23/10/2017. Diagnosis and Monitoring of Primary Immunodeficiencies. What are Primary Immunodeficiencies? How do we organize them???

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1 Diagnosis and Monitoring of Primary Immunodeficiencies Marianna Tzanoudaki, MD PhD Department of Immunology & Histocompatibility, Specialized Referral Centre for Primary Immunodeficiencies - Paediatric Immunology, Aghia Sophia Children s Hospital, Athens, Greece What are Primary Immunodeficiencies? Congenital defects of the Immune response As Immune Response Mechanisms are highly variable... >300!?!?!? How do we organize them??? PIDs are highly variable too! MORE than 300 monogenic syndromes recognized Recent classification of PIDs In Summary 1

2 Rough outline of the Immune Response and PIDs Tregs Tregs T Th T Th Combined SCIDIDs + associated or syndromic features digeorge, Wiskott Aldrich, HIES (Job), Ataxia /Telanghiectasia and PIDs and PIDs Tregs Tregs T Th Primary Antibody deficiencies tk (ruton s agammaglobulinaemia-xla), CVID, Specific Antibody deficiency, Transient hypogammaglobulinaemia of infancy, etc. T Th Diseases of Immune Dysregulation Familial HLH, XLP, ALPS, APECED, IPEX T IRAK-4, other TLR related deficiencies, Chronic Mucocutaneous Candidiasis Congenital neutropenias, LAD, Chronic Granulomatous Disease, Chemotaxis defects, MSMD and PIDs Th Tregs Congenital defects of phagocyte number/ function, Defects of innate immunity How do we diagnose PIDs??? 2

3 Suspicion! asic Lab tests Sophisticated lab tests Genetics asic steps to PID diagnosis NOT only infections!!! (Failure to thrive, Autoimmunity, Malignancies..) Serum Igs Family History!! (look for co-sanguinity!!!!) Complement Microbiology Full lood Count! Rule out other diseases Flow Cytometry!!! (among others ) is this old fashioned??? The Next Generation Sequencing Revolution! Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) widely available! Continuously increasing number of novel PID mutations Easy diagnosis of atypical PID cases May be more cost effective than complex FC assays Reasons why FC based assays are valuable WES/WGS May miss large deletions/ translocations What is left for Flow Cytometry??? Significance of new mutations has to be proved. Polyphen2 SHIFT may help predict impact on protein function However still Headache from the VUS Genotype /Phenotype correlation in not always strong (variable penetrance) Genes may undergo epigenetic modifications WES/WGS not readily available in every country Not covered by public insurance in many countries Not affordable by everyone Where to start from??? Refer to published algorithms! Know the basic pathways of the Immune System!!! Know the syndromes!!! 3

4 CD19 CD3 CD8 CD20 CD3 asic Steps (Step 1) Ask for Clinical Information and the patient s history! Review the rest of the Lab tests. asic Steps (Step 2) Look for (and count!) the basic lymphocyte subsets CD3 CD19 CD16/56 Τ Β ΝΚ Know what you are looking for!!! Severe Combined Immunodeficiency (SCID) Complete Di George sy. asic Steps (Step 2) Look for (and count!) the basic lymphocyte subsets CD3 CD19 CD16/56 Τ Β ΝΚ asic Steps (Step 2) Look for (and count!) the basic lymphocyte subsets CD3 CD19 CD16/56 Τ Β ΝΚ GATA2 def Severe Combined Immunodeficiency (SCID) Complete Di George sy. XLA: X-Linked Agammaglobulinaemia (ruton s) Severe Combined Immunodeficiency (SCID) Complete Di George sy. XLA: X-Linked Agammaglobulinaemia (ruton s) 12,5% 3,2% CD3 CD4 CD3 No T cells CD19 HLADr No cells Intact T cells Probably XLA (ruton s agammaglobulinaemia) CD16 74% (also No cells): It a SCID HLADr 4

5 CD20 CD3 Τ-Β- SCID ΝΚ What kind of SCID??? Think of the underlying mechanisms! Τ/ΝΚ progenitor CD8 SC Β progenitor Thymus RAG1/RAG2 NHEJ (Artemis Cernunnos LIG4 DNA-PKcs) CD4 γ jak3 α β SC IL-2, IL-4,IL-7, IL-9,IL-15,IL21 IL-15 IL-7 Thymus ΝΚ CD8 CD4 CD3 12,5% 3,2% 74% T--NK+ SCID RAG1/RAG2?? Artemis?? CD19 CD16 HLADr asic Steps (Step 3) Look for additional markers on/in the lymphocytes Expression of markers related to underlying mechanisms Enumeration of specific cell subsets Protein detection Markers related to underlying mechanisms Mostly activation/ maturation markers. Tests for clonality Characteristic of certain syndromes Directly define the defect 5

6 CD3 CD45RO Additional markers on lymphocytes CD19 CD16/56 CD3 Τ HLADR Β ΝΚ TCRγδ TCRαβ IgDIgM IgDIgM Τ Τ Β Β Β CD3 CD4 CD3 CD8 Τ4 Τ Τ8 Τ HLADR CD27 CD27 CD45RA Activated T: HLADR+ (<10%) Naïve CD4+: CD45RA+ CCR7+ Memory Β: CD27+ (>10%) Memory switched IgD-IgM-CD27+ (>5%) TCRγδ Clonality (skewed Vβ repertoire) Transitional / CD21low Example for additional markers on lymphocytes 88,5% 85,2% 3,7% CD3 CD4 CD8 CD19+:0,5% - CD3-CD16/56+: 10% HLADr CD45RA Normal 11m infant Normal adult maria SCID infant F-HLH infant kinnas 6

7 IgD IgD RESULT (% on CD4+) Low Normal High Normal RESULT (% on CD4+) Low Normal High Normal IgM IgM CD4Vb 5.3 CD4 Vb 7.1 CD4 Vb 3 CD4 Vb 9 CD4 Vb 17 CD4 Vb 16 CD4 Vb 18 CD4 Vb 5.1 CD4 Vb 20 CD4 Vb 13.1 CD4 Vb 13.6 CD4 Vb 8 Vβ repertoire CD4 Vb 5.2 CD4 Vb 2 Normal RESULT (% on CD8++) CD4 Vb 12 CD4 Vb 23 CD4 Vb 1 CD4 Vb 21.3 CD4 Vb 11 CD4 Vb 22 CD4 Vb 14 CD4 Vb 13.2 CD4 Vb 4 CD4 Vb 7.2 one Marrow pro- asic cell development pathway pre- Lomg lived plasma cell Transitional Germinal Center Th Spleen Marginal Zone Naïve Follicular Short lived plasma cell CD4Vb 5.3 CD4 Vb 7.1 CD4 Vb 3 CD4 Vb 9 CD4 Vb 17 CD4 Vb 16 CD4 Vb 18 CD4 Vb 5.1 CD4 Vb 20 CD4 Vb 13.1 CD4 Vb 13.6 CD4 Vb 8 CD4 Vb 5.2 CD4 Vb 2 CD4 Vb 12 CD4 Vb 23 Skewed in SCID CD4 Vb 1 CD4 Vb 21.3 CD4 Vb 11 CD4 Vb 22 CD4 Vb 14 CD4 Vb 13.2 CD4 Vb 4 CD4 Vb 7.2 IgG, IgA or IgE Memory Germinal Center Isotype switch Affinity maturation (Somatic Hypermutations) IgM asic markers for the study of cell isotype switch patient patient Marker asic immunophenotypic markers for Peripheral lood cell characterization Role Naïve Transitional Naïve Follicular Memory switched cell subset Exhausted Plasmablasts Marginal Zone like CD27 CD27 CD19 CD21 CD27 CD23 co stimulation low + forms complex with CD21 and CD81- Complement Receptor- Co stimulation med + + low med ++ TNF-R familyco stimulation FcεR- IgE coated Ag capture in GC C CD24 GPI anchored- Cell adhesion IgM ++ low/ IgD low low CD27 CD27 CD38 cyclic ADP ribose hydrolase-??? There is a need for extra cells markers to Classify the defect ie EuroCLASS for Common Variable Immunodeificiency (CVID) Predict the level of the defect Avoid pitfalls in subset definition CD21low cells (CD21loCD38-) Transitional cells (IgD-IgM- CD24++CD38++) Plasmablasts (CD27+CD24- CD38++) ON TOTAL MEMORY How do you define class switched memory? CD27+IgD-IgMis different from CD27+IgD- e careful with normal values! 7

8 IgD CD21 CD27 CD21 CD21low Transitional cells Normal CVID with granulomata CD21lo CD38 CD19+ CD38 HELPS DEFINE TRANSITIONAL REGION Plasmablasts Example IgM CD38 They increase the % of the Memory switched region Does this patient have so many memory switched cells? Most of them are plasmablasts! Examples of lymphocyte subsets related to certain PIDs Markers helping the detection/enumeration of subsets related to certain diseases CD21lowCD38- Double Negative T (DN= TCRαβ+ CD4-CD8-) inkt cells CVID ALPS (Autoimmune Lymphoproliferative sy.) XLP (X-linked Lymphoproliferative sy.) 8

9 CD8 HLADR HLADR CD8 Vα24-PE CD25 CD25 Vα24-PE CD25 CD25 TCRαβ+ Double Negative T cells (DN= TCRαβ+ CD4-CD8-) ALPS patient TCRαβ+ CD3+ cells XLP and inkt cells A CD3+ cells CD4 CD4 DN DN Vβ11-FITC Vβ11-FITC XLP patient CD45RA CD45RA Protein Detection Examples of Protein detection by FC for PID diagnosis WASP in T Wiskott Aldrich Syndrome Foxp3 in T (Tregs) IPEX syndrome CD25 on T (Tregs) IPEX like syndrome Perforin in act. CD8+ or NK CD18, CD11a,b,c on polys FHL 2 LAD SAP in CD8+T/XIAP in ly. XLP1/XLP2 CD40L on act.cd4+ X linked Hyper IgM gp91 (7D5) on polys X Linked CGD CTLA4 in Tregs CTLA4 or LRA def tk in monos XLA Some cases may be straightforward Others concern a certain minor subset (eg Tregs ) CD25def /IPEX like patient Patient with Wiskott Aldrich sy. CD4 CD4 WASP-PE WASP-PE CD127 CD127 9

10 CD25 CD25 and others are hard to recognize Pattern of expression! Caution!!! Intensity! Fox p3 FoxP3 IPEX patient The epitope Your MoAb recognizes may be intact!!! (while the rest of the molecule is defective ) You have seen that the cells subsets are there You have counted them Up to now You have seen that the cells subsets are there You have counted them Up to now You have seen that their phenotype is normal Are you sure that the Immune System is healthy? Good looking cells aren t necessarily functional! Examples from the clinical practice A 2 yo boy with liver abscess A 7 yo boy with persistent mucosal and nail Candidiasis and a bad breath A 5 yo girl with Leishmaniasis, disseminated T lymphohyperplasia and autoimmune cytopenias A 3 yo girl in a coma. Death of a sibling from HLH. ALL with normal Ig levels, and NO findings on thorough P immunophenotype! 10

11 How cells of the Immune System usually respond to stimuli The basic concept of functional tests Regulatory pathways Signal transduction pathways Phosphoproteins Receptor Stimulus eg. Antigen, cytokine, cell ligand, chemokine PAMP etc Cytokine release Mitosis nuclear/transcription factors Effector function such as Oxidative burst Degranulation/ cytotoxicity Apoptosis Regulatory pathways nuclear/transcription factors The defect may be at ANY level!!! Signal transduction pathways Phosphoproteins Effector function such as Receptor Stimulus eg. Antigen, cytokine, chemokine PAMP etc Cytokine release Mitosis Oxidative burst Degranulation/ cytotoxicity Apoptosis Regulatory pathways nuclear/transcription factors How we can check their function Signal transduction pathways 2. Check for activation (eg. Phosphorylation) of specific mediators or 3. Chose the Effector function to check Receptor 1.Chose a Stimulus: Antigen, cytokine, chemokine,pamp (LPS) PHA, PWM, ConA, PMA etc Cytokine production/release Mitosis Oxidative burst Degranulation/ cytotoxicity Apoptosis Upregulation of surface molecules.. Examples of functional tests Example 1: DHR test for the diagnosis of Chronic Granulomatous Disease (CGD) 11

12 What is CGD? What is CGD? X-linked CGD NADPH e - O 2 - H 2 O 2 HOCl X-linked CGD NADPH e - e - O 2 - H 2 O 2 HOCl rac 47 rac Autosomal CGD NADP+ Autosomal CGD NADP+ What is CGD? The DHR test Granulomata X-linked CGD e - HOCl H 2 O 2 catalase e - O - 2 NADPH X-linked CGD NADPH e - O 2 - HOCl H 2 O 2 R Autosomal CGD rac NADP Autosomal rac CGD NADP+ DHR 123 Unstimulated (Negative control) The DHR test PMA stimulated (test) Unstimulated (Negative control) The DHR test PMA stimulated (test) 37 ο C 37 ο C 12

13 Unstimulated (Negative control) The DHR test PMA stimulated (test) Normal DHR test result RC lysis 37 ο C Unstimulated PMA Stimulated DHR pitfalls Which PMA concentration? If using elevated PMA concentration This would be considered autosomal CGD PMA Stimulated GCD patient the boy with the liver abscess X-CGD carrier the boy s mother Stimulation with 17 μg/ml PMA (10X) MFI: 55.3 Stimulation with 1.7 μg/ml PMA MFI: 9.99 Effect of PMA concentration on the DHR Fluorescence pattern. DHR pattern of a p47 heterozygote was normalized when increased PMA concentration was used. DHR pitfalls DHR pitfalls Normal unstimulated Normal PMA stimulated p47 homozygote Upon presentation 3 weeks later 5 weeks later MPO deficiency Systemic corticosteroids DHR fluorescence patterns in various situations (Stimulation with 1.7 μg/ml PMA) p47 heterozygote The effect of infection on DHR pattern (atypical Mycobacteria infection, before, and after initiation of treatment) 13

14 Example 2: Th17 detection for the diagnosis of Chronic Mucocutaneous Candidiasis (CMC) or of the Hyper IgE/Job s sy (HIES) STAT 3 Τ STAT1 TH17 in CMC/ HIES STAT 3 STAT3 STAT3 IL 17 MCP1 STAT5 IL17 production: Immunity against Candida / IL-6, IL-10, IL11, Staphylococcus IL-21,IL22, IL23 STAT1 STAT 3 TH17 in CMC/ HIES STAT 3 STAT3 STAT5 STAT3 HIES: No STAT3 No IL17 Staphyloccocal infections STAT1 STAT 3 TH17 in CMC/ HIES STAT 3 STAT3 STAT5 STAT3 CMC: STAT1 GOF block of STAT3 related transcription Candida infections Τ IL 17 MCP1 Τ IL 17 MCP1 TH17 in CMC/ HIES HIES or CMC the boy with the Candidiasis and the bad breath Example 3: IL12/IFNγ axis study for the diagnosis of Mendelian Susceptibility for Mycobacterial Disease (MSMD) and some other syndromes Intracellular IL-17 detection in PMA/ionomycin stimulated, FA treated T cells 14

15 The IL12/IFNγ axis Mendelian Susceptibility for Mycobacterial Disease- MSMD IFN-γ R STAT1 IL-12 IFN-γ R STAT1 IL-12 IFN-γ Tyk2 IL-12 R IFN-γ Tyk2 IL-12 R Mainly infections from Mycobacteria, Leishmania, Salmonella How to test the IL12/IFNγ axis efficiency How to test the IL12/IFNγ axis efficiency PPD, LPS or CG PPD, LPS or CG PPD, LPS or CG IL-12 IFN-γ Patient Measure IL-12, IFNγ (intracellularly, or in the culture supernatant) PPD stimulated T cells How to test the IL12/IFNγ axis efficiency Patient The IL12/IFNγ axis MC LPS + IL12 IFN-γ R STAT1 IL-12 LPS + IFNγ IFN-γ Tyk2 IL-12 R IL12 (blue), IFNγ (violet) and TNFα (yellow) levels in culture supernatant, measured with a Cytometric ead Array Method 15

16 The patient was the girl with Leishmaniasis, disseminated T lymphohyperplasia and autoimmune cytopenias Defect downstream of the IL12R UT MSMD gene panel: No mutations found! How cells of the Immune System usually respond to stimuli Regulatory pathways Signal transduction pathways Phosphoproteins Receptor Stimulus eg. Antigen, cytokine, cell ligand, chemokine PAMP etc Cytokine release Mitosis WES: STAT3 GOF mutation! (explaining autoimmunity and lymphohyperplasia) nuclear/transcription factors Effector function such as Oxidative burst Degranulation/ cytotoxicity Apoptosis Familial Hemophagocytic Lymphohistiocytosis Syndromes (FHL). asic mechanisms Example 4: Degranulation test for the diagnosis of Familial Hemophagocytic Lymphohistiocytosis syndromes (FHL) Defective Cytotoxicity Cytotoxicity CD8+ or NK Target Cell Perforin deficiency (FHL2) Granule formation deficiency (Griscelli s., Chediak Higashi s.) Degranulation deficiency Phagocyte Hemophogacytosis Lymphohyperplasia Familial Hemophagocytic Lymphohistiocytosis Syndromes (FHL). asic mechanisms Familial Hemophagocytic Lymphohistiocytosis Syndromes (FHL). asic mechanisms Defective Cytotoxicity Cytotoxicity CD8+ or NK Target Cell Perforin deficiency Granule formation deficiency (Griscelli s., Chediak Higashi s.) Degranulation deficiency Defective Cytotoxicity Cytotoxicity CD8+ or NK Target Cell Perforin deficiency Granule formation deficiency (Griscelli s., Chediak Higashi s.) Degranulation deficiency Phagocyte Can be revealed by Cytotoxicity Assays (need for target cells) Phagocyte Can be revealed by Degranulation Assays 16

17 Degranulation assays: asic concept eg. PMA, +/- CD28 beads, anti-cd3, Degranulation assays: asic concept CD107 CD107 CD8+ or NK surface CD107 NON detectable on resting cells CD8+ or NK surface CD107 detectable after stimulation CD107 degranulation test Is there more in Phospho-flow? Resting CD8+ cells CD107a+b-FITC CD107a+b-FITC IL-10 Studying STAT phosphorylation status may help identify receptor defects IL-10R Stimulated CD8+ cells CD107a+b-FITC The girl had grey hair! The comatose girl Griscelli type 2 mutations were found. Coma probably CD107a+b-FITC due to intracranial HLH STAT3 Patient 17

18 Thank you!!! Our Department FC Technicians Virginia Polaki Anastasia Limioti Younger Cytometrists Eliana Gkika Eleni Ploumi Pediatricians Manolis Liatsis Sophia Tantou Head of the Department Maria Kanariou FC veteran Effie Vrachnou Acknowledgements International Centers Europe Hôpital Necker-Enfants Malades, Paris Great Ormond Street for Children NHS Trust, London Centre of Chronic Immunodeficiency (CCI), Freiburg UCL Institute of Child Health, London Sanquin Research at CL, Amsterdam University Children's Hospital, Zürich USA Childrens Hospital, Harvard Medical School Laboratory of Host Defenses, Immunopathogenesis Section, ethesda 18