43 Immunodeficiencies II Immunology 297 September 9 2015

#43 Immunodeficiencies II Immunology 297 September 9, 2015 Ikuo Tsunoda, MD, Ph. D. Department of Microbiology and Immunology LSUHSC E-mail: itsunoda@hotmail. com Homepage: http: //tsunodalaboratory. web. fc 2. com/

Thymic defects with additional congenital anormalies (Table 2. 3 a) • Failure of thymus to undergo normal development affects the development of all populations of T cells • Di. George syndrome, described by Dr. Angelio Di. Geroge in 1968 • Deletion of region on chromosome 22 q 11. 2 can result in complete absence of a thymus: can be explained by a deletion of a transcription factor, T box-1 (TBOX 1) • The result is immunodeficiency, nearly complete T cell defect, and lack of T cell-dependent B cell activation • Normal level of serum immunoglobulin • Defective development of structures that develop from the third and fourth pharyngeal pouches during fetal life: absent parathyroid glands, abnormal development of great vessels

Pharyngeal Pouches https: //www. youtube. com/watch? v=Wi. E 7 LJu 3 AL 4

Pharyngeal pouches. paired evaginations of embryonic pharyngeal endoderm, between the pharyngeal arches, extending toward the corresponding ectodermally lined pharyngeal grooves; during development they evolve into epithelial tissues and organs, such as thymus and thyroid glands. SYN: branchial pouches, endodermal pouches.

Di. George syndrome • 22 q 11. 2 deletion syndrome = velocardiofacial syndrome = Di. George syndrome Microdeletion; a submicroscopic loss of a segment of DNA of varying size, typically several kilobases long • Incidence; 1 in 2000 - 4000 live births • Craniofacial and cardiovascular anomalies, immunodeficiency, short stature and hypocalcaemia Mouse chromosome 16 • Cognitive and behavioral impairments and a high risk for developing schizophrenia • Mouse models of the 22 q 11. 2 microdeletion

Di. George syndrome • Also can result in facial abnormalities including dysplasia of ears and mouth, and abnormally long distance between the eyes. Differing phenotypes occur because the mutation can (but does not always) affect the development of multiple organs: thymus parathyroid gland heart outflow vessels facial deformity CATCH 22 syndrome: cardiac, abnormal faces, thymic hypoplasia, cleft palate, hypocalcemia

Immune disorders involving the thymus Nude (nu/nu) mice : • Hairless mouse spontaneously generated in a mouse facility • Athymic or vestigial thymus, so very few T cells. • In both mice and humans, mutations in FOXN 1 (also known as WHN), transcription factor selectively expressed in skin and thymus • FOXN 1 is necessary for the differentiation of thymic epithelium and the formation of a functional thymus • B cell development is normal

Winged helix deficiency (nude) (Table 2. 9) Nasal dystrophy, alopecia of scalp, eyebrows, eyelashes

FOX: forkhead box The crystal structure of the forkhead domain: “winged box” owing to its doublewing structure like a butterfly

SCID mouse and nude mouse Nude mice no thymus, B cells+, few T cells defect in the gene for Whn, a transcription factor required for terminal epithelial cell differentiation, normal bone marrow SCID mice Defect in bone marrow, not in the thymus

Reciprocal grafts of thymus and bone marrow between SCID and nude mice Nude bone marrow precursors develop normal in a scid thymus Transplanting a scid thymus into nude mice leads to T-cell development SCID bone marrow cannot develop T cells, even in a wild-type recipient

Hyper Ig. E syndrome (HIES, Table 2. 5) • Autosomal dominant hyper-Ig. E recurrent infection syndrome • Davis et al. (1966) called the disorder 'Job’s syndrome' because of phenotypic similarity to the biblical figure Job: 'Satan. . . smote Job with sore boils from the sole of his foot unto his crown' (Job 2: 7) • Defect in STAT 3, which is activated downstream of IL-6 and IL-23 • Deficient Th 17 differentiation • Defective neutrophil recruitment • Bacteria and fungal infections http: //www. omim. org/entry/147060? search=147060&highlight=147060

Epithelium Neutrophil Dendritic cell http: //downloads. info 4 pi. org/pdfs/Inmunocytes-against-Candida---The-importance-of-our-TH 17 -army. pdf

HIES: Hyper Ig. E syndrome

Table 3. Predominantly antibody deficiency CVID

• Immunoglobulin levels in newborn infants fall to low levels at about 6 month of age • Ig. G is actively transported across the placenta from the mother during gestation • After birth, Ig. G production does not begin for 6 months • Ig. G levels are low from the age of 3 months to 1 year

Endothelial cells of fetal capillary Syncytiotrophoblast Maternal blood Villi to the fetal circulation

Fc. Rn: neonatal Fc receptor for Ig. G

Syncytiotrophoblasts are bathed in maternal blood and internalize serum containing maternal Ig. G. Fc. Rn is expressed in the internal vesicles of the syncytiotrophoblast. On acidification in the endosome, Fc. Rn binds to maternal Ig. G and transcytoses it to the fetal circulation where it is released at physiological p. H.

B cell immunodeficiencies • Encapsulated bacteria (Haemophilus influenza, Pneumococcus spp. , etc. . ) have polysaccharide capsules that are not bound by pattern recognition receptors on macrophages and neutrophils, and therefore are not phagocytosed directly • Thus, antibody and complement are critical for binding the bacteria and initiating opsonization. • Antibody is also critical for some viruses, particularly those that infect the gut (enteroviruses)

X-linked agammaglobulinemia (Table 3. 1 a) • The first description of an immunodeficiency disease was by Ogden C. Bruton (1952) in a child that failed to produce antibody • The disease was later termed: Bruton’s X-linked agammaglobulinemia (XLA) • Recurrent bacterial infection, such as Streptococcus pneumoniae, and chronic viral infections, such as hepatitis B and C, poliovirus, echovirus, coxsackie viruses and adenovirus Primary role for secretory Ig. A in host defense (T cells are normal) • Disease is caused by a mutation in a protein tyrosine kinase important for B cell signaling: Btk: Bruton’s tyrosine kinase • During the first 6 to 9 months, remain well by maternally transmitted Ig. G antibodies, thereafter, repeated infections with extracellular organisms, unless given prophylactic antibiotics or γ-globulin therapy

Btk function in B cell development Btk signaling from the pre-B cell receptor is required for development. CD 79 a, Table 3. 1 d CD 79β, Table 3. 1 e Table 3. 1 a Table 5. 5 b Table 2. 14

BTK gene is important for B-cell development • In XLA male, B cell development arrest • In females, one of the two X chromosomes in each cell is permanently inactivated. Choice of inactivation is random • In females, each cell has one active chromosome and one inactive chromosome. If the mutation is on the inactive copy, then there is no effect. • If the wild-type btk gene is on inactivated chromosome, no development. All B cells have the normal X chromosome. • In monocytes, equal mixture of normal and BTK mutant X chromosomes (BTK gene is required only for B cell development) in carrier

(A) Among lymphocyte-gated cells, only CD 20+ B cells are Btk+ (B) Monocytes are also Btk+, while neutrophils are Btk- (all) CD 20+ B cells are Btk protein+ No B cells Btkcells B cells and monocytes are Btk protein+ Btk+ cells

Many immunodeficiencies are X-linked (Table 5. 3 a) (Table 8. 16) (Table 2. 1 a) (XSCID, Table 1. 1 a) (Btk-, Table 3. 1 a) (Table 3. 3 a, Table 1. 3) • Most gene defects in PIDs are recessive • Many are caused by gene mutations on the X chromosome • All male with defective X chromosome gene show the disease, as males have only one X chromosome • Female carriers with one defective X chromosome are usually healthy

Hyper Ig. M syndrome (Table 3. 3) • Normal B- and T-cell development, normal or high levels of serum Ig. M (as high as 10 mg/ml; normal levels are 1. 5 mg/ml) • Limited Ig. M antibody responses against antigens that require T-cell help • Produce only very low levels of other classes of antibody because of problems with Ig class switching. • Patients are highly susceptible to infection with extracellular pathogens • At least six different gene mutations cause hyper Ig. M syndrome • Most common form: X-linked hyper Ig. M syndrome = CD 40 ligand deficiency (Table 3. 3 a)

X-linked hyper Ig. M syndrome • Opportunistic infection, increased risk of malignancy • Deficiency in CD 40 ligand (CD 154) on activated CD 4+ T cells • Class switching and formation of memory B cells both require contact with CD 40 L on helper T cells • B cells develop normally, but cannot become fully activated by most antigens • Treatment: bone marrow transplant, intravenous Immunoglobulin (IVIG) administration 1. 2. Antigen bound by surface immunoglobulin on the B cell is internalized and returned to the cell surface as peptides bound to MHC class II Helper T cells recognize the peptide and activate B cells via interaction between CD 40 L on T cells and CD 40 on B cells

• Interaction of B cells and helper T cells leads to CD 40 L expression on T cells and IL-4, 5, and 6 production, which drive the proliferation and differentiation of B cell into plasma cells and memory cells • The second signal required to activate antibody production against thymusindependent antigens is provided by recognition of microbial constituent or by extensive membrane Ig. M cross-linking

Hyper Ig. M syndrome Most common form: X-linked hyper Ig. M syndrome Formation of germinal centers requires B cell activation by helper T cells

FIGURE 46. 2 The role of the CD 40 ligand (CD 154) in B cell class switching. The CD 154 gene is mutated in X-linked hyper immunoglobulin M (Ig. M) syndrome. Thus, this is a T cell, not a B cell, defect. IL, interleukin. (From Allen RC, Armitage RJ, Conley ME, et al. CD 40 ligand gene defects responsible for Xlinked hyper Ig. M syndrome. Science. 1993; 259: 990) Fundamental Immunology 6 th edition

Selective Ig. A deficiency (Table 3. 4 g) • The most common inherited form of immunoglobulin deficiency : 1 in 333 reported among some blood donors • Normal numbers of s. Ig. A-expressing B cells, but decreased synthesis or release of Ig. A • Low serum Ig. A < 50 μg / ml (normal 2 to 4 mg / ml) • Normal or elevated levels of Ig. M and Ig. G • The absence of Ig. A predisposes to some types of infections (particularly respiratory), but many with an Ig. A deficiency are outwardly “normal”. • The cause of selective Ig. A deficiency is unknown • Some patients have mutations in TACI (one of the three types of receptors for BAFF and APRIL) (Table 3. 2 g)

Table 4. Diseases of immune dysregulation

Some deficiencies can lead to lymphoproliferative diseases (Table 4. 1) • Familial hemophagocytic lymphohistiocytosis (FHL or FHLH) is caused by an inherited deficiency of perforin • Hemophagocytosis: ingestion of red blood cells by macrophages • Pfp-/- mice infected with some types of viruses result in a disease similar to FHL because the immune system is uncontrolled demonstrates that perforin plays an important role in regulating the immune response • Hemophagocytic lymphohistiocytosis (HLH) =Hemophagocytic syndrome (HPS) =Macrophage activation syndrome

• In FHL, mutations prevent NK cells and cytotoxic T cells from releasing their cytoplasmic granules, which leads to uncontrolled proliferation of lymphocytes and macrophages • These cells phagocytose blood cells and release huge amounts of proinflammatory cytokines • Cytokine burst explains the inflammation, fever and systemic illness • T cell and macrophage infiltration in liver, spleen, lymph nodes, bone marrow, and central nervous system • T cells and macrophages respond strongly to microbes to compensate for the CTL and NK cell defects?

• • • Anemia Thrombocytopenia Hemophagocytosis in bone marrow, spleen, lymph node Increased cytokine release: interferonγ, TNF, IL-6, IL-10, macrophage colony -stimulating factor (M-CSF) Treatment: control the cytokine burst by chemotherapy and immunotherapy with etoposide, corticosteroids and cyclosporine, followed by bone marrow transplantation

Hemophagocytic lymphohistiocytosis (HLH) Hemophagocytic syndrome (HPS)

Genetic hemophagocytic lymphohistiocytosis =primary hemophagocytic syndrome • Familial hemophagocytic lymphohistiocytosis (FHL) (Table 4. 1. 1) FHL 1: unidentified gene on chromosome 9 FHL 2: perforin (PRF 1) mutation FHL 3: Munc 13 -4 (UNC 13 D) mutation FHL 4: syntaxin 11 (STX 11) mutation FHL 5: syntaxin binding protein (STXBP) 2 (Munc 18 -2) mutation All four proteins are involved in the granule-mediated cytotoxic pathway of lymphocytes • FLH with hypopigmentation (Table 4. 1. 2) Chediak-Higashi syndrome 1: LYST (=CHS 1) mutation Griscelli syndrome 2: RAB 27 A mutation LYST and RAB 27 A: role in vesicle trafficking in CTL Hermansky-Pudlak syndrome 2 (HPS 2), AP 3 B 1 mutation • (X-linked lymphoproliferative syndrome: SH 2 D 1 A mutation) NK cell inhibition leads to severe EB virus infection and sustained proliferation of CTL (Table 4. 2 a)

Chediak-Higashi syndrome • A defect in LYST (=CHS 1), which is involved in intracelluar vesicle formation and trafficking • Reported by Moises Chediak (1954) and Ototaka Higashi (1956) • A failure of lysosomes and phagosomes to fuse properly • Phagocytes have enlarged granules and impaired intracellular killing ability • Partial albinism, abnormal platelet function, severe http: //www. jpeds. or. jp/saisin_100604. html immunodeficiency http: //www. nurs. or. jp/~academy/igaku/t 1241. htm Ototaka Higashi, 東音高

IPEX: immune dysregulation, polyendocrinopathy, enteropathy, Xlinked syndrome (Table 4. 3 a) • Mutations in the forkhead box P 3 (FOXP 3) gene on Xp 11. 23 • FOXP 3 is expressed on CD 4+CD 25+ regulatory T cells (Tregs) • Lack of (and/or impaired function of) CD 4+CD 25+ T regs • Fatal X-linked recessive immunologic disorder • Multisystem autoimmunity: early onset type I diabetes mellitus, severe enteropathy with watery, often bloody diarrhea associated with eosinophilic inflammation and an eczematous dermatitis • Recurrent infections caused by Enterococcus and Staphylococcus species

CD 4+ regulatory T cells (Treg) • Suppress effector T cells to prevent autoimmunity • Multiple Treg populations • Foxp 3+ Treg; FOXP 3 (forkhead box P 3)+, CD 4+, CD 25 (IL-2 receptor α-chain)+, CTLA-4 (CD 152)+, GITR (glucocorticoid-induced TNF receptor)+, CD 127 (IL-7 Rα) -

Sakaguchi et al. Cell. 2008, 133: 775 -87

FOXP 3 mutations in IPEX • Immune dysregulation; autoantibody • Polyendocrinopathy; diabetes, thyroiditis • Enteropathy; diarrhea (autoimmune enteropathy) • X-linked; male patients Scurfy mice • Lack Foxp 3 and Treg • Scaly skin rash • Diarrhea • Lymphadenopathy, lymphocytic infiltration in organs • Affect male mice Sakaguchi et al. Cell. 2008, 133: 775 -87

DEREG mice / Foxp 3 -DTR mice • Foxp 3+Treg express diphtheria toxin receptor (DTR) • Administration of diphtheria toxin (DT) into mice depletes Treg selectively • DT administration lead to splenomegaly, lymphadenopathy, and inflammation in organs Lahl et al. , J Exp Med, 2007, 204, 57 -63

Mechanisms of suppression by Treg Sakaguchi et al. Cell. 2008, 133: 775 -87 “How many mechanisms do regulatory T cells need? ” Dario A. A. Vignali Eur. J. Immunol. 2008. 38: 901– 937

Case 17 Autoimmune Polyendocrinopathy. Candidiasis-Ectodermal Dystrophy (APECED) = Autoimmune polyglandular syndrome (APS)-1 (Table 4. 4 a)

Autoimmune regulator (AIRE) gene encodes a transcription factor involved in the presentation of tissuerestricted antigens during Tcell development in the thymus

Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) = Autoimmune polyglandular syndrome (APS)-1 (Table 4. 4 a) • Mutation in AIRE • Triad: Addison disease, hypoparathyroidism, mucocutaneous candidiasis

ALPS 1 A ALPS 1 B Animal Model • The recessive lymphoproliferation (lpr) phenotype and the generalized lymphoproliferative disease (gld) phenotype are mouse models of aberrant T-cell accumulation • In lpr mice, a splicing defect in the Fas gene results in greatly decreased expression of Fas. • In gld mice, a point mutation in the C terminus of Fasl impairs its ability to interact successfully with its receptor • These mutations lead to a failure of apoptosis and complex immune disorders in lpr and gld mutant mice that are analogous to the human disorders ALPS 1 A and ALPS 1 B.

Table 5. Congenital defects of phagocyte number, function or both OMIM# 601604 MSMD: Mendelian susceptibility to mycobacterial disease

Immunodeficiencies of phagocytic cells Phagocyte immunodeficiencies can be grouped into 4 types: 1. Table 5. 1. Defect of neutrophil function » Phagocyte number↓ (neutropenia): Development defect in gene required for myeloid progenitor cell differentiation 2. Table 5. 2. Defects of motility » Phagocyte adhesion ↓ 3. Table 5. 3. Defects of respiratory burst » Killing ↓ 4. Table 5. 4. Mendelian susceptibility to mycobacterial disease (MSMD) » Interferon-γ signaling pathway ↓: susceptibility to Mycobacteria and Salmonella

Leukocyte adhesion deficiency (LAD) (Table 5. 2) • The most significant problem is the inability of cells to attach to the vascular endothelium and migrate to sites of inflammation • LAD type 1 (LAD-1): deficient expression of β 2 integrins due to CD 18 gene mutations • LAD type 2 (LAD-2): absence of sialyl Lewis X: ligand on neutrophils required for binding to E-selectin and P-selectin on endothelium [Caused by FUCT 1 (fucose transporter gene) mutation: the failure to transport fucose into the Golgi complex results in failure to synthesize sialyl Lewis X] • LAD type 3, deficiency of Kindlin-3, required for firm adhesion

LAD type 1 • Leukocyte adhesion deficiency (LAD) results from mutation in the CD 18 molecule – the b 2 chain of the integrin family • The result is a loss of surface expression of key integrin molecules: Abnormal functions: endothelium adherence, neutrophil chemotaxis, phagocytosis, cytotoxicity Mac-1 p 150: 95

Defects in phagocytic cells are associated with persistence of bacterial infection 5. 1: Defects of neutrophil differentiation 5. 2: Defects of motility LAD 6. 2 a, b: IRAK 4, My. D 88 deficiency 5. 3: Defects of respiratory burst CGD Table 4. 1. 2: FLH syndromes with hypopigmentation

Autoinflammatory diseases are clinical disorders marked by abnormally increased inflammation, mediated predominantly by the cells and molecules of the innate immune system, with a significant host predisposition.

Complement deficiencies Defects in elimination of apoptotic cells, associated with SLE MBL: mannose-binding lectin Defects in the membrane-attack components are associated only with Neisseria infections (meningitis, gonorrhea)

= http: //tsunodalaboratory. blog. fc 2. com/blog-entry-155. html http: //tsunodalaboratory. blog. fc 2. com/blog-entry-155. htm

We have added a new category in Table 9 in which “Phenocopies of PID” are listed. This has resulted from our understanding and study of conditions that present as inherited immunodeficiencies, but which are not due to germline mutations and instead arise from acquired mechanisms. Examples include somatic mutations in specific immune cell populations that give rise to the phenotype of autoimmune lymphoproliferative syndrome (ALPS), and also autoantibodies against specific cytokines or immunological factors, with depletion of these factors leading to immunodeficiency.

Therapeutic approaches for PIDs • Two aims • Minimize and control infections • Replace the defective or absent components of the immune system by adoptive transfer or transplantation • Passive immunization with pooled gamma globulin • X-linked agammaglobulinemia • Bone marrow transplantation • SCID with ADA deficiency, Wiskott-Aldrich syndrome, bare lymphocyte syndrome, LAD • T cell depletion from bone marrow and HLA matching to prevent graft-versus-host disease • Enzyme replacement therapy • ADA and PNP deficiency • Bovine ADA conjugated to polyethylene glycol to prolong its serum half-life • Gene replacement • Difficulties in purifying self-renewing stem cells (ideal target) • Lack of method for introducing genes into cells to achieve stable, long-lived, and high-level expression

Bone marrow donor and recipient must share at least some MHC molecules to restore immune function • Major difficulties in bone marrow transplantation result from MHC polymorphism • MHC alleles expressed by the thymic epithelium determine which T cells can be positively selected • T cells and antigen presenting cells are derived from the graft

GVHD and HVGD in bone marrow transplantation • GVHD: Mature T cells from graft can attack host cells by recognizing their MHC antigen • This can be prevented by T-cell depletion of the donor bone marrow • HVGD: if the recipient has competent T cells, these can attack the bone marrow cells, causing transplant rejection • Bone marrow recipients are treated with irradiation that kills their own lymphocytes, thus making space for the grafted bone marrow of mature T cells, and minimizing the threat of HVGD • There is little problem in SCID patients because of immunodeficiency

Questions X-lined hyper Ig. M syndrome is caused by a defect in T cells, but not in B cells. Explain this hyper Ig. M syndrome with a figure showing an interaction between B cells and T cells, and using patients’ data below Flow cyotmetry can be useful for detection of X-lined agammaglobulinemia patients and carriers. Explain this flow cytometric analyses of (A) dot plots of CD 20 and Bruton’s tyrosine kinase (Btk) expression in lymphocyte-gated cells and (B) histograms of Btk expression in CD 14+ monocytes from normal control (top), a male patient (middle) and his mother (bottom).

Questions Explain genetic mutations and immunodeficiencies in SCID and nude mice and compare and contrast these two immunodeficient mice ( in the Exam, I recommend using the following figure ? ? Explain immunopathology of hemophagocytic lymphohistiocytosis (FHL or FHLH, hemophagocytic syndrome, HPS), using the following key words: perforin, anemia, macrophages, cytokines

CD 19 is expressed from pre-B cells until the terminal differentiation to plasma cells BTK(+) B cells? BTK(-) pre-B cells? All B cells in carrier should be Btk+?