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 Generationof 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

1. Antibodies are specific for individual epitopes2. Membrane bound form is present on a B-cell3. Ag binding to B cell stimulates it to secrete Ab

Location and production of ImmunoglobulinsHumoral Immunity - immunity dominated by antibodies that can be transferred to another person

Fab

Fc

Antibody structure

Heavy (5 classes), Light (2 classes), Constant (C) and Variable (V)Disulfide bonds link heavy chainsFab- fragment antigen bindingFc- fragment crystallizable

Immunoglobulin Isotypes or Classes

pentamers

Monomers or dimers

N-linked carbohydrateHinge regionDisulfide bondsMonomer as BCRMultimeric forms-Light chainIg domain

Figure 2-5Immunoglobulin (Ig) domain

~100 amino acid domain - very stableTwo types Ig domains- variable and constantAntigen 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 light-chain 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 2-8Poliovirus

VP1- blue, contains several epitopes (white) that can be recognized by human antibodies

PocketsAntibodies bind a Range of Structures

Extended surfacesGrooves Molecule DNA 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.

Ag

1

2

3

4

Isolate serum

Ab-1, Ab-2Ab-3, Ab-4

Ag

1

2

3

4

Polyclonal antibodies

1 2

34

Isolate B-cellsSpleen

+

Myeloma cells

3 41 2

HybridomaCells

1 2 3 4

Ab-1

Polyclonal vs Monoclonal Antibodies

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

Urine

Inputwindow

Pregnancywindow

Controlwindow

Reactionzone

Mouse Monoclonalanti-HCG

Ab-enzyme conjugate

Immobile Polyclonal

anti-HCG Ab+ dye

substrate

Immobile Goat Anti-mouse Ab

+ dye substrate

Pregnancy test

http://mcb.berkeley.edu/courses/mcb150/lecture5/Preg%20Test%20movie.swf

Rh diseaseRh disease (Erythroblastosis Fetalis) - Hemolytic disease of a newborn - occurs in Rh- woman 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 (sheep-

based) 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

-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

Light Chain Variable - V, JConstant - C

Heavy ChainVariable - V, D, JConstant - C

V-variable, J-joining, D-diversity gene segments; L-leader sequences

Figure 2-14

Gene rearrangements during B-cell development

V-variable, J-joining, D-diversity gene segments; L-leader sequences

- 30 V & 4 pairs J & C (light chain) chs22

– 40 V & 5 J & 1 C (50% have 2x V) (light chain) chs2

H – 65 V & 27 D & 6 J chs14

CDR1 and CDR2 CDR3

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

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

* Only one light chain loci gives rise to one functional polypeptide!

10,530 = 3,369,600 Ig molecules 120*)(200* + X

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)

• V(D)J recombinase: all the protein components that mediate the recombination steps

• RAG complex: Recombination Activating Genes (RAG-1 and RAG-2) 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

Mechanism of Somatic Recombination

JunctionalDiversity

Recombination +

http://www.blink.biz/immunoanimations/#

Figure 2-19

Figure 2-18 part 2 of 3Junctional Diversity

• 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

Figure 2-21Alternative Splicing of Primary Transcript to

generate IgM or IgD

Summary Biosynthesis of IgM in B cells

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

Mature B cell

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

• 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

Chromosome 22 2 14

* *

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

Immature B cell Mature naive B cell(expressing BCR -

IgM and IgD)

1. Development before antigen

2. Development after antigen

plasma cell(expressing BCR and secretes Antibodies)

Developmental stages of B cells

Processes occurring after B cells encounter antigen

• Processing of BCR versus Antibody1. Plasma cells switch to secreted Ab2. Difference occurs in the c-terminus of the heavy chain3. Primary transcript RNA is alternatively processed to

yield transmembrane or secreted Ig’s

• Somatic Hypermutation1. Point mutations introduced to V regions2. 106 times higher mutation rate3. 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 CDROccurs on both gene copies - but only one expresses proteinAID - Activation induced Cytidine DeaminaseUNG - Uracil-DNA glycosylase

Process of Affinity Maturation

Hypermutation

IgG

IgM

IgM

Hypermutation leads to different B-cells

Mutant BCRs have various affinities

Higher affinity BCR’s are preferentially selected to mature

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 isotypeSequential switchingAID is importantAID 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 +)

IgG

IgAIgE

IgMantigen-dependent

isotype switching

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

Figure 2-31 part 2 of 2Immunoglobulin classes

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 complement2. IgG crosses placenta3. Receptors for constant regions (Fc Receptors)

- IgG (FcG receptors): mac, neutrophils, eosinophils, NK cells, others- IgE (FcE receptors): mast cells, basophils, others

IgM(plasma cells in lymph nodes,

spleen, and bone marrow and circulate in

blood/lymph)

Low affinity binding to antigen

via multiple binding sites

Exposure of constant region

Activate complement

Killdirectly

Phagocytose

Hypermutation and affinity maturation

Two binding sites sufficient for

strong binding

Isotype switching to IgG

Initial Immune Response mediated by IgM

IgG(lymph nodes,

spleen, and bone marrow)

Circulates in blood and lymph (most abundant Ab in internal

fluids)

Extravasation,Higher affinity

binding to antigen

Multiple effector

functions

Recruit phagocytes

Neutralize antigens

Activate complement

Monomeric IgAPlasma cells in lymph nodes, spleen, bone

marrow

Secreted into Blood

Dimeric IgAlymphoid tissue associated with

mucosal surfaces

Secreted into Gut lumen & body

secretions

Effector functions1. Mainly neutralization2. Minor opsonization

and activation of complement

IgA most made of any Ab

IgEPlasma cells in lymph nodes or germinal centers

Bind strongly to Mast cells via Fc

receptor

Cross-linking of receptor bound

Ab releases histamine and

other activatorsInflammation- Expulsion of large pathogens- Allergies

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