, i.e. are resident as well. Finally, another interconnected population of Bsm of the B1 lineage is present in spleen and bone marrow. In the bone marrow, all Bsm individually dock onto VCAM1+ stromal cells, resting in terms of activation, proliferation and mobility. The discrete B cell memory of bone marrow may be key to rapid secondary humoral responses to systemic antigens.


Statistics
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Software and code
Policy information about availability of computer code Data collection Raw sequencing data for single cell transcriptome analysis were processed using cellranger-2.1.1. mkfastq and count commands. Raw sequencing data for single cell immune profiling (B cell receptor sequences (BCR) for IgG heavy and light chain) were processed using cellranger-3.0.2. mkfastq and vdj commands. The high-confidence contig sequences of the isotype-switched memory B cells were reanalyzed using the HighV-QUEST database at IMGT web portal for immunoglobulin (IMGT 1.2.8 , database release LIGMDB_V12 ) to retrieve the V-, J-and D-genes as nucleotide and amino acid CDR3 sequence. Lineage trees were computed using GLaMST (w/o versioning). The single cell transcriptome data was further analyzed using R 3.5.0., and Seurat R package 2.3.4. Bulk Ig repertoire analysis was performed using MIGEC-1.2.4a.

Data analysis
Statistics and data analysis was performed in R (version 3.5). Additionally analyses were performed using Java. Modelling of random cell positioning, in a modification of the previously published approach (Zehentmeier et al, Eur J Immunol 2014.). Code will be made available on request.
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Sequencing data have been deposited at GEO (https://www.ncbi.nlm.nih.gov/geo/; superseries GSE140133). Cytometry and histological data will be made vailable by the authors upon reasonable request.

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Life sciences study design
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Sample size
Sample size was determined on the basis of expected effect size. In brief, t-tests with simulated data was performed for experiments for figures 1a-c . Data for sequencing was performed with minimal sample size of 3 (bulk receptor repertoires) and 2 (single cell RNA and Ig repertoire). Data collection for lab mice from different immunizations (Suppl. Fig1a) was performed by sample sharing of mice used for other experiments. Sample collection from wild mice was part of pest control measures and performed over the time period licenced, number of pet mice was chosen accordingly. No sample size calculations were performed prior to data collection.
Data exclusions No data were excluded during analysis.

Replication
Quantification of antigen-specific memory B cells of mice and for survival of memory B cells during Cyclophosphamide treatment were pooled from 2 independent experiments each. Quantification of switched memory B cells was obtained from mice of various different backgrounds to ascertain general validity of findings. Preferential co-lozalisation of cells in histology was tested against random distribution 1000 times. Difference in distribution of BCR clonotypes to spleen and bone marrow or to specific populations within those were tested against random distribution of observed sequences 1000 times. Distribution of clonally related BCRs to different subsets was tested against random distribution 1000 times. All attempts to replication of results was successful.
Randomization Study design did not necessitate randomization, internal controls were used as appropriate (i.e. comparing Bsm of different organs or different populations within the same animals, blocking antigen specific staining). To confirm non-random distribution of cells in situ or BCR clones, the statistical significance of observed vs simulated random distribution was determined.

Blinding
Blinding was not performed, since mostly paired samples of the same animals had to be compared due to experimental design.

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Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals C57BL/6J/N mice were purchased from Charles River (Sulzfeld, Germany) or Janvier Labs (Le Genest-Saint-Isle, France). Mice expressing GFP under the control of the Prdm1 promoter (Blimp1-GFP) 43 were bred at the DRFZ animal facility. C57BL/6 and Blimp1-GFP mice were housed under specific pathogen-free conditions with 12h light/dark cycle at 18-21°C with food and water ad libitum. Pet mice were obtained as adult animals at pet shops in Berlin.

Wild animals
Feral mice (male and female of uncertain age) were caught as free-living animals as part of a pest control measure at nonresidential farm buildings in Altlandsberg, Brandenburg, Germany. Live traps were placed and controlled by a trained veterinarian. Mice were anesthetized with isofluran prior to sacrifice and organ harvest.

Field-collected samples
The study did not involve laboratory experiments with field collected samples. Mice caught in the field were sacrificed on site and measurements were obtained on the day of sacrifice.

Ethics oversight
All animal experiments were performed according to institutional guidelines and licensed under German animal protection regulations by the Landesamt für Gesundheit und Soziales Berlin and Landesamt für Arbeitsschutz (pet and lab mice), Verbraucherschutz und Gesundheit Brandenburg (feral mice).
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Flow Cytometry
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