Anti cd19 microbeads

EBV-specific T cell responses were analyzed using an IFN-γ enzyme-linked immunospot (ELISpot) assay as previously described (10 (link), 54 (link)). Briefly, splenocytes were depleted of human CD19⁺ cells using anti-CD19 microbeads (Miltenyi Biotec). The CD19-depleted fraction was stimulated with autologous LCLs at a ratio of 1:4 for 18 h, and incubation with R10 and phorbol myristate acetate (PMA)-ionomycin served as negative and positive controls, respectively. Each condition was performed in duplicates. Spots were counted on an ELISpot reader system (ELR02; Autoimmun Diagnostika GmbH).

Single cell suspensions of murine spleens were prepared by dispersion through a 70 μm cell strainer (BD Biosciences, Breda, The Netherlands), and erythrocytes depleted by lysis. For analysis of splenic myeloid cell populations, spleens were digested for 1 hour at 37°C by incubation with collagenase D (2 mg/ml; Roche, Woerden, The Netherlands) and DNase I (2000 U/ml; Sigma-Aldrich) before dispersion. B cells were purified from spleens by using anti-CD19 MicroBeads (Miltenyi Biotec, Leiden, The Netherlands) following the manufacturer’s protocol. Purity was routinely ~95–98%. After a typical CD19 MACS sort, circa 83% of all contaminating cells were CD3⁺ T cells, 7% CD11b⁺CD11c⁺ cells, 2% CD11b⁺ CD11c^- cells, and 8% other cells. To determine cytokine secretion of splenic B cell subsets, CD19⁺ B cells were subsequently sorted by flow cytometry for follicular B cells (CD23⁺CD21^low) and marginal zone B cells (CD23^-CD21^hi) which resulted in purities of > 98%. CD4⁺ T cells were purified from spleens by negative selection and depleted of CD25-expressing cells using anti-CD25 MicroBeads (Miltenyi Biotec).

Single-cell suspensions from BM of 3× NP-CGG immunized female C57BL/6 mice were prepared and CD19⁺ cells were enriched by magnetic cell sorting using anti-CD19 microbeads (Miltenyi Biotech). Ex vivo IgG1⁺/IgG2b⁺CD19⁺CD38⁺GL7⁻CD138⁻IgM⁻IgD⁻ memory B cells were isolated by FACS (Influx cell sorter (BD Bioscience)) and applied to the 10× Genomics platform using the Single Cell 5′ Library & Gel Bead Kit (10× Genomics) following the manufacturer’s instructions. The amplified cDNA was used for simultaneous 5′ gene expression (GEX) and murine BCR library preparation. BCR transcripts were amplified by Chromium Single Cell V(D)J Enrichment Kit for murine B cells (10× Genomics). Upon adapter ligation and index PCR, the quality of the obtained cDNA library was assessed by Qubit quantification, Bioanalyzer fragment analysis (HS DNA Kit, Agilent) and KAPA library quantification qPCR (Roche). The sequencing was performed on a NextSeq500 device (Illumina) using either a High Output v2 Kit (150 cycles) with the recommended sequencing conditions (read1: 26nt, read2: 98nt, index1: 8 nt, index2: n.a.) or a Mid Output v2 Kit (300 cycles for BCR repertoire analysis (read1: 150 nt, read2: 150 nt, index1: 8 nt, index2: n.a., 20% PhiX spike-in).

Murine plasmacytoid BMDCs were generated from the bone marrow of tibiae and femora. Bone marrow cells were plated on bacterial petri-dishes overnight in culture medium (RPMI 1640, 10% heat-inactivated FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin (PAA Laboratories GmbH, Pasching, Austria) and 50 μM 2-ME (Invitrogen, Carlsbad, USA)) to remove adherent cells. Non-adherent cells were directly plated on 6 well plates at a density of 4.5x10⁶ cells/well and cultivated for 7–8 days in complete medium in the presence of FLT3 ligand (R&D Systems Europe, Ltd., Abingdon, United Kingdom) to mature the cells. The medium was additionally supplemented with sodium pyruvate 1%, NEA 1%, L-Glutamine 1% (PAA Laboratories GmbH, Pasching, Austria). FACS analysis demonstrated that the majority of cells obtained were CD45R/B220 high and CD11b low.
Human peripheral blood mononuclear cells (PBMCs) were prepared from blood of volunteers by Ficoll (Biochrom AG, Berlin, Germany) density gradient centrifugation. Human B-cells were positively selected via magnetic cell separation using anti-CD19 microbeads (Miltenyi Biotec). FACS analysis demonstrated that 95% of the cells obtained were CD20⁺ B-cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood of healthy volunteers by Ficoll gradient centrifugation, and B cells were purified from PBMCs by using anti-CD19 MicroBeads (Miltenyi Biotec, Leiden, The Netherlands) following the manufacturer’s protocol.