Disturbed canonical nuclear factor of κ light chain signaling in B cells of patients with common variable immunodeficiency Baerbel Keller, PhD, Zoltan.

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Presentation transcript:

Disturbed canonical nuclear factor of κ light chain signaling in B cells of patients with common variable immunodeficiency Baerbel Keller, PhD, Zoltan Cseresnyes, PhD, Ina Stumpf, Claudia Wehr, MD, Manfred Fliegauf, PhD, Alla Bulashevska, PhD, Susanne Usadel, MD, Bodo Grimbacher, MD, Marta Rizzi, MD, Hermann Eibel, PhD, Raluca Niesner, PhD, Klaus Warnatz, MD Journal of Allergy and Clinical Immunology Volume 139, Issue 1, Pages 220-231.e8 (January 2017) DOI: 10.1016/j.jaci.2016.04.043 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 BCR-mediated IκBα degradation in B cells from patients with CVID. A, IκBα expression in B cells from representative HDs and CVID 21norm and 21low patients. B, Basal MFI of IκBα. C, MFI of IκBα with and without stimulation. D, Ratio of the MFI of IκBα after and without stimulation. E, Kinetics of IκBα degradation. P values (*P < .05, **P < .01, and ***P < .001) derive from comparison of the 3 B-cell populations at each time point by using ANOVA. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 BCR-mediated activation of p65. A, Representative fluorescence-activated cell-sorting plots and statistical analysis of the MFI p65(pS529) ratio. B, Representative pictures and distribution of the percentage of p65 (yellow) nuclear translocation in CD21+ B cells of HDs (n = 6) and CD21+ (red) or CD21low B cells of CVID 21low patients (n = 6), as described in detail in this article's Online Repository. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Phospho-IκBα levels in patients' B cells. A, Statistics of the MFI of phospho-IκBα in unstimulated B-cell subpopulations. B, Ratio of the MFI of phospho-IκBα after treatment with anti-IgM, MG132 and anti-IgM + MG132 to the MFI of untreated cells in B cells of HD CVID 21low patients. *P < .05, **P < .01 and ****P < .0001. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 CD40-induced NF-κB activation. A, Fluorescence-activated cell-sorting plots and statistics of the MFI of IκBα and its ratio after stimulation. B, MFI of IκBα after stimulation with decreasing amounts of CD40L in 5 HDs and 6 CVID 21low patients. C, Representative pictures of p65 nuclear translocation (yellow) with CD21 costaining (red) and distribution of the percentage of p65 in HDs (n = 6) and CVID 21low patients (n = 6). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 TLR9-mediated IκBα degradation. Statistics of the MFI of IκBα without and after CpG stimulation and the ratio thereof. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 NF-κB target gene expression and survival in B cells of patients with CVID. A and B, Representative fluorescence-activated cell-sorting plots and statistics of the ratio of the MFI of Bcl-xL (Fig 6, A) and IκBα (Fig 6, B). C, Percentage of Annexin V−/4′-6-diamidino-2-phenylindole (DAPI)− B cells. D, Ratio of the MFI of CD69, ICAM-1, and CD25 with and without anti-IgM stimulation. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 NF-κB signaling in HIV-derived CD21low B cells. A, Ratio of IκBα degradation in CD27−/IgD+ CD21+ or CD21low B cells after BCR stimulation in 6 HDs and 6 patients with HIV. B, IκBα degradation after stimulation with decreasing amounts of CD40L in 4 HDs and 5 patients with HIV. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Automatic algorithm for the segmentation and classification of individual CD21+ and CD21low B cells, respectively. A, DIC images were segmented after shadow casting them from 8 different angles. The objects were filtered by size to exclude cellular debris. The average size of a human B cell is known to be 80 μm2. We set the condition that objects of less than 20 μm2 are debris. These objects have been excluded from further analysis. B, By using an intensity-thresholding algorithm, fluorescence images of the nuclear staining was segmented, and then the objects were filtered by size to exclude debris and other artifacts: only nuclei with a size of between 15 and 100 μm2 were taken into account. Cell clusters from DIC images of a size of greater than 180 μm2 were separated according to the number of contained cell nuclei. C, Results of object segmentation from the DIC images and Hoechst fluorescence images were merged together to further analyze only the objects having plausible B-cell size and only 1 nucleus of plausible size. Each cell received a cell ID for further tracking. D, By using simultaneous IgM fluorescence staining, the identified cells were verified to be B cells, whereas non–B-cell contaminants were excluded from further analysis. Non–B-cell contaminants represented 4.4% ± 1.3% of all imaged cells in patients with CVID and 6.7% ± 1.5% in HDs. Internal intensity references (ie, maximum fluorescence intensity, as well as background and background noise within an image) were used to counteract possible artifacts induced by means of variable acquisition and/or staining conditions typical for confocal microscopy. E, Only the IgM+ cells were evaluated as far as their CD21 signal is concerned. As previously described, the maximum fluorescence intensity, as well as background and background noise of CD21 staining within an image, were used as reference, to exclude artifacts caused by variable fluorescence signal between images. Only a signal greater than maximal intensity (Imax)/2 was considered to indicate CD21+ cells (corresponding to 2× the background noise); at less than this value, IgM+ cells were considered to be CD21low cells. For HDs, we could find only CD21+ IgM+ cells, whereas for the patients with CVID, values varied between 20% and 40%. Thus our results are in accordance with results of previous studies. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Automatic approach to evaluate the frequency of p65 nuclear distribution from confocal fluorescence images. A, Raw p65 fluorescence images were multiplied either by the mask of the whole-cell areas (from segmented DIC images) or by the nuclear mask (from segmented Hoechst images) to generate 2 images: 1 identifying the total p65 signal in the whole cell body and 1 identifying p65 signal only within the nucleus of each segmented B cell (IgM+). The 2 images are merged in 1 exemplary image of B cells of an HD after IgM treatment over 2 hours. The ratio between the 2 values for each cell defines the contribution of nuclear p65 to total p65 within a cell and thus is an indicator of p65 nuclear translocation. B, All these ratio values were summarized for each condition into a table that allows us to retrieve the source and characteristics of each cell (CD21+ or CD21low, IgM+ or IgM−) by using the cellular ID. C, From each table, we generated a graph showing the frequency of the nuclear p65 ratio values per condition. The background was not excluded, and we assumed it can vary strongly between different experiments. However, the bimodal nature of the distribution indicating cells not showing any p65 nuclear translocation and cells with p65 nuclear translocation is not influenced by any experimental parameter. Thus we approximated all distributions using 2-peak Gaussian functions to achieve minimum reduced c2 values (best approximation of the experimental data) to determine the percentage of B cells showing nuclear p65 translocation of the total number of B cells per condition. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Impaired Ca2+ mobilization but intact phosphorylation of PLCγ2 in CD21+ and CD21low B cells of patients with CVID. A, Representative fluorescence-activated cell-sorting plot of anti-IgM–induced Ca2+ mobilization in CD21+ B cells of an HD and CD21+ and CD21low B cells of a patient with CVID. B, Phosphorylation of PLCγ2 without and after stimulation with anti-IgM. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 PMA-induced NF-κB activation is not affected in patients with CVID. A, Representative fluorescence-activated cell sorting plots and statistical analysis of the MFI of IκBα after PMA stimulation in B cells of HDs (n = 11) and CVID 21low patients (n = 9). B, Degradation of IκBα after PMA stimulation in naive B cells of a CARD11-deficient patient and 1 HD. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Reduced upregulation of B-cell activation markers in patients with CVID. Fluorescence-activated cell-sorting plots for CD69, ICAM-1, and CD25 expression without and after stimulation with anti-IgM in B-cell subpopulations of 1 representative HD and 1 CVID 21low patient. Journal of Allergy and Clinical Immunology 2017 139, 220-231.e8DOI: (10.1016/j.jaci.2016.04.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions