Online supplement:
IL-17-producing T Helper Cells and Th17 Cytokines in Human Airways Exposed to Endotoxin
Pernilla Glader1, Margaretha E. Smith1, Carina Malmhäll2, Barbro Balder1, Margareta Sjöstrand2, Ingemar Qvarfordt1, Anders Lindén1
1
Lung Immunology Group, Department of Internal Medicine/Respiratory Medicine and Allergology, Sahlgrenska Academy at University of
Gothenburg, 2 Krefting Research Centre, Department of Internal Medicine Sahlgrenska Academy at University of Gothenburg
Corresponding author
Pernilla Glader, Ph.D.
Lung Immunology Group
Department of Internal Medicine/Respiratory Medicine and Allergology
Sahlgrenska Academy at University of Gothenburg
Box 480
SE 405 30 Gothenburg
Sweden
e-mail : pernilla.glader@gu.se
Tel: +46 31 342 33 86
Gene
Primer sequence
Reference
IL-17A
Fwd: 5´ TGG AAT CTC CAC CGC AAT G 3´
Primer design: TIB MOLBIOL
Rev: 5´ CAG GAC CAG GAT CTC TTG CT 3´
Fwd: 5´ GCA GGC TTG ACA AGT CCA ACT 3´
IL-22
WILSON (1)
Rev: 5´ GCC TCC TTA GCC AGC ATG AA 3´
RORC2 Fwd: 5´ TGA GAA GGA CAG GGA GCC AA 3´
WILSON (1)
Rev: 5´ CCA CAG ATT TTG CAA GGG ATC A 3´
ROR-
Fwd: 5´ CGG TGC CTT TGA CTC TCA GAA CAA CAC CG 3´
MANEL (2)
Rev: 5´ TCT TTC CAA ATT CAA ACA CAA AGC 3´
HPRT
Fwd: 5´ ATC AGA CTG AAG AGC TAT TGT AAT GAC CA 3´ Primer design: TIB MOLBIOL
Rev: 5´ TGG CTT ATA TCC AAC ACT TCG TG 3´
Table E1 . Nucleotide sequence of the specific primers used in PCR amplification
References:
1
2
Wilson, N.J., et al., Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol, 2007.
8(9): p. 950-7.
Manel, N., D. Unutmaz, and D.R. Littman, The differentiation of human T(H)-17 cells requires transforming growth factor-beta and
induction of the nuclear receptor RORgammat. Nat Immunol, 2008. 9(6): p. 641-9.
Symptom
Nausea
Headache
Mucus
Cough
Myalgia
Chest pain
Wheezing
Number of subjects with
no symptoms
7
7
7
7
8
10
10
Number of subjects with
mild symptoms
4
5
4
4
3
2
1
Number of subjects with
moderate symptoms
1
0
0
1
1
0
1
Number of subjects with
severe symptoms
0
0
1
0
0
0
0
Table E2. Symptom assessment of all 12 study subjects, during the first 12h after bronchoscopy I. Moderate and severe symptoms were reported
by one subject only.
Recovery, ml
Cells/ml, x106
Viability, %
% Neutrophils
12h VEH
101 (95-109)
0.09 (0.04-0.17)
87 (78-93)
12 (4-38)
12h E
98 (90-105)
1.52 (0.75-2.97)
97 (94-98)
71 (56-84)
24h VEH
100 (91-115)
0.073 (0.05-0.115)
88 (77-92)
6 (4-22)
24h E
95 (89-102)
1.21 (0.37-1.72)
96 (94-98)
68 (52-78)
Number of
Neutrophils/ml,
x106
0.013
(0. 003-0.445)
0.996
(0.526-2.503)
0.007
(0.002-0.02)
0.782
(0.219-1.288)
% Macrophages
51 (41-65)
18 (12-30)
57 (40-75)
21 (13-27)
Number of
Macrophages /ml,
x106
0.047
(0.026-0.09)
0.309
(0.131-0.392)
0.037
(0.03-0.069)
0.204
(0.1-0.306)
% Lymphocytes
12 (6-23)
9 (3-12)
15 (6-36)
8 (6-19)
Number of
Lymphocytes/ml,
x106
0.011
(0.005-0.024)
0,109
(0.066-0.161)
0.008
(0.005-0.056)
0.085
(0.040-0.229)
Table E3. Recovery of BAL sample, total cells count/ml BAL and differential counts. Data presented as median values and ranges.
12h VEH
12h E
24h VEH
24h E
% CD3+ of total
BAL cells
% CD4+ of
CD3+
% IL23R+ of % IL23R+ of % CCR6+ of
CD3+CD4+
CD3+CD8+
CD3+CD4+
% CCR6+ of
CD3+CD8+
8 (5-23)
5 (2-11)
12 (6-29)
7 (5-16)
71 (51-76)
56 (44-75)
55 (17-75)
65 (52-80)
7 (4-19)
2 (0-10)
8 (5-12)
7 (2-8)
15 (7-18)
11 (4-24)
11 (3-20)
5 (4-13)
4 (0-4)
0 (0-1)
2 (1-5)
1 (0-7)
46 (23-51)
27 (18-46)
41 (36-49)
30 (19-31)
Table E4. T-cell populations in BAL. Data presented as median values and ranges.
% CD45R0+
of
CD3+CD4+
95 (93-97)
95 (91-97)
96 (92-98)
94 (88-98)
% CD45R0+
of
CD3+CD8+
97 (77-99)
93 (64-99)
93 (84-96)
87 (80-90)
Figure E1. IL-17 containing CD4+ T-cells lack surface expression of IL-23R. Plots are gated on CD4+CD3+ BAL cells. Donors are numbered 26,
27, 29, 32, 33 and 34. Donor number+N means vehicle side of the lung and donor number +E means endotoxin side of the lung.