Figure 5.
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).

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).

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