1. Haploinsufficiency of TNFAIP3 (A20) by germline mutation is involved in autoimmune lymphoproliferative syndrome 
Masatoshi Takagi, MD, PhD, Shohei Ogata, MD, PhD, Hiroo Ueno, MD, Kenichi Yoshida, MD, PhD, Tzuwen Yeh, BSc, Akihiro Hoshino, MD, PhD, Jinhua Piao, DDS, PhD, Motoy Yamashita, MD, Mai Nanya, PhD, Tsubasa Okano, MD, Michiko Kajiwara, MD, PhD, Hirokazu Kanegane, MD, PhD, Hideki Muramatsu, MD, PhD, Yusuke Okuno, MD, PhD, Yuichi Shiraishi, MD, PhD, Kenichi Chiba, BA, Hiroko Tanaka, BS, Yuki Bando, MD, PhD, Motohiro Kato, MD, PhD, Yasuhide Hayashi, MD, PhD, Satoru Miyano, PhD, Kohsuke Imai, MD, PhD, Seishi Ogawa, MD, PhD, Seiji Kojima, MD, PhD, Tomohiro Morio, MD, PhD 
Journal of Allergy and Clinical Immunology 
Volume 139, Issue 6, Pages (June 2017)
DOI: /j.jaci
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Patients' characteristics. A, Bilateral cervical lymphadenopathy identified by means of ultrasound echo imaging. B, Massive hepatosplenomegaly detected by means of computed tomography. Spleen index = 29 cm2. C, Skin rash. Right panel, Histopathology of skin biopsy. CD4+ or CD8+ cells were identified by using 3,3′-diaminobenzidine staining. HE, Hematoxylin and eosin staining. D, Liver biopsy specimens. Upper left, Lower magnification of HE staining; upper middle, higher magnification of HE staining; upper right, elastica van Gieson staining. Lower panels show immunostaining with the indicated antibodies.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
Increased DNT cell counts and genetic analysis of TNFAIP3 (A20). Flow cytometric analysis of patient-derived lymphocytes is shown. For DNT cells, CD8 and CD4 double staining was performed in T-cell receptor αβ–expressing cells.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
WES and the variant filtering strategy were used to narrow down the most promising causative mutations. SNP, Single nucleotide polymorphism; VAF, variant allele frequency.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5. Fig 4
Genetic analysis of the patient. A, Sequence electropherogram showing a TNFAIP3 (A20) mutation in PBMCs. Exon 7 was amplified by using PCR. Direct sequencing was performed in the sense and antisense directions. Blue letters indicate deleted nucleotides. B, Sequence electropherogram showing a TNFAIP3 (A20) mutation in PBMNCs. TA-cloned PCR products were sequenced. C, Schematic protein structure of TNFAIP3 (A20). OUT, OUT-like cysteine protease domain; Znf-A20, A20-like zing finger domain. D, Sequence electropherogram of a nail. The sequence from the sense strand is shown. E, Western blot analysis of control (Cont.) and patient (Pt.)–derived activated T cells.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6. Fig 5
NF-κB signaling pathway activation in patient-derived cells. A, Western blot analysis of control (Cont.) and patient (Pt.)–derived activated T cells under normal culture conditions without stimulation. B, Flow cytometric analysis of NF-κB phosphorylation. Control and patient-derived PBMCs were stimulated with 10 ng/mL TNF-α for 15 minutes. Unstimulated and stimulated samples were analyzed. MFI, Mean fluorescence intensity. C, Western blot analysis of control and patient-derived activated T cells. Cells were harvested at the indicated time points after stimulation with 10 ng/mL TNF-α. D, Nuclear accumulation of NF-κB. Cells were harvested at 15 minutes after stimulation with 10 ng/mL TNF-α. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. E, MAPK activation in response to TNF-α stimulation as in Fig 5, C.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7. Fig 6
Impaired deubiquitinase function of mutant A20. Cells were collected at 48 hours after transfection, and equal amounts of whole-cell lysates were subjected to immunoprecipitation by using TRAF6 antibody. High-molecular-weight ubiquitin aggregates (top) of the precipitates were detected by means of immunoblotting with anti-HA antibody. Cell lysates were also blotted with anti-TRAF6, anti-green fluorescent protein (GFP), and anti–β-actin antibodies to monitor the amounts of precipitant and loading amounts and to evaluate transfection efficiency. MT, Mutant type.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8. Fig 7
A, Control (Cont.) and patient (Pt.)–derived PBMCs were directly added to the carboxyfluorescein succinimidyl ester dye and stimulated with a CD3/CD28 antibody for 36 hours or exposed to PHA. The left panel indicates proliferation of CD4 cells, and the right panel indicates proliferation of CD8 cells. The numbers on individual histograms indicate the percent of proliferated cells. B, Cell proliferation assay. Live (left graph) and dead (right graph) cell numbers are shown. Numbers of live and dead cells were counted daily by using a hemocytometer after Trypan blue staining. Initially, cells were seeded at a density of 1 × 106 cells/mL. One half of the medium supernatant was replaced on day 3. C, Western blot analysis of control and patient-derived activated T cells. *P < .05 (Mann-Whitney U test).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions