Molecular mechanisms of functional natural killer deficiency in patients with partial DiGeorge syndrome Peilin Zheng, PhD, Lenora M. Noroski, MD, Imelda.

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Presentation transcript:

Molecular mechanisms of functional natural killer deficiency in patients with partial DiGeorge syndrome Peilin Zheng, PhD, Lenora M. Noroski, MD, Imelda C. Hanson, MD, Yuhui Chen, PhD, Michelle E. Lee, BSc, Yu Huang, MSc, Michael X. Zhu, PhD, Pinaki P. Banerjee, PhD, George Makedonas, PhD, Jordan S. Orange, MD, PhD, William T. Shearer, MD, PhD, Dongfang Liu, PhD Journal of Allergy and Clinical Immunology Volume 135, Issue 5, Pages 1293-1302 (May 2015) DOI: 10.1016/j.jaci.2015.01.011 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 CrkL silencing disrupts both lytic granule polarization to the IS and pVav-1 accumulation. A, Confocal microscopy of WT, CrkL short hairpin RNA (shRNA) knockdown (KD), and scrambled shRNA (Control) lentivirus-transduced CD16-KHYG-1 cells in conjugate with K562 cells. Cells were stained with antibodies to visualize perforin (green), F-actin (blue), β-tubulin (red), and pVav-1 (cyan). B-D, Quantification of the distance between the MTOC and the IS (Fig 1, B) and total intensity of pVav-1 (Fig 1, C) and F-actin (Fig 1, D) at the IS. E, Confocal images of IL-2–activated primary NK cells transfected with either CrkL small interfering RNA (KD) or control small interfering RNA (Control) in conjugate with K562 cells. Cells were stained with antibodies to visualize CrkL (blue), perforin (green), β-tubulin (red), and pVav-1 (cyan). F and G, Quantitative analysis of the distance between the MTOC and the IS (Fig 1, F) and total intensity of pVav-1 at the IS (Fig 1, G) in small interfering RNA–treated NK cells stratified by CrkL MFI values. Bright-field images are shown on the left. Error bars represent SDs. Scale bars = 3.0 μm. Data are from 3 independent experiments. NS, Not significant. Journal of Allergy and Clinical Immunology 2015 135, 1293-1302DOI: (10.1016/j.jaci.2015.01.011) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 CrkL silencing inhibits activating receptor CD16 and integrin clustering. A, Confocal images of WT, CrkL short hairpin (shRNA; KD), and scrambled shRNA (Control) CD16-KHYG-1 cells activated on lipid bilayers carrying anti-CD16–Alexa Fluor 568 (red) and ICAM-1–Alexa Fluor 647 (blue). Fixed and permeabilized NK cells were stained for perforin, followed by Alexa Fluor 488–conjugated secondary antibody (green) to visualize lytic granules. B, Quantification of the total intensity of polarized perforin at the lipid bilayer focal plane. C-E, Quantification of the size (Fig 2, C), total intensity (Fig 2, D), and number (Fig 2, E) of anti-CD16 microclusters on lipid bilayers carrying ICAM-1 and anti-CD16. F-H, Quantification of the size (Fig 2, F), total intensity (Fig 2, G), and number (Fig 2, H) of ICAM-1 microclusters on lipid bilayers. Error bars represent SDs. Scale bars = 3.0 μm. Data represent 2 to 6 independent experiments. Journal of Allergy and Clinical Immunology 2015 135, 1293-1302DOI: (10.1016/j.jaci.2015.01.011) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Decreased cytotoxicity of NK cells from patients with pDGS. A, The natural cytotoxicity of human NK cells from 5 patients with pDGS and 3 healthy control subjects was measured with 51Cr-labeled K562 cells at 37°C for 4 hours. B, ADCC was measured with 51Cr-labeled Raji cells in the presence of rituximab. Error bars represent SDs. Journal of Allergy and Clinical Immunology 2015 135, 1293-1302DOI: (10.1016/j.jaci.2015.01.011) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Defects in lytic granule polarization, activating receptor clustering, and signaling in NK cells from patients with pDGS. A, Confocal images of NK cells from healthy control subjects (Control) and patients with pDGS (pDGS) in conjugate with K562 cells. Cells were stained with antibodies against perforin (green), phalloidin (blue), and β-tubulin (red). B, Quantification of the distance between the MTOC and the IS in primary NK cells. Error bars represent SDs. Data are from 3 independent experiments. C, Confocal images of NK cells from control subjects and patients with pDGS stimulated on bilayers carrying anti-CD16–Alexa Fluor 568 (red) and ICAM-1–Alexa Fluor 647 (blue). Cells were stained with anti-perforin–Alexa Fluor 488 (green) and anti–pVav-1–Alexa Fluor 405 (cyan). Data represent 4 independent experiments. D-G, Quantification of total fluorescent intensity of anti-CD16 (Fig 4, D), ICAM-1 (Fig 4, E), perforin (Fig 4, F), and pVav-1 (Fig 4, G) on bilayers carrying ICAM-1 and anti-CD16. Journal of Allergy and Clinical Immunology 2015 135, 1293-1302DOI: (10.1016/j.jaci.2015.01.011) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Defective integrin signaling in NK cells from patients with pDGS. A, Flow cytometric analysis of activated LFA-1 (mAb 24+) on primary NK cells with or without K562 stimulation at 5 minutes. B, Kinetics of activated LFA-1 (mAb 24+) at different time points. C, Percentage of intermediate LFA-1 (KIM127+) on primary NK cells with or without K562 stimulation at 5 minutes. D, Percentage (left) and MFI (right) of activated (mAb 24+) LFA-1 after mixing with K562 target cells for 5 minutes. Data are pooled from 5 control subjects and 6 patients with pDGS. E, Percentage (right) and MFI (right) of intermediate (KIM127+) LFA-1 after mixing with K562 target cells for 5 minutes. Data are pooled from 5 control subjects and 4 patients with pDGS. Journal of Allergy and Clinical Immunology 2015 135, 1293-1302DOI: (10.1016/j.jaci.2015.01.011) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Reconstitution of CrkL restores NK cell function in patients with pDGS. PBMCs from 7 patients with pDGS were transduced with GFP (Control virus) or CrkL-GFP (CrkL virus) lentivirus. A, MFI of perforin and granzyme B in GFP+ NK cells in response to K562 stimulation. B, Percentage of IFN-γ+ and TNF-α+ transduced NK cells in response to phorbol 12-myristate 13-acetate and ionomycin stimulation. C and D, Quantification of CD107a total intensity on the plasma membrane. Transduced NK cells were activated on bilayers carrying anti-CD16–Alexa Fluor 568 and unlabeled ICAM-1 (Fig 6, C) or anti-NKG2D–Alexa Fluor 568 in the presence of unlabeled ICAM-1 and anti-2B4 (Fig 6, D). Data were pooled from 2 independent experiments. P values are for the paired t test (Fig 6, A and B) or the Student t test (Fig 6, C and D). Journal of Allergy and Clinical Immunology 2015 135, 1293-1302DOI: (10.1016/j.jaci.2015.01.011) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 A new model for the adaptor protein CrkL in controlling NK cell activation. A, NK cell activation is triggered by activating receptor (blue bars) signaling. Activating receptors accumulate toward the center of the IS. The adaptor protein CrkL links cytoskeletal scaffold proteins to guanine nucleotide exchange factor Vav-1. Tyrosine phosphorylated Vav1 (p-Vav1) is a central regulator of T-cell and NK cell IS. pVav-1 promotes polarization of MTOC and lytic granules to the IS, as well as activating receptor clustering by promoting F-actin reorganization. Integrin LFA-1 is in open conformation because of activating receptor triggering (red bars). B, After CrkL silencing (or CrkL haploinsufficiency in patients with pDGS, red X), activation strength is reduced (blue bars). In the absence of CrkL, Vav-1 phosphorylation is greatly reduced, less F-actin reorganization occurs, and MTOC and lytic granules cannot polarize to the IS. The majority of integrin remains inactive (closed) because of less activating receptor clustering and signaling. Journal of Allergy and Clinical Immunology 2015 135, 1293-1302DOI: (10.1016/j.jaci.2015.01.011) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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