Hans D. Ochs University of Washington & Seattle Children ’ s Hospital Seattle, Washington

1. Hans D. Ochs University of Washington & Seattle Children’s Hospital Seattle, Washington Immunodeficiency Syndromes: When to suspect How to screen Confirm And what can be done Almaty 2017

2. Goals • A brief review of Immunology and Immunodeficiency • Overview of basic tests of the immune system • Modern testing approaches in the practice of clinical immunology: • Screening Tests • Gene Sequencing • Flow Cytometry Testing • Test Immunizations

3. 4 Compartments of the Immune System Innate Immunity Adaptive Immunity Complement B Cells “Air Force” “Land Mines” Y Y Y Y Y Y Y Host Defense T Cells Phagocytes “The Generals” “The Assassins” “The Psychologists” “The Marines” Cytokines & Chemokines Neutrophils Macrophages

4. Relative Incidence of Primary Immunodeficiencies Cellular Immunodeficiency (7%) Combined Immunodeficiency (23%) Antibody Deficiency (53%) Complement Deficiency (1%) PMN Dysfuction (14%) Other (2%) Skoda-Smith and Barrett, Contemporary Pediatrics 17:156-165

5. 4 Compartments of the Immune System innate Immunity adaptive Immunity T Cell Complement Phagocytic B Cell • Recurrent disseminated • Neisserial infections. • Pyogenic bacterial • infections. • Angioedema of face, hands, • feet, or GI tract. • Autoimmune symptoms • (Lupus). • History suggestive of • autosomal dominant • inheritance. • Soft tissue abscesses or • lymphadenitis. • Infection with catalase(+) • organisms (Staph aureus, • Serratia, E. coli, Aspergillus) • Poor wound healing. • Delayed separation of the • umbilical cord. • Chronic gingivitis and • periodontal disease. Colitis • Mucosal ulcerations. • Recurrent bacterial sino- • pulmonary infections or • sepsis, particularly with • encapsulated organisms. • Chronic or recurrent • gastroenteritis (Giardia • and Enterovirus common). • Chronic enteroviral menigo- • encephalitis. • Arthritis, Crohn’s • Unexplained bronchiectasis. • Pneumocystis carinii • pneumonia. • Fungal infections. • GVHD (rash, abnormal LFT’s, • Autoimmune disorders • and chronic diarrhea. • Recurrent, severe, or • unusual viral infections. • Failure to thrive. Combined

6. 3 year old boy with recurrent pyogenic bacterial infections, systemic lupus erythematosus (SLE), nephritis, CH50 = 0 • . CH50 - tests entire complement cascade (U$ 83.-) L A B S • CBC/Differential ($40) • Quantitative IgG, IgM, IgA, • and IgE ($127) • Anti-tetanus titers ($57) • Anti-pneumococcal titers pre- • & 4 wks post-Immunization • CBC/Differential ($40) • T & B cell subset analysis • ($202) • DTH reaction to Candida & • Tetanus • CBC/Differential ($40) • Neutrophil oxydative burst • assay - AKA NBT assay ($86) Complement Phagocytic B Cell T Cell • Recurrent disseminated • Neisserial infections. • Pyogenic bacterial • infections. • Angioedema of face, hands, • feet, or GI tract. • Autoimmune symptoms • (Lupus). • History suggestive of • autosomal dominant or • recessive inheritance. • Recurrent bacterial sino- • pulmonary infections or • sepsis, particularly with • encapsulated organisms. • Chronic or recurrent • gastroenteritis (Giardia • and Enterovirus common). • Chronic enteroviral menigo- • encephalitis. • Arthritis • Unexplained bronchiectasis. • Pneumocystis carinii • pneumonia. • Fungal infections. • GVHD (rash, abnormal LFT’s, • and chronic diarrhea. • Recurrent, severe, or • unusual viral infections. • Failure to thrive. • Soft tissue abscesses or • lymphadenitis. • Infection with catalase(+) • organisms (Staph aureus, • Serratia, E. coli, Aspergillus) • Poor wound healing. • Delayed separation of the • umbilical cord. • Chronic gingivitis and • periodontal disease. • Mucosal ulcerations. D E F E C T T Y P E Combined

7. The 2 Goals of allImmunologic Testing • NUMBERS • FUNCTION

8. Testing - Complement • Number: • Individual complement protein levels (C2, C3, C4, etc.) • Function: • CH50 test

9. Y Y Y Y Y Y Y Classical Alternative CH50 Immune Complex Microbes C1 C3(H2O), Bb • The amount of • complement (serum dilution) that causes lysis of 50% of antibody coated Sheep RBC’s. Tests only the Classical pathway. C4 C2 C3b, Bb C4b, 2a C3b, Bb, P C3 C3a C3b C5 C5a C5b, 6, 7, 8, 9 Membrane Attack Complex - Cell Lysis

10. Y Y Y Y Y Y Y Classical Alternative AH50 Immune Complex Microbes C1 C3(H2O), Bb • The amount of • complement (serum dilution) that causes lysis of 50% of non-sensitized rabbit RBC’s. Tests only the Alternative pathway. C4 C2 C3b, Bb C4b, 2a C3b, Bb, P C3 C3a C3b C5 C5a C5b, 6, 7, 8, 9 Membrane Attack Complex - Cell Lysis

11. C-Deficiencieswhat can be done? • Hyper-immunization (PPS, neisseria) • Antibiotic prophylaxis (+/-) • IVIG prophylaxis (+/-) • Replacement/prophylaxis C1-esterase inhibitor • Check family members

12. Immunodeficiency Algorithm • 9 month old male with purulent cervical lymphadenitis and a history of pneumonia at age 6 months. A maternal grand-uncle died of acute lung infection at age 2 years in the 1950ies. • CBC/Differential ($40) • Neutrophil oxidative burst • assay - AKA NBT assay ($86) L A B S • CBC/Differential ($40) • Quantitative IgG, IgM, IgA, • and IgE ($127) • Anti-tetanus titers ($57) • Anti-pneumococcal titers pre- • & 4 wks post-Immunization • CBC/Differential ($40) • T & B cell subset analysis • ($202) • DTH reaction to Candida & • Tetanus • CH50 - tests entire • complement cascade ($83) Phagocytic B Cell T Cell Complement • Recurrent disseminated • Neisserial infections. • Pyogenic bacterial • infections. • Angioedema of face, hands, • feet, or GI tract. • Autoimmune symptoms • (Lupus). • Hisory suggestive of • autosomal dominant • inheritance. • Pneumocystis carinii • pneumonia. • Fungal infections. • GVHD (rash, abnormal LFT’s • and chronic diarrhea. • Recurrent, severe, or • unusual viral infections. • Failure to thrive. • Recurrent bacterial sino- • pulmonary infections or • sepsis, particularly with • encapsulated organisms. • Chronic or recurrent • gastroenteritis (Giardia • and Enterovirus common). • Chronic enteroviral menigo- • encephalitis. • Arthritis • Unexplained bronchiectasis. • Soft tissue abscesses or • lymphadenitis. • Infection with catalase(+) • organisms (Staph aureus, • Serratia, E. coli, Aspergillus) • Poor wound healing. • Delayed separation of the • umbilical cord. • Chronic gingivitis and • periodontal disease. • IBD • Mucosal ulcerations. D E F E C T T Y P E Combined

13. Phagocytes • Soft tissue abscesses or • lymphadenitis. • Catalase(+) organisms • (Staph aureus, • Serratia marcesans, • Aspergillus) -> CGD • Poor wound healing’ • IBD. • Delayed separation of the • umbilical cord, • Chronic gingivitis and • periodontal disease • -> LAD (1/2/3) Delves and Roitt, NEJM 343:37-49

14. Neutrophil Defects • Neutropenia – congenital, severe (Kostmann-HAX1; ELA2; XLN-WAS) • Cyclic neutropenia (ELA2) • LAD1; 2; 3 (CD18; Fructose transporter; Kindlin3) • CGD (gp91phox; p22; p47; p67phox) • WHIM (warts, hypog., myelokathexis mutation of CXCR4 (R for CXCL12)

18. Chronic Granulomatous Disease(CGD)Diagnostic Approach Killing defect in vitro Endotoxin stimmulated NBT slide test Quantitative Dihydrorhodamine – Flow test

22. Neutropenia (Primary)

24. AdhesionDefects: LAD 1 • Poor wound healing. • celulitis, no pus formaion • Delayed separation of the • umbilical cord. • Chronic gingivitis and • periodontal disease. • Mucosal ulcers. Typhlitis • . Neutrophilia • Adhesion defect • Lack of CD18 Delves and Roitt, NEJM 343:37-49

26. Testing - Phagocytes • Number: • CBC/differential (neutropenia) • Function: • Neutrophil oxidative burst (CGD) • Adhesion: LAD1,2,3 [CD18 Expression (LAD1)]

27. B Cells Delves and Roitt, NEJM 343:37-49 • Unexplained bronchiectasis. • Recurrent bacterial sinopulmonary infections. • Chronic or recurrent gastroenteritis (Giardia, • Cryptosporidium, etc.). • Chronic enteroviral menigoencephalitis. • Arthritis -> XLA

28. Immunodeficiency Algorithm 18 month old boy admitted with acute pneumonia and sepsis, due to Strep pneumoniae. He started to have upper respiratory infections at age 11 month. On examination, he had no tonsillar tissue. A maternal uncle died of paralytic polio after oral polio vaccine. • CBC/Differential ($40) • Quantitative IgG, IgM, IgA, • and IgE ($127) • Anti-tetanus titers ($57) • Anti-pneumococcal titers pre- • & 4 wks post-Immunization • CBC/Differential ($40) • T & B cell subset analysis • ($202) • DTH reaction to Candida & • Tetanus L A B S • CBC/Differential ($40) • Neutrophil oxidative burst • assay - AKA NBT assay ($86) • CH50 - tests entire • complement cascade ($83) B Cell Complement Phagocytic T Cell • Recurrent bacterial sino- • pulmonary infections or • sepsis, particularly with • encapsulated organisms. • Chronic or recurrent • gastroenteritis (Giardia • Crohn’s disease • and Enterovirus common). • Chronic enteroviral menigo- • encephalitis. • Arthritis • Unexplained bronchiectasis. • Recurrent disseminated • Neisserial infections. • Pyogenic bacterial • infections. • Angioedema of face, hands, • feet, or GI tract. • Autoimmune symptoms • (Lupus). • Hisory suggestive of • autosomal dominant • inheritance. • Soft tissue abscesses or • lymphadenitis. • Infection with catalase(+) • organisms (Staph aureus, • Serratia, E. coli, Aspergillus) • Poor wound healing. • Delayed separation of the • umbilical cord. • Chronic gingivitis and • periodontal disease. • Mucosal ulcerations. • Pneumocystis carinii • pneumonia. • Fungal infections. • GVHD (rash, abnormal LFTs, • and chronic diarrhea. • Recurrent, severe, or • unusual viral infections. • Failure to thrive. D E F E C T T Y P E Combined

29. XLA, the prototype of Ab-deficiency • Gene: Btk • Function: allows B cell development • Diagnosis: low IGs, low # B, mutation of Btk • new screening test by flow • At least 5 other genes (a.r.) • Improved tx: self infusion at home (IV or SC) • Unsolved problems: chron. Lung disease progressive neurodegeneration of unknown ethiology

30. 5 Generations of XLA patients, carriers

31. Testing – B cells & Antibodies • Number: • CBC/differential (lymphopenia) • Lymphocyte subsets (T/B/NK) • Quantitative IgG, IgM, IgA, IgE • Function: • Vaccine titers (protein & carbohydrate antigens)

32. Flow-Based Assays • Basic Subset Analysis – T/B/NK • Detailed Immunophenotyping • Disease-specific Testing • Functional Testing

33. Tagging Cells for Flow Cytometry Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y MacrophagesB Cells T Cells

34. Flow Cytometry - Mechanism Detector Cells Counter Laser Fluorescence (T Cells)

35. B cell Immunophenotyping Maturation “Memory” (CD24hi/CD38lo+) Maturation CD19, CD27, CD24, CD38, CD10 Transitional (CD24hi/CD38hi) Mature naive (CD24low/CD38+) IgA Switched Memory (IgA+/IgDlow/IgMlow) Memory Unswitched Memory (CD27+/IgD+/IgM+) Memory CD19, CD27, IgM, IgA, IgG IgG Switched Memory (IgG+/IgDlow/IgMlow) Switched Memory (CD27+/IgDlow/IgMlow)

36. 1000 100 800 85.8 80 600 60 Side Scatter % of Max 400 40 200 20 46.9 0 0 0 200 400 600 800 1000 1 10 100 1000 10000 FSC-Height XIAP-FITC 100 Ungated Lymphocytes Frequency: 100 Normal Control (Father) 80 60 % of Max 6.8 93.2 40 20 0 0 1 2 3 4 10 10 10 10 10 FL1-H: SAP- FITC 100 80 Ungated Lymphocytes Frequency: 100 60 % of Max XIAP(-) Patient 98 1.96 40 20 0 0 1 2 3 4 10 10 10 10 10 1000 FL1-H: SAP- FITC 100 800 13.8 80 600 60 Side Scatter % of Max 400 40 200 20 57.1 0 0 0 200 400 600 800 1000 1 10 100 1000 10000 FSC-Height XIAP-FITC Disease-Specific Flow Tests WASp BTK SAP XIAP FOXP3 (WAS) (XLA) (XLP1) (XLP2) (IPEX) Normal Patient

37. T Cells Generals Assassins Psychologists • Pneumocystis jirovecii pneumonia • Other fungal infections • GVHD/Autoimmunity (rash, diarrhea, LFT’s, etc.) • Recurrent, severe, or unusual viral infections (CMV, • Adenovirus, EBV, Metapneumovirus, etc.) • Failure to thrive

40. Testing – T cells • Number: • CBC/Differential (lymphopenia) • Lymphocyte subsets (T/B/NK) • Function: • T cell proliferation to mitogens and antigens • Vaccine titers

41. T cell/(sever)combined Immunodeficiency,a medical Emergency!what can be done? • Early diagnosis is important (NB screening) • IVIG, subcutaneous IG • Prophylactic antibiotics/antiviral PCP prophylaxis • No live vaccines • Hematopoietic stem cell transplantation • Gene therapy (+/-)

42. The Evolution of PIDD Knowledge • ADA Deficiency first PIDD molecularly defined in 1972 (Giblett et al.) • Approximately 300 molecularly defined disorders of the immune system • SCID – 21 genes 5th edition (2005) 3rd edition (1983) 1st edition (1973)