1. IMMUNOGLOBULINS STRUCTURE AND FUNCTION
BSc Public Health
5th week, 2014

2. IMMUNOGLOBULINS Definition
Glycoprotein molecules that are present on B cells (BCR) or produced by plasma cells (usually referred to as antibodies) in response to an immunogen (antigen that provokes immune response)

3. B CELL ACTIVATION
Gene rearrangement in an immature B cell leads to the expression of membrane-bound IgM and IgD on the mature B cell surface. After encounter with an antigen this isotype is produced in a secreted form by the plasma cell (IgM largely while IgD only is small amounts). All isotypes (IgA, IgD, IgE, IgG and IgM) can be made in two forms: the bounded immunoglobulins serving as BCRs, and soluble secreted antibodies by the plasma cells. (The difference b/w membrane bound and secreted is a hydrophobic part and hydrophilic parts at the carboxy terminus, respectively).

4. Immunoglobulin STRUCTURE
2x Heavy chain (light blue)
2x light chain (dark blue)
Variable regions  antigen binding
Constant regions
disulfide bond
carbohydrate CL VL
CH2
CH3
CH1
hinge region VH

5. mIg sIg BCR (B cell receptor) Antibody
Transmembrane domain
Associated chains for signaling
Cytoplasmic domain
SOLUBLE (freely circulating)
MEMBRANE BOUND!
Antigen binding  effector functions
Produced by plasma cells
Antigen recognition  B cell activation

6. ANTIBODY DOMAINS AND THEIR FUNCTIONS
Antigen recognition
Variable domain Ag Ag
Constant domains
Effector functions

7. B CELL ACTIVATION
B cell
BCR oligomerization results in B cell activation, proliferation and differentiation

8. ANTIGEN BINDING Antigen Binding Fragment (Fab) Complement binding site
Placental transfer
Constant fragment (Fc)
Binding to Fc receptors on phagocytic cells

9. HYPERVARIABLE REGIONS
B cell development in the red bone marrow  DNA recombination (somatic gene rearrangement) of gene segments encoding variable domains of heavy and light polypeptide chains is responsible for generation of B cells with highly variable specificity
Epitope
CDR1
CDR2
CDR3
Light chain
Heavy chain
CDR = complementarity determining region = hypervariable region

10. DIFFERENT VARIABLE REGIONS  DIFFERENT ANTIGEN-BINDING SITES  DIFFERENT SPECIFICITIES
the same applies to TCRs!

11. Sequence variability of H/L-chain constant regions
ISOTYPE (CLASS)
Sequence variability of H/L-chain constant regions
IgG - gamma (γ) heavy chains
IgM - mu (μ) heavy chains
IgA - alpha (α) heavy chains
IgD - delta (δ) heavy chains
IgE - epsilon (ε) heavy chains

12. PHASES OF B CELL RESPONSE
Gene rearrangement in an immature B cell leads to the expression of membrane-bound IgM and IgD on the mature B cell surface. After encounter with an antigen this isotype is produced in a secreted form by the plasma cell (IgM largely while IgD only is small amounts). All isotypes (IgA, IgD, IgE, IgG and IgM) can be made in two forms: the bounded immunoglobulins serving as BCRs, and soluble secreted antibodies by the plasma cells. (The difference b/w membrane bound and secreted is a hydrophobic part and hydrophilic parts at the carboxy terminus, respectively).

13. ISOTYPE SWITCHING
Isotype Switching during B Cell Development During the initial stages of a B cell’s primary response to antigen, it produces and secretes IgM. Later in the primary response or during subsequent responses, different heavy chain isotypes may be expressed by the progeny of the original IgM-producing clone. Such “switching” occurs at the DNA level, resulting in the production of an Ig protein with the same V region but a different C region. Thus, over the lifetime of a B cell clone, it may produce antibodies of the same specificity but different isotypes.

14. MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES
IgG1-IgG4
IgA1-IgA2

15. FLEXIBILITY OF ANTIBODIES

16. ANTIBODY PRODUCTION DURING THE
PRIMARY AND THE SECONDARY IMMUNE RESPONSES
Level of antibodies
secondary response against antigen A
Primary response against antigen A
primary response against antigen B
Days
napok
Antigen A
Antigen A and B

18. EFFECTOR FUNCTIONS OF ANTIBODIES
Antibody-mediated immune responses
Fab part: NEUTRALIZATION
Fc part:
OPSONIZATION followed by
opsonized phagocytosis (macrophage; IgG)
ADCC (NK cell; IgG)
mast cell degranulation (parasite, allergy; IgE)
COMPLEMENT ACTIVATION

19. NEUTRALIZATION

20. Complement binding site Binding to Fc receptors
Antigen binding
Complement binding site
Placental transfer
Binding to Fc receptors

21. OPSONIZED PHAGOCYTOSIS
Flagging a pathogen
Antigen binding fragment (Fab) binds the pathogen  the Fc region is accessible for Fc-receptors of phagocytic cells, facilitating (speeding up) the process of phagocytosis

22. Opsonization facilitate and accelerate the recognition of the pathogens by phagocytes
Main opsonins:
antibodies
Complement molecules
Acute-phase proteins (CRP, SAP)
Phagocytes must express receptors for the opsonins:
IgG  FcγRI
C3b  CR1

23. Antibody Dependent Cellular Cytotoxicity
(ADCC)

24. MAST CELL DEGRANULATION
FcεRI +
IgE
Mast cells, basophils and activated eosinophils in mucosal surfaces play a role in the defense against parasites and express the FcεRI.
FcεRI has such high affinity that the IgEs, specific for many different antigens- cannot dissociate.
Upon antigen binding and FcεRI cross-linking the mast cell is activated (degranulation) inflammatory mediators released, acting on vessel permeability- swelling, pain etc, and acting on smooth muscle cells  Sneezing, coughing, vomiting, diarrhea.
Directly killing the parasite by toxic granule content or Indirectly flushing it.
An unnecessary response to an innocuous substance (pollens etc.) are an unfortunate side effect of the highly specialized and powerful antibodies.
(A) High-affinity FcRs on the surface of the cell bind antibodies before it binds to antigen. (mast cell)
(B) Low-affinity FcRs bind multiple Igs that have already bound to a multivalent antigen. (macrophage, NK cell)

25. Complement binding site Binding to Fc receptors
Antigen binding
Complement binding site
Placental transfer
Binding to Fc receptors

26. Complement binding site Binding to Fc receptors
Antigen binding
Complement binding site
Placental transfer
Binding to Fc receptors
FcRn on the placenta facilitate the transfer of maternal IgG to the body of the fetus

27. PRODUCTION OF IMMUNOGLOBULINS
IgG transport is so efficient that at birth babies have as high a level of IgG in their plasma as their mothers
These transfers are a form of passive immunization. The babies protection by IgG and IgA is against those pathogen that the mother has mounted
The children are most vulnerable during the first year of life (esp.3-12m) when maternal IgGs have disappeared but the de novo synthesis is at low level

28. Pathological consequences of placental transport of IgG
(hemolytic disease of the newborn)
Passive anti-D IgG
anti-Rh
IgM
Rhesus incompatibility: In case a fetus is Rh+ (meaning he expresses the D antigen on his RBCs surface) and the mother is RH- (no D antigens and no anti-D antibodies) after the first delivery when some fetal RBCs mix with maternal circulation, the mother will initiate a primary immune response towards the D antigen. These antibodies as it is the first immune response will be of IgM isotype and therefore not able to pass the placenta. However, with time, isotype switching might take place that will result in the production of IgG antibodies against the D antigen. These are now able to pass through the placenta, thus, in the second pregnancy if the fetus is Rh+ his RBCs will be attacked by maternal anti-D IgG antibodies, causing the mild to severe ‘hemolytic disease of the newborn’ (destruction of red RBCs anemia)
To avoid that, i.m. anti-D IgGs are administered to the mother. These will bind any D antigens on fetal RBCs that entered the mothers circulation and prevent her from developing anti-D antibodies. The IgGs eliminate the RBCs coated with D antigens through opsonisation and elimination by macrophages and granulocytes.
… One might say, well IgGs pass the placenta, then these anti D antibodies can attack the fetus RBCs- true, but the clinical course of such an event is benign and requires no treatment.

29. SECRETORY IgA AND TRANSCYTOSISJ C S s
‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR (the secretory component) J C S s J C S s
MUC US J C S s J C S s
IgA and pIgR are transported to the apical surface in vesicles J C S s
Epithelial
cell
pIgR and IgA are
internalised
Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa J C S s
plasma cells located in the submucosa
produce dimeric IgA