X-linked Hyper IgM Syndrome

The Case of Dennis Fawcett, cont.

X-linked Hyper IgM Syndrome

FACS Analysis

of peripheral blood lymphocytes

Dennis' anti-A and anti-B titers measured

11% react with antibody to CD19 (B cell marker)

DPT Booster Injection

and Typhoid Vaccine

14 days later: no Ab

Dennis has RBC of Type O. Why does he have Ab to A and B RBC?

87% react with anti-CD3

(T cell marker)

normal results, except...

diptheria toxoid, pertussis, tetanus toxoid

to tetanus toxoid or typhoid O and H antigens

2% react with anti-CD56

(NK cell marker)

Bacteria in the intestine have Ag closely related to A and B Ag.

Antibody-mediated bacterial killing

X-linked Hyper IgM Syndrome

All of CD19 cells have

surface IgM and IgD,

none with IgA or IgG

Question 3

Question 6

Dennis' anti-A titer was 1:3200 and anti-B titer was 1:800, both very elevated. Anti-A and anti-B Ab IgM class only.

Why did Dennis make antibodies to blood group A and B antigens but not to tetanus toxoid, typhoid O and H, and streptolysin antigens?

• Intestinal bacteria have an Ag closely related to A and B Ags. Dennis creates a cross-reactive Ab for A and B Ags.

• Dennis' body has no antigens comparable to tetanus, typhoid, and streptolysin antigens. He cannot create them naturally because of his CD40L malfunction.

Neutralization

What Ab can be produced by someone with a CD40L deficiency?

Agglutination

• Coat pathogens in antibodies such that they are not a threat

• Precipitation of antibody-antigen products. Product can be flushed from the system before bacteria attacks cells

ADCC

Opsonization

Dennis has an X-linked immunodeficiency and yet there is no informative family history. Is he a new mutation or is his mother a carrier of the defect in the CD40L gene? Can we test his sister to determine whether she is a carrier? Would it be useful to examine the T cells of Dennis' mother and sister for nonrandom X inactivation?

IgM in response to T-cell independent Ag

no other Ab isotypes

• Promotes antigen phagocytosis by macrophages and neutrophils

Treatment

• Antibody is bound to surface of target cell and Fc receptor of NK cells
• Allows attacking cell to recognize and destroy antigen

cannot make any Ab to T-cell dependent Ag: vulnerable to many bacteria

Oxidation

Dennis is treated with intravenous gamma globulin, one injection 600 mg/kg body weight per month

Dennis' brother and sister do not have the disease. No family history.

• Chemical reaction releases oxidative material , kills bacterial cell

Activated T cells do not bind soluble CD40

T-cell independent Ag

gamma globulins are a class of globulins, identified by serum protein electrophoresis

a bag of blood plasma, which contains gamma globulin

Complement is Antibody-mediated

significant portion are Igs

What is X inactivation?

dosage compensation for 2 Xs in females and 1 X in males

The classical pathway of the complement system is antibody-dependent

• First, antibody (IgM or IgG) binds to antigen
• C1 complex binds to two antibody Fc sites

T cells activated (marked by CD25 presence)

mother

father

females inactivate 1 X early in development

Flow cytometric analysis: soluble CD40 protein

Ag binding to mIg provides both signals for B cell activation

normal individual: two pop of cells, CD40 and non-CD40 binding

generally a random event, with females expressing a mosaic of maternal and paternal X chromosomes

patient: CD40 fluorescence coincides with nonspecific control (no CD40 binding)

Antibody-mediated complement system leads to the creation of MAC and lysis of bacteria

brother

Dennis

sister

seen phenotypically in female calico cats with both orange and black genes: random inactivation

Dennis remained free of infection!

Hyper IgM

nonrandom X inactivation at the cell level is characteristic of female carriers, indicative of a defective X chromosome

no T cell help required

TI-1:PAMP activation through TLR provides signal 2

TI-2: cross-links mIg and CD21 (via complement C3d)

Case Study: Dennis Fawcett

Dennis Fawcett

White Blood Cell Count

• Doctors expect high WBC count
• Count is normal: 4200/microliter
• 26% neutrophils (low)
• 56% lymphocytes (normal)
• 28% monocytes (elevated)

• 5 years old
• Severe acute infection of ethmoid sinuses
• Recurrent since he was 1
• Had pneumonia from infection with Pneumocystis carinii when 3 years old
• treated successfully with antibiotics
• Group A Beta-hemolytic streptococci cultured from nose and throat

Successfully treated with IV antibiotics.

Serum tested for antibodies to streptolysin O, an antigen secreted by streptococci.

No antibodies to streptococcal antigen found.

Question 4

Most IgM is in the blood and less than 30% of IgM molecules get into the extravascular fluid. On the other hand, over 50% of IgG molecules are in the extravascular space. Furthermore we have 30 to 50 times more IgG molecules than IgM molecules in our body. Why are IgG antibodies more important in protection against pyogenic bacteria?

CD40 ligand

B Cell Development

Lymph node biopsy

Serum Igs

pyogenic bacteria are found in extravascular areas and must be opsonized by IgG molecules to aid with clearance

various pus-producing bacteria:

Staphylococcus aureus

leading cause of hospital-acquired infections

skin infections, respiratory disease, and food poisoning

Neisseria meningitidis

reside in back of nose and throat, may spread to meninges (type B outbreak on campus)

CD40/CD40L interaction induces cytokine expression

Poorly organized structures with absence of secondary follicles and germinal centers

B-cell activation is a two-step process

• 1. Antigen binding
• 2. CD40/CD40L interaction between B cell and T helper cell

Overview of B Cell Development

IgG: 25mg/dl (very low)

IgA: undetectable

IgM: 210mg/dl (elevated)

Antigen-Independent Phase

Sites of B Cell Activation

Secondary Lymphoid Organs

B Cell Activation Requires Two Signals

• Ig-gene rearrangement
• IgM expression
• negative selection (central tolerance)

• cortex: rich in B cells
• paracortex: rich in T cells

1. Antigen Binding

Antigen-Dependent Phase

• cross-linking of mIg

Normal lymph node

HXIM lymph node

• entering Ag percolates, foci of activated B cells form

Germinal Centers

MHC/TCR interaction induces expression of CD40L on T helper cells

2. CD40/CD40 ligand interaction

• Antigen activation in SLO
• Class switching
• Affinity maturation
• Differentiation

• germinal centers develop; activated B and T cells

• An example: production of IgE
• CD40/CD40L interaction, along with TCR/MHC interaction, induces production of IL4
• IL-4 induces class switching from IgM to IgE (and IgG1)
• CD40/CD40L engagement also leads to upregulation of B7 expression on B cell. Increases co-stimulation of B and T cells and further upregulation of IL-4 synthesis.

• T helper cell binds MHC Class II-peptide

Three important events:

1. affinity maturation

• T cells secrete activating cytokines recognized by B cells
• cytokines deliver differentiation, proliferation, and survival signals to B cells

CD40/CD40L interaction provides second signal for B-cell activation

2. class switching

3. plasma and memory cell formation

CD40/CD40L interaction induces cytokine production in T cell

Class Switching

VDJ unit combines with any C gene segment

Question 5

Class Switching

CD40 activation leads to isotype switching

Ig Isotypes

mediated by switch recombinase and activation-induced cytodine deaminase (AID)

• mediated by B cell interaction with T helper cell (CD40/CD40L necessary)

Newborns have difficulty in transcription of the CD40 ligand gene. Does this help to explain the susceptibility of newborns to pyogenic infections?

What does that antibody do?

switch regions upstream of C segments

• allows the variable heavy chain to associate with a constant region of any isotype

• Each human Ig has specialized functions and a unique distribution

IgM

in this example, end up with IgE, with an IgG intermediate

Question 1

first antibody type developed during response

Dennis' B cells expressed IgD as well as IgM on their surface. Why did he not have any difficulty in isotype switching from IgM to IgD?

• There is no switch region between the constant M and D regions. No recombination is necessary for IgD transcription because recombination occurs between switch regions.

• effector functions of antibodies secreted determined by isotype of heavy chain constant domain

activate complement system

• Mature, naive B cells express both IgM and IgD. The role of IgD is unknown, but it is necessary for Ag interaction.

• 5 major types

found in blood and lymph

• cytokines determine the class produced

• Yes. Without CD40L, newborns cannot create many of the necessary antibodies for opsonization
• They have, however, inherited IgG from their mother, which confers passive immunity during early life

• CD40 crosslinkage activates protein tyrosine kinases such as Lyn and Syk, creating a signaling pathway leading to production of transcription factors
• Together with cytokine regulation, results in class switching recombination, transcription of mRNA, and IgE synthesis

IgD

IgG

unclear

IgG1: opsonization

• determined by class of secreting plasma B cell, Fc portion mediates effector function

transport across placenta

diffusion into extravascular sites

IgA

IgE

The case of Subject III-3

Cyclosporin A, a drug widely used for immunosuppression in graft recipients, also inhibits the transcription of the CD40L gene. What does this imply for patients taking this drug?

mucosal immunity

sensitization of mast cells

main Ig in mucosal secretions

immunity to parasites

allergies

• Graft recipients taking Cyclosporin A will also have inhibited class switching and will be unable to make all isotypes upon new infection.

X-linked Hyper IgM syndrome

Dennis was given monthly injections of gamma globulin. What's another possible treatment?

Pedigree of a Family with X-Linked Hyper IgM Syndrome

what's going on in this pedigree?

Diagnosis

bone marrow transplant

2 maternal uncles had died at ages 6 months and 2 years of protracted diarrhea

Pyogenic Bacteria

Opportunistic Infections

Subject III-3 received a bone marrow transplant from his sister.

inability to counter cryptosporidium infection

Question 2

Dennis is susceptible to pyogenic and opportunistic infections

Pyogenic (pus-forming) bacteria are resistant to phagocytosis unless opsonized with antibody and complement.

• Why? These bacteria are gram-positive, more resistant to destruction

Dennis' ethmoiditis caused by Streptococcus pyogenes, a pyogenic bacterium

• Opportunistic infections: bacteria, viruses, fungi that normally reside in our bodies cause infections when cell-mediated immunity is defective.

Dennis' pneumonia was caused by Pneumocysitis carinii

• Common opportunistic infection in AIDS and cancer patients

Question 2. Normal mice are resistant to Pneumocystis carinii. SCID mice, which have no T or B cells but normal macrophages and monocytes, are susceptible to this infection. In normal mice, Pneumocystis carinii are taken up and destroyed by macrophages. Macrophages express CD40. When SCID mice are reconstituted with normal T cells they acquire resistance to Pneumocystis infection. This can be abrogated by antibodies to the CD40 ligand. What do these experiments tell us about this infection in Dennis?

activation of macrophages by T cells via CD40/CD40L

histopathology of the lung shows P. carinii infection

Thank you!

Failure to develop leukocytosis

Outcome

bone marrow transplantation

intravenous immune globulin

Microsatellite Typing before and after Bone Marrow Transplantation of the the patient. Samples also displayed from the patient's mother, father, and his donor sister.

Why are CD40 ligand-deficient males unable to develop leukocytosis?

allele 2 associated with the CD40 mutation (10 bp deletion)

Subject III-3 changed blood type for O- to A+ (blood type of his sister)

ability to create Ab to vaccines

leukocytosis: rise in WBC count during inflammation

activated macrophages release GM-CSF: granulocyte-macrophage cell-stimulating factor, which normally activates neutrophils and causes them to divide

develops into neutropenia (neutrophil deficiency)

Neutropenia

neutrophils are usually the first responders to a site of inflammation

most abundant WBC

they phagocytose bacteria and release antimicrobial granules

neutropenia can lead to severe sores and blisters in mouth and throat

Thomas, Caroline, et al. "Correction of X-Linked Hyper-IgM Syndrome by Allogenic Bone Marrow Transplantation," New England Journal of Medicine

why?

infested with many bacteria, neutrophils normally protect oral mucosa

Treatment:

give patients GM-CSF to account for lack of macrophage secretion