Fig. 3: CEACAM1 is required for BCR signaling.

a–e Indicated cell lines were transduced with control or CEACAM1 shRNA, followed by stimulation with anti-IgM (1 μg/mL). a Shown are normalized Ca²⁺ signals. ****P < 0.0001 by two-way ANOVA. Data were representative of at least three independent experiments. b, c Immunoblots show effects of CEACAM1 knockdown on BCR signaling components. Data were representative of three independent experiments. d, e Immunoblots show CEACAM1 knockdown (top panels) and its effect on Ca²⁺ signals (bottom panels). f Top panel, immunoblots of splenocytes from wild type (+/+) or Ceacam1-deficient (−/−) mouse with indicated antibodies. Bottom panel, Ca²⁺ signals of indicated splenic B220⁺ B cells. Data from (d–f) are representative of at least two independent experiments. g CEACAM1 mRNA expression levels in CD19+ sorted MCL cells from peripheral blood (PB) or lymph nodes (LN) analyzed from the GSE70910 dataset⁴⁴. Closed circles or squares represent individual patient samples. Horizontal bars from each group indicate mean mRNA expression. P value is from a two-sided unpaired t-test with Welch’s correction. h CEACAM1 expression is correlated with ibrutinib response. Left panels, FACS plots showing surface CEACAM1 expression on indicated MCL and MZL cell lines. Isotype, negative isotype antibody on Z-138 cells. Right panels, Indicated cell lines were treated with indicated doses of ibrutinib for 4 days, and viable propidium iodide (PI)-negative cells were assessed by flow cytometry. Line graphs showing means of normalized PI-negative fractions from three independent experiments. Error bars, SD. Fifty-percent inhibition concentration (IC₅₀) values were calculated by GraphPad Prism v8. i, j ITIM tyrosine residues are required for BCR signaling. JEKO-1 cells transduced with control (gNTC) or CEACAM1 gRNA (gCC1), followed by reintroducing WT CEACAM1 (4 L), CC1-4L-Y493F/Y520F mutant (YY/FF), or short cytoplasmic tail (4S). Controls or reconstituted cell lines were verified for CEACAM1 expression by FACS using B1.1 antibody (i) and stimulated with 1 μg/mL of anti-IgM for 5 min, followed by immunoblotting with indicated antibodies, including the CEACAM1 E1 antibody (j). Data from (i, j) are representative of at least two independent experiments. Source data are provided as a Source Data file.