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

Th-cell phenotypes

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

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)

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

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

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

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

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

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

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

Correction of defect in Th17 cells in two CGD patients after successful BMT 10 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

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

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

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

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