1. Disturbances in the homeostasis of Th17 lymphocytes in patients with hyper IgE syndrome (HIES) and chronic granulomatous disease (CGD)
Horvath R.1, Lastovicka J.1, Polouckova A.1, Sedlacek P.2, Bartunkova J.1, Sediva A.1, Spisek R.1
1Department of Immunology, Charles University, 2nd Medical School and Faculty Hospital Motol, Prague, Czech Republic
2Department of Pediatric Hematology and Oncology, BMT unit, Charles University, 2nd Medical School and Faculty Hospital Motol, Prague, Czech Republic

2. Th-cell phenotypes

3. Th17 cell lineage development
APC
IL23-APC
RORγT
STAT-3
Th17
Pre-Th17
IL-17
IL-22
IL-21
IL-6
TGF-β
Naive CD4

4. Th 17 cell functions
Production of IL-17 cytokines family (IL-17, IL-21, IL-22) which leads to the chemoattraction of neutrophils
Accumulating data suggest that Th17 are highly pro - inflammatory and that Th17 cells with specificity for self-antigens lead to severe autoimmunity- (psoriasis, Crohn´s disease, multiple sclerosis)

5. Physiological role of Th17 cells in humans
Initially, from studies in mice, Th17 cells were thought to play an important role in host defense against extracellular pathogens, which are not efficiently cleared by Th1-type and Th2- type immunity
However, identity of pathogens cleared by Th17 was unknown
Direct evidence for understanding physiological target of Th17 cells came from studies of patients with mutations in STAT-3, a critical transcription factor for the differentiation of Th17 cells

6. Defective Th17 cells in Hyper IgE syndrome
Primary immunodeficiency caused by mutations in STAT-3 transcription factor, NEJM 2007
Dermatitis, boils, cyst-forming pneumonias, retained primary dentition, bone abnormalities and elevated serum IgE levels
Abnormal and devastating susceptibility to a narrow spectrum of infections, most commonly Staphylococcus aureus and Candida albicans
Abrogated numbers of Th17 cells, Nature 2008, JEM 2008
specific stim. with candida
nonspecific stimulation
specific stim. with stp.aureus

7. Defective Th17 cells in Hyper IgE syndrome
Studies in Hyper IgE point to a critical role of Th17 cells in the response against candidal and staphylococcal infections
However, there are other diseases with similar spectrum of dominant pathogens where the characteristics of Th17 have not been tested
We thus decided to test Th17 cells compartment in chronic granulomatous disease

8. Clinical mimicry of HIES with other primary immunodeficiencies – Chronic granulomatous disease (CGD)
Primary immunodeficiency
Mutations in the NADPH oxidase system
Profound defect of respiratory burst in myeloid cells
Recurrent infections, organ granulomas
Dominant susceptibility to staphylococcal and candidal infections

9. Aim of the study Patients and methods
Analyze and compare the characteristics of Th17 compartment in HIES and CGD patients
Patients and methods
4 patients from 3 families with HIES
7 patients with CGD (2 patients underwent allo-BMT)
Mutations in STAT-3 and NADPH oxidase - genetics
FACS, ELISA

10. Results – Th17 numbers in CGD and HIES
absent Th17 cells in HIES
high frequencies of Th17 in CGD B 10 2 3 4 5
4.12 10 2 3 4 5
1.67 10 2 3 4 5
2.32
3,5
p<0,05 3
p<0,05
2,5
p<0,05
0.51
0.13
1.98
% of IL-17+ CD4+ cells 2 10 2 3 4 5 10 2 3 4 5 10 2 3 4 5
1,5 10 2 3 4 5
5.91 10 2 3 4 5
3.5 10 2 3 4 5
1.25 1
IFN gamma - PE
0,5
0.49
0.078
Controls
HIES
CGD
2.87 10 2 3 4 5 10 2 3 4 5 10 2 3 4 5 10 2 3 4 5
6.51 10 2 3 4 5
6.97 10 2 3 4 5
1.91 14
p<0,05
p=0,39
0.76
0.36
1.55 12 10 2 3 4 5 10 2 3 4 5 10 2 3 4 5
p=0,05
Controls
HIES
CGD 10
% of IFN gamma+ CD4+ cells 8
IL-17 A647 6 4 2
Controls
HIES
CGD

11. Results – cytokine production
Th17 effector cytokines
Polarization cytokine A B
IL-17
IL-21
IL-23
200
400
600
800
1000
1200
pg/ml
Controls
HIES
CGD
p<0,05
1200
p<0,05
high levels of IL-17 and IL-21 in CGD
2500
p=0,06
extremely elevated levels of IL-23 in HIES
p<0,05
p=0,29
1000
2000
p<0,05
p=0,14
800
1500
pg/ml
600
pg/ml
elevated levels of IL-23 in CGD
1000
400
low levels of IL-17 and IL-21 in HIES
500
200
Controls
HIES
CGD
Controls
HIES
CGD

12. Correction of defect in Th17 cells in two CGD patients after successful BMT10 2 3 4 5
1.83
CGD after BMT
normalized numbers of Th17 cells after BMT
p=0,52
p=0,24
post-BMT 3
2,5 2
% of CD4 pos. T cells
1,5 1 ,5
0.12
IFN gamma - PE
pre-BMT
p#2 10 2 3 4 5 10 2 3 4 5
8.64
IFN-g 14 12
Controls
% of CD4 pos. T cells 10 8
HIES 6
CGD 4
0.84 2 10 2 3 4 5
IL-17 A647
pre-BMT

13. Results – cytokine production after allo-BMT
Th17 effector cytokines
Polarization cytokine
p=0,24
1400
1200
CGD after BMT
p=0,33
p=0,12
decreased production of IL17,21 and 23 after BMT
post-BMT
IL-17
1000
IL-23
800
600
1000
pg/ml
400
800
200
600
pre-BMT
IL-21
pg/ml
400
2000
200
1750
1500
pre-BMT
post-BMT
1250
1000
pg/ml
750
500
Controls
250
HIES
pre-BMT
CGD

14. Possible theoretical explanations
Healthy
Th17
APC
IL-23
IL-17,22,21 etc
Neutro
Lympho
Mono
Eradication
Candida
S.aureus
HIES
Th17
APC
IL-23
IL-17,22,21 etc
Candida
S.aureus
STAT3 mutation
In-efficient immune cells attraction
Pathogen persistence
CGD
Th17
APC
IL-23
IL-17,22,21 etc
Neutro
Lympho
Mono
In-efficient eradication
Candida
S.aureus
NADPH mutation
Pathogen persistence

15. Conclusions
We identified disturbances in the homeostasis of Th17 lymphocytes in HIES and CGD patients
Absent Th17 cells in STAT-3 deficient HIES patients
Significantly higher frequencies of Th17 cells in CGD
Increase of Th17 cells in CGD is likely to be secondary as a result of defect in neutrophils
BMT leads to the normalization of elevated Th17 cell numbers and coresponding IL-17 production
Positive pro-inflammatory loop caused by Th17 cells contributes to the formation of granulomas
These findings confirm the critical role of Th17 lymphocytes in the elimination of candidal and staphylococcal infections 15

16. Acknowledgements
Dpt. of Immunology, Charles University, Prague, Czech Republic
Lastovicka Jan
Polouckova Andrea
Rozkova Daniela
Kayserova Jana
Budinsky Vit
Sochorova Klara
Kytka
Minarik Ivos
Fucikova Jitka
Jaresova Irena
Sediva Anna
Bartunkova Jirina
Spisek Radek
Dpt. of Pediatric Hematology and Oncology,
BMT Unit, Charles University, Prague, Czech Republic
Formankova Renata
Keslova Petra
Stary Jan
Sedlacek Petr