CD4+ Tregs (CD4+CD25+) and CD8+ Tregs (CD8+CD25+CD103+) from Cd19creRosaIL-2 and littermate mice and Tconv cells (Thy1.1−CD44loCD62Lhi) from Foxp3Thy1.1 mice were isolated from spleens and LN by negative selection with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher Scientific) followed by cell sorting (BD FACSAria III). Antigen-presenting cells were sourced by digesting Rag−/− spleens. For the suppression assay, 1 × 105 Tregs were plated per well in complete RPMI along with 5 × 104Rag−/− splenocytes preincubated with 1 µg/ml anti-CD3 (Thermo Fisher Scientific). CD4+ and CD8+ Tconv cells (responders) were labeled with CellTrace Violet (Thermo Fisher Scientific), plated at the indicated ratios with Tregs, and incubated for 3 d at 37°C. Suppression was calculated by comparing the proliferative index of the sample with the proliferative index of Tconv cells cultured in identical conditions except without cocultured Tregs.
Magnisort streptavidin negative selection beads
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The MagniSort Streptavidin Negative Selection Beads are superparamagnetic beads coated with streptavidin. They are designed for the negative selection of biotinylated cells or molecules from a heterogeneous sample.
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Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Flt3l, by Thermo Fisher Scientific (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (2 mentions)
Tcrb bio, by BioLegend (2 mentions)
Cd11b bio, by BioLegend (2 mentions)
Ter119 bio, by BioLegend (2 mentions)
Ril 4, by Thermo Fisher Scientific (2 mentions)
Ril 21, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Hepes, by Lonza (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced sybr green reaction mix, by Bio-Rad (1 mentions)
Cfx manager software 2, by Bio-Rad (1 mentions)
Facsmelody cell sorter, by BD (1 mentions)
22 protocols using magnisort streptavidin negative selection beads
1
Treg Suppression Assay in Mice
2
Induction of Regulatory T Cells by Kynurenine-Treated Dendritic Cells
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LPS-unprimed and LPS-primed cDCs treated or not with L -kynurenine, as described above, were cultured with CD4+ T cells isolated from the spleens of Foxp3YFP mice. Briefly, CD4+ T cells were isolated via cell separation by two steps. A first step was meant to enrich the CD3+ cell fraction, using biotin mouse monoclonal antibodies against B220 (a marker of pDCs and B cells) and CD11c (a marker of DCs). After this step, cells were incubated with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher) followed by depletion of B220+CD11c+. B220– CD11c– collected cell fractions were incubated with magnetic anti-mouse CD4 beads (Miltenyi Biotec) to select CD4+ T cells. The purity of CD4+ T cells was verified by FACS analysis by cell staining with anti-B220, anti-CD3, anti-CD11c, anti-CD4, and anti-CD8 specific antibodies. CD4+ T cells (2 × 105) were activated with 5 μg/ml anti-CD3 mAb (clone OKT3) and co-cultured for 4 days with unprimed and primed cDCs (5 × 104) treated or not with L -kynurenine for 36 h (19 (link)). Treg cells were evaluated by FACS, analyzing the induction of Foxp3 expressing YFP.
3
Bone Marrow Cell Isolation and FACS Analysis
4
Isolating Bone Marrow Dendritic Cell Progenitors
5
Isolation and Purification of Bone Marrow Progenitors
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Bone marrow (BM) progenitors were isolated as described (Bagadia et al. 2019 (link)) and depleted of CD3-, CD19-, CD105-, TER-119-, Ly-6G-, and B220-expressing cells by staining with the corresponding biotinylated antibodies, followed by depletion with MagniSort streptavidin-negative selection beads (Thermo Fisher). The remaining lineage– BM cells were then stained with fluorescent antibodies before sorting. CD117hi BM progenitors were identified as lineage–CD117hi cells, CDPs were lineage– Siglec-H−CD117intCD135+CD115+MHCII−CD11c− BM cells, pre-cDC1s were lineage–Siglec-H−CD117intCD135+MHCIIint-negCD11c+CD24+ BM cells, and pre-cDC2s were lineage–Siglec-H−CD117−CD135+CD115+MHCII−CD11c+ BM cells.
Cells were sorted into Iscove's modified Dulbecco's medium supplemented with 10% FBS, 1% penicillin streptomycin solution, 1% sodium pyruvate, 1% MEM nonessential amino acid, 1% L-glutamine solution, and 55 µM β-mercaptoethanol (complete IMDM).
Cells were sorted into Iscove's modified Dulbecco's medium supplemented with 10% FBS, 1% penicillin streptomycin solution, 1% sodium pyruvate, 1% MEM nonessential amino acid, 1% L-glutamine solution, and 55 µM β-mercaptoethanol (complete IMDM).
6
Isolation and Quantification of Murine Basophils
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Single-cell suspensions of bone marrow (BM) cells (in PBS containing 2% FCS and 2 mM of EDTA) were stained with biotin-conjugated anti-Ly6G (1A8) and anti-CD19 (6D5) antibodies (BioLegend, San Diego, CA, USA) before removing positive cells with MagniSort™ streptavidin-negative selection beads following the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). After this basophil enrichment, primary BM basophils were selected for CD200R3 expression and sorted using a BD FACSMelody™ cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). RNA extraction was performed by using Trizol reagent as described in the manufacturer’s protocol (Invitrogen). cDNA was synthesized with SuperScript™ III First-Strand Synthesis System (Invitrogen). Quantitative PCR was performed with SsoAdvanced SYBR green reaction mix (Bio-Rad, Hercules, CA, USA) using the M_B2m_1 KiCqStart™ primer pair for mouse β2-microglobulin as housekeeping gene (Sigma-Aldrich, Merck) and the following primers for Mcpt8 quantification: Forward primer: 5′-GTGGGAAATCCCAGTGAGAA-3′ and Reverse primer: 5′-TCCGAATCCAAGGCATAAAG-3′ (12 (link)). Quantitative PCR was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and, following amplification, Ct values were obtained using the CFX Manager™ software 2.1 (Bio-Rad, Hercules, CA, USA).
7
Basophil Enrichment from Mouse Splenocytes
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Mouse splenocytes were harvested as described above and resuspended in PBS containing 2% FCS and 2 mM of EDTA. Splenocytes were then depleted from B, NK, and T cells by magnetic selection through the use of biotinylated anti-CD19 (6D5), anti-NK1.1 (PK136), anti-CD8α (53-6.7), anti-CD4 (RM4-5) (BioLegend) and MagniSort™ streptavidin-negative selection beads, following manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). This basophil-enriched cell suspension was then stained with a FITC-conjugated anti-IgE antibody (23G3, Southern Biotech, Birmingham, AL, USA) in FACS buffer, washed, incubated on poly-l -lysine-coated coverslips for 20 min at 37°C, washed in PBS, fixed with a fixation buffer (BioLegend), and permeabilized with PZB. The nuclei were stained with 360 nM of 4′,6-diamidino-2-phenylindole (DAPI) in PZB and washed in PBS. Coverslips were then mounted in Shandon Immu-Mount (ThermoFisher Scientific, Waltham, MA, USA) on superfrost slides (VWR, Radnor, PA, USA) and incubated overnight at 4°C. Image acquisition was realized with a Zeiss, Oberkochen, Germany LSM 780 confocal microscope, and image analysis was done with the Zen 2012 (Blue edition) service pack 2 software.
8
Isolation and Sorting of Dendritic Cell Progenitors
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For sorting experiments, BM was isolated as described above and depleted of CD3-, CD19-, CD105-, Ter119-, and Ly6G-expressing cells by staining with the corresponding biotinylated antibodies followed by depletion with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher). All remaining BM cells were then stained with fluorescent antibodies prior to sorting. Gates used to define MDPs, CDPs and pre-cDC1s were a combination of previously established markers21 (link) and those identified in this study. MDPs were identified as Lin−CD117hiCD135+CD115+CD11c−MHCII−. CDPs were identified as Lin−CD117intCD135+CD115+CD11c−MHCII−. Pre-cDC1s were identified as either Lin−CD117intCD135+Zbtb46-GFP+ or as Lin−CD117intCD135+CD226+. Pre-cDC2s were identified as Lin−CD117loCD135+CD115+. Lineage markers included CD3, CD19, CD105, CD127, NK1.1, Ter119, and Ly6G. For retroviral reporter assays and in vitro CRISPR/Cas9 deletion CD117hi cells were sorted. A FACSAria Fusion was used for sorting and cells were sorted into cIMDM. Sort purity of >95% was confirmed by post-sort analysis before cells were used for further experiments. For culture experiments, DC progenitors were cultured at 37⁰C in 200 µL total volume of cIMDM supplemented with 100 ng/mL Flt3L (Peprotech) for five days before further analysis.
9
Splenic Follicular B Cell Isolation
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Splenic B cells were enriched by magnetic depletion, removing non-B cells using the following antibodies coupled to biotin: CD11b (M1/70), CD11c (N418), CD4 (GK1.5), CD5 (53-7.3), CD8α (53-6.7), Gr1 (RB6-8C5), NK1.1 (PK136) as well as Ter119 (TER-119) (Biolegend), and the MagniSort Streptavidin Negative Selection Beads according to the manufacturer’s instructions (ThermoFisher). Alternatively, splenic CD19+B220+CD93–IgM+CD1d+ Fo B cells were FACS-isolated on a FACSAriaIII (BD Biosciences).
10
Pre-B Cell Isolation and RNA-Seq Analysis
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Pre-B cells were isolated from the bone marrow of recipient mice that had been transplanted 3 weeks earlier with sgTfap4/Eµ-MYC/Cas9 or sgControl/Eµ-MYC/Cas9 FLCs. B cells were enriched from bone marrow of all long bones by staining with a cocktail of biotinylated antibodies against TER119 (Ly76), MAC-1 (M1/70), GR-1 (RB6-8C5) for 20 min on ice, washed, then incubated with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher Scientific) according to the manufacturer’s protocol to remove undesired erythroid and myeloid cells. The supernatant was transferred into a fresh tube, washed, and resuspended in 100 µl of a cocktail of fluorochrome-conjugated antibodies against B220 (RA3-6B2), IgM (5.1), c-KIT (2B8) and 1 × 106 live (PI negative) pre-leukaemic pre-B cells (B220+ sIgM− c-KIT−) were FACS sorted, centrifuged, and resuspended in QIAzol Lysis Reagent (Qiagen) and stored at −80 °C. Total RNA was extracted using the QIAgen miRNeasy Mini Kit according to the manufacturer’s instructions with optional on-column DNAase digestion. An input of 100 ng of total RNA was used to prepare mRNA libraries and indexed using the TruSeq RNA samples Prep Kit (illumina) according to the manufacturer’s protocol. The samples were sequenced on an Illumina NextSeq using paired-end sequencing. RNA-sequencing analysis are detailed in the Supplementary Material .
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