NOTCH2 is increased and inducible after in vivo exposure to high BAFF and alloantigen and associated with increased responsiveness to surrogate antigen and Notch ligand. (A) BCR activation assay comparing B cells from BAFF Tg BM only mice vs WT BM only recipients. Peripheral blood was collected at day 42 post-BMT, and B cells were stimulated with anti-IgM (10 µg/mL) for 4 days; frequency of 7-AAD−B220+Ki-67+ cells was analyzed by intracellular staining and flow cytometry; n = 6 (BM only) and n = 8 (BAFF Tg BM only). (B) Representative flow cytometric plot showing coexpression of the activation marker GL7 and NOTCH2 on a subset of B cells. Pregating was on 7-AAD−CD19+ cells. (C) Proportion of NOTCH2+GL7+ B cells in mice with cGVHD manifestations. Blood samples were obtained on day 36 after allo-BMT. GL7 and NOTCH2 expression was assessed by flow cytometry, and B cells were pregated on 7-AAD−CD19+ cells; n = 10 (BM only), n = 8 (BM + Sp), and n = 5 (Syn). (D) NOTCH2 expression by flow cytometric analysis of gated 7-AAD−CD19+ B cells after BCR stimulation. Blood samples were obtained on day 80 after allo-BMT. After lysis of red blood cells, the cells were stimulated with anti-IgM (1 or 5 µg/mL) overnight (for 18 hours). Expression of NOTCH2 was analyzed by flow cytometry, and median fluorescence intensity was used to indicate the level of NOTCH2 expression; n = 6 (WT BM only) and n = 8 (BAFF Tg BM only). (E-F) B-cell response to the NOTCH ligand Delta-like 1 (DL1) after exposure to soluble BAFF with or without BCR stimulation. Blood samples were obtained on day 105 after allo-BMT, red blood cells were lysed, and cells were added to a cell culture plate that had been seeded with OP9 cells lacking or expressing DL1 before culturing for 48 hours in the presence or absence of anti-IgM (10 μg/mL). Intracellular staining for Ki-67 was used to assess response to stimulation. Representative flow cytometric profiles are shown in panel E, with the results summarized in panel F. Numbers in panel E are frequencies of 7-AAD−B220+Ki-67+ cells; n = 6 (WT BM only) and n = 8 (BAFF Tg BM only). (G) Frequency of marginal zone (MZ) B cells as assessed by flow cytometry. Cells were pregated on Zombie−CD19+CD93− cells, with additional gating on CD21highCD23low cells to identify MZ B cells. Syn BAFF Tg transplant recipients only received BAFF Tg BM cells. Data were combined from 3 individual experiments. Splenocytes were analyzed at day 84 or 92 post-BMT; n = 10 (BM only), n = 8 (BM + Sp), n = 12 (BAFF Tg BM only), n = 9 (BAFF Tg BM + Sp), and n = 5 (Syn BAFF Tg). Statistical analysis was performed by unpaired Student t test with Welch’s correction (A), ordinary 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (C,G), or ordinary 2-way ANOVA (D,F) using GraphPad Prism 8 software. *P < .05, **P < .01, ***P < .001, ****P < .0001. NS, not significant (P > .05).