1

X-LINKED AGAMMAGLOBULINEMIA(BRUTON’S AGAMMAGLOBULINEMIA)

SUSAN R. S. GOTTESMAN

CASE REPORT

“Sam always gets sick; now he has a bad earache andfever.”

Day 1

Sam is a four-year-old boy who is brought to you inthe emergency room (ER) by his distraught parentsbecause he has a fever, has been vomiting in the lastfew hours, and seems slightly disoriented (confused).His mother relates that initially Sam complained of aright-sided earache that was only transiently relieved withacetaminophen. Over the next few days, he developeda fever and became less active and more irritable anddid not want to eat. Yesterday he began complainingof a headache “all over his head” and this morning hebegan vomiting. His mother states that Sam has a historyof frequent ear infections (otitis media) with effusionbut is concerned that this time it is more than just anearache.

Sam has a generally normal appearance and size butthat he has an acute illness marked by fever, withdrawalfrom his surroundings, and extreme irritability, that is,

Immunology: Clinical Case Studies and Disease Pathophysiology, By Warren Strober and Susan R. S. GottesmanCopyright © 2009 John Wiley & Sons, Inc.

crying spells unabated by his mother’s attempts to calmhim down. He has a temperature of 40◦C, his skin is hotto the touch, and he appears to be mildly dehydrated (asevidenced by dryness around his mouth and eyes). Histhroat is injected (red) but without exudate (pus) and hisright ear has an opacified tympanic membrane with a lossof the light reflex; his left ear is normal. He has nuchalrigidity (neck stiffness when his head is flexed). Youevaluate him for other signs of meningeal irritation andfind that he has a positive Kernig’s sign (pain when lowerleg is extended with his hip flexed at 90◦) and a positiveBrudzinski’s sign (involuntary flexion of the knees andhips when you flex his neck).

His lungs are clear to auscultation and his abdomen hasnormal bowel sounds, is nondistended, and is nontender.His joints are nontender and mobile.

You are alarmed at the evolution of Sam’s initialcomplaint and his neurologic signs. Suspecting that hehas an infectious meningitis you immediately perform alumbar puncture (after checking his eyes for papilledemawith an ophthalmoscope) to confirm the diagnosis and toattempt to identify the organism. The cerebrospinal fluid(CSF) is obtained and blood and CSF samples are sentfor emergency analysis and to the microbiology lab forculture.

15

COPYRIG

HTED M

ATERIAL

16 CHAPTER 1 X-LINKED AGAMMAGLOBULINEMIA (BRUTON’S AGAMMAGLOBULINEMIA)

Results obtained quickly are as follows:

Cerebrospinal fluid: Result Reference Range

Opening pressure 180 mm Hg 50–80 mm Hg

Appearance Cloudy Clear

White blood cells (WBC) 1078/μL <5/μL

Neutrophils 75% <25%

Red blood cells (RBC) 0 0

Protein 300 mg/dL 20–45 mg/dL

Glucose 3 mg/dL >50 mg/dL

Peripheral WBC count

and differential: Absolute Count Reference Range

Total WBC 24,000/μL 5500–15,000

Neutrophils 75% 18,000/μL 1500–8500

Lymphocytes 19% 4,560/μL 2000–8000

Monocytes 5% 1,200/μL 300–650

Eosinophils 1% 240/μL 100–500

In view of the above history and laboratory findings, whatis your initial diagnosis and treatment plan?

Sam shows all the typical signs and symptoms of meningitis.These include headache, vomiting, and disorientation aswell as physical evidence of meningeal irritation (e.g.,nuchal rigidity). These clinical findings and the elevatedopening pressure on entry into the meningeal space, alongwith the presence of a cloudy CSF containing WBC, pointunmistakably to infection of the meninges. Moreover, thepredominance of neutrophils in the CSF, along with a lowglucose, strongly suggests a bacterial meningitis, a type ofmeningitis that must be treated urgently to preventpermanent neurologic defects or even death.

Recurrent otitis media is fairly common in youngchildren and may occasionally lead to meningitis, as itapparently did in Sam’s case. Viral meningitis is the morecommon form, although bacterial meningitis does sometimesoccur in normal children as well.

Sam’s peripheral blood cell count is high due to agranulocytosis. Granulocytosis is an increase in cells of thegranulocytic series, of which the neutrophils are by far themost prominent. This again suggests a bacterial infectionand one that has spread to the peripheral blood. Given thislikelihood, you start Sam on intravenous ceftazidine, anantibiotic that provides good broad-spectrum coverage.

Day 2

The next morning, after almost 24 h of intravenous cef-tazidine, you walk into Sam’s hospital room to find that heis calm, smiling faintly, and beginning to eat. His parents

are much relieved and are already asking when Sam canbe taken home. The laboratory reports that Streptococcuspneumoniae was isolated from both his blood and CSF butantibiotic sensitivities are not yet available; you thereforecontinue the ceftazidine therapy. When they become avail-able, you may elect to change the antibiotic on the basis ofthe sensitivities of the isolated streptococcal organism.

Now that the urgent problem of bacterial meningitisis resolving, you obtain a more complete history from theparents. The mother mentions again that Sam seems to getsick quite often, definitely more often than his playmates.She relates that Sam had skin abscesses at 9 and 10 monthsof age, three episodes of pneumonia documented by X-rayfindings, multiple episodes of diarrhea, and one episode ofleft knee swelling. His parents take him to the pediatricianon a regular basis.

This history of multiple, severe, and in some casesunusual infections is disturbing and you decide that furtherinvestigation is needed. You consider the possibility thatSam has a primary immunodeficiency disorder and referthe family to an allergist/immunologist for more definitiveworkup once he has recovered from the acute infection.

Subsequent Workup Three Weeks Later

Three weeks later, Sam has fully recovered from the menin-gitis and has completed the recommended antibiotic treat-ment (the streptococcus was sensitive to ceftazidine). Atthis point he is seen by an allergist/immunologist who con-firms that Sam is within the expected range for heightand weight and has a normal appearance. On examina-tion, however, he notices that Sam lacks tonsillar tissue,quite an abnormal finding in a 4-year-old child. There is nolymphadenopathy (enlargement of lymph nodes) or hep-atosplenomegaly (enlargement of liver and spleen). He alsochecks Sam’s blood count and finds that it has returned tonormal.

From questioning the parents, the allergist/immuno-logist learns that Sam is an only child and that both parentsare healthy. The pregnancy was uneventful and Sam was afull-term baby of normal size. The parents do not remem-ber any fevers or infections during the first 6–8 months ofhis life. In relating the family history, Sam’s mother statesthat her two sisters are healthy but that she had a brotherwho died at age 18. She recalls that the brother was alwayssick and had repeated pneumonias resulting in chronic lungdisease. Her male cousin died of polio after receiving anoral polio vaccine during childhood. Sam’s father’s fam-ily history is negative for family members with multipleinfections.

The allergist/immunologist concludes that Sam’s his-tory and the family history are sufficiently suggestive towarrant an immunodeficiency workup.

What information does one seek in such a workup?

CASE REPORT 17

The initial goal of an immunodeficiency workup is todetermine if the patient (a child in this case) has a B-celldeficiency, a T-cell deficiency, a combined T- and B-celldeficiency, a phagocytic cell deficiency, or a complementdeficiency. By deficiency we mean a decrease in the amountor function of these immunologic elements. If a defect isdetected, one would then attempt to define the specificunderlying molecular abnormality. Sam’s age suggests thata congenital deficiency needs to be considered; his maternalfamily history raises suspicion of an inherited disorder.

Tests may be organized to investigate each arm ofthe immune system (see the introduction to this unit andbelow). In addition, specific assays either provide quantita-tive information or measure functional activity. In general,the results of quantitative assays are more quickly available.Given the fact that Sam has a sufficiently strong personaland family history to justify this workup, most of thesetests are initiated at the outset. As the results are collectedand evaluated, it will become obvious that some give pre-dictable results. For learning purposes, we will present theresults of this evaluation arranged according to the immunesystem being probed.

Humoral Immunity Studies (Mainly Involving BCells)

Serum Immunoglobulin Levels: QuantitativeAssay. Blood is drawn from Sam for determinationof serum immunoglobulin levels. These are measuredby a spectrometric assay called nephelometry, with theexception of IgE, which, because of its normally lowlevel, requires a more sensitive method: radioimmunoassay(RIA) or enzyme linked immunosorbent assay ELISA (seeunit V for detailed assay descriptions).

Result

Reference Range

IgG 140 mg/dL 923 ± 256 mg/dL

IgM 32 mg/dL 65 ± 25 mg/dL

IgA 48 mg/dL 124 ± 45 mg/dL

IgE 1.5 μg/dL 2.0–10 μg/dL

What information do you obtain from overall immunoglob-ulin levels?

Overall immunoglobulin levels are used as a grossindication of the individual’s ability to respond to antigenicexposure with a humoral response and to be capable of asecondary response (IgG) and as an indicator of allergicreactions (IgE). As the levels are the result of priorexposure, it is not unexpected that immunoglobulin levelsincrease with age. They generally reach adult levels atpuberty. Thus, Sam’s levels are compared with those ofother four-year-olds.

Sam shows abnormally low levels of all the immunoglob-ulin subclasses.

Specific Antibody Responses to AntigenicChallenge: Functional Assays

Retrospective. Studies of response to previouschallenge. Blood is drawn for the determination ofmeasles, mumps, rubella, and varicella serum antibodytiters with the knowledge that Sam had been administeredvaccines for these organisms at approximately 12 monthsof age. These antibodies are measured by ELISA usingthe specific antigens. This allows detection of low levelsof antibody, as one would expect in response to a specificantigen, and allows titers to be obtained.

Result. Titers to all four organisms come back as “unde-tectable” or at the lowest limit of detection.

Prospective. Response to a new challenge. Ina more definitive test of ability to mount an antibodyresponse, Sam is administered a standard dose of diphtheriaand tetanus (DT) antigens as well as pneumococcal anti-gens to test his capacity to mount an antibody response toprotein (DT) or to polysaccharide (pneumococcal) antigens[see Case 24 and Coico and Sunshine (Chapter 10) fordescription of T-dependent and T-independent antigens].Blood is drawn just prior to antigen administration andagain at two and four weeks after immunization. Serumantibody titers to each specific antigen are measured at thethree time points. Although Sam would already have beengiven three doses of DT as part of the routine vaccinationschedule, a normal individual will exhibit at least a 5- to10-fold rise in titer to DT following this immunizationprotocol and a 4- to 5-fold rise in titer to one or more ofthe pneumococcal subtypes. The data are obtained abouttwo weeks after the submission of the sera.

Result. Sam is unable to mount a significant antibodyresponse to either DT or pneumococcal antigens.

Conclusion of Humoral Immunity Studies.The low “across-the-board” Ig levels and poor antibodyresponses point to a severe deficiency in B-cell function.However, from this information one cannot state definitivelythat this is the only immune defect present or that the Bcells are the culprits.

T- and B-Cell StudiesAnalysis of Lymphocyte Subsets in the

Peripheral Blood: Quantitative Studies by FlowCytometry. Sam’s peripheral blood cells are stainedwith fluorochrome-bound antibodies recognizing CDmarkers characteristic of particular lymphocyte subsets.The number of cells staining with each antibody isquantitated using a flow cytometer (see Unit V).

18 CHAPTER 1 X-LINKED AGAMMAGLOBULINEMIA (BRUTON’S AGAMMAGLOBULINEMIA)

Result.

Reference Range Absolute Count

CD3 (pan T-cellmarker)

85% 55–82% 1755/μL

CD4 (marker ofhelper T cell)

55% 33–57% 1053/μL

CD8 (marker ofcytotoxic Tcell)

31% 8–34% 725/μL

CD19 (B-cellmarker)

0% 6–22% 0

CD16/56 (NKcell markers)

15% 6–31% 350/μL

Combining the Data. The lymphocyte subset quantifica-tion studies suggest that Sam’s inability to mount an anti-body response is due to the absence of mature B cells.

In Vitro Proliferation Studies: NonspecificFunctional Assays. Sam’s cells are cultured withvarious stimulators for three days, after which their levelof proliferation is quantitated by their ability to incorporatetritium-labeled thymidine. Agents that stimulate T-cellproliferation include the mitogens phytohemagglutinin(PHA) and concanavalin A (ConA) and the T-cell-specificantibody combination anti-CD3 and anti-CD28. B cellsare stimulated to proliferate by incubation with pokeweedmitogen (PWM), Staphylococcus aureus Cowen type I,and the B-cell specific antibodies anti-μ heavy chain oranti-CD40.

Sam’s cells are cultured with the anti-CD3/anti-CD28,which is designed to mimic the manner in which T cells arenormally stimulated, and with anti-CD40, which is designedto mimic the normal interaction between T helper cells andB cells.

Result.

Anti-CD3/anti-CD28 stimulation: the proliferationresponse of Sam’s T cells is normal as comparedto simultaneously run control cells.

Anti-CD40 stimulation: the proliferation of Sam’s cellsis significantly decreased as compared to simultane-ously run control cells.

These phenotyping and proliferation studies reveal the pres-ence of a B-cell defect, corroborating the humoral immunitystudies which focused on B cells. Given the fact that Samhas no detectable levels of B cells in the peripheral blood,stimulation with B-cell specific mitogens or anti-CD40 wasexpected to be undetectable. It is also not surprising thatSam is unable to mount an antibody response given theabsence of mature B cells (and tonsillar tissue). The results

additionally demonstrate that Sam has normal T- and NKcell numbers and that Sam’s T cells can mount a normalproliferative response to stimulation via the specific recep-tors that would normally be involved in a T–B collaboration.Thus, his T cells appear to be normal.

Cell-Mediated Immunity Studies (Mainly Involv-ing T Cells)

Delayed-Type Hypersensitivity (DTH) Test:In Vivo T-Cell Functional Assay. An antigen towhich Sam has been previously exposed is injectedintracutaneously on the forearm and development ofredness and induration (swelling) is evaluated at 48 h.Since Sam has been immunized with tetanus toxoid inthe past, tetanus toxoid is injected intracutaneously in theforearm area.

Result. At 48 h, a 1 cm area of redness and swelling isnoted.

DTH is a classic test for cell-mediated immunity but infact only measures the interaction between T helper cellsand APCs (along with nonspecific vascular changes, etc.).Additional tests of cell-mediated immunity include studies ofcytotoxic T-cell function; however since all evidence, bothclinical and laboratory, suggests that this arm of Sam’simmune system is intact, they will not be performed in thiscase (see differential diagnosis section below and Unit V).

Nonspecific Immunity and Complement Studies.For completeness, Sam’s blood is also sent to the laboratoryfor the determination of complement levels and the abilityof his white cells to generate reactive oxygen species orhydrogen peroxide. The latter is a function of phagocytesand is impaired in chronic granulomatous disease.

Complement Studies: Quantitative Assay ofComplement Components.

Result.

Reference Range

CH50 180 μg/ml 150–310 μg/ml

C3 95 mg/dL 85–201 mg/dL

C4 30 mg/dL 16–47 mg/dL

Phagocytic Cells: Functional Assays ofNeutrophils.

Nitroblue Tetrazolium (NBT) Test. Yellow NBTis converted to blue/violet formazan within normal neu-trophils due to reduction of NBT by O−

2 , thus measuringtheir ability to generate reactive oxygen species.

Result. Sam’s cells exhibit blue/violet crystals followingincubation with NBT.

DIFFERENTIAL DIAGNOSIS OF SAM’S IMMUNODEFICIENCY 19

Flow Cytometric Analysis of PMA-StimulatedNeutrophils Loaded with Dihydrorhodamine. Whendye is oxidized by H2O2, it exhibits increased fluorescence,demonstrating the neutrophils’ ability to generate H2O2

when stimulated.

Result. Sam’s cells exhibit normal flow patterns.

Complement levels and neutrophil functions are normal.

Based on the above results, what is your overall conclu-sion concerning the functioning of Sam’s immune systemand location of possible defect?

Overall, the above results show that, despite Sam’s normalperipheral white blood cell and normal lymphocyte counts,he has low to undetectable numbers of B cells and hasreduced or absent B-cell function. The fact that the B-celldefect was not reflected in the white count is not unexpectedgiven the fact that B cells normally account for only aminority of peripheral lymphocytes. The absence of matureB cells goes along with the absence of tonsils since the latteris a secondary lymphoid organ in which the majority of cellsare B cells. Finally, T-cell numbers and function, both invitro and in vivo, are normal, strongly suggesting the B-celldefect is not secondary to a T-cell defect. The fact that the Bcells are not just nonfunctional but are absent in theperiphery points to an intrinsic inability of the B-cellprecursors to differentiate or to a block in the differentiationof mature B cells from precursors.

Why was Sam healthy until the age of six months?

Neonates are protected against infection by the presence ofmaternal antibodies obtained during gestation and nursing.IgG crosses the placenta and IgG and IgA especially arecomponents of breast milk (Case 4; see also Coico andSunshine, Chapter 4 for function of different isotypes.). Thisis essentially a natural form of passive immunity andprotects the infant until its immune system is developedenough to generate its own immune response. Theseprinciples also underlie the vaccination schedule in infants(Coico and Sunshine, Chapter 20).

Follow-Up Visit

Based on the laboratory results, the allergist/immunologistconcludes that Sam has a B-cell disorder. Since this has ledto an inability to produce normal levels of immunoglobu-lins and mount normal antibody responses, he starts Sam onintravenous immunoglobulin (IVIG) therapy administeredevery four weeks as a replacement for his own inability toproduce immunoglobulins. After a single IVIG administra-tion Sam’s trough IgG level (the level obtained four weeksafter the initial dose and just prior to the second dose) is400 mg/dL. His trough level after six months of therapyis 550 mg/dL. Since studies have shown that no addi-tional protection is achieved with trough IgG levels higherthan 400 mg/dL, the six-month result indicates that optimal

therapy has been achieved. IgM and IgA levels remain lowbecause the IVIG preparation does not contain significantamounts of these immunoglobulins. One year later, Samshows definite clinical improvement; he has been withoutinfections since IVIG therapy was started.

DIFFERENTIAL DIAGNOSIS OF SAM’SIMMUNODEFICIENCY

The differential diagnosis of Sam’s immunodeficiency startswith the immunologic studies conducted above that showthe presence of a B-cell defect. However, even before suchdata are obtained, certain features of Sam’s clinical courseallow informed speculation concerning his diagnosis. Whatare these features?

As discussed in the introduction to this unit, whenconsidering the history of a patient with possibleimmunodeficiency, it is useful to take note of the infectiousagents and whether they are most consistent with thepresence of a defect of the humoral response (antibody) asopposed to a defect of cell-mediated immunity or phagocyticcell immunity. Thus one needs to consider if the organismsinvolved are bacterial (extracellular) as opposed to viral orfungal (intracellular).

It was mentioned above that, although Sam had a historyof infections early in life, his first several months wereinfection free. This points to a primary immunodeficiencyrather than a secondary immunodeficiency, but one whichmay have been masked by immune elements such asmaternal immunoglobulins that Sam received from hismother in utero. Second, Sam’s infections appeared, at leastby history, to be due mainly to extracellular “high grade”bacterial pathogens that could cause infections in bothnormal and immunocompromised individuals, rather than“low grade” intracellular pathogens such as certain viruses,fungi, and parasites. This suggests the presence of ahumoral abnormality rather than a cell-mediated immunedefect, since the former is usually associated with infectionswith highly virulent, high grade bacteria. However, thisinfectious history does not rule out a complement disorder,which is also marked by infections with such pathogens.Finally, Sam’s infection history is inconsistent with adisorder of the innate immune system such as a neutrophilor NK cell disorder, since these disorders would also beexpected to give rise to infection with low grade pathogens.A deficiency in humoral immunity may be due to a defect inB cells. Alternatively, T helper cells or the interactionbetween T and B cells may be at fault.

In evaluating where Sam’s defect lies, it is helpful tolook at a scheme of B-cell development and function andconsider where the block may occur (Fig. 1.1).

How does Sam’s workup allow one to pinpoint theproblem?

Sam’s immunologic workup revealed a reduction in all theserum immunoglobulins and an absence of response to

20 CHAPTER 1 X-LINKED AGAMMAGLOBULINEMIA (BRUTON’S AGAMMAGLOBULINEMIA)

Lymphoidstem cell

Pre-Bcell

ImmatureB cell

4

Bone marrow

Pro-Bcell

MatureB cell

Periphery

CD4+TH

IgM

IgG

IgE

7

86

5

IgA8

5

4 4

IgM

X-linked agammaglobulinemia; � heavy-chain deficiency; �5 (�-like) deficiency

Key:

Common variable immunodeficiency

Bare lymphocyte syndrome37

5

Selective IgA deficiency64

X-linked hyper-IgM syndrome8

Primary lymphoid organs

Figure 1.1. Scheme ofB-cell development and func-tion and locations of blocksin development with variousimmunodeficiency disorders.

immunization. The absence of all immunoglobulin isotypessuggests that the block occurs at a stage prior to thedevelopment of isotype-specific plasma cells. Specifically,this eliminates a selective IgA deficiency and hyper-IgMsyndrome. The latter is unlikely because Sam’s IgM levelsare low and because some forms of hyper-IgM syndromeresult from T-cell abnormalities (see Case 4).

Working our way to the left in Figure 1.1, we encountercommon variable immunodeficiency (CVID) and barelymphocyte syndromes. Bare lymphocyte syndrome, in whichthe cells lack the expression of one of the human leukocyteantigen (HLA) classes, would lead to a defect in DTH andhumoral immunity combined, since both depend on class IIMHC expression. Lack of class I MHC antigen expressionwould affect T cytotoxic activity and spare T helper cellstimulation.

It is somewhat more difficult to clinically distinguishbetween CVID and X-linked agammaglobulinemia (XLA),which might both be part of your differential diagnoses. LikeXLA, CVID is dominated by a humoral immune defect

manifested by the inability to mount normal antibodyresponses, and the two have similar infectious histories.However, CVID usually manifests later than XLA andindeed was initially called “acquired agammaglobulinemia”because it has its onset in adulthood more often than inchildhood (see Case 5). Four years of age would beparticularly young for CVID. In addition, patients withCVID not infrequently manifest various types of autoimmunemanifestations leading to chronic gastrointestinal (GI)disorders, hematopoietic abnormalities, and liverabnormalities that are never seen in XLA. Importantly, inCVID, mature B cells are present, although the activationand differentiation to antibody-producing cells are blocked.In Sam, the defect is earlier and prevents the development ofmature B cells. As you will note, we have worked our wayback to the earliest stages of B-cell development to localizethe point of Sam’s block (Fig. 1.2).

A final differential must be made among the various typesof B-cell abnormalities that result in a block in early B-celldevelopment at the pro-B cell stage and thus lead to the

CLINICAL ASPECTS OF X-LINKED AGAMMAGLOBULINEMIA 21

Figure 1.2. Remain-ing developing andmature lymphoid popu-lations in XLA. Gray cellsare populations missingin XLA patients. Barsindicate positions ofdevelopmental blocks.

Lymphoidstem cell

Pre-Bcell

ImmatureB cell

MatureB cell

Pre-Tcell

Bone marrow

Periphery

Thymus

CD4+TH

CD8+T

IgM

IgG

IgE

IgA

Pro-Bcell

IgM

Key: Grey cells are absent. Stripped cells are present in reduced numbers.

Primary lymphoid organs

absence of mature B cells in the periphery: XLA, μ heavychain deficiency, and λ5 deficiency (see the section onnormal B-cell development below for events leading toB-cell maturation). The distinction among these diseases onclinical or even on immunologic grounds can be quitedifficult. However, both μ heavy chain deficiency and λ5chain deficiency show autosomal recessive patterns ofinheritance and are quite rare. The family history of healthyfemales and affected male relatives on Sam’s mother’s sideis classic for an X-linked Mendelian genetic disease and isthus more consistent with XLA. Another difference is that μ

heavy chain deficient patients generally display even lowerlevels of IgG than XLA patients and have undetectableperipheral B cells. Both XLA and λ5 deficiency patientsgenerally have very small numbers of circulating B cells, butin XLA the B cells have an immature phenotype, and in λ5deficiency they have a mature phenotype.

In the end, distinction among these early B-cell defectsrequires confirmation by molecular analysis. This is usuallydone by analysis of patient monocytes, another cell thatordinarily expresses Bruton’s tyrosine kinase (Btk) (see

below). Identification of the mutation may be necessary forscreening of future pregnancies for the occurrence ofaffected fetuses.

CLINICAL ASPECTS OF X-LINKEDAGAMMAGLOBULINEMIA

XLA is characterized by susceptibility to infection withpyogenic bacterial organisms, particularly encapsulatedGram-positive and Gram-negative organisms such asHaemophilus influenzae, S. pneumoniae, S. aureus , andPseudomonas species. It thus results in severe recurrentacute infections starting at approximately six months of agewhen maternal antibodies transmitted to the child via theplacenta in utero are no longer present in sufficient amount.Upper and lower respiratory infections are especiallycommon, but life-threatening sepsis and meningitis (as inthe present case) also occur. A particularly serious andlong-term problem is recurrent pneumonia because it can

22 CHAPTER 1 X-LINKED AGAMMAGLOBULINEMIA (BRUTON’S AGAMMAGLOBULINEMIA)

lead to bronchiectasis, pulmonary fibrosis, and pulmonaryfailure. As previously noted, a clue to the early diagnosisand treatment of XLA (and a clinical pearl) is the absenceof lymph node enlargement and underdeveloped tonsils ina young child who presents with what seem to be normalchildhood infections.

Organisms that enter the bloodstream through the GItract are a particular problem for patients with B-cell defi-ciencies. Thus, immunization with live poliovirus may leadto infection rather than protection. This was the probablescenario in Sam’s mother’s cousin who died of poliomyeli-tis following “vaccination.” A particularly ominous type ofinfection that can occur in XLA (as well as in CVID) isenteroviral infection of the central nervous system (CNS).This infection can lead to a poorly understood type ofautoimmune encephalomyelitis that results in progressiveloss of CNS function characterized by motor and intellec-tual deterioriation and culminating in death. It is refractoryto treatment since it is usually recognized when the infec-tious phase is over and the CNS is sterile. In some casesXLA is associated with a dermatomyositis-type disorder. Aspreviously mentioned, in sharp contrast to CVID patients,XLA patients do not manifest the array of autoimmunedisorders mentioned above. Other viral and fungal infec-tions are well handled by XLA patients.

NORMAL B-CELL DEVELOPMENT

In the fetus, B-cell development first begins, along with allhematopoiesis, in the liver and then transfers to the bonemarrow, the major site of development at birth. The orderlyprocess of hematopoiesis is dependent on the interaction ofprecursor cells with their microenvironment, such as, bonemarrow stromal cells and osteoblasts, in ways which arestill being elucidated. In order to produce mature B cells,the first decision point is at the level of a pleuripotentialhematopoietic stem cell in the bone marrow which commitsto the lymphoid lineage. The next decision point is thedetermination as to whether differentiation will progressalong the T/NK pathway or the B-cell pathway. We willpick up the story of B-cell development at the level of theprogenitor B cell (pro-B), as illustrated in Figure 1.3. Notethat most of the experimental data have been obtained onmice, and there are differences between human and mouseB-cell development. The nomenclature also varies (evenfrom investigator to investigator) within one species. Wewill focus on human data where available.

During normal B-cell development, the pro-B cellinitiates immunoglobulin synthesis with the rearrangementand transcription of the μ heavy-chain gene. This occursfirst with the joining of the DHJH segments, followed

Pro-B cell

VH–DH JH rearrangement

Nonlymphoidlineages

T/NKlineages

DH–JHrearrangement

Td T+

Rag 1/2+

Rag 1/2+

Largepre-B cell

Pre-BCR

Cycling

Noncycling

Expression of pre-BCR

Small pre-Bcell

Decreasedexpression of pre-BCR

Light-chainrearrangement

Lymphoidstem cell

Pluripotent hematopoietic

stem cell

ImmatureB cell

Figure 1.3. B-celldevelopment in thebone marrow. Patientswith XLA do not expandtheir pre-B cell popu-lation due to lack ofsignaling and the cellsdo not survive.

PATHOGENESIS OF XLA AND RELATED DISORDERS 23

by the joining of VH segments. Accomplishing this taskrequires the expression of a variety of transcription factorsand recombination enzymes (Rag 1 and 2), endonucleases,and TdT (terminal deoxynucleotidyl transferase) similarto those operating in the developing T cell (see Case2). The productive rearrangement of the μ heavy-chaingene culminates in the expression of μ protein on thecell surface plasma membrane and/or in the internalcytoplasmic membranes in association with a “surrogate”light chain. The surrogate light chain is the product oftwo genes, VpreB and λ5 (also called λ-like in humans).The heavy chains and surrogate light chains assembleinto an Ig-like complex and become associated with twotransmembrane signal-transducing molecules, Igα and Igβ

(CD79a and CD79b). The molecular complex thus formedis called the pre-B-cell receptor (pre-BCR) and the cell isnow termed a precursor (pre)-B cell. The down-regulationof the recombination enzymes, further differentiation,continued survival, and, in humans, the proliferation of thepre-B cell requires stimulation via the pre-BCR and theactivation of a pre-BCR-dependent signaling cascade. Thisincludes the activation of a key cytosolic tyrosine kinaseenzyme known as Bruton’s tyrosine kinase, which is a Tecfamily protein tyrosine kinase (PTK) (Fig. 1.4).

The activation of Btk and its role in B-cell developmentis illustrated in Figure 1.5. A description of this biochemicalpathway is as follows:

Activation, possibly by cross-linking, of the pre-BCR causesphosphorylation of the cytoplasmic tails of Igα and Igβ ontheir ITAM domain (immunoreceptor tyrosine-basedactivation motif).These then activate the Src family kinases,particularly Lyn and then Syk and Blk. This allows theformation of a docking station for Syk, which then recruitsand phosphorylates SLP-65 and Btk at its PH (Pleckstrinhomology) domain. The adaptor protein SLP-65 (also knownas BLNK or BASH) functions as a scaffold, bringing Btk tothe membrane. This activated SLP-65 molecule further

PH = Pleckstrin homology TH = Tec homology SH3, SH2, SH1 = Src homology kinase domainsPR = Proline rich domain

SH3 SH2 SH1

TH

PHPR

PR

SH2 SH1TH

P P

SH3

PH

Figure 1.4. Structure of Bruton’s tyrosine kinase, a Tec fam-ily protein tyrosine kinase. Upon activation by phosphorylationmolecule unwinds into a linear strand.

recruits Btk and phospholipase Cγ2 (PLC-γ2). Btk activatesPLC-γ2, which then hydrolyzes PIP2 (phophatidylinositolbisphosphate) to IP3 (inositol triphosphate) and DAG(diacylglycerol). This opens calcium channels and activatesprotein kinase C. In humans, SLP-65 and Btk control bothdifferentiation and proliferation of the pre-B cell, whereas inmice, although differentiation appears dependent on thesemolecules, proliferation involves different pathways andappears dependent on IL-7 signaling. Proliferation involvesthe activation of the RAS and MAPK (mitogen-activatedprotein kinase) pathways. Thus in humans, mutations in Btkand the much more rare mutations in SLP-65 or λ5 result ina severe phenotype; mice with mutations in these moleculesor “knockouts” demonstrate milder and “leakier” B-celldeficiency (see Unit V for description of knockout mice).Recent evidence suggests that, after activation, Btk andSLP-65 down-regulate the pre-BCR, thereby limitingpre-B-cell proliferation and possibly functioning as tumorsuppressor molecules.

Activated Btk again functions in the mature B cell andmay integrate pathways important for its survival,proliferative response, and modulation of the signalthreshold necessary for B-cell activation via the BCR. Inaddition, Btk is expressed in mast cells and may play a rolein their ability to degranulate in response to surface receptorcross-linking. Btk expression is restricted to the B-celllineage, myeloid (granulocytic) cells, and erythroid cells.

The pre-B cell has now gone from a larger proliferatingcell to a small pre-B cell. It has exited the cell cycle anddown-regulated pre-BCR expression and is no longer syn-thesizing surrogate light chain molecules. Re-expression ofthe enzymes required for immunoglobulin gene rearrange-ment, Rag 1 and 2, initiates light chain gene rearrangementand protein synthesis. This light chain must be able to asso-ciate with the μ heavy chain on the surface for the cell toescape apoptosis. The cell is now the immature B cell and issubject to negative selection for elimination of autoreactivecells.

PATHOGENESIS OF XLA ANDRELATED DISORDERS

X-linked agammaglobulinemia, or Bruton’s agammaglob-ulinemia, was described by Ogden Bruton in 1952 and,indeed, was the first description of an immunodeficiencydisorder. Dr. Bruton, a colonel in the U.S. army at WalterReed Hospital in Washington DC, identified the first casewhen he noticed that a patient with recurrent infections hada very low gamma globulin band in the recently developedprotein electrophoresis test. The disease has a frequency of3–6 per million. It was not until 1993 that the responsibleprotein was identified and its gene mapped to the long armof the X chromosome at Xq21.2–22. It was subsequentlynamed for Dr. Bruton, Bruton’s tyrosine kinase.

24 CHAPTER 1 X-LINKED AGAMMAGLOBULINEMIA (BRUTON’S AGAMMAGLOBULINEMIA)

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Figure 1.5. The activation of Btk and its role in B-cell development 1 Activation of pre-BCR by unknown mechanism(? crosslinking by ligand) brings 2 Src family kinases, Lyn, Fyn and Blk to the ITAMS on Igα and Igβ as a result of theirphosphorylation on their ITAMS. This results in 3 a docking site for Syk which 4 recruits and phosphorylates SLP-65 and Btk.5 BtK in turn associates with and activates PLCγ. 6 Activated PLCγ2 hydrolyzes PIP2 to DAG + IP3 7 which opens Ca++ channels

in the membrane. SLP-65 and BtK also activate differentiation and proliferation, the latter through RAS and MAPK pathways and thendown-regulate the pre-BCR. A , B , and C denote the three pathways affected by SLP-65 - Btk activation.

In the presence of a mutated Btk and the consequentabsence of signaling of the pre-B cell via the pre-BCR, thepre-B cell undergoes apoptosis or fails to mature. The B lin-eage cells thus accumulate in the marrow at the pro-B cellstage with few pre-B cells identifiable. Instead of the largercycling cells normally seen at the beginning of the pre-Bcell stage, those pre-B cells which are present are small.This block in proliferation and differentiation leads to anabsence of mature B cells in the periphery; no or few circu-lating immature B cells; tonsils and lymph nodes that lackgerminal centers; and the absence of plasma cells. Recentlyit has been shown that bone marrow B cells in XLA patientsdo produce antibodies, but these antibodies have alteredreactivity, leading in some cases to autoantibody activity.Thus, intact Btk function may also be necessary for deletionof B cells that react to autoantigens.

Over 300 different mutations that give rise to XLAhave been identified in the BTK gene. These range frompoint mutations with single amino acid substitutions to dele-tions and stop codons. It should be noted that XLA is oneof several immunodeficiency disorders caused by a muta-tion in a tyrosine kinase enzyme. The other two thus faridentified are mutations in JAK3 , which causes a form of

SCID, and mutations in ZAP70 , which results in a type ofT-cell deficiency.

Several other genetic abnormalities, μ chain deficiency,λ5 (also called λ-like in humans) deficiency, and Igα defi-ciency are similar to XLA in that they also lead to a defectin BCR expression at the pro/pre-B-cell stage of B-celldevelopment. This emphasizes the fact that expression ofthe pre-BCR and its signaling are necessary for maturationof the B cell beyond the pro/pre-B-cell stage. These auto-somal recessive abnormalities, which have been describedin only a handful of patients, are clinically similar to XLA,although μ chain deficiency is a somewhat more severedisease with an earlier onset and a more profound lack ofIg. The exclusive accumulation of pro/pre-B cells in thebone marrow of patients with these genetic abnormalitiesallows definition of its phenotype as CD19+CD34+ B cells,defines the bone marrow as the site of B-cell maturation innonavian species (birds have the bursa of Fabricius for mat-uration of B cells), and suggests the existence of little orno extramedullary B-cell maturation. Recall that the mousemodel for XLA (the xid mouse), which is also due to a Btkmutation, is characterized by much less severe symptoma-tology, suggesting that in murine B cells, non-Btk signaling

REFERENCES 25

can partially compensate for Btk signaling or that Btk func-tions are more restricted in mice.

Bonus Question. What would you observe in a female car-rier of XLA who is heterozygous for glucose-6-phosphatedehydrogenase (G6PD) isozyme?

The B cells of a female carrier of XLA would express only asingle isozyme of G6PD, whereas her T cells, red bloodcells, fibroblasts, and all other non-B cells would express amixture of the two G6PD isozymes. G6PD is an enzyme that,like Btk, is encoded by a gene on the X chromosome. Allthose pre-B cells whose active X chromosome had thedefective BTK gene would die in the marrow or fail tomature past the pro-B-cell stage, leaving only those B cellswith the normal BTK gene to fully mature. Consequently themature B cells would express only one G6PD isozyme, theone encoded by the X chromosome not bearing the defectiveBTK gene. In contrast, cells that do not depend on Btk forsurvival (non-B cells) would randomly inactivate one Xchromosome (lyonization principle) and thus give rise to anon-B-cell population with a mixture of G6PD isozymes.

TREATMENT OF X-LINKEDAGAMMAGLOBULINEMIA

XLA and related B-cell deficiencies are logically and effec-tively treated by the administration of IVIG. Such treat-ment usually prevents serious lower respiratory infectionand thus prevents the development of bronchiectasis andother chronic pulmonary problems. IVIG therapy has sup-planted intramuscular IG therapy because it results in higherlevels of serum IgG and because it avoids the local skinand muscle problems associated with the injection of intra-muscular injection of IG. IVIG contains only low amounts

of IgM and IgA, and thus IVIG therapy does not restorethe levels of these immunoglobulins. In addition, IgA nor-mally functions at the mucosal surface and would not beeffectively if replaced in this manner. If infection occursin spite of IVIG therapy, it is important to treat the patientearly with appropriate antibiotics. As with any genetic dis-order resulting from the absence of a single protein, genereplacement therapy is the hope of the future.

As these patients live longer, the possibility ofan increased incidence of malignancies, particularlyhematologic malignancies, arises. Interestingly, gastriccarcinoma, also frequent in CVID patients, is reported at ahigher than expected rate in XLA patients.

REFERENCES

Espeli M, Rossi B, Mancini SJC, Roche P, Gaunthier L, SchiffC (2006): Initiation of pre-B cell receptor signaling: Commonand distinctive features in human and mouse. Sem Immunol18:56–66.

Geier JK, Schlissel MS (2006): Pre-BCR signals and the controlof Ig gene rearrangements. Sem Immunol 18:31–39.

Kawakami Y, Kitaura J, Hata D, Yao L, Kawakami T (1999):Functions of Bruton’s tyrosine kinase in mast and B cells. JLeukocyte Biol 65:286–290.

Nomura K, Kanegane H, Karasuyama H, Tsukada S, AgematsuK, Murakami G, Sakazume S, Sako M, Tanaka R, Kuniya Y,Komeno T, Ishihara S, Hayashi K, Kishimoto T, Miyawaki T(2000): Genetic defect in human X-linked agammaglobulinemiaimpedes a maturational evolution of pro-B cells into a laterstage of pre-B cells in the B-cell differentiation pathway. Blood96:610–617.

Yang WC, Collette Y, Nunes JA, Olive D (2000): Tec kinases: Afamily with multiple roles in immunity. Immunity 12:373–382.