GC reaction is impaired in NOTCH2 knock-in mice.

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GC reaction is impaired in NOTCH2 knock-in mice. GC reaction is impaired in NOTCH2 knock-in mice. A, Representative images of spleen sections from WT control and ICN2 knock-in mice stained for GC marker PNA 14 days after immunization with NP65-CGG. The black squares on left column (4×) highlight GCs, which are shown at 20× amplification in the right column. Scale bars, 100 μm (4×) and 20 μm (20×). B, Same experiment performed in panel A for BCL6+/B220+ staining. C, The number of GCs per spleen (y-axis) from immunohistochemistry for PNA+ clusters shown in A. D, The number of GCs per spleen (y-axis) from immunohistochemistry of BCL6+B220+ clusters shown in B. C and D, The range bars represent the mean values and SEM and the P values are shown on top. E, The surface area occupied by GCs in the spleens of immunized and induced ICN2 and control mice is shown for PNA staining and is represented by their area in μm2 (y-axis). F, The same as in panel E for BCL6/B220 staining. E and F, The average of the means for each group is shown. Each column corresponds to an individual mouse; each point is an individual GC; P values are shown on top. SEM and P values are shown. G, Average of GC area (μm2) of WT (black) and ICN2 (green) mice from IHC of paraffin-embedded spleen slides stained with PNA+ and BCL6+/B220+ antibodies. The mean values are 262 ± 36 versus 91 ± 10 μm2 for PNA+ GC, and 177 ± 30 versus 84 ± 13 μm2 for BCL6+/B220+. Data are shown for immunized CγCre-WT (n = 5) and CγCre-ICN2 (n = 8) mice. The statistical significance of this difference is shown based on unpaired two-tailed t test (P = 0.0015 and P = 0.0336, respectively). H, Flow-cytometry analysis of B220+GL7+CD95+ labeled splenic GC B-cell populations. Cells were gated for B220+, GL7 is on the y-axis and CD95(Fas) is on the x-axis. The percentage of double positive cells corresponds to GC B cells (black box). I, The percentage of B220+ gated GL7+/CD95+ GC B cells among total splenocytes is shown from the spleens of immunized WT (n = 5) and ICN2 induced (n = 8) mice. In the presence of ICN2, the abundance of B220+GL7+CD95+ GC B cells was reduced 3-fold as compared with WT animals (1.7% vs. 0.6% mean GC B cells vs. total splenocytes, P = 0.0003 unpaired two-tailed t test). See Supplementary Figs. S3 and S4 for additional data. Ester Valls et al. Cancer Discov 2017;7:506-521 ©2017 by American Association for Cancer Research