Figure 1.
Figure 1. NOTCH2 ligation heightens ex vivo active cGVHD B-cell responses to minimal BCR engagement by surrogate antigen. B cells were magnetically purified to >95% from viably frozen PBMCs from HCT patients who at the time of sample collection had active cGVHD (Active; n = 9) or no cGVHD (No; n = 6). After plating B cells onto OP9 stromal cell monolayers or OP9-DL1 cells that express the NOTCH ligand DLL1, cultures were either treated with the γ-secretase inhibitor DAPT (10 μM in DMSO) to block Notch activation, or with DMSO alone. After 30 minutes, agonistic anti-IgM Ab was added to the appropriate wells at a concentration of 0.625 μg/mL. The cells were cultured for 72 hours, harvested, and flow cytometry analysis performed to assess Ki-67 expression. (A) Representative flow cytometry histograms for Ki-67 expression are shown for healthy donor (Healthy), no cGVHD (No), or active cGVHD (Active) donor B cells cultured in the presence of 0.625 μg/mL anti-IgM and plated on OP9 parental cells (top panels) or OP9-DL1 cells (bottom panels). The effect of DAPT treatment on active cGVHD B cells is also shown (right panels). (B-C) Frequency of Ki-67+ B cells for all patients assessed in each group where the B cells were stimulated with 0.625 μg/mL anti-IgM (B) or were not stimulated through the BCR (C). P values were determined using a nonpaired Student t test for intergroup comparisons, and paired Student t test for same group comparisons. (D) Representative flow cytometry histograms showing BLNK expression as assessed by intracellular flow cytometry in B cells from active cGVHD patients stimulated as described for panel A, with plating on OP9 cells or OP9-DL1 cells. (E) Median fluorescence intensity (MFI) expression for BLNK in B cells from n = 4 active cGVHD patients stimulated as described for panel A and cultured on OP9-DL1 cells. In some cultures, DAPT (10 μM) was added to inhibit NOTCH activation, or R406 (0.1 μM) was added to inhibit SYK. DMSO alone used as the vehicle control in parallel. P values were determined using a paired Student t test.

NOTCH2 ligation heightens ex vivo active cGVHD B-cell responses to minimal BCR engagement by surrogate antigen. B cells were magnetically purified to >95% from viably frozen PBMCs from HCT patients who at the time of sample collection had active cGVHD (Active; n = 9) or no cGVHD (No; n = 6). After plating B cells onto OP9 stromal cell monolayers or OP9-DL1 cells that express the NOTCH ligand DLL1, cultures were either treated with the γ-secretase inhibitor DAPT (10 μM in DMSO) to block Notch activation, or with DMSO alone. After 30 minutes, agonistic anti-IgM Ab was added to the appropriate wells at a concentration of 0.625 μg/mL. The cells were cultured for 72 hours, harvested, and flow cytometry analysis performed to assess Ki-67 expression. (A) Representative flow cytometry histograms for Ki-67 expression are shown for healthy donor (Healthy), no cGVHD (No), or active cGVHD (Active) donor B cells cultured in the presence of 0.625 μg/mL anti-IgM and plated on OP9 parental cells (top panels) or OP9-DL1 cells (bottom panels). The effect of DAPT treatment on active cGVHD B cells is also shown (right panels). (B-C) Frequency of Ki-67+ B cells for all patients assessed in each group where the B cells were stimulated with 0.625 μg/mL anti-IgM (B) or were not stimulated through the BCR (C). P values were determined using a nonpaired Student t test for intergroup comparisons, and paired Student t test for same group comparisons. (D) Representative flow cytometry histograms showing BLNK expression as assessed by intracellular flow cytometry in B cells from active cGVHD patients stimulated as described for panel A, with plating on OP9 cells or OP9-DL1 cells. (E) Median fluorescence intensity (MFI) expression for BLNK in B cells from n = 4 active cGVHD patients stimulated as described for panel A and cultured on OP9-DL1 cells. In some cultures, DAPT (10 μM) was added to inhibit NOTCH activation, or R406 (0.1 μM) was added to inhibit SYK. DMSO alone used as the vehicle control in parallel. P values were determined using a paired Student t test.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal