Congenital agammaglobulinemia results from defects of early B cell development

B lymphocytes develop in the bone marrow from the hematopoietic stem cell (HSC), through various stages of maturation (see Fig. 9.w1) during which time they rearrange their immunogloulin genes to generate the pre-B cell receptor (see Fig. 9.w2). Defects in the expression and/or signaling through the pre-BCR cause congenital agammaglobulinemia with lack of circulating B lymphocytes.

X-linked agammaglobulinemia (XLA) is the prototype of these disorders, and was described by Dr Bruton in 1952. Affected males suffer from recurrent pyogenic infections. They lack serum IgA, IgM, IgD and IgE, and IgG levels are extremely low, usually <100 mg/dL. Circulating B lymphocytes are absent or markedly reduced (<1% of peripheral lymphocytes). Tonsils are absent and lymph nodes are unusually small. XLA is caused by mutations of the Bruton tyrosine kinase (BTK) gene, that encodes an enzyme involved in signaling through the pre-BCR and the BCR (Fig. 16.1). BTK mutations cause an incomplete, but severe, block at the pre-B cell stage in the bone marrow (Fig. 16.2). The BTK protein is also expressed by other cells (including monocytes and megakaryocytes), but its defect does not affect development of these cell types.

Fig. 16.1 Congenital agammaglobulinemia

Congenital agammaglobulinemia results from defects of proteins that participate at signaling through the pre-B cell receptor (pre-BRC). This is composed of the μ heavy chain, the surrogate light chains V-preB and λ5, and the signal transducing molecules Igα and Igβ. Signaling through the pre-BCR triggers activation of tyrosine kinases such as Fyn, Syk, and BTK, and involves the adaptor molecule BLNK. Ultimately, these signals converge on activation of the phospholipase C-γ (PLC-γ) and induction of calcium flux. The proteins whose mutations result in a known form of congenital agammaglobulinemia in humans are boxed in red.

Fig. 16.2 B cell maturation in X-linked immunodeficiencies

In X-LA, affected male infants have no B cells and no serum immunoglobulins, except for small amounts of maternal IgG. In IgA deficiency, IgA-bearing B cells, and in some cases IgG2- and IgG4-bearing B cells, are unable to differentiate into plasma cells. People with HIgM lack IgG and IgA. In CVID, B cells of most isotypes are unable to differentiate into plasma cells. Black bars denote points of inhibition.

For the first 4–6 months of life, males with XLA are protected by the maternally-derived IgG that has crossed the placenta, but once this supply of IgG is exhausted, they develop recurrent bacterial infections. Patients with XLA are also at risk of enteroviral infections (such as Echovirus) that may cause encephalitis. If immunized with attenuated poliovirus vaccine, they may develop paralytic poliomyelitis. Treatment of XLA is based on regular administration of immunoglobulins (IgG).

More rarely, congenital agammaglobulinemia is inherited as an autosomal recessive trait, due to mutations of other genes that encode for components of the pre-BCR or of the adaptor molecule BLNK (see Fig. 16.1). In all of these cases, there is a severe block in B-cell development at the pre-B cell stage in the bone marrow. The clinical phenotype is virtually identical to that of XLA.

Defects in terminal differentiation of B cells produces selective antibody deficiencies

Terminal maturation of B lymphocytes is marked by their differentiation into antibody-secreting plasma cells. Generation of plasma cells is markedly reduced in patients with CVID (see Fig. 16.2), who typically develop progressive hypogammaglobulinemia in the second and third decades of life. CVID is the most common primary immunodeficiency (1:10 000 affected individuals in the general population), characterized by extensive clinical and immunologic heterogeneity. Some patients have a reduced number of circulating B cells, and especially of CD27⁺ memory B lymphocytes; others show impaired function of T lymphocytes. CVID is usually sporadic, and the underlying molecular defect remains unknown in most cases. However, in some families CVID is inherited as an autosomal dominant or an autosomal recessive trait. A minority of CVID patients carry mutations in genes that play a key role in T-B cell interaction and B cell signaling (Fig. 16.3).

Fig. 16.3 Mutations associated with CVID

Common variable immunodeficiency may associate with mutations of proteins involved in B cell activation. These include co-stimulatory components of the B cell receptor (CD19, CD81, CD21), ICOS ligand (ICOS-L), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and the B-cell activating factor receptor (BAFF-R). These molecules deliver survival, activation and differentiation signals in mature B lymphocytes. (APRIL, a proliferation inducing ligand; BCMA, B cell maturation antigen; HSPG, heparan sulfate proteoglycan.)

CVID is characterized by reduced levels of specific antibody isotypes

Individuals with CVID have impaired antibody production in response to immunization or to natural infections and there is a virtual absence of plasma cells in lymphoid tissues and in the bone marrow. They suffer from recurrent infections of the respiratory tract (sinusitis, otitis, bronchitis, and pneumonia) sustained by common bacteria (non typeable H. influenzae, S. pneumoniae, etc.); lack of mucosal antibodies results in increased risk of gastrointestinal infection due to Giardia lamblia (Fig. 16.4). They are also highly prone to autoimmmune manifestations (cytopenias, inflammatory bowel disease), granulomatous lesions, lymphoid hyperplasia, and tumors (especially lymphomas). Treatment is based on immunoglobulin replacement therapy and antibiotics. Immunosuppressive and anti-inflammatory drugs may be needed in patients with autoimmune or inflammatory complications.

Fig. 16.4 Giardia lamblia

Numerous Giardia lamblia parasites can be seen swarming over the mucosa of the jejunum of a patient with CVID.

Defects of class switch recombination (CSR)

Class switch recombination (CSR) is the mechanism by which the μ chain of immunoglobulins is replaced by other heavy chains, resulting in the production of IgG, IgA, and IgE. The process occurs in germinal centres and is accompanied by affinity maturation as described in Chapter 9.

Deficiency of CD40L (X-linked) or more rarely of CD40 (autosomal recessive) results in failure of CSR, with very low or undetectable levels of IgG, IgA, and IgE and normal to increased levels of serum IgM (see Fig. 16.2). In the past, this condition was also known as ‘hyper-IgM syndrome’. In the lymph nodes, primary follicles are present, but germinal centers are absent (Fig. 16.5). Binding of CD40L to CD40 is also important to promote interaction between activated T cells and dendritic cells or monocytes/macrophages. This promotes T cell priming, production of IFNγ and activation of macrophages, that are important in the immune defense against intracellular pathogens. Consistent with this, the clinical phenotype of CD40L and of CD40 deficiency is characterized not only by recurrent bacterial infections, but also by increased risk of early-onset opportunistic infections (Pneumocystis jiroveci pneumonia, cytomegalovirus infection, protracted and watery diarrhoea due to Cryptosporidium). Neutropenia and severe liver disease are frequent. Therefore, CD40L and CD40 deficiency are not pure antibody deficiency, but rather represent examples of combined immunodeficiency. Treatment of these disorders is based on administration of immunoglobulins and antibiotics, but often requires hematopoietic stem cell transplantation (HSCT).

Fig. 16.5 CD40 ligand deficiency

Lymph nodes from patients with CD40 ligand deficiency show primary follicles, but lack germinal centers.

In B cells, signaling through CD40 promotes transcription of the gene encoding for activation-induced cytidine deaminase (AID), a DNA-editing enzyme that replaces deoxycytidine residues with deoxyuracil in the DNA of the immunoglobulin heavy chain switch regions. The resulting mismatch in the DNA is recognized by the enzyme uracil N-glycosylase (UNG) that removes the deoxy-uracil residues, leaving abasic sites that are resolved by means of DNA repair mechanisms. These DNA modifications trigger both CSR and somatic hypermutation. Both AID and UNG mutations cause severe deficiency of IgG, IgA, and IgE production; furthermore, the immunoglobulins (almost entirely IgM) produced by these patients have low affinity for the antigen. Clinically, these immunodeficiency diseases are characterized by recurrent bacterial infections. Dramatic expansion of germinal centers (leading to tonsil and lymph node enlargement), and the lack of susceptibility to opportunistic infections distinguish hyper-IgM syndrome due to AID and UNG mutations from the forms due to defects of CD40L or CD40. Treatment of AID and UNG deficiency is based on administration of immunoglobulins.

Severe combined immunodeficiency (SCID) can be caused by many different genetic defects

SCID includes a heterogeneous group of genetic disorders that affect various stages of T lymphocyte development or function (Fig. 16.6). The main pathophysiology mechanisms of SCID (and the associated diseases) are:

Fig. 16.6 Gene defects causing SCID

There is a wide range of gene defects that affect T cell development or maturation and cause combined immunodeficiency in humans. Some of these defects also affect B and/or NK cell development. The diagram indicates the main stages that are affected by each deficiency. (γc, common γ chain; ADA, adenosine deaminase; AK2, adenylate kinase 2 (causing reticular dysgenesis); CORO-1A, coronon-1A; DNA-PKcs, DNA-protein kinase catalytic subunit; IL-7R, interleukin 7 receptor; JAK3, janus-associated kinase 3; LIG4, DNA ligase IV; MHC II, major histocompatibility class II antigens (due to mutation of various transcription factors); PNP, purine nucleoside phosphorylase; RAG, recombinase activating gene; STIM1, stromal interaction molecule 1.)

While severe T cell abnormalities are a hallmark of all forms of SCID, some of these diseases also involve abnormalities of B and/or NK lymphocytes. In particular, some forms of SCID are characterized by absence of T lymphocytes, but presence of B lymphocytes (T⁻B⁺ SCID), whereas others show absence of both T and B lymphocytes (T⁻B⁻ SCID). Both of these groups of SCID include forms with or without natural killer (NK) lymphocytes.

SCID has a prevalence of approximately 1:50 000 live births, and is more common in males, reflecting the existence of X-linked SCID (X-SCID), the most common form of SCID in humans. This disease is due to mutation of the gene encoding for the common gamma chain (γc), shared by several cytokine receptors, namely those for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

Accordingly, patients with X-SCID have a T⁻B⁺NK⁻ phenotype.

Among the autosomal recessive forms of SCID in humans, the most common are represented by defects of RAG1 or RAG2, and by adenosine deaminase (ADA) deficiency. The recombinase activating genes (RAG) 1 and 2 are lymphoid-specific genes that initiate the process of V(D)J recombination, that is required for both T and B lymphocyte development. Therefore, mutations of RAG1 and RAG2 genes cause T⁻B⁻NK⁺ SCID.

ADA is a ubiquitously expressed enzyme involved in purine metabolism. Also purine nucleoside phosphorylase (PNP) is involved in the same metabolic pathway (Fig. 16.7). Lack of ADA results in accumulation of adenosine, deoxyadenosine, and their phosphorylated derivatives. Among them, dATP is particularly toxic; it inhibits the enzyme ribonucleotide reductase, that is required for DNA synthesis and hence for cell replication.

Fig. 16.7 Possible role of adenosine deaminase and purine nucleoside phosphorylase deficiency in SCID

It is thought that deficiencies of ADA and PNP lead to accumulations of dATP and dGTP, respectively. Both of these metabolites are powerful inhibitors of ribonucleotide reductase, which is an essential enzyme for DNA synthesis.

Lymphopenia (typically, less than 3000 lymphocytes/μL), and marked reduction of the T cell count in particular, is a hallmark of SCID. However, some infants with SCID have circulating T cells, occasionally even in normal numbers. This may reflect the presence of genetic defects that are permissive for T cell development (as in late defects in T cell development, or in patients with hypomorphic mutations in SCID-causing genes), but more often is due to engraftment of maternal T cells. Transplacental passage of maternally-derived T cells is common in pregnancy, but these cells are rejected by the immune system of the fetus. In contrast, maternally-derived T cells persist and expand in infants with SCID, and may cause tissue damage (graft-versus-host disease, GvHD) upon recognition of paternally-derived HLA alloantigens expressed by the patient’s cells.

The thymus of SCID infants is very small and typically devoid of lymphoid elements (Fig. 16.8); lymph nodes are often absent or – when present – contain mostly stromal cells. Although B cells are normally present in some forms of SCID, antibody responses are profoundly impaired and immunoglobulin levels are usually reduced.

Fig. 16.8 Thymus of SCID

Note that the thymic stroma has not been invaded by lymphoid cells and no Hassall’s corpuscles are seen. The gland has a fetal appearance.

Clinically, SCID is apparent in the first months of life. Interstitial pneumonia (due to Pneumocystis jiroveci or to viral infections: cytomegalovirus, syncytial respiratory virus, adenovirus, parainfluenzae virus type 3), protracted diarrhea leading to failure to grow, and persistent candidiasis (Fig. 16.9) are common clinical findings; however, other infections (meningitis, sepsis) are also possible. Use of live vaccines in SCID infants often leads to severe consequences and should be strictly avoided; in particular, administration of rotavirus vaccine may cause intractable diarrhea, and immunization with BCG may lead to disseminated infection.

Fig. 16.9 Candida albicans in the mouth of a patient with SCID

C. albicans grows luxuriantly in the mouth and on the skin of patients with SCID.

The DiGeorge anomaly arises from a defect in thymus embryogenesis

The thymic epithelium is derived from the third and fourth pharyngeal pouches by the sixth week of human gestation. Subsequently the endodermal anlage is invaded by lymphoid stem cells, which undergo development into T cells.

A congenital defect in the organs derived from the third and fourth pharyngeal pouches results in the DiGeorge anomaly. The T cell deficiency is variable, depending on how badly the thymus is affected; only in <1% of the patients, the T cell deficiency is so severe to cause SCID. Infants with DiGeorge anomaly have distinctive facial features (Fig. 16.10). They also have congenital malformations of the heart or aortic arch and neonatal tetany due to hypocalcemia resulting from the hypoplasia or aplasia of the parathyroid glands.

Fig. 16.10 DiGeorge anomaly

Note the wide-set eyes, low-set ears, and shortened philtrum of upper lip.

The majority of patients with DiGeorge anomaly have partial monosomy of 22q11.2 or 10p. Patients with DiGeorge anomaly who present with a SCID phenotype may be treated by thymus transplantation. Thymic tissues derived from unrelated infant donors at the time of heart surgery is treated to remove all lymphoid cells (to avoid graft-versus-host disease) and is implanted into muscular tissue of the affected patients. In spite of the complete HLA mismatch, lymphoid progenitors derived from hematopoietic stem cells of the patients, colonize the transplanted thymic tissue, mature there and are then exported to the periphery.

Defective function of regulatory T (Treg) cells causes severe autoimmunity

Regulatory T cells (CD4⁺ CD25⁺) suppress immune responses to self-antigens in the periphery.

Mutations of the FOXP3 gene cause Immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX) syndrome, a severe, X-linked form of autoimmunity. Males with IPEX syndrome present in the first months of life with intractable diarrhoea, insulin-dependent diabetes and skin rash. Severe infections may follow because of breakage of the cutaneous and mucosal barriers. There is a lack of functional CD4⁺ CD25⁺ FOXP3⁺ lymphocytes. Activated, self-reactive T lymphocytes infiltrate target organs (Fig. 16.11) and there are high levels of autoantibodies against insulin, other pancreatic antigens and enterocytes. In typical cases, the disease evolves rapidly. Treatment with immunosuppressive drugs is required to control autoimmune manifestations, however stem cell therapy is the only curative approach.

Fig. 16.11 Gut mucosa of a patient with IPEX

The gut mucosa of patients with IPEX shows villous atrophy and severe infiltration by T cells, identified by staining for CD3.

Impaired apoptosis of self-reactive lymphocytes causes autoimmune lymphoproliferative syndrome (ALPS)

Apoptosis of autoreactive lymphocytes is important to preserve immune homeostasis. Interaction between FAS ligand (FasL), expressed by activated lymphocytes, and FAS (CD95), triggers intracellular signaling that ultimately results in activation of caspases and cell death. Mutations of FAS are the predominant cause of autoimmune lymphoproliferative syndrome (ALPS), with lymphadenopathy, hepatosplenomegaly and autoimmune cytopenia. There is an increased risk of malignancies (especially B-cell lymphomas), that occur in 10% of the patients with FAS mutations. Patients with ALPS have an increased number of ‘double negative’ T lymphocytes that express the αβ form of the TCR, but do not express CD4 or CD8 molecules. ALPS is most often inherited as an autosomal dominant trait, and is due to dominant-negative mutations that interfere with the signal transducing activity of FAS trimeric complexes. A rare variant of FAS is due to FasL mutations. In a few patients, mutations of caspase-8 and of caspase-10 have been also identified. Treatment is based on use of immunosuppressive drugs.

Congenital defects of lymphocyte cytotoxicity result in persistent inflammation and severe tissue damage

The cytotoxic activity of T cells and NK cells depends on the expression of cytolytic proteins that are assembled into granules and transported through microtubules to the lytic synapse that is formed upon contact with target cells. Familial hemophagocytic lymphohistiocytosis (FHL) includes a group of disorders characterized by impairment of the mechanisms of transport, docking or release of the lytic granules. Deficiency of perforin (see Fig. 10.12) is the most common form of FHL. In these diseases, persistence of the pathogen (most often, a virus) causes expansion of CD8⁺ T cells that, while unable to mount a cytotoxic response, secrete increased amounts of TH1 cytokines, and IFNγ. Excessive amounts of IFNγ trigger macrophage activation, causing phagocytosis of blood elements and tissue damage. The disease is usually fatal, and treatment is based on immunosuppressive drugs (to reduce immune activation) and HSCT.

Similarly, X-linked proliferative syndrome (XLP) results from a failure to control the normal proliferation of TC cells following an infection with Epstein–Barr virus (EBV), which causes infectious mononucleosis.

Affected males appear normal until they encounter EBV, when they develop either:

The defective gene on the X chromosome encodes an adapter protein of T and B cells called SAP or the SLAM-associated protein. SLAM is expressed on the surface of T and B cells. Its intracellular tail interacts with the adapter protein, SAP, which is required for cytolytic activity of T and NK cells. Furthermore, SAP is important also for the function of follicular helper T cells (T_FH), that govern trafficking of B lymphocytes to the germinal centers. Defective function of T_FH cells accounts for the hypogammaglobulinemia of XLP. Treatment is based on HSCT.

Chromosomal breaks occur in TCR and immunoglobulin genes in hereditary ataxia telangiectasia

Hereditary ataxia telangiectasia (AT) is inherited as an autosomal recessive trait. Affected infants develop a wobbly gait (ataxia) at about 18 months and ultimately are wheel-chaired. Dilated capillaries (telangiectasia) appear in the eyes and on the skin by 6 years of age (Fig. 16.12). AT is accompanied by a variable T cell deficiency. About 70% of patients with AT are also IgA deficient and some also have IgG2 and IgG4 deficiency.

Fig. 16.12 Ocular telangiectasias

Ocular telangiectasias in a patient with ataxia telangiectasia.

The number and function of circulating T cells are greatly diminished, so cell-mediated immunity is depressed. Patients develop severe sinus and lung infections. Their cells exhibit chromosomal breaks, usually in chromosome 7 and chromosome 14, at the sites of the T cell receptor (TCR) genes and the genes encoding the heavy chains of immunoglobulins.

The cells of patients with AT are very susceptible to ionizing irradiation because the defective gene in AT encodes a protein involved in the repair of double-strand breaks in DNA. This defect leads to increased risk of malignancies, especially lymphoma and leukemia.

T cell defects and abnormal immunoglobulin levels occur in Wiskott–Aldrich syndrome

The Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency disease. Affected males with WAS:

The malfunction of cell-mediated immunity gets progressively worse. The T cells have a uniquely abnormal appearance, as shown by scanning electron microscopy, reflecting a cytoskeletal defect. They have fewer microvilli on the cell surface than normal T cells. Similar defects of cytoskeleton reorganization are observed in the patients’ monocytes and dendritic cells, with severe impairment of filopodia formation and of migration in response to chemokines. Patients with WAS have also a severe defect of natural killer (NK) cytolytic activity which accounts for the higher rate of herpes virus infections.

The Wiskott–Aldrich Syndrome protein (WASp) plays a critical role in cytoskeleton reorganization. In T and NK cells, it participates at formation of the immunological synapse, favoring tight interaction of T lymphocytes with dendritic cells and B cells, and of NK cells with target cells.

Because of the severity of the disease, and because expression of the WAS gene is restricted to the hematopoietic system, definitive treatment is based on HSCT.

Deficiency of STAT3 causes impaired development and function of TH17 cells in hyper-IgE syndrome

Hyper-IgE syndrome (HIES) can be inherited as an autosomal dominant or recessive trait; however, the clinical and immunological features of these forms are distinct. Autosomal dominant HIES is due to heterozygous mutations of the signal transducer and activator of transcription 3 (STAT3) gene. This is a transcription factor that is activated in response to activation of the JAK-STAT signaling pathway through cytokine and growth factor receptors that contain the gp130 protein. Biological responses to IL-6 and IL-10 are depressed, and development of TH17 cells is impaired, resulting in poor secretion of IL-17 and IL-22. This causes impairment in immune defense against bacterial and fungal infections; in addition, production of antibacterial molecules (e.g. defensins) by epithelial cells is also affected. The clinical phenotype includes eczema, cutaneous, and pulmonary infections sustained by S. aureus (with formation of pneumatoceles), and Candida spp. Patients with STAT3 deficiency also show defective shedding of primary teeth, scoliosis, higher risk of bone fractures, joint hyperextensibility, and characteristic facial traits.

In contrast, the autosomal recessive HIES syndrome is due to mutations of the dedicator of cytokinesis 8 (DOCK8) gene, that encodes for a protein involved in cytoskeleton reorganization. Patients with DOCK8 deficiency suffer from severe infections from early life. Viral infections due to CMV, HPV, and HSV, and allergies are particularly common. There is also an increased risk of malignancies. In vitro proliferation of T cells to mitogens is markedly reduced. Immunoglobulin levels are variable, but IgG is often increased whereas IgM is low. The clinical and immunological phenotype of DOCK8 deficiency indicates that this is a combined immunodeficiency.

Chronic granulomatous disease results from a defect in the oxygen reduction pathway

Patients with CGD have defective NADPH oxidase, which catalyzes the reduction of O₂ to •O₂^– by the reaction: . They are therefore incapable of forming superoxide anions (O₂^–) and hydrogen peroxide in their phagocytes following the ingestion of microorganisms.

As a result, microorganisms remain alive in phagocytes of patients with CGD. This gives rise to a cell-mediated response to persistent intracellular microbial antigens, with formation of granulomas.

Children with CGD develop pneumonia, infections in the lymph nodes (lymphadenitis), and abscesses in the skin, liver, and other viscera. Infections due to Staphylococcus aureus are particularly common, however patients with CGD are uniquely prone to fungal (in particular Aspergillus and Candida) and mycobacterial infections. Treatment of CGD requires regular use of antibacterial and antifungal prophylaxis and aggressive management of infections. HSCT may provide a definitive cure.

Enzyme defects in CGD

The NADPH oxidase complex has many subunits. In resting phagocytes, the membrane contains two proteins (gp91^phox and p22^phox) that compose a phagocyte-specific flavocytochrome.

• one of 91 kDa, encoded by a gene on the short arm of the X chromosome; and

• one of 22 kDa, encoded by a gene on chromosome 16.

When phagocytosis occurs, the p47^phox and p67^phox cytosolic proteins become phosphorylated, move to the membrane of the phagosome, and bind to flavocytochrome. The complex formed acts as an enzyme, NADPH oxidase, catalyzing the NADPH oxidation reaction, production of oxygen radical, and activation of lytic enzymes (cathepsin G, elastase), thereby permitting intracellular killing of bacteria and fungi (Fig. 16.w1).

Fig. 16.w1 NADPH oxidase

In its inactive state, the NADPH oxidase includes membrane-associated proteins (gp91^phox, p22^phox) and cytosolic components (p47^phox, p67^phox, p40^phox, Rac1/2). Phagocytosis provides a stimulus for the cytosolic components to associate and move to the membrane of the phagosome. Once the cytosol components are associated with the membrane components, the oxidase becomes catalytically active, allowing formation of superoxide anion (O₂⁻) by transferring an electron to O₂. Superoxide is converted to hydrogen peroxide (H₂O₂) and eventually formation of hypochlorous acid (HClO⁻) and of hydroxyl anion (OH⁻) ensues. Both HClO⁻ and OH^– have microbicidal activity.

(Adapted from Rosenzweig SD, Uzel G and Holland SM. Phagocyte disorders. In: Steihm ER, Ochs HD, Winkelstein JA Eds., Immunological disorders in infants and children, 5th Edition, Elsevier, 2004, pp.618–651.)

The most common form of CGD (75% of all cases) is X-linked and involves a defect in the 91-kDa chain of cytochrome b₅₅₈. Four types of CGD are autosomal recessive and result from defects in the 22-kDa chain of the cytochrome b₅₅₈, or from defects in p47^phox, p67^phox.protein, or in the p40^phox component that also stimulates phagocytosis-induded NADPH oxidase activity.

Diagnosis of CGD is most commonly performed by flow cytometry, evaluating dihydrorhodamine-123 (DHR-123) oxidation upon in vitro activation. In the past, diagnosis of CGD was made by demonstrating inability of phagocytes to reduce nitroblue tetrazolium (NBT) dye after a phagocytic stimulus. NBT is a pale, clear, yellow dye taken up by phagocytes when they are ingesting a particle. When NBT accepts H⁺ and is reduced as a result of NADPH oxidation it forms a deep purple precipitate inside the phagocytes; precipitation does not occur in the phagocytes of patients with CGD (Fig. 16.w2).

Fig. 16.w2 Nitroblue tetrazolium test

In normal polymorphs and monocytes, reactive oxygen intermediates (ROIs) are activated by phagocytosis, and yellow NBT is converted to purple-blue formazan (1). Patients with CGD cannot form ROIs and so the dye stays yellow (2).

(Courtesy of Professor AR Hayward.)

LAD is due to defects of leukocytes trafficking

The receptor CR3 in the phagocyte membrane that binds to C3bi on opsonized microorganisms is critical for the ingestion of bacteria by phagocytes. This receptor is deficient in patients with type 1 LAD (LAD1), who consequently develop severe bacterial infections, particularly of the mouth and gastrointestinal tract. CR3 is composed of two polypeptide chains:

In LAD1, there is a genetic defect of the β chain, encoded by a gene on chromosome 21.

Two other integrin proteins share the same β chain as CR3 – namely lymphocyte functional antigen (LFA-1) and p150,95 (see Chapter 6); these proteins are also defective in LAD1.

LFA-1 is important in cell adhesion and interacts with intercellular adhesion molecule-1 (ICAM-1) on endothelial cell surfaces and other cell membranes. Because of the defect in LFA-1, phagocytes from patients with LAD1 cannot adhere to vascular endothelium and cannot therefore migrate out of blood vessels into areas of infection. As a result patients with LAD1 cannot form pus efficiently and this allows the rapid spread of bacterial invaders.

When leukocytes in the circulation enter an area of inflammation their speed of movement is greatly retarded by the interaction of selectins with their ligands (see Fig. 6.6). E-selectin interacts with Sialyl Lewis^x (SLeX), a fucosylated molecule that is expressed on the surface of neutrophils and monocytes. In LAD2 a genetic defect of intracellular fucose transporter prevents fucosylation of membrane glycoproteins, including SLeX. Consequently, the leukocytes of patients with LAD2 cannot roll on the endothelium and fail to extravasate and reach inflamed tissues. Since fucose metabolism is important also in the central nervous system, patients with LAD2 also show mental retardation and dysmorphisms in addition to infections.

A third form of LAD (LAD3) is due to impaired integrin signaling that also involves platelets. These patients suffer from severe infections and increased bleeding. All forms of LAD are characterized by marked elevation of the leukocyte count in peripheral blood (leukocytosis); this reflects the response of the bone marrow to inflammatory stimuli (with increased production of myeloid cells) and inability of the leukocytes to leave circulation and reach peripheral tissues.

Macrophage microbicidal activity is impaired by defects in IFNγ signaling

The destruction of intracellular microorganisms that flourish in macrophages depends on the activation of microbicidal activity in macrophages by IFNγ. When microorganisms are taken up by macrophages, the macrophages secrete IL-12, which then binds to the IL-12 receptor on T cells and induces secretion of IFNγ.

Children with genetic defects in the genes encoding IL-12, the IL-12 receptor (IL-12R), or the IFNγ receptor suffer from recurrent infection with non-pathogenic mycobacteria and, to a lesser extent, with salmonella. These various defects are inherited as autosomal recessive or autosomal dominant traits. Treatment with IFNγ is beneficial in patients with IL-12 and IL-12R mutations, whereas HSCT is the treatment of choice for patients with mutations in IFNγ receptor.

Defects of TLR-signaling cause susceptibility to pyogenic infections

Toll-like receptors (TLR) are a series of molecules that are expressed at the cell surface or at the membrane of endosomes, and mediate recognition of pathogen-associated molecular patterns, such as lipopolysaccharide, glycolipids, single- or double-stranded RNA (see Fig. 6.20). The classical pathway of TLR activation involves the adaptor molecules MyD88 and the intracellular kinases IRAK-4 and IRAK-1. Activation of this pathway upon binding of TLRs to their ligands, results in the induction of NFκB and production of inflammatory cytokines (IL-1, IL-6, TNFα, IL-12). Mutations of IRAK4 and MyD88 cause severe and invasive pyogenic infections early in life, often without significant inflammatory response. Infections tend to become less frequent later in life, when the adaptive immune system has matured.

TLR-3, -7, -8 and -9 can activate an alternative pathway that involves the adaptor molecule UNC-93B, resulting in the induction of type 1 interferons (IFNα -β). Mutations of TLR3 and UNC-93B cause selective susceptibility to herpes simplex encephalitis due to infection by HSV-1. Severe viral infections are also observed in patients with complete deficiency of STAT1, a transcription factor that is activated following binding of type 1 interferon to the specific receptor, resulting in expression of IFN-dependent genes.

Immune complex clearance, inflammation, phagocytosis, and bacteriolysis can be affected by complement deficiencies

Deficiencies of the classical pathway components, C1q, C1r, C1s, C4, or C2, result in a propensity to develop immune complex diseases such as systemic lupus erythematosus.

Deficiencies of C3, factor H, or factor I result in increased susceptibility to pyogenic infections – this correlates with the important role of C3 in the opsonization of pyogenic bacteria.

Deficiencies of the terminal components C5, C6, C7, and C8, and of the alternative pathway components, factor D and properdin, result in remarkable susceptibility to infection with the two pathogenic species of the Neisseria genus: N. gonorrhoeae and N. meningitidis. This clearly demonstrates the importance of the alternative pathway and the macromolecular attack complex in the bacteriolysis of this genus of bacteria.

All of these genetically-determined deficiencies of complement components are inherited as autosomal recessive traits, except:

Hereditary angioneurotic edema (HAE) results from C1 inhibitor deficiency

The C1 inhibitor (C1INH) is responsible for dissociation of activated C1, by binding to C1r2C1s2. Deficiency of C1INH results in HAE (Fig. 16.14), that is inherited as an autosomal dominant trait. Patients have recurrent episodes of swelling of various parts of the body (angioedema):

Fig. 16.14 Hereditary angioneurotic edema

This clinical photograph shows the transient localized swelling that occurs in this condition.

C1INH inhibits not only the classical pathway of complement, but also elements of the kinin, plasmin, and clotting systems.

The edema is mediated by two peptides generated by uninhibited activation of the complement and surface contact systems:

Fig. 16.15 Pathogenesis of hereditary angioneurotic edema

C1 inhibitor is involved in inactivation of elements of the clotting, kinin, plasmin and complement systems, which may be activated following the surface-dependent activation of factor XII (Hageman factor). The points at which C1 inhibitor acts are shown in red. Uncontrolled activation of these pathways results in the formation of bradykinin and C2 kinin, which induce edema formation.

The effect of these peptides is on the postcapillary venule, where they cause endothelial cells to retract, forming gaps that allow leakage of plasma (see Chapter 6).

There are two genetically determined forms of HAE:

The distinction between type I and type II is important because the diagnosis of type II disease cannot be made by quantitative measurement of serum C1 inhibitor alone. Simultaneous measurements of C4 must also be done. C4 is always decreased in the serum of patients with HAE because of its destruction by uninhibited activated C1.

C1INH deficiency is not always genetically-determined, but may be acquired later in life. In particular, patients with autoimmune diseases or with B cell lymphoproliferative disorders (chronic lymphocytic leukemia, multiple myeloma, or B cell lymphoma) may produce autoantibodies to C1INH. C1INH brands are available for intravenous use to treat or prevent acute attacks of angioedema.