PRIMARY IMMUNODEFICIENCY DISEASES

EKTA JAJODIA

Define immunodeficiency Classification B cell/Antibody deficiency T Cell/ cellular deficiency Phagocyte deficiency Complement deficiency Application of flow

cytometry Diagnostic evaluation of PID

DEFINITION

It is the absence or failure of normal function of one or more elements of the immune system

Results in immunodeficiency disease

Immunodeficiency Diseases

Primary: Usually congenital, resulting from genetic defects in some components of the immune system.

Secondary (Acquired): as a result of other diseases or conditions such as:

» HIV infection» malnutrition» immunosuppression

PRIMARY IMMUNODEFICIENCIES

Primary immunodeficiencies are inherited defects of the immune system

They are classified according to IUIS , 2013

INTERNATIONAL UNION OF IMMUNOLOGICAL SOCIETIES

(IUIS:2013)Combined immunodeficiencies

Combined immunodeficiencies with associated or syndromic features

Predominantly Ab deficiency

Disease of immune dysregulation

Defects in phagocytosis

Defects in innate immunity

Autoinflammatory disorders

Complement deficiencies

The immune system functional compartments

The B-lymphocyte/ Antibody system The T-lymphocyte/ cellular system The Phagocytic system The Complement system

Clinical Manifestations of the Primary Immunodeficiency

Diseases INFECTIOUS DISEASES AUTOIMMUNE DISEASES GASTROINTESTINAL DISEASE HEMATOLYMPHOID DISEASES

INFECTIOUS DISEASES

An increased susceptibility to infection is the hallmark of the primary immunodeficiency diseases (PID)

In most patients, this is manifested by recurrent infections

HEMATOLYMPHOID DISEASES

Anemia, thrombocytopenia, or leukopenia are seen frequently in patients with PID

Patients have increased chances of malignancy especially of lymphoid organs

CELLULAR DEFICIENCIES

CELLULAR DEFICIENCIES

SCID and Combined immune deficiency

Wiskott-Aldrich syndrome

Hyper-IgM syndrome

Ataxia-telangiectasia

Di-George syndrome

Other primary cellular immunodeficiencies

SEVERE COMBINED IMMUNODEFICIENCY (SCID)

Fatal PID

Combined absence of T and B lymphocytes

13 different genetic defects that can cause SCID

MOST COMMON TYPESSCID

XSCID (X linked )

Adenosine deaminase (ADA)

T-B+NK- phenotype SCID

Deficiency of common gamma chain of TCR (X-SCID)

Deficiency of Janus kinase 3

Deficiency of common gamma chain of TCR (X-SCID)

MC form

Common gamma chain (γc ) – a component shared by TCR and other growth factor receptors

Mutation in gene encoding γc

Result in T-B+NK- phenotype

XR – so only males are affected

Deficiency of Janus kinase 3 Mutation in gene encoding Jak3

Required for function of γc So phenotype is T-B+NK- ( same as X-SCID)

But this is AR – can affect both boys and girls

T-B+NK+ phenotype SCID

Deficiency of  α chain of IL-7 receptor

Deficiency of CD3 chains

Deficiency of CD45

Deficiency of  α chain of IL-7 receptor 3rd MC cause

Mutation in gene encoding IL-7Rα – a component of growth factor receptor

T-B+NK+ phenotype

However, B cells do not function due to lack of T cells

AR

Deficiency of CD3 chains

CD3 is a receptor complex on T cells and c/o : CD3δ , CD3ε and ζ-chain 

3 forms of SCID are due to mutations in genes encoding these 3 chains of CD3 complex

Deficiency of CD45 T-B+NK+ phenotype

AR

T-B-NK- phenotype SCID

Adenosine deaminase deficiency

Reticular dysgenesis

Adenosine Deaminase deficiency 2nd MC SCID Mutation in gene encoding ADA enzyme

ADA is essential for T-cell function : Its absence cause accumulation of toxic metabolites within lymphocytes that cause cells to die

T-B-NK- phenotype

T-B-NK+ phenotype SCIDRAG1 and RAG2 gene mutation

Artemis deficiency

Cernunnos deficiency

Ligase 4 deficiency

Life threatening infections : most dangerous organisms are-

1. Pneumocystis jiroveci2. Chicken pox3. CMV4. Herpes simplex

Live vaccines should not be given to SCID patients : they may contract infection from vaccine viruses

So if family history of SCID is +ve : avoid live vaccines

Diagnosis

Easiest way to diagnose: Absolute lymphocyte count(ALC)

Normally ALC >4000/cu mm; 70% of which are T cells

SCID have ALC < 1500/cu mm

If ALC found low Repeat test again If low again specific tests to be done to count

T cells and measure T cell function

COMBINED IMMUNODEFICIENCIES

Group of rare genetic disorders that result in combined immunodeficiency but do not reach a clinical severity level to qualify as SCID

7 types

Bare lymphocyte syndrome

Purine nucleosidase phosphorylase deficiency

ZAP70 deficiency

CD25 deficiency

Cartilage hair hypoplasia

Coronin 1A deficiency

MHC classI deficiency

HYPER IgE SYNDROME

Aka Job syndrome/ Buckley syndrome Characterised by :

1. Recurrent eczema2. Skin abscesses: particularly by S.aureus3. Lung infection4. Eosinophilia5. Increased IgE levels

• AD• STAT3 mutation• Connective tissue and

skeletal abnormality• Typical facial appearance,

hyper extensibility of joints, bone fractures after minor trauma

TYPE1

• AR• DOCK8 mutation• Recurrent and severe viral

infection esp by herpes and molluscum

• Do not have connective tissue or skeletal abnormality

TYPE2

Diagnosis

Increased IgE levels Normal IgG,A,M Increased peripheral blood eosinophils HIES scoring system by National Institute of

Health(NIH) :Score : 0-15= unaffected 16-39= possibly affected 40-59= probably affected >60 = definitely affected

Scoring system is esp for the diagnosis of Type 1 HIES

Definitive diagnosis : genetic analysis of STAT3 and DOCK8 genes

WISKOTT ALDRICH SYNDROME

TRIAD :

1.Increased tendency to bleed: due to small, dysfunctional and decreased number of platelets

2.Recurrent infection

3.Eczema

Associated with WAS gene mutation

Was gene produces WAS protein (WASp)

If mutation is severe: complete absence of WAS protein : known as classic WAS

If mutation is mild : some mutated WAS protein present : known as milder form of WAS

Diagnosis1. Platelet abnormality : decreased number and

small size : characteristic

2. Increased IgE

3. Sequencing of WAS gene to identify mutation : definitive diagnosis

4. Determine WAS protein expression in blood cells

HYPER IgM SYNDROME

Inability to switch from production of Ab of IgM type to Abs of IgG, A or E types

Normal B cells can produce IgM on their own but require Help from T cells to switch from IgM to IgG,A,E

HIGM results from defect in interaction between T and B cells

Genetic defects

CD40L def.

CD40 def.

AID def.

UNG def

NEMO defect

CD40L (CD154) : deficiency of this ligand is the most common form of HIGM syndrome

XR So only boys are affected

Defect in NEMO gene : Known as ectodermal dysplasia

Associated with sparse hair and conical teeth

DIAGNOSIS Characteristic : failure to express CD40L on

activated T cells – can be assessed by flow cytometry

CD40 L deficiency is due to mutation in CD40L gene

If gene is normal and CD40L is deficient : not HIGM syndrome

So, for exact diagnosis : demonstration of CD40L gene mutation

ATAXIA TELANGIECTASIA Mutation in ATM gene (11q) This gene is required for cell repair after

DNA damage 2 important presenting features:

• Abnormality in cerebellum• Can be confused with

cerebral palsy(CP)• In AT neurologic

deterioration occurs with age (but not in CP)

• Needs wheelchair by 10-12 yrs age

ATAXIA

• Dilated and corkscrew shaped vessels esp in white of eyes

TELANGIECTASIA

DIAGNOSIS

Clinical feature is very important but difficult to diagnose at early age as telangiectasia occurs only by 5 yrs of age

Most imp test : AFP levels in blood – 95% have increased levels

Other tests:Absence of ATM protein on western blot

Abnormal DNA sequence (mutation) of ATM gene

Increased chromosomal breakage after exposure of blood cells to X rays

Increased CA 125

DI GEORGE SYNDROME

Defect : microdeletion in 22q11.2

So aka : 22q11.2 syndrome Aka : velocardiofacial syndrome, conotruncal

anomaly face syndrome

MC microdeletion syndrome 2 imp gland abnormality

• Thymic hypoplasia• T-cell number and

maturation defect• So, increased

susceptibility to infections

THYMU

S GLAND

• Underdeveloped and hypoparathyroidism occurs

• Hypocacemia occurs

PARATHYROID GLAND

DIAGNOSIS

FISH analysis to identify 22q11.2 deletion

OTHER PRIMARY CELLULAR IMMUNODEFICIENCIES

Chronic mucocutaneous candidiasis

Cartilage hair hypoplasia

X-linked lymphoproliferative syndrome 1 & 2

Veno occlusive disease

Schimke syndrome

X-linked immune dysregulation and polyendocrinopathy syndrome (IPEX)

Hoyeraal-Hereidarson syndrome (dyskeratosis congenita)

Immunodeficiency with centromeric instability anomalies (ICF)

Comel-netherton syndrome(C-NS)

Chronic mucocutaneous candidiasis

Severe and persistent candida infection of mucous membrane, scalp, skin and nails

MC abnormality : negative delayed hypersensitivity skin test to candida Ag despite widespread candidial infection

2 important gene defects

STAT1 IL-17

Hereditary form of CMC

APECED syndrome : autoimmune polyendocrinopathy candidiasis ectodermal dysplasia syndrome

Associated with AIRE gene defect

• X-linked lymphoproliferative syndrome 1 & 2

XLP1: SH2DIA

XLP2: XIAP• X-linked immune dysregulation

and polyendocrinopathy syndrome (IPEX)

FOX P3

• Immunodeficiency with centromeric instability anomalies (ICF)

DNMT3B

• Schimke syndromeSMARCL1

ANTIBODY DEFICIENCIES

ANTIBODY DEFICIENCIES

Agammaglobulinemia

Common variable immune deficiencies

Selective IgA deficiency

IgG subclass deficiency

Specific antibody deficiency

Transient hypogammaglobulinemia of infancy

Other Ab deficiency disorders

AGAMMAGLOBULINEMIA

• Aka bruton’s/congenital agammaglobulinemia

• BTK gene defect (on X chr)• Pre B cells cannot develop into immature

B cells• So no Ab formation

XLA

• Both boys and girls are affected• Gene defect : μ heavy chain, λ5, Igα, Igβ,

BLNK• These encode proteins that work with

BTK to convert pre B to mature B cellsARA

COMMON VARIABLE IMMUNE DEFICIENCY

• Since it is relatively frequent

COMMON

• Since degree and type of Ig deficiency varies from patient to patient

VARIABLE

• Granulomatous, lymphoproliferative, interstitial lung disease

GLILD

DIAGNOSIS

Low levels of serum Igs : IgG, IgA, IgM CHARACTERISTIC

Normal number of B-cells, but these cells fail to mature into plasma cells – can be assessed by flow cytometry based immunophenotyping of

B-cells

Diagnostic criteria by European Society of immunodeficiency (ESID)

Plasma IgG levels<2 SD below mean for age + marked decrease in eithe IgA or IgM

Age of onset of immunodeficiency >2yrs of age

Absent isohemeagglutinins or poor response to vaccines

Defined causes of hypoagammaglobulinemia have been excluded

SELECTIVE IgA DEFICIENCY Undetectable level of IgA in blood and

secretions, but no other Ig deficiency

IgA

Present in serum

Present in secretions (known as secretory IgA)

SECRETORY IgA

Composed of:

•IgA dimer•J-chain•Secretory piece

DIAGNOSIS

Undetectable levels of IgA (<5-7mg/dl)

Normal levels of other Igs

Normal B and T lymphocytes

•Nomal IgA levels in adults: 50-200mg/dl

•In 20% cases associated with low levels of IgG2 or IgG4 subclass

Subjects >4 years of age with serum IgA consistently <7mg/dl but normal IgG and IgM levels are considered Selective IgA deficient

Defect – impaired differentiation from naïve B-cells to IgA producing plasma cells

Patients have anaphylactic reactions during infusion of blood products due to sensitisation to IgA which behaves as foreign antigen

IgG SUBCLASS DEFICIENCY

IgG is 2nd most common circulating protein 4 IgG subclasses : IgG1,2,3,4

When 1 or more of these subclasses are persistently low and total IgG is normal, a subclass defiency is present

IgG(60-70%) > IgM> IgA> IgD > IgE

IgG1(20-30%) > IgG2 (5-8%) > IgG3 (1-3%) > IgG4

MC is IgG2 or IgG3 subclass deficiency

May be associated with :1. IgA deficiency2. WAS3. A-T

TRANSIENT HYPOGAMMAGLOBULINEMIA OF INFANCY

Unborn baby makes no IgG At 6 months of pregnancy, maternal IgG via

placenta goes to fetus

This increases during the last trimester of pregnancy

At term, baby has IgG level equal to that of mother

This IgG dissapears completely by 6 months of age

Fetus doesn’t get any maternal IgM, A or E as they do not cross placenta

So, if IgM is found: s/o in utero infection

Between 3-6 months of age :• Maternal IgG starts falling• Infant’s IgG starts forming• As a result IgG levels are low: k/a physiologic

hypogammaglobulinemia of infancy

In some infants, period of hypogammaglobulinemia is more severe and

prolonged beyond 6 months of age : k/a Transient hypogammaglobulinemia of infancy

Definition :1. Infants >6 months2. IgG< 2 SD below the mean for age (typically

<400mg/dl)

Mostly IgG correction occurs by 2 yrs of age

OTHER AB DEFICIENCY DISORDERS

Antibody deficiency with normal or elevated Igs

Selective IgM deficiency

Immunodeficiency with thymoma (Good’s syndrome)

Transcobalamin II deficiency

Drug induced Ab deficiency

Warts, hypogammaglobulinemia, infection and myelokathexis syndrome (WHIM)

Kappa chain deficiency

Heavy chain deficiency

Post meiotic segregation disorder(PMS2)

Unspecified hypogammaglobulinemia

PHAGOCYTE DEFICIENCY

CHRONIC GRANULOMATOUS DISEASE

Phagocytes (neutrophils and monocytes) form phagosome within cell

Formation of NADPH oxidase complex

generate burst of reactive oxygen species

activates proteases which destroy ingested bacteria

Here, mutations occur which affect formation of NADPH oxidase

2 different types of CGD:

1.X-linked : MC form only boys affected mutation in CYBB gene

2. AR forms : Mutations in CYBA, NCF1, NCF2 or NCF4 genes

INFECTIONS

Staphylococcus

aureus

Burkholderia cepacia complex

Serratia marcescensNocardia

Aspergillus

DIAGNOSIS Any patient of any age with a CGD type infection

should be tested for CGD unless there is a good reason not to

Previously used test – Nitroblue Tetrazolium slide Test (NBT)

Most accurate test – Dihydrorhodamine reduction test (DHR) : Measures amount of H2O2 produced in phagocytes

NITROBLUE TETRAZOLIUM SLIDE TEST Neutrophils make reactive

oxygen species which reduces colorless NBT dye into a blue colored formazan salt

In CGD these reactive oxygen species are not formed,

so dye is not reduced to blue color

OTHER PHAGOCYTIC CELL DISORDERS

Neutropenias

Phagocyte killing defects

Leukocyte adhesion deficiencies

Specific granule deficiency

Glycogen storage disease typeIb

Beta-actin deficiency

Chediak higashi syndrome

Griscelli syndrome

Neutropenia

<500cells/microl (normally >2000cells)

DIFFERENT FORMS OF NEUTROPENIA

•Severe congenital neutropenia (Kostmann syndrome) HAX1 gene

•Cyclic neutropeniaELA2 gene

•Benign chronic neutropenia•Immune neutropenia------

Leukocyte adhesion deficiencies

LAD1• CD18 mutation

LAD2• Mutation in

enzymes that attach fucose to proteins

LAD3• FERMT3 mutation

CHEDIAK HIGASHI SYNDROME (CHS) Mutation in LYST gene

Characteristics: 1. Giant granules within neutrophils2. Partial albinism3. Frequent infections

Most patients reach a stage known as : accelerated phase/lymphoma like syndrome – triggered by EBV

Here, the defective WBCs divide uncontrollably and invade many organs

GRISCELLI SYNDROME

•MYO5A gene defectGS TYPE1

•RAB27A gene defect•a PID disorder•Partial albinism, recurrent infection, neutropenia and thrombocytopenia

GS TYPE2

•Melanophilin (MLPH) gene defectGS TYPE3

Difference from CHS :

1. Morphology of neutrophils : Normal in GS (giant granules in CHS)

2. Leukocyte specific protease activity : Normal in GS (low in CHS)

COMPLEMENT DEFICIENCIES

Complement deficiencies

Classical pathway : Activated by Abs that are bound to Ags

Lectin pathway : initiated by Mannose binding lectin (MBL), Ficolins and collectin; associated with enzyme- MASPs (MBL- associated serine proteases)

Alternative pathway : initiated by fragments of C3, Factor B, Factor D, and properdin

Terminal pathway : forms MAC – membrane attack complex

Components are - C3, C5,6,7,8,9

Terminal complement complex (TCC) : fluid phase of MAC

Found in circulation when complement is activated

Useful lab marker for complement activation

Deficiency of classical pathway

Def. in C1q, r, s, C2, C4 : ass with SLE and RA

Deficiency in C1 esterase inhibitor: it inactivates classical pathway component to prevent from uncontrolled activation

Its def. causes hereditary angioedema

Deficiency in lectin pathway

Def. in MBL, M-ficolin, L-ficolin, H- ficolin, MASPs :

Increased bacterial infection

Deficiency of alternative pathway

Factor B and Factor D def : very rare

Properdin : only X-linked complement protein

Synthesised by monoctes, granulocytes and T cells

Def. causes increased susceptibility to Neisseria, esp; N. meningitidis

Factor H : it is alternative pathway control protein

If def. – uncontrolled activation of alternative pathway and so depletion of C3

Associated with atypical HUS(aHUS) and age related macular edema (AMD)

APPLICATION OF FLOW CYTOMETRY

This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA – phorbol myristate acetate) can be oxidized by hydrogen peroxide, produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry.

Rho 123 is excitable at 488nm and emits at 515 nm

How to diagnose PIDs? First Step:

Think of PID Second Step:

Think of PID Third Step:

Think of PID

Careful look at the CBC

Initial laboratory screening of PID CBC including TLC, DLC, Platelets (size) Quantitative serum Immunoglobulins (nephelometry)-

IgG, IgA, IgM +/- IgE (ELISA) Lymphocyte subset analysis by flowcytometry for

Quantitation of total T cells (CD3+) T cell subsets (CD4+, CD8+) B cells (CD19+, CD20+) NK cells (CD16+, CD56+) HLA-DR to rule out MHC II deficiency Dihydrorhodamine to rule out CGD

Isohemagglutinin titres IgG antibodies to

Known exposure (VZ) Known Immunizations (tetanus, diphtheria, Hib, meningococcus, polio,

rubella).

Comprehensive Laboratory Evaluation B cell deficiency Quantitative IgG, IgM, IgA, IgE IgG subclass B cell numbers in peripheral blood (CD19, CD20) Isohemagglutinin titres Antibody response to test immunizations, e.g. D/T,

Pneumovax/Typhum Vi. Antibody response to neoantigens e.g., KLH, bacteriophage

ØX174 In vitro IgG synthesis by mitogen-stimulated PBL or purified B

cells cultured in the presence of anti-CD-40 and lymphokines In vitro proliferation of B cells in response to anti-CD40 and IL-4

Biopsies: rectal mucosa, lympho nodes (if appropriate) Molecular and mutation analysis (e.g. Btk, µ heavy chain)

Comprehensive Laboratory Evaluation T cell deficiency Absolute lymphocyte count T cell, T subset, and NK cell enumeration (CD3, CD4, CD8; also CD16,

56) Delayed-type hypersensitivity skin tests (only in older children and

adults) (Candida, tetanus toxoid, mumps). In vitro proliferation of lymphocytes to mitogens (PHA, ConA), allogenic

cells, and specific antigens (Candida, tetanus toxoid). Production of cytokines by activated lymphocytes (ELISA or flow) Expression of activation markers (e.g. CD40L, CD69) and lymphokine

receptors (e.g. IL-2Rγc, IFN- γR) after mitogenic stimulation Enumeration of MHC I and MHC II expressing lymphocytes Chromosome analysis (probe for 22q11) Enzyme assays (ADA, PNP). Caution: ? Recent transfusion Biopsies: Skin, lymph node, thymus (if appropriate) Molecular and mutation analysis (e.g. CD40L, γc chain, Jak 3, ZAP-70)

Comprehensive Laboratory EvaluationDeficiency of Phagocyte system

Absolute neutrophil count (serially to rule out cyclic neutropenia) WBC turnover Anti-neutrophil antibody Bone marrow biopsy Chemotaxis, assessment of adhesion in vivo and in vitro CD11/CD18 assessment for LAD1 (flowcytometry) Bombay phenotype/Sialyl Lewis-X/ CD15 (LAD2) Phagocytosis (Baker’s yeast) NBT Slide test; metabolic bursts (DHR flowcytometry);

chemiluminescence; bacterial assays Enzyme assays: MPO, G6PD, Glutathione peroxidase, NADPH

Oxidase Mutation analysis (e.g. gp91phox; p22phox; p47phox; p67phox; β integrin)

Complement deficiency

CH50 : The CH50 is a screening assay for the activation of the classical complement pathway

Sheep RBCs are coated with rabbit anti sheep RBC Ab. To it test serum is added to see if complement can lyse these RBCs

If complement is absent : CH50 will be 0 If complement is decreased : CH50 will decrease AH50 Analysis of quantity and function of C components Chemotactic activity of complement split products, e.g., C3a,

C5a

CONCLUSION Immunodeficiency disorders are fairly infrequent

Some are transient with improvement over time

More severe forms of immunodeficiency are associated with shortened life span without bone marrow transplantation

A genetic cause has been identified for a substantial portion of these disorders

Treatment options incude:Prophylactic antibioticsIV immunoglobulinStem cell or bone marrow transplantationNew biologicalsGene therapy

Outcomes for children with immunodeficiency dependent depends on the following:Timely recognitionAdequate therapy and surveillanceThe nature of the underlying disease

Which type of PID is associated with this cartoon

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