Transcript Document

Figures replaced or skipped
for Ch 2
• Figure 2.3 - Enzyme digestion of Ab’s
• Flow cytometry - Fig 2.13
• Monoclonal Ab Fig 2.12 was replaced
Chapter 2
Antibody Structure and the Generation
of B-Cell Diversity
• Molecular and structural basis of antibody diversity
• How B cells develop and function in the body
• How B cells are activated and participate in adaptive immunity
Location and production of Immunoglobulins
Humoral Immunity - immunity dominated by antibodies
that can be transferred to another person
1. Antibodies are specific for individual epitopes
2. Membrane bound form is present on a B-cell
3. Ag binding to B cell stimulates it to secrete Ab
Antibody structure
Fab
Fc
Heavy (5 classes), Light (2 classes), Constant (C) and Variable (V)
Disulfide bonds link heavy chains
Fab- fragment antigen binding
Fc- fragment crystallizable
Immunoglobulin Isotypes or Classes



pentamers
N-linked carbohydrate
Hinge region
Disulfide bonds
Monomer as BCR
Multimeric forms
k l - Light chain
Ig domain


Monomers
or dimers
Immunoglobulin (Ig) domain
Figure 2-5
~100 amino acid domain - very stable
Two types Ig domains- variable and constant
Antigen Binding site - VH and VL
Heavy chain of IgG - four domains - VH, CH1, CH2, CH3
Figure 2-6
Anti-parallel -strands - contribute to the structural part
Loop region - in the V domain contribute to variability
Hypervariable Regions (CDR) of Antibodies
HV - hypervariable regions
CDR - complementarity
determining regions
Framework region -  strand region
that has reduced variability
Amino Acid Sequence Variability in the V domain
110 amino acid lightchain V domain
Framework regions can
have variability but
variability is much higher
in the HV regions
Physical Properties of Antigens-Antibody Binding
Epitope - part of the antigen bound by Ab
Antigen is usually carbohydrate or protein
• Lock and Key concept
• Variety of structures and sizes recognized by Ab’s
• Affinity vs Avidity - terms describing binding strength of an
antibody for its epitope
Epitopes
• Epitope (antigenic determinant) is the part of
the antigen bound by Ab
• Most antigens have multiple epitopes
(multivalent)
• Usually carbohydrate or peptide.
Fig. 2.9
Mechanisms of Epitope Recognition
• Linear and discontinuous epitopes
• Multivalent Antigens
• Polymeric Antibodies
• Affinity vs Avidity - terms describing binding
strength of an antibody for its epitope
• Epitope binding mechanisms
Figure 2-9
Figure 2-26 part 1 of 2
First Ab to be made during during an immune response to an
antigen
Monomers disulfide bonded together via the J-chain (joining chain)
Figure 2-30
Monomers disulfide bonded to the J-chain
Dimeric in mucosal lymphoid tissue - secreted into
the gut to prevent pathogens binding to gut cells and
can act as an antitoxin
Monomeric made by B-cells in lymph nodes/spleen
and is not J-chain dependent
Antibody-antigen interaction
• Non-covalent binding:
– Electrostatic
– Hydrogen bonds
– Van der Waals force
– Hydrophobic forces
• Affinity: Strength of interaction between epitope
and one antigen-binding site
• Avidity: Strength of the sum of interactions
between antibody and antigen
Figure 2-8
Poliovirus
VP1- blue, contains
several epitopes (white)
that can be recognized
by human antibodies
Antibodies bind a Range of Structures
Pockets
Molecule
Grooves
DNA
Extended surfaces
Lysozyme
Examples of Problematic Ab binding to various
structures
Pocket:
Penicillin - can bind to RBC surface proteins to create a foreign
epitope. IgE binds to drug-RBC protein complex and initiates an
inflammatory response
Groove:
DNA - Systemic Lupus Erythematosus (Lupus) - autoimmune
disease in which antibodies are made against DNA and other
molecules leading to inflammatory reactions in joints, skin and
kidney
Extended Surface:
Lysozyme/Ovalbumin - allergies to hen egg components - more
common in children under 5 - “desensitization” protocol can help
Haptens
Small molecules that are not
immunogenic by
themselves, but can bind
immunoglobulins or TCRs.
Haptens can induce an
immune response when linked
to a larger protein.
Polyclonal vs Monoclonal Antibodies
1
4 A
g
3
2
Isolate B-cells
Spleen
1
2
4
3
Myeloma
cells
+
Isolate serum
Polyclonal
antibodies
1
4 A
g
3
2
Ab-1, Ab-2
Ab-3, Ab-4
Hybridoma
Cells
1
2
3
4
1
2
3
4
Ab-1 Ab-2 Ab-3 Ab-4
Monoclonal
antibodies
Crossreactivity
Occurs when an Antiserum is raised against
antigen A but also reacts with antigen B
Antigen A and B share epitopes
Antigen A and B have similar
(but not identical) epitopes
Antibody Structure Summary
•
Produced by B-cells
•
Y shape, Four polypeptide chains, Ig domains
•
Constant and Variable regions
•
5 classes - IgG, IgM, IgD, IgA, IgE
•
CDR, Hypervariable regions
•
Epitope recognition
Examples of Commercial uses for Ab’s
•
•
•
•
Pregnancy test
Rh disease therapy
Antitoxin serum - Antivenin, rabies, etc
Anti-cancer monoclonals - will be
covered later in the course
Pregnancy test
Input
window
Reaction
zone
Pregnancy
window
Control
window
Urine
Mouse
Monoclonal
anti-HCG
Ab-enzyme
conjugate
Immobile
Polyclonal
anti-HCG Ab
+ dye
substrate
Immobile
Goat Antimouse Ab
+ dye
substrate
http://mcb.berkeley.edu/courses/mcb150/lecture5/Preg%20Test%20movie.swf
Rh disease
Rh disease (Erythroblastosis
Fetalis) - Hemolytic disease of
a newborn - occurs in Rhwoman carrying Rh+ fetus
RhoGAM - human plasma
with anti-Rh+ (D antigen)
antibodies. Works by binding
any fetal RBC’s before the
mother is able to produce an
immune response and form
anti-D IgG
Antitoxin serum
• Serum can be used in the prophylaxis and/or treatment of rabies,
botulism, diphtheria, gas gangrene, snake and spider bites
• Antivenin Crotalidae Polyvalent (ACP) - horse serum based
antivenom - gold standard for snake bites but can cause “serum
sickness”
• New types of antitoxins
– Fragmented antivenin (CroFab) where only the Fab fragment (sheepbased) is used
– Humanized antibodies derived from mouse sources
Generation of Ig diversity in B cells
Unique organization
- Only B cells can express Ig protein
- Gene segments
k l - Light chain
, , , ,  - Heavy Chain
present on three chromosomes
The Gene Rearrangement
Concept
• Germline configuration
• Gene segments need to be
reassembled for expression
• Sequentially arrayed
• Occurs in the B-cells precursors
in the bone marrow (soma)
• A source of diversity BEFORE
exposure to antigen
V-variable, J-joining, D-diversity gene segments; L-leader sequences
Light Chain
Variable - V, J
Constant - C
Heavy Chain
Variable - V, D, J
Constant - C
Gene rearrangements during B-cell development
V-variable, J-joining, D-diversity gene segments; L-leader sequences
Figure
2-14
l - 30 V & 4 pairs J & C (light chain) chs22
k – 40 V & 5 J & 1 C (50% have 2x V) (light chain) chs2
H – 65 V & 27 D & 6 J
chs14
V-region of light
chain consists of
one V and one J
segment
C-region is encoded
by one C segment
Variability comes
from the V segment
CDR1 and CDR2
CDR3
D-diversity
CDR1 and CDR2
CDR3
Two recombination events needed to combine 3 segments to make the
Variable region
Random Recombination of
Gene segments is one
factor contributing to
diversity
(200* + 120*)
X
10,530 = 3,369,600 Ig molecules
* Only one light chain loci gives rise to one functional polypeptide!
Mechanism of Recombination
• Recombination signal sequences (RSSs) direct recombination
- V and J (L chain)
- V D J (H chain)
• RSS types consist of:
- nonamer (9 base pairs)
- heptamer (7 base pairs)
- Spacer
- Two types: [7-12-9], [7-23-9]
• RSS features:
- recognition sites for recombination enzymes
- recombination occurs in the correct order
(12/23 rule)
Mechanism of Somatic Recombination
• V(D)J recombinase: all the protein components that mediate the
recombination steps
• RAG complex: Recombination Activating Genes (RAG-1 and RAG2) encode RAG proteins only made in lymphocytes
• Recombination only occurs through two different RSS bound by two
RAG complexes (12/23 rule)
• DNA cleavage occurs to form a single stranded hairpin and a break at
the heptamer sequences
• Enzymes that cut and repair the break introduce Junctional Diversity
Recombination
+
Junctional
Diversity
http://www.blink.biz/immunoanimations/#
Figure 2-19
Junctional Diversity
Figure 2-18 part 2 of 3
• Nucleotides introduced at recombination break in the coding joint
corresponding to CDR3 of light and heavy chains
- V and J of the light chain
- (D and J) or (V and DJ) of the heavy chain
• P nucleotides generate short palindromic sequences
• N nucleotides are added randomly - these are not encoded
• Junctional Diversity contributes 3 x107 to overall diversity!
Generation of BCR (IgD and IgM)
• Rearrangement of VDJ of the heavy chain brings the gene’s promoter
closer to C and C
• Both IgD and IgM are expressed simultaneously on the the surface of
the B cell as BCR - ONLY isotypes to do this
• Alternative splicing of the primary transcript RNA generates IgD and
IgM
• Naïve B cells are early stage B cells that have yet to see antigen and
produce IgD and IgM
Alternative Splicing of Primary Transcript to
generate IgM or IgD
Figure 2-21
Summary Biosynthesis of IgM in B cells
Mature B cell
Figure 2-23
•
Long cytoplasmic tails
interact with intracellular
signaling proteins
•
Disulfide-linked
•
Ig and Ig - invariant
- Transmembrane proteins
- Dual-function
1) help the assembled Ig
reach the cell surface from
the ER
2) signal the B cell to divide
and differentiate
Principle of Single Antigen Specificity
• Each B cell contains two copies of the Ig locus (Maternal and
Paternal copies)
• Only one is allowed to successfully rearrange - Allelic Exclusion
l
*
Chromosome 22
k
H
2
14
*
• All Igs on the surface of a single B cell have identical specificity
and differ only in their constant region
• Result: B cell monospecificity means that a response to a pathogen
can be very specific
DNA hybridization of Ig genes can diagnose B-cell leukemias
Peripheral blood from
healthy patient is made
up of mostly neutrophils
Peripheral blood from a
leukemia patient has an
abnormally high
proportion of B-cells.
Cancer cells are derived
from one clonal line of
B-cell which has V and
C chains rearranged next
to each other
Generation of B cell diversity in Ig’s before Antigen
Encounter
1. Random combination of V and J (L chain) and V, D, J (H chain)
regions
2. Junctional diversity caused by the addition of P and N nucleotides
3. Combinatorial association of Light and Heavy chains
(each functional light chain is found associated with a different
functional heavy chain and vice versa)
Concept of Combinatorial Association
Developmental stages of B cells
1. Development
before antigen
Immature B cell
2. Development
after antigen
Mature naive B cell
(expressing BCR IgM and IgD)
plasma cell
(expressing BCR and
secretes Antibodies)
Processes occurring after B cells encounter antigen
•
Processing of BCR versus Antibody
1. Plasma cells switch to secreted Ab
2. Difference occurs in the c-terminus of the heavy chain
3. Primary transcript RNA is alternatively processed to
yield transmembrane or secreted Ig’s
•
Somatic Hypermutation
1. Point mutations introduced to V regions
2. 106 times higher mutation rate
3. Usually targets the CDR
•
Affinity maturation - mutant Ig molecules with higher affinity
are more likely to bind antigen and their B cells are
preferentially selected
•
Isotype switching
RNA processing to generate BCR or Antibody
MC - membrane coding
SC - secretion coding
Somatic Hypermutation
(random introduction of point mutations)
Mutations occur throughout the V domain - especially CDR
Occurs on both gene copies - but only one expresses protein
AID - Activation induced Cytidine Deaminase
UNG - Uracil-DNA glycosylase
Process of Affinity Maturation
Hypermutation leads to different B-cells
IgM
Hypermutation
Mutant BCRs have various affinities
Higher affinity BCR’s are preferentially selected to mature
IgG
IgM
Isotype switching
1. IgM is the first Ab that is secreted in the IR
2. IgM is pentameric and each H chain can bind complement proteins
3. Isotypes with better effector functions are produced by activated B
cells
4. Rearrangement of DNA using SWITCH regions
- all C genes preceded by switch sequence (except 
- start from the  gene and any other C gene (plus sequential)
5. Regulated by cytokines secreted by T cells
Switch regions flank each C gene (except delta)
Mu to any other isotype
Sequential switching
AID is important
AID deficiency ---> Hyper IgM syndrome (antibodies not made
after IgM and IgD )
Stages at which Isotype Switching occurs
antigen-independent
stem cell
pre-B cell
immature B cell
(IgM +)
mature B cell
(IgM +, IgD +)
antigen-dependent
IgM
isotype switching
IgG
IgE
IgA
Does Isotype Switching occur in one B cell?
1. Activated B cell resides in the Germinal
Center
-some individuals will mature directly into
plasma cells
2. Some B cells in the germinal center divide and
undergo hypermutation and/or isotype
switching
3. After this stage they cannot divide and the
higher affinity ones are selected
4. These cells can mature to plasma cells
5. End result: The B cell makes a different
antibody isotype but with the same specificity
Immunoglobulin classes
Figure 2-31 part 2 of 2
1. C regions determine the class of antibody and their effector
function
2. Divided into Subclasses based on relative abundance in serum
3. Each class has multiple functions
1. IgM and IgG can bind complement
2. IgG crosses placenta
3. Receptors for constant regions (Fc Receptors)
- IgG (FcG receptors): mac, neutrophils, eosinophils,
NK cells, others
- IgE (FcE receptors): mast cells, basophils, others
Initial Immune Response mediated by IgM
IgM
(plasma cells in
lymph nodes,
spleen, and bone
marrow and
circulate in
blood/lymph)
Low affinity
binding to antigen
via multiple
binding sites
Two binding sites
sufficient for
strong binding
Hypermutation
and affinity
maturation
Exposure of
constant region
Activate
complement
Phagocytose
Isotype switching to IgG
Kill
directly
IgG
(lymph nodes,
spleen, and
bone marrow)
Circulates in
blood and lymph
(most abundant
Ab in internal
fluids)
Extravasation,
Higher affinity
binding to
antigen
Recruit
phagocytes
Multiple
effector
functions
Neutralize
antigens
Activate
complement
Monomeric IgA
Plasma cells in
lymph nodes,
spleen, bone
marrow
Dimeric IgA
lymphoid tissue
associated with
mucosal surfaces
Secreted into
Blood
Secreted into Gut
lumen & body
secretions
IgA most made of any Ab
Effector functions
1. Mainly
neutralization
2. Minor
opsonization
and activation
of complement
IgE
Plasma cells in
lymph nodes or
germinal centers
Bind strongly to
Mast cells via Fc
receptor
Inflammation
- Expulsion of large pathogens
- Allergies
Cross-linking of
receptor bound
Ab releases
histamine and
other activators
IgD
• Very low concentration in serum
• Primarily found with IgM on naïve
mature B cells
• Function is not clear
Figure 2-32
Summary: Generation of B-cell diversity
• Diversity before Antigen exposure (Antigen Independent)
- Random Recombination
- Junctional Diversity
- Combinatorial association
• Diversity after Antigen exposure (Antigen Dependent)
- Switch to secreted Ab
- Somatic Hypermutation
- Affinity Maturation
- Isotype Switching
• Immunoglobulin Classes
- Properties
- Effector functions