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PRODUCTION OF IMMUNOGLOBULINS
BEFORE BIRTH
AFTER BIRTH
breast milk
IgA
100%
(adult)
maternal IgG
IgM
IgG
IgA
0
3
month
6
9
1 2 3 4 5 adult
year
Ig. Concentration
level
of antibodies
secondary response against
Szekunder
’lasyecondary resp
antigen A
primary response against
antigen
A response
primer
IgG
IgA
IgE
IgM
IgM
primary
response against
antigen B
5
„A” antigA
én
Antigen
10
15
20
25
30
„A” és „B”
Antigen
A and B
antigén
napok
days
napok
Polyclonal antibody response
Ag
Polyclonal
antibody
Immunserum
Set of B-cells
Ag
Activated B-cells
Antibodyproducing
plasma-cells
Antigen-specific antibodie
Ig isotype
Serum
concentration
Characteristics, functions
Trace
amounts
 Major isotype of secondary
(memory) immune response
 Complexed with antigen activates
effector functions (Fc-receptor
binding, complement activation
 The first isotype in B-lymphocyte
membrane
 Function in serum is not known
Trace
amounts
 Major isotype in protection against
parasites
 Mediator of allergic reactions (binds
to basophils and mast cells)
3-3,5 mg/ml
 Major isotype of secretions (saliva,
tear, milk)
 Protection of mucosal surfaces
12-14 mg/ml
1-2 mg/ml
 Major isotype of primary immune
responses
 Complexed with antigen activates
complement
 Agglutinates microbes
 The monomeric form is expressed in
B-lymphocyte membrane as antigen
binding receptor
STRUCTURE OF
IMMUNOGLOBULINS/ANTIBODIES
Heavy chain (H)
VH
VL
CH
Light chain (L)
CL
Antigen
Antigen
binding
antigénkötés
s
VL
s
s
s
s
s
DEGRADATION
TRANSPORT
s
ss
ss
Constans
domains
konsta
ns dom ének
effektor funkc iók
Effector functions
CH2 ss
s
s
CH3 ss
s
s
s
BINDING TO CELLS
s
s
CL
s
COMPLEMENT ACTIVATION
s
CH1
s
va riábilis
d om ének
Variable
domains
s
s
S
s
S
s
s
s
s
VH
Antibodies with different isotypes differ in their
Binding affinity, effector functions and their
Transport.
Carbohydrate antigens are usually recognized
By IgM type antibodies.
Differences in transport makes all the differece:
Antibodies spec. to blood group antigens
Structures of the ABO blood group antigens
Defined by specific enzymes inherited co-dominant genes (Mendelian rules)
Donors and recipients for blood transfusion
-
+
+
+
-
-
+
+
-
+
-
+
-
-
-
-
Rhesus (Rh) blood group antigen (D)
POLYPEPTIDE TYPE ANTIGEN
extracellular space
cytoplasm membrane
intracellular space
IgG type antibody
- incomplete
no direct agglutination
but human immunglobulin-reactive
2. antibody can cause agglutination
indirect agglutination
Pathological consequences of placental transport of IgG
(hemolytic disease of the newborn)
Effects of agglutination in vivo
ABO incompatibility
intravascular haemolysis
(complement mediated haemolysis)
Rh incompatibility
haemolytic disease of the
newborn (erythroblastosis
fetalis)
(opsonisation of red blood cells, which
are then phagocytosed by macrophages
and granulocytes)
Rh profilaxis
ANTIBODY DIVERSITY
AMINO ACID SEQUENCE OF IMMUNOGLOBULINS
Multiple myeloma (MM)
Plasma cell tumors – tumor cells reside in the bone marrow
Produce immunoglobulins of monoclonal origin, serum concentration 50-100mg/ml
Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification
Gel electrophoresis
L
H
Reduction
50 kDa
Heavy chain
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
25 kDa
Light chain
Variable
Constant
GENETIC BACKGROUND OF ANTIBODY DIVERSITY
VH
VL
S–S
VH
VL
S–S
Mechanism of the generation of variability?
Different rules for encoding the variable and constant regions?
Symmetric molecule  two identical VH and VL  both chromosomes encode the same
sequence?
DOGMA OF MOLECULAR BIOLOGY
CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE
1 GEN = 1 PROTEIN
THEORIES
1 GEN
Gen
High rate of somatic mutations in the V-region
V
C
Many GENES (10 000 – 100 000)
V1 C1
Protein
V2 C2
Vn Cn
MOLECULAR GENETICS OF IMMUNOGLOUBLINS
How can the bifunctional nature of antibodies be explained genetically?
In 1965, Dreyer & Bennett proposed that for a
single isotype of antibody there may be:
• A single C region gene encoded in the GERMLINE and separate from the V
region genes
• Multiple choices of V region genes available
• A mechanism to rearrange V and C genes in the genome so that they can
fuse to form a complete Immunoglobulin gene.
This was genetic heresy as it violated the then accepted notion
that DNA was identical in every cell of an individual
The Dreyer - Bennett hypothesis
V
V
V
V
V
V
V
V
V
V
V
V C
V
V
A single C region gene is
encoded in the germline and
separated from the multiple V
region genes
C
A mechanism to rearrange V and C genes
in the genome exists so that they can
fuse to form a complete Immunoglobulin
gene
Find a way to show the existence of multiple V genes and rearrangement to
the C gene
Approach
V
V
V
V
V
V
V
V
V
V
V
V
V C
Germline DNA
C
Rearranged DNA
V
Tools:
• A set of cDNA probes to specifically distinguish V regions from C regions
• DNA restriction enzymes to fragment DNA
• Examples of germline (e.g. placenta) and mature B cell DNA (e.g. a
plasmacytoma/myeloma)
Experiment of Susumi Tonegawa 1975 Basel
DNA-extraction
Digestion by restriction enzyme
Gel electrophoresis
Southern blot
Kb
Liver cell
V-probe
6,0
4,0
1,5
V
C-probe
C
C
6.0 Kb
V
4.0 Kb
V
C
1.5. Kb
V
C
B-cell
B-cell
CONCLUSION
V and C genes get close to each other in B-cells only
V
V
V
C
B-CELL
There are many variable genes but only one constant gene
V
V
V
V
C
GERM LINE
Ig gene sequencing complicated the model
The structures of germline VL genes were similar for Vk, and Vl,
However there was an anomaly between germline and rearranged DNA:
VL
CL
~ 95aa
~ 100aa
L
L
VL CL
~ 208aa
Where do the extra 13 amino
acids come from?
L
VL
~ 95aa
JL
CL
~ 100aa
Some of the extra amino
acids are provided by one
of a small set of J or
JOINING regions
Further diversity in the Ig heavy chain
L VH
DH
JH
CH
The heavy chain was found to have further amino acids (0 – 8) between the JH
és CH genes
D (DIVERSITY) region
Each heavy chain requires 2 recombination events
JH to DH , VH to JHDH,
L VL
JL
CL
Each light chain requires 1 recombination events
VL to JL
IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE
SEGMENTS
ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS
Chromosome 2
kappa light chain gene segments
Chromosome 22
lambda light chain gene segments
Chromosome 14
heavy chain gene segments
HOW MANY IMMUNOGLOBULIN GENE SEGMENTS
Gene segments
Light chain
kappa
Heavy chain
lambda
Variable (V)
40
30
65
Diversity (D)
0
0
27
Joining (J)
5
4
6
SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS
Vκ Jκ
B-cell 2
Vκ
Vκ
5 Jκ
40 Vκ
Vκ
Vκ
Germ line
Jκ Jκ Jκ
Jκ
During B-lymphocyte
development
Vκ
B-cell 1
DNA
Vκ
Vκ
Jk Jκ Jκ Jκ
EXPRESSION OF THE KAPPA CHAIN
Vκ
P
Vκ J
pA
J
E
Cκ
J
E
Cκ
Vκ-Jκ
Leader
Vκ J
Primary RNA transcript
Vκ J
Cκ
AAAA
mRNA
Translation
Vκ J
Cκ
Protein
Efficiency of somatic gene rearrangement?
SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS
65 VH
VH1
VH2
27 D
VH3
D
D
D
6 JH
D
JH JH JH JH
During B-cell development
VH1
VH2
VH3
VH1
D
D JH JH
VH2
D
D JH JH
VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES
B cells of one individual
2
3
1
4
V-Domains
C-Domains
VH
D
JH
VL
VH-D-JH
JL
VL-JL
ORDER OF REARRANGEMENTS OF IMMUNOGLOBULIN GENE
SEGMENTS
D – J recombination
V – DJ recombination
VDJ – δ transcription
Surrogate light chain
V – J recombination
VJ – k (or VJ - l) transcription
δ translation
k or l translation
B-sejt
mIgD
mIgM
Secreted IgM
Estimates of combinatorial diversity
Taking account of functional V D and J genes:
65 VH x 27 DH x 6JH = 6,480 combinations
40 Vk x 5 Jk = 200combinations
30 Vl x 4 Jl = 120 combinations
= 320 different light chains
If H and L chains pair randomly as H2L2 i.e.
6,480 x 320 = 2,073,600 possibilities
Due only to COMBINATORIAL diversity
In practice, some H + L combinations do not occur as they are unstable
Certain V and J genes are also used more frequently than others.
There are other mechanisms that add diversity at the junctions between genes
- JUNCTIONAL diversity
GENERATES A POTENTIAL B-CELL REPERTOIRE
THE RESULT OF SOMATIC GENE REARRANGEMENTS
1. Combination of gene segments results in a huge number of various variable regions of
the heavy and light chains expressed by different B-cells
SOMATIC GENE REARRANGEMENT
2. Successful somatic rearrangement in one chromosome inhibits gene rearrangement in
the other chromosome
ALLELIC EXCLUSION
3. One B-cell produces only one type of heavy and one type of light chain
COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE
4. The B-cell pool consist of B-cells with differently rearranged immunoglobulin genes
INDEPENDENT OF ANTIGEN
OCCURS DURING B-CELL DEVELOPMENT IN THE BONE
MARROW
Evidence for allelic exclusion
ALLOTYPE- a polymorphism in the Heavy chain C region of Ig
Allotypes can be identified by staining B cell surface Ig with antibodies
B
a
AND b
b
B
B
Y
Suppression of H chain rearrangement by pre-B
cell receptor prevents expression of two
specificities of antibody per cell
B
Y
b
Y
B
Y
a
a/b
b/b
Y
Y
a/a
a
Allelic exclusion is needed for efficient clonal selection
Antibody
S. typhi
S. typhi
All daughter cells must express the same Ig specificity
otherwise the efficiency of the response would be compromised
Suppression of H chain gene rearrangement helps to prevent the emergence of
new daughter specificities during proliferation after clonal selection
Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell
Y
Y Y
S. aureus
Y
Y
Y
Y
Anti
S. aureus
Antibodies
B
S. aureus
Anti
brain
Abs
Y
Y
Y
Y
Self antigen
expressed by
e.g. brain cells
Y Y
B
Anti
S. aureus
Antibodies
Suppression of H chain gene rearrangement
ensures only one specificty of Ab expressed per cell.
Prevents induction of unwanted responses by pathogens
Allelic exclusion is needed to prevent holes in the repertoire
One specificity of Ag
receptor per cell
IF there were two specificities
of Ag receptor per cell
Anti-brain Ig
Anti-brain Ig
AND
anti-S. Aureus Ig
B
B
Exclusion of anti-brain B cells
i.e. self tolerance
B
Deletion
OR
B
BUT anti S.Aureus B cells will
be excluded leaving a
“hole in the repertoire”
Anergy
B
S. aureus
SYNTHESIS OF IMMUNOGLOBULINS
Secreted Ig
Membrane Ig
Golgi
ER
H and L chains are synthesized
on separated ribosomes
CHAPERONES
Leader sequence
Ribosome
mRNA
B – CELL ACTIVATION
RECEPTOR MEDIATED CELL ACTIVATION
Ligand
Ligand
Cross - linking
Conformational change
SIGNAL
SIGNAL
CROSS – LINKING OF THE RECEPTOR INITIATES A SIGNALING
CASCADE
ligand
kinase activation
phosphorylation
recruitment of adaptors
SIGNAL
Activation of transcription
factors
Gene transcription
(review)
BCR signaling
SIGNALING UNITS OF THE B-CELL RECEPTOR
Ig-a/CD79a
Ig-b/CD79b
a b
Y
ITAM
Y
Ig domain + CHO
Y
Y
ITAM
ITAM: YxxL x7 YxxI
ITAM: Immunoreceptor Tyrosine-based Activation Motif
KINETICS OF LYMPHOCYTE ACTIVATION
ANTIGEN
SIGNAL1.
Resting limfocita
lymphocyteGG
Nyugvó
00
Co-receptor
Adhesion molecule
Cytokines
SIGNAL2.
Effector cell Memory cell
G1
Transport
Membrane change
RNA and protein synthesis
M
proliferation
G2
S
DNA synthesis
Lymphoblast
Resting lymphocyte G0
PTK activation
RNA synthesis
Free Ca++
Protein synthesis
Protein phosphorylation
DNA synthesis
0 10sec 1min 5min
1hr
6 hrs
12 hrs
24 hrs
THE CO-STIMULATORY ROLE OF CR2 (CD21) COMPLEMENT
RECEPTOR IN B – LYMPHOCYTES
C3d
ANTIGEN
Antigenic
determinant
CD21
CD19
TAPA=CD81
B-CELL
Y
Y
Enhanced B-cell activation
THE NEURAMIC ACID RECEPTOR CD22 INHIBITS ACTIVATION
THROUGH THE A B-CELL RECEPTOR
Mannose
Tissue cells
Bacterium
Neuraminic (sialic)
acid
Antigen
B Cell
CD22
Inhibited B cell activation
B cell differentiation is „helped” by T-cells
T SEJT (CD4+ helper)
ANTIGÉN
CITOKINEK
B SEJT
PLAZMA SEJT
IZOTÍPUS VÁLTÁS ÉS AFFINITÁS ÉRÉS CSAK T SEJT SEGÍTSÉGGEL MEGY VÉGBE
HOGYAN LÁTJÁK A T SEJTEK AZ ANTIGÉNT?
1) I read that monocytes have a phagocytic role.
When is it? Don't they need to be activated and
become macrophages to be able to phagocyte?
2) When do monocytes differentiate into macrophages?
And when do they differentiate into dentritic cells?
3) I saw that sometimes macrophages are considered
as innate immunity, and sometimes as acquired immunity
What is the difference within the macrophages?
(when are they considered as innate, and when as
4) About the phagoctose process: what are ROI and NO?
Aren't lysosomes enough to "digest" the antigen?
5) About the structure of the thymus: why is there
macrophages and dentritic cells into it? What is their role?
6) The recognition by toll receptors in unavoidable. But these toll
receptors are part of the innate immunity. So are they used to attach
the antigen to phagocytose it. And then, the antigen presentation
enable the acquired immunity to make a receptor/antibody
more precise for this specific antigen?
7) About the cytokines. I read that the IFN gamma are
produced by T helper, and they activate the
macrophages. Aren't the macrophages activated before?
8) Finally, I didn't understand few slides: Lecture 3-4:
slides 31, 43, 44, 45