Supplementary Tables and Figures DOCK8 functions ...

Supplementary Tables and Figures DOCK8 functions as an adaptor that links Toll-like receptor–MyD88 signaling to B cell activation

Haifa H. Jabara1,15, Douglas R. McDonald1,15, Erin Janssen1, Michel J. Massaad1, Narayanaswamy Ramesh1, Arturo Borzutzky1, Ingrid Rauter1, Halli Benson1, Lynda Schneider1, Sachin Baxi1,, Mike Recher1, Luigi Notarangelo1, Rima Wakim2, Ghassan Dbaibo2, Majed Dasouki3, Waleed Al-Herz4, Isil Barlan5, Safa Baris5, Necil Kutukculer6, Hans Ochs7, Alessandro Plebani8, Maria Kanariou9, Gerard Lefranc10, Ismail Reisli11, Kate Fitzgerald12, Douglas Golenbock12, John Manis13, Sevgi Keles11,14 Reuben Ceja14, Talal Chatila14 and Raif S. Geha1

1

Division of Immunology, Children’s Hospital and Department of Pediatrics, Harvard

Medical School, Boston, MA, 2American University of Beirut, Beirut, Lebanon, 3

Departments of Pediatrics & Internal Medicine, Division of Genetics, Endocrinology &

Metabolism, University of Kansas Medical Center, Kansas City, KS, 4Allergy and Clinical Immunology Unit, Department of Pediatrics, Al-Sabah Hospital, Kuwait, 5Division of Pediatric Allergy/Immunology, Marmara University, Istanbul, Turkey, 6Department of Pediatric Immunology, Ege University, Izmir, Turkey, 7Department of Pediatrics, University of Washington, Seattle, WA, 8Department of Pediatric Hemato-Oncology, Spedali Civili, Brescia, Italy, 9Agia Sophia Children's Hospital, Athens, Greece, 10 University of Montpellier, Montpellier, France, 11 Division of Pediatric Allergy and Immunology, Meram Medical Faculty, Selcuk University, Konya, Turkey, 12University of Massachusetts, Worcester, MA 14

13

Department of Transfusion Medicine, Children’s Hospital, Boston, MA,

Division of Allergy and Immunology and Department of Pediatrics, University of California

Los Angeles, Los Angeles, CA.

15

H.H.J. and D.R.M. contributed equally to this work.

Supplementary Table 1 Genotype of patients with homozygous mutations in DOCK8

Patient Mutation 1 2 3 4 5 6 7 8 9 10

c.[3235-?_3390+?del]+[3235-?_3390+?del] (includes deletion of exon 28) c.[4070C>A]+[4070C>A]; p.[S1357*]+[ S1357*] (mutation in exon 33) c.[1-?_156+?del]+[1-?_156+?del] (includes deletion of exons 1 and 2) c.[3391-?_4473+?del]+ [3391-?_4473+?del] (includes deletion of exons 29-36) Point mutation in splice site IVS17 -1 G/C Large deletion c.[1490T>A]+[1490T>A]; p.[L497*]+[L497*] (mutation in exon 14) c.[1-?_156+?del]+[1-?_156+?del] (includes deletion of exons 1 and 2) c.[1641_1642del]+[ 1641_1642del]; p.[E546fs]+[E546fs] (mutation in exon 15) Deletion of exon 39

Reference Unpublished Unpublished Unpublished Unpublished ARH008 (ref. 24) ARH004 (ref. 24) Unpublished Unpublished Unpublished ARH009 (ref. 24)

The numbering of exons and nucleotides is according to sequence NM_203447.3. The nomenclature used is based on the recommendations of the Human Genome Variation Society

Supplementary Table 2 Clinical and laboratory characteristics of DOCK8 deficient patients Clinical characteristics EczSkin Food ema infections allergy

Pt.

Sex

Age yrs

1

M

4

S/P infect . +

2 3 4

M M F

12 6 8

+ + +

+ + +

5 6 7 8 9

F M F F F

10 10 15 9 3.5

+ + + + +

+ + + + +

10

F

15

+

+

Abbreviations: eosinophils;

Pt: C.

+

C. albicans Molluscum Herpes zoster Molluscum HPV Molluscum HSV HPV Molluscum HSV C. albicans HPV, HSV Herpes zoster

Patient; albicans:

S/P

infect:

Candida

Eos./ 3 mm

Laboratory data IgE IgM IU/ml mg/dL

IgG mg/dL

+

650

2,731

50

1,430

+ + +

1,290 21,940 11,020

810 6,985 1,340

22 48 66

1,440 1,100 533

+ + + +

5,100 4,260 2,250 1,400 3,240

10,500 5,000 20,500 8,643 17,960

62 29 21 18 26

1,500 1,154 1,210 3,240 918

+

1,000

19,100

26

1,568

sinopulmonary

albicans;

infections;

Molluscum:

Eos:

Molluscum

contagiosum; HPV: human papilloma virus; HSV: herpes simplex virus. For all patients, values for circulating eosinophils and serum IgE were above, serum IgM concentration was below, and serum IgG concentration was within the range for age-matched controls.

Supplementary Table 3 Heterozygous STAT3 mutations in HIES patients. Patient 1 2 3 4 5 6

STAT3 mutation R84Q R382Q V463del Y657C Y705C Y705N

Affected STAT3 domain Proximal coiled-coiled domain DNA-binding domain DNA-binding domain SH2-domain Phosphotyrosyl tail segment Phosphotyrosyl tail segment

*

0.7

IgM anti-TT OD 405

0.6 0.5 0.4 0.3 0.2 0.1 0.0

C

Pt

Supplementary Figure 1. Decreased serum levels of IgM antibody response to tetanus toxoid (TT) in DOCK8 deficient patients. Serum IgM anti-TT levels were determined by ELISA on 5 patients and 5 controls, and expressed as O.D. units. *P< 0.05 (Student’s t-test).

b

a 10

Patient 10

103

103 69.7

84.4 0.8

6.78

IgD

102

102

101

100 100

4

101

16.5

101

102

103

104

100 100

1

101

102

103

104

% CD19+IgD+CD27+ cells

Control 4

*

7 6 5 4 3 2 1 0

C

Pt

CD27 Supplementary Figure 2. Decreased circulating MZ-like B cells in DOCK8 deficient patients. (a) Representative FACS analysis of IgD+CD27+ MZ-like B cells in PBMCs from DOCK8 deficient patients and age matched controls, gating on CD19+ B cells. (b) Percentage of IgD+CD27+ MZ-like B cells in circulating B cell populations of DOCK8 deficient patients and controls (n=3 each; mean and s.e.). *P< 0.001 (Student’s t-test).

a Total B cells

CD27

10

Naive B cells 10

4

4

103

103

102

102

101

101

10

0

100

101 102

103 104

100 100

101 102

103 104

CD19

3

10

0 α−CD40 Total Naive Total + IL21 Pt C

10

IgG (µg/ml)

20

IgM (µg/ml)

[3H]Td (c.p.m. X 103)

b 2 1 0

Total Naive Total Pt C

5

0

Total Naive Total Pt C

Supplementary Figure 3. FACS analysis of purified normal total and naïve B cells and their response to anti-CD40 plus Il-21. (a) Representative FACS analysis of purified total CD19+ B cell and naive CD19+CD27- B cell populations isolated from the same normal subject. (b) Proliferation and secretion of IgM and IgG by highly purified total and naïve B cells from controls (n=3 each) and purified total B cells from DOCK8 deficient patients (n=2 each) in response to anti-CD40 + IL-21. Columns and Bars represent mean and s.e.

Medium 105 0.45

Control

Propidium iodide

1.52

105 0.91

104

104

103

103

102 0

102 0

92.6 0 102

Patient

CpG

5.41 103

104

105 1.12

105 2 .5

103

103

102 0

102 0

8.88 103

104

105

10.8 103

104

105 1.91 104

0 102

85.8 0 102

104

87.5

2.43

4.35

79.1 0 102

105

14.6 103

104

105

AnnexinV-FITC Supplementary Figure 4. CpG stimulation of PBMCs does not cause increased apoptosis and cell death in DOCK8 deficient B cells. FACS analysis of Annexin V and propidium iodide (PI) staining by gated CD19+ B cells after 3 days of stimulation of PBMCs with CpG. Similar results were obtained in two additional experiments.

30000 20000 10000 0

α-CD40 + IL21

-

+ C

-

*

8000

IgG (ng/ml)

[3H]Td (c.p.m.)

*

+

6000 4000 2000 0

α-CD40 + IL21

AD-HIES

-

+ C

-

+

AD-HIES

Supplementary Figure 5. Response of PBMCs from AD-HIES patients with STAT3 mutations to stimulation with anti-CD40 plus IL-21. Proliferation (Left panel) and IgG production (right panel) by PBMCs from controls (proliferation n=4; IgG n= 6) and from AD-HIES patients (n=6 each) in response to anti-CD40 + IL21; the small horizontal lines indicate mean. *P