Humoral Immunodeficiencies

focus on B cell defects

1. H umoral I mmunodeficiencies Shobhita Katiyar JULY 20, 2016

2. PID : distribution Distribution of identified PIDs in ESID registry database

3. B cell development

4. Surface receptors on developing B cell

5. CD 19, CD21 and CD 81

6. Clinical cytometry society,2008 B cell development

7. J Clin Immunol 2015

8. B cell immunodeficiency: Warning signs

9. Question: Which is the most common antibody produced in the body? IgG IgM IgA IgE IgD

10. Question: Which is the most common antibody in circulation in the body? IgG IgM IgA IgE IgD

11. Agammaglobulinemias

12. Agammaglobulinemias

13. X Linked Agammaglobulinemia (XLA)

14. First recognized human immune deficiency Discovered in 1952 by Colonel Ogden Bruton Case report : 8-year-old boy, recurrent infections over a 4-year period - Majority of infections: pneumococcus - Bruton attempted t o vaccinate → no γ globulin was produc tion - Treated with monthly intramuscular injections of human γ globulin with significant clinical improvement - No family history Subsequent cases revealed a similar clinical phenotype with an X-linked pedigree Agammaglobulinemia.Pediatrics , 1952 , Clin Exp Immunol , 2000 . Introduction

15. Epidemiology Incidence Unknown because general population screening for the disorder is not done (1/3 new mutation ) Prevalence 1 /10,000 in the general population Variable ( 1/379,000 in USA, 1/100,000 in Norway ) Clinical and Molecular Allergy ,2008

16. Pathophysiology C ause : mutations in the human BTK gene – halts B cell development In 1993 , two groups of investigators independently / simultaneously discovered mutated gene in XLA. European group called atk gene → ammaglobulinemia tyrosine kinase American group called bpk gene → B-cell pro-genitor kinase A compromise was reached with the term ; ‘ Btk ( Bruton's tyrosine kinase ) ’ in honor of Dr. Bruton Immunological Reviews 2005 Cell 1993

17. Btk in B cell signaling

18. Btk gene & protein Contains 19 exons → 37 kb of DNA Intracellular signal transduction molecule - Member of Tec family ; 75 kDa cytoplasmic tyrosine kinase National Library of Medicine , 2012

19. BTK protein consists of 5 functional domains - Pleckstrin Homology ( PH) domain - Tec homology (TH) domain - Src homology 3 (SH3) domain - Src homology 2 (SH2) domain - Catalytic kinase (SH1) domain Mutations in all domains of the BTK gene have been shown to cause XLA . protein-protein interactions Catalytic activity Journal of Hematology and Oncology, 2013

20. Question: Which cell population will you gate for analyzing Btk expression in a suspected patient?

21. Btk expression in hematopoietic cells

22. BTK required for pre-BCR signalling BTK is found in cytoplasm, moves to membrane during B-cell activation, where upon it is phosphorylated by a number of different kinases. Association of the BTK SH2 domain with the B cell linker protein (BLNK) is required for the activation of PLC gamma by BTK Immunology,2003

23. BTK → phosphorylates a range of downstream targets → activation of cellular pathways (vital B-cell processes) Nature reviews

24. Btk Mutation > 600 different mutations in the BTK gene have been found 90 % : Single base pair substitution & insertion or deletion < 5 bp No clear correlation has been found between mutation location and clinical phenotype. 55% of males have no family history of XLA - De novo causing mutation : 15%-20% - Mother is a carrier of a disease-causing mutation : 80%-85% Female carriers of XLA can be identified by the presence of either; - non-random X chromosome inactivation in their B cells or - mutated gene (if known in the family) National Library of Medicine , 2012 .

25. Clinical Manifestations The Indian Journal of Pediatrics,2016

26. Diagnosis Family history Clinical manifestations Intermittent neutropenia can occur at the onset of an acute infection Low serum IgG , IgM and IgA level Peripheral blood CD19 B-cell counts < 2 % Laboratory investigation by Prenatal diagnosis: d etection of the mutated gene in chorionic villus or amniocentesis samples Confirmation by demonstrating; - absence of BTK protein in monocytes (flowcytometry) - detection of a mutation in BTK in DNA (sequence analysis)

27. Flowcytometry Btk expression in monocytes

28. Treatment Early diagnosis and treatment would improve the survival Intravenous immunoglobulin (IVIG) and antibiotic prophylaxis are conventional treatments → increased survival rate Genetic counselling, carrier detection, and prenatal diagnosis Gene therapy

29. Blood, 2004

30. Take home Early diagnosis and treatment - IVIG, ATB prophylaxis Defining genetic etiology Long-term follow-up : Infection

31. Common variable immunodeficiency

32. CVID Term coined in 1971 by WHO committee to separate less well defined antibody deficiency syndrome from others Causes of CVID: largely obscure; no universally accepted definition A group of antibody deficiencies that lack a more specific genetic or phenotypic classification Patients with antibody deficiency (no secondary causes for it) lacking uniform genetic defect and clinical features

33. Epidemiology Prevalence 1 in 25,000 to 1 in 50,000 Most patients are diagnosed between the ages of 20 and 40 years, approximately 20% are under the age of 20 Affects both sexes equally, boys > girls in children Blood,2012

34. CVID: Diagnostic Criteria

35. Genetics 90% sporadic cases – unknown defect 10% Familial - AD with variable penetrance(80%) - AR (20%)

36. CVID: genetic defects Arthritis Research & Therapy 2012

37. ICOS Deficiency

38. Other genetic mutation include: CD20, IKZF1,LRBA, NFKB1,NFKB2,PRKCD,TWEAK and MSH5 ↓ Cellular or humoral immune defect The Lancet,2008 Mutations

39. Mutations

40. Other genetic mutation include: CD20, IKZF1,LRBA, NFKB1,NFKB2,PRKCD,TWEAK and MSH5 Monocyte/dendritic cell defects ↓ NK cells Reduced TRECs/KRECs Signalling abnormalities: Stimulation of B cells/ plasmacytoid dendritic cells via TLR 7,8,9 is inhibited → ↓ switched memory B cells Other defects

41. Serum immunoglobulin levels and electrophoresis Full blood count : absolute count, platelet count and size Renal and liver functional tests Serum IgG antibodies to exposure and immunisation antigens Lymphocyte – T, B and NK cells; B memory( isotype , switched and naïve)→ flowcytometry Urinary protein electrophoresis Investigations for specific diseases – e.g. fungal or mycobacterial antigens Base line respiratory tests including computerised tomography British Journal of Haematology,2009 CVID : Lab approach

42. Clinical Cytometry,2008 Analysis : flowcytometry B cell subset analysis

43. Clinical Cytometry,2008 Analysis : flowcytometry T cell subset analysis

44. British journal of Hematology,2009 Clinical manifestations

45. Blood, 2008

46. Autoimmunity in CVID Others include Neutropenia Pernicious anaemia Anticardiolipin Ab Antiphospholipid syndrome Diabetes mellitus Juvenile Idiopathic Arthritis Uveitis Multiple sclerosis Systemic lupus erythematosus Autoimmune thyroid disease Lichen planus Vasculitis Vililago American Society of Hematology,2012

47. Q: An 8 year old boy p/w lower limb predominant arthritis of 4 m duration. It was not controlled with NSAIDs alone and was started on SSZ f/b Mtx in combination for it. Again, there was incomplete response and he also developed bloody diarrhoea 2 months later. He was investigated for possible PID with autoimmunity. No family history. Hb = 8gm%, TLC = 14000, N90 L6, Plt = 3.3 Lac STP = 5.6 gm , Alb. = 3.2 gm ,  IgG , A and M Imp: Panhypogamamglobulinemia Diagnosis & next step?

48. Treatment Primary treatment is antibody replacement: IVIG or SCIG IVIG: 400 – 600 mg/Kg q3-4 weeks SCIG: 100 – 150 mg/Kg/week Ch. Lung Disease and IBD: Higher doses Trough levels: 7gm/L Infection prevention: Antibiotic prophylaxis Autoimmune phenomena: Steroids, IS, Rtx Severe hematological changes (chronic transfusion need, leukopenia , Thrombocytopenia) & secondary malignancies - Stem cell transplantation

49. Selective IgA deficiency

50. Introduction IgA: first described in serum in 1953; Selective IgA deficiency: first reported in 1964 Incidence – Caucacians > Asians No well defined genetic susceptibility → AD , AR and sporadic Most abundant Ab isotype produced in body Subclasses: IgA 1 and IgA 2 → locus α 1 and α 2 on chromosome 14 Circulating IgA : monomeric - predominantly IgA 1 - Produced in BM from plasma cell Secretory IgA : dimeric - predominantly IgA 2 - Produced locally in the mucosal tissue

51. Definition Selective IgA Deficiency Male / Female > 4 yrs. of age Serum IgA < 7 mg/ dL Normal serum IgG and IgM Other causes of hypogammaglobulinemia have been excluded Normal IgG antibody response response to vaccination Partial IgA Deficiency Serum IgA > 7 mg/ dL but 2 SD below for normal of that age