Mb19 1

For in vivo differentiation experiments, CHILPs were highly purified by magnetic beads enrichment followed by FACS sorting. Briefly, single-cell suspension of leukocytes obtained from bones was stained with biotinylated antibodies against CD11b (M1/70, eBioscience), CD19 (MB19-1, Biolegend), TER-119 (TER-119, eBioscience). Stained cells were passed through LD depletion column (Miltenyi Biotec) according to manufacturer’s instructions. The enriched Lineage negative fraction was stained with fluorescently labeled antibodies (as described above) and then sorted using a BD FACSAria I or BD FACSAriaIII cell sorter (BD Biosciences). Highly purified CLP (Lin^neg CD127⁺ Id2⁻ CD135⁺) or CHILPs (Lin^neg CD127⁺ Id2⁺ CD135⁻ α4β7⁺ CD25⁻) from PBS or B16-Flt3L injected Id2^Gfp/+ donors were injected intravenously into Rag2^−/−Il2rg^−/− recipient mice. Each recipient mouse received between 150 to 2000 cells. Five to ten weeks after transfer, recipient mice were sacrificed and donor-derived cells were analyzed in the indicated organs. Engraftment index was calculated by dividing the number of ILCs retrieved in the host by the number of progenitors cells injected in the same mouse.

Flow cytometry analysis was performed according to the methods described previously [28 (link)]. Briefly, the cells isolated from the MLN (the MLN cells) were incubated with an Fcγ receptor-blocking mAb (CD16/32; 2.4G2, BD Biosciences) for 15 minutes at 4°C. For surface antigen detection, the cells were labeled with monoclonal antibodies against Gr-1 (RB6-8C5, BD Biosciences), F4/80 (BM8, BD Biosciences), αβTCR (H57-597, BD BioLegend), γδTCR (GL3, BioLegend), NK1.1 (PK136, BioLegend), CD4 (RM4-5, BD Biosciences), CD44 (IM7, BD Biosciences), CD25 (PC61, BioLegend), B220 (RA3-6B2, BioLegend), and CD19 (MB19-1, BioLegend) for 30 min at 4°C.
For intracellular cytokine staining, the cells isolated from the MLN (the MLN cells) were stimulated with ionomycin (1 mg/mL; Sigma-Aldrich) and PMA (25 ng/mL; Sigma-Aldrich) for 5 h at 37°C, with brefeldin A (10 mg/mL; Sigma-Aldrich) added after 1 h. Then, the cells were fixed and permeabilized with fixation/permeabilization working solution for 20 min at 4°C followed by incubation with monoclonal antibodies against IFN-γ (XMG1.2, BD Biosciences), IL-17A (TC11-18H10.1, BD Biosciences), and IL-10 (JES5-16E3, BD Biosciences). The cells were analyzed using a Cytofix/Cytoperm Plus Kit.

Tumors were analyzed by flow cytometry using the following cell surface markers for (a) B cells: RA3-6B2, anti-B220; R2/60, anti-CD43; II/41, anti-IgM; 11/26C, anti-IgD; MB19-1, anti-CD19; 53-7.3, anti-CD5 and B3B4, anti-CD23; 7E9, anti-CD21 (BioLegend, Fell, Germany); and (b) T cells: GK1.5, anti-CD4; H57-597, anti-TCRb (all from eBioscience, Vienna, Austria) and 53-6.7, anti-CD8; (from BD Pharmingen, San Diego, CA, USA). Biotinylated antibodies were monitored with streptavidin-RPE (DAKO, Vienna, Austria) or streptavidin-PE-Cy7 (BD Phamingen). Using a FACS^Vantage cell sorter (Becton Dickinson, Heidelberg, Germany) premalign pre/pro-B cells (B220⁺/IgM⁻) or immature (IgM⁺/IgD^low) and mature (IgM⁺/IgD^high) B cells were isolated and sorted from bone marrow and spleen, respectively. Flow cytometry data were analyzed using Cyflogic free ware and FlowJo (Ashland, OR, USA).