Extended Data Figure 2: Ifitm3 is essential for the development of B1 and germinal center B-cells.

a, Hardy fractions of B-cell subsets isolated from bone marrow of Ifitm3^+/+ and Ifitm3ˉ^/ˉ littermates analyzed by flow cytometry (n=3). b, Surface expression of IgM, CD20, CD19, IgD, CD2 and CD21 measured by flow cytometry in enriched bone marrow (Gr-1⁻, Nk1.1⁻ and B220⁺) and splenic B-cells (CD3⁻ and B220⁺) from Ifitm3^+/+ or Ifitm3ˉ^/ˉ mice (n=7; mean±s.d.). Mean fluorescence intensities (MFI) values for individual measurement compared by two-tailed t-test. c, Ca^2+-mobilization from cytoplasmic stores in response to BCR (IgM)-engagement was measured in Ifitm3^+/+ and Ifitm3ˉ^/ˉ splenic B cells. Ca²⁺ release was induced by addition of 10 μg ml⁻¹ anti-mouse IgM 60 seconds after acquisition of background fluorescence. Ca²⁺ release was measured over 300 seconds with cell permeant Rhod-2 dye; MFI compared between replicates (n=3). d, Ca²⁺ mobilization in response to BCR-engagement measured upon CRISPR-Cas9-mediated deletion of IFITM3 in Jeko1 mantle cell lymphoma (MCL) cells. Ca²⁺-release upon addition of 10 μg ml⁻¹ of polyclonal F(ab’)₂ anti-human IgM was measured for 300 seconds with cell permeant Fluo-4 dye; MFI compared between replicates (left; n=3). Surface expression of CD19 following deletion of IFITM3 in Jeko1 MCL cells, MFIs for CD19 indicated (right; n=3). e, Jeko1 MCL cells were electroporated with non-targeting RNP (Cas9-gRNA ribonucleoproteins, gNT) or IFITM3-targeting RNP complex (gIFITM3). Following electroporation, MCL cells were treated with vehicle (DMSO) or 25 nmol l⁻¹ of Dasatinib for 3 hours. Cells were stimulated with 10 μg ml⁻¹ of anti-human IgM F(ab’)2 for indicated timepoints and subjected to co-immunoprecipitation with an anti-CD19 antibody. Immunoblots were performed to measure levels of CD19-tyrosine phosphorylation, and binding of LYN to CD19. Levels of IFITM3, Src-pY⁴¹⁶ and Lyn were assessed in whole cell lysates (10% input) with β-actin as loading control. (n=3; gel source data Supplementary Fig. 1). f-h, Relative fractions (left) and absolute cell counts (right) of total B-1 (f), B-1a (g) cells in the peritoneal cavity and marginal zone B cells (h) in spleen of Ifitm3^+/+ and Ifitm3^−/− littermates (n=5) are shown (means ± s.d.; two-tailed t-test).