Immunoglobulin isotype switching, or class switching, is a complex process.After antigen has been encountered and the mature B cell is activated, therearranged immunoglobulin variable (V) region can become progressivelyassociated with different constant (C)-region genes by a form of somaticrecombination (see Fig. 11.2). This class switch recombination requires signals from T lymphocytes as well as engagement of the appropriate biochemical machinery in B lymphocytes. In the preceding case of X-linkedhyper IgM syndrome (see Case 11) a failure of isotype switching resulted froma defect in the gene that encodes the cell-surface molecule CD40 ligand(CD40L, also known as CD154), which is expressed by T cells. The engage-ment of CD40 ligand on activated CD4 T cells with its receptor, CD40, on Bcells is required to initiate immunoglobulin class switching. CD40 ligand isencoded on the X chromosome and so this type of hyper IgM syndrome isseen only in males.

An inability to undergo isotype switching can also be caused by a defect in theB cell’s own biochemical pathways. The biochemical events underlying isotype switching have only recently been clarified. While studying a culturedB-cell line that was being induced to undergo class switching from IgM to IgAsynthesis, immunologists in Japan observed the marked upregulation of anew enzyme, activation-induced cytidine deaminase (AID). This enzyme

An intrinsic B-cell defect prevents immunoglobulin

isotype switching.

71

Activation-induced

Cytidine Deaminase

(AID) Deficiency

CASE 12

Somatic hypermutation

Antibody isotypes

Isotype switching

Topics bearing onthis case:

case12 Final.qxd 11/5/07 12:00 Page 71

converts cytidine to uridine, and it is now known to trigger a DNA repairmechanism that causes isotype switching. The contribution of AID to classswitching was confirmed by ‘knocking out’ the gene encoding AID by homol-ogous recombination in mice; the mutant animals developed hyper IgM syn-drome and were unable to make IgG, IgA, or IgE.

In humans, hyper IgM syndrome is also encountered in females, in whom ithas an inheritance pattern in many families that suggests autosomal reces-sive inheritance (Fig. 12.1). The defective gene of the autosomal recessive form was mapped in several informative families to the short arm ofchromosome 12, in a region that corresponds to the region containing theAID gene in mice. This prompted the search for a link between hyper IgMsyndrome and AID deficiency in humans, and several cases of the autosomalrecessive form were found to have mutations in the AID gene.

At 3 years old Daisy Miller was admitted to the Boston Children’s Hospital with

pneumonia. Her mother had taken her to Dr James, a pediatrician, because she had

a fever and was breathing fast. Her temperature was high, at 40.1°C, her respiratory

rate was 40 per minute (normal 20), and her blood oxygen saturation was 88%

(normal >98%). Dr James also noticed that lymph nodes in Daisy’s neck and

armpits (axillae) were enlarged. A chest X-ray was ordered. It revealed diffuse

consolidation (whitened areas of lung due to inflammation, indicating pneumonia)

of the lower lobe of her left lung and she was admitted to the hospital.

Daisy had had pneumonia once before, at 25 months of age, as well as 10 episodes

of middle-ear infection (otitis media) that had required antibiotic therapy. Tubes

(grommets) had been placed in her ears to provide adequate drainage and

ventilation of the ear infections.

In the hospital a blood sample was taken and was found to contain 13,500 white

blood cells mml–1, of which 81% were neutrophils and 14% lymphocytes. A blood

culture grew the bacterium Streptococcus pneumoniae.

Because of Daisy’s repeated infections Dr James consulted an immunologist. He

tested Daisy’s immunoglobulin levels and found that her serum contained 470 mg

dl–1 of IgM (normal 40–240 mg dl–1), undetectable IgA (normal 70–312 mg dl–1), and

40 mg dl–1 of IgG (normal 639–1344 mg dl–1). Although Daisy had been vaccinated

against tetanus and Haemophilus influenzae, she had no specific IgG antibodies

against tetanus toxoid or to the polyribosyl phosphate (PRP) polysaccharide

antigen of H. influenzae. Because her blood type was A, she was tested for anti-B

antibodies (isohemagglutinins). Her IgM titer of anti-B antibodies was positive at

1:320 (upper limit of normal), whereas her IgG titer was undetectable.

Daisy was started on intravenous antibiotics. She improved rapidly and was sent

home on a course of oral antibiotics. Intravenous immunoglobulin (IVIG) therapy

was started, which resulted in a dramatic decrease in the frequency of infections.

Analysis of Daisy’s peripheral blood lymphocytes revealed normal expression of

CD40 ligand on T cells activated by anti-CD3 antibodies, and normal expression of

CD40 on B cells (Fig. 12.2). Nevertheless, her blood cells completely failed to

secrete IgG and IgE after stimulation with anti-CD40 antibody (to mimic the effects

of engagement of CD40 ligand) and interleukin-4 (IL-4), a cytokine that also helps to

stimulate isotype switching, although the blood cells proliferated normally in

The case of Daisy Miller: a failure of a critical B-cell

enzyme.

Case 12: Activation-induced Cytidine Deaminase (AID) Deficiency72

affected female

normal female

normal male

proposita

Fig. 12.1 A pedigree of a family with AIDdeficiency. As the parents of the affectedchild (the proposita) show no signs ofdisease themselves, the defective AID genemust be recessive. They both carry a singlecopy of this gene and, as they are firstcousins, the most likely source of thedefective gene is their shared grandfather orgrandmother. If one of these wereheterozygous for AID deficiency, they couldhave transmitted the defective gene to boththeir sons. The fact that the affected child isa girl, and that neither her father nor anyother males in her extended family showsigns of disease, indicates that the gene is carried on an autosome and not the X chromosome.

Five-year-old girl with

repeated ear infections

Defective CD40L or AID?

Order DNA test

High IgM; IgG very low;

no IgA. Hyper IgM

syndrome?

case12 Final.qxd 11/5/07 12:00 Page 72

response to these stimuli (Fig. 12.3). cDNAs for CD40 and for the activation-

induced cytidine deaminase (AID) were made and amplified by the reverse

transcription–polymerase chain reaction (RT–PCR) on mRNA isolated from blood

lymphocytes activated by anti-CD40 and IL-4. Sequencing of the cDNAs revealed a

point mutation in the AID gene that introduced a stop codon into exon 5, leading to

the formation of truncated and defective protein. The CD40 sequence was normal.

It is now apparent that there are two distinct phenotypes of hyper IgM syn-drome, resulting from four different genetic defects. One phenotype, whichresults from defects in the genes encoding CD40 or CD40 ligand (see Case 11),manifests itself as susceptibility to both pyogenic and opportunistic infections.The other phenotype, which results from defects in the AID gene or the geneencoding uracil-DNA glycosylase (UNG), resembles X-linked agammaglobu-linemia in that these patients have increased susceptibility to pyogenic infec-tions only.

When CD40 and the IL-4 receptor on B cells are ligated by CD40 ligand andIL-4, the AID gene is transcribed and translated to produce AID protein. Atthe same time, transcription of cytidine-rich regions at isotype-switch sites isinduced, which involves separation of the two DNA strands at these sites.AID, which can deaminate cytidine in single-stranded DNA only, then proceeds to convert the cytidine at the switch sites to uridine. As uridine is

Activation-induced cytidine deaminase (AID)

deficiency.

Case 12: Activation-induced Cytidine Deaminase (AID) Deficiency 73

Numberof B cellsbindinganti-CD40antibody

Numberof T cellsbindinganti-CD40Lantibody

300100

80

60

40

20

0 0

800

640

480

320

160

0

200

160

120

80

40

0

Normal individual Patient 1

Anti-CD40 log fluorescence intensity

Anti-CD40L log fluorescence intensity100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

Fig. 12.2 Flow cytometric analysisshowing normal expression of CD40 andCD40 ligand in a patient with AIDdeficiency. Top row: CD40 measured bythe binding of flurorescently tagged anti-CD40 antibodies to CD40 on B cellsfrom (left panel) a normal individual and(right panel) a patient with AID deficiency.Bottom row: measurement of CD40 ligand(CD40L) on T cells from (left panel) a normalindividual and (right panel) the same patientactivated in vitro with the mitogen phorbolester (PMA) and ionomycin.

IgE

(pg

ml–1

)

Normalindividual

CD40Ldeficient

Patient

30,000

20,000

10,000

0

MediumIL-4Anti-CD40 + IL-4

Fig. 12.3 Comparison of isotypeswitching, as judged by IgE secretion, ina normal individual, a patient with CD40Ldeficiency, and a patient with AIDdeficiency. IgE production from peripheralblood mononuclear cells in vitro wasmeasured after no stimulation (‘medium’;negative control), stimulation with IL-4 alone,or stimulation with anti-CD40 and IL-4. Theanti-CD40 and IL-4 together cancompensate to some degree for the lack ofstimulation of B cells by CD40 ligand on Tcells, but cannot compensate at all for thedefect in AID.

case12 Final.qxd 11/5/07 12:00 Page 73

not normally present in DNA, it is recognized by the enzyme uracil-DNA glycosylase, which removes the uracil base from the rest of the nucleotide.The damaged DNA strand is now recognized by DNA repair endonucleases,which excise the damaged region. The repair of these breaks in the switchregions results in class switching.

Interestingly, AID is not only required for class switching but also for somatic hypermutation in B cells. This is the process that underlies the production of antibodies of increasingly higher affinity for the antigen as animmune response proceeds. When a B cell is activated by the combination ofbinding of its surface IgM and signals from other cells, especially T cells (forexample via CD40–CD40-ligand interaction), not only cell division and classswitching are initiated. In addition, point mutations are introduced into theDNA that codes for the immunoglobulin V region. A process of selection thenoccurs. Cells expressing mutated surface immunoglobulin with a strongeraffinity for antigen compete best for binding the available antigen and receivestronger signals via this cell-surface antigen receptor; they consequently proliferate. Cells with lower-affinity immunoglobulins are less likely to bindand be stimulated by antigen, and if they do not receive these stimulatory signals, they die. This leads to selection of cells with mutations that result inhigh-affinity antibodies, a process referred to as ‘affinity maturation’ of theantibody response. B cells in mice and humans that lack functional AID areunable to generate these mutations after activation and therefore cannotundergo affinity maturation. The only response these B cells can make to theactivating signals is to proliferate. This results in the accumulation of IgM+ Bcells in the lymphoid organs, giving rise to an enlarged spleen (splenomegaly)and enlarged lymph nodes (lymphadenopathy) (Fig. 12.4).

Daisy and other patients with hyper IgM syndrome caused by amutation in AID do not seem to suffer from opportunistic infections,such as Pneumocystis carinii and Cryptosporidium, which arecharacteristic of the X-linked hyper IgM syndrome. Why?

A 3-year-old girl presents with hyper IgM syndrome, recurrentbacterial infection and a history of Pneumocystis carinii pneumonia. HerCD40L and AID genes are normal. What is the potential gene defect inthis patient?

A 6-year-old girl presents with hyper IgM syndrome, recurrentbacterial infection and no history of opportunistic infections. Her CD40and AID gene sequences are normal. What is the potential gene defect inthis patient?

Can you think of a simple test to distinguish hyper IgM syndromecaused by an intrinsic B-cell defect from that caused by CD40 liganddeficiency without recourse to DNA sequencing?

Dr James noted that Daisy had enlarged lymph nodes. Patients withX-linked hyper IgM syndrome do not have enlarged lymph nodes. Can youexplain the difference?

5

4

3

2

1

Questions.

Case 12: Activation-induced Cytidine Deaminase (AID) Deficiency74

Fig. 12.4 Histology of a lymph node froma patient with AID deficiency stained withhematoxylin and eosin. Note the largefollicles.

case12 Final.qxd 11/5/07 12:00 Page 74