Lecture 09.05.2018

B-cells and antibodies Monika Raulf

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Antibodies 1) Structure 2) Development 3) Function

Topics

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What is an antibody? An antibody is a glycoprotein, which can specific bind on a particular substance, i.e. on its antigen (antigenic determinants or epitopes).

Based on its unique structure every antibody molecule can bind specifically and selectively the appropriate antigen. All antibodies have nevertheless the same overall structure, and they are summarized under the term immunoglobulins (Ig).

Antibodies are produced by B lymphoid lineage cells, termed plasma cells, in response to stimulation with an immunogen (infection or immunisation). They bind and neutralise pathogens or prepare them for the exposure and destruction by phagocytes.

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The structure of a typical antibody molecule I 2 heavy chains (CH), 4-5 domains 2 light chains (CL), 2 domains

The chains are linked to one another (CH-CH und CH-CL) by disulfide bonds.

Every chain has a variable (N-terminal) and 1-4 constant (C-terminal) domain.

The variable domains build the antigen binding side (2 per Ab). The constant domains mediate the antibody function (e.g. receptor binding)

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The single domains can additionally bear carbohydrate structures

The single domains and chains are twisted around each other.

The structure of a typical antibody molecule II

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Fc fragment

pFc´

Peptides

F(ab´)2

IgG

Fab

Fab

Fc

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The binding of an antigen I

Antibody binding always between epitope (antigen) and paratope (antibody); complementarity-determine region (CDR)

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The binding of an antigen by an antibody is never covalent.

The binding of an antigen II

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Antibodies (Ig) in the blood serum may be grouped according to their physicochemical properties and antigenic characteristic.

• Ig are found in plasma, urine, spinal fluid, lymph nodes and spleen.

• Ig constitute approximately 1-2 % of the total serum proteins in healthy men.

• Antibodies are in the γ Globulin fraction of serum. During electrophoresis they are the slowest migrating fraction.

• γ Globulins are serum proteins that show the lowest mobility toward anode during electrophoresis when the pH is neutral.

• γ Globulins comprise 11.2 to 20.1 % of the total serum content in men.

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Antiserum • is a preparation of serum containing antibodies specific

for a particular antigen, i.e. immunogen • contains a heterogeneous collection of antibodies that

bind the antigen used for immunisation

Polyclonal antiserum • is a serum that possesses antibodies synthesised by

numerous different B-cell clones following stimulation by an antigen. Different epitopes on the antigen molecule stimulate this multiplicity of antibodies

Serology

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Monoclonal antibody

Antigen injection Polyclonal serum

B cells from spleen + Myelome cell line PEG fusion

Growth and cloning 3 Hybride 5 Hybride

Hybrid * Hybrid *

YYY

YYY

YYY

YYY

YYY

YYY

YYY

YYY

Propagation

Assays for clone secreting high affinity specific antibody

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Antibody titer • is the amount or level of circulating antibody e.g. in patients

with an infectious disease. • Titer is an approximation of the antibody activity in each unit

volume of a serum sample. • The term is determined by preparing serial dilutions of antibody

to which a constant amount of antigen is added. • The end point is the highest dilution of antiserum in which a

visible reaction with antigen, e.g. agglutination, can be detected.

• Reciprocal of the serum dilution e.g. 1:1240 • The titer only provides an estimate of antibody activity. For

absolute amounts of antibody, quantitative precipitation or other methods must be employed.

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Anti-immunoglobulin antibodies

• are antibodies specific for immunoglobulin constant domains which render them useful for detection of bound antibody molecules in immunoassay.

• are produced by immunizing one species with immunoglobulin antibodies derived from another.

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Cross-reacting antibody reacts with epitopes on an antigen molecule different from the one that stimulated its synthesis.

fotolia.com

A)

Partial cross-reactivity (in part the same epitopes

are recognized by Ig)

B)

Cross-reactivity between A and B (A contains more Ig-binding epitopes than B)

C)

Cross-reactivity versus co-sensitization

Co-sensitization Ig recognized different epitopes;

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Cross-reaction versus co-sensitisation Detection using in vitro IgE-inhibition

Solid phase

allergen A

IgE

Allergen A + patients serum

Addition of 2. allergen B

Competitive binding

(inhibition)

2. anti-IgE

100 % binding reduced binding

inhibition Monika Raulf 16

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Development of the antibody diversity I

Possibilities:

1) Germline theory: Each antibody has its own specific gene But: 1x1011 different antibodies exist

2) Somatic diversification or recombinatorial germ-line theory is a hypothesis to explain the enormous variability and postulated that variable- and constant-region of the Ig-genes are separated and rejoined.

The human body consists of ~ 1x1014 (100 billion) cells, 3.5x1013 of them are tissue cells (including 1010 neurone (nerve cells), rest are blood cells

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C J D V

Development of the antibody diversity II

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Somatic recombination I

Combinatorial diversity: In human: κ-Locus on chromosome 2: Light chain κ: ∼40 VL-segments* 5 JL-segments 1 CL-segment

∼ 40 x 5 x 1= 200

λ-Locus on chromosome 22: Light chain λ: ∼ 30 VL-segments 4 JL-segments 4 CL-segments ∼ 30 x 4 x 4= 480

= 680 light chains *doubling is possible

19

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Combinatorial diversity: In human on chromosome 14

Heavy chain: ∼ 65* VH-segments ∼ 27 DH-segments 6 JH-segments x CH-segments ∼ 65 x 27 x 6 = X x 10530

X x 10530 heavy chains

Somatic recombination II

* Number of functional VH-gene segments

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Junctional diversity At the junction of the gene segments further nucleotides are included or deleted (P- and N-nucleotides). It occurs when gene segments join imprecisely, an the amino acid sequence may vary and affect variable region expression, which can alter codons at gene segment junctions.

2/3 of the nucleotide inserts are unproductive.

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Somatic hypermutation

Affinity maturing of the antigen bonding sides takes place in the secondary lymphatic organs (only in activated B-cells, which already bound an antigen).

Mutations occur in the whole variable region of the antibody.

Mutations appear cumulative in certain regions (hot spots).

.

Antibodies producing cells, whose antibodies will change negatively caused by the mutations, die by apoptosis.

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Summary Antibodies can be established against nearly every optional substance

To appropriate the essential antibody diversity for that, three mechanisms exist:

1) Combinatorial diversity (Somatic recombination)

2) Junctional diversity (Imprecise conjunction)

3) Somatic hypermutation (Affinity maturing in germinal centres)

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Antibody classes - Isotypes I

Immunoglobulins differ from each other • in the number of the constant domains • in the position and number of the disulfide bonds • in the number and location of the carbohydrate side chains • in the occurrence of a hinge region

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IgM generates pentamers

• Increase of the affinity Avidity

Antibody classes - Isotypes II

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IgM compensate its low affinity with high avidity

Affinity - Avidity

Affinity = Binding strength between one antigen binding side and the antigen.

Avidity = Binding strength between more antigen binding sides and one multivalent antigen.

Association Dissociation

Affinity

Association

Avidity

Dissociation

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IgA generates dimers

• Secretion of the antibody

(Garland Science 2005)

Antibody classes - Isotypes III

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Human immunoglobulins and their properties

Ig IgG IgM IgA IgD IgE Serum concentration (mg/dl) 800-1700 50-190 140-420 0.3-0.4 <0.001

Total Ig (%) 85 5-10 5-15 <1 <1 Complement fixation + ++++ - - -

Principal biological effect

Resistance-opsonin,

secondary response

Resistance-precipitin, primary

response

Resistance prevents

movement across mucous

membranes

? Parasite defence; Allergy;

Anaphylaxis

Principal site of action Serum Serum Secretion ?; receptor for B-cells Mast cells

Molecular weight (kd) 154 900 160 (+ dimer) 185 190 Serum half-life (days) 23 5 6 2-3 2-3 Antibacterial lysis + +++ + ? ? Antiviral lysis + + +++ ? ? H-chain class g µ a d e

Subclass g1g2g3g4 a1a2

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cµ = IgM, cδ = IgD, cγ = IgG, cε = IgE, cα = IgA

Ψ = Pseudogene

The expression of the genes of the constant region changes during der maturation of the B-cell = Change of class (Isotype-Switch).

Antibody classes - Isotypes IV

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Heavy chain class (isotype) switching is the mechanism whereby a B-cell changes the class of antibodies (or isotype); from IgM to IgG, IgE, or IgA without altering the antigen-binding specificity of the antibody.

Helper T-lymphocyte cytokines (e.g. IL-4) and CD40 ligand regulate heavy chain class switching which involves B-cell VDJ segment recombination with downstream heavy chain gene segments.

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During the Switch the gene segments located between the switch regions are removed Maturation just in one direction

Antibody classes - Isotypes V

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• Specific DNA-sequences (Switch regions) regulate the recombination

• The isotype-switch takes place in the lymph node (germinal centre)

• It requires a specific milieu of cytokines, which activate the switch factors (transcription factors)

Antibody classes - Isotypes VI

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Isotype-switch Heavy chain

JH1-6 Cµ Cδ Cγ 3 Cγ1 ωCε Cα1 Cγ2 Cγ4 Cε Cα2

IL-4

IL-5

IFN-γ

TGF-β

= induces

= inhibits

= increases the synthesis

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Isotype-switch

During B-cell development an isotype-switch takes place.

Primary IgM is formed, later on further Ig-classes, which then also undergo an affinity maturation.

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IgG

IgA

IgM

Incubation time Acute phase Early antibodies

Convalescence Late- or protection-antibodies

Begin of disease Contact

Process of antibody titers

The incubation time of pathogens is different. She can be between 2 and 6 days (influenza) and 60-180 days (hepatitis B).

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Immunoglobulin function

is to link an antigen to its elimination mechanism

(effector system). Antibodies induce complement

activation and cellular elimination mechanisms that

include phagocytosis and antibody-dependent cell-

mediated cytotoxicity (ADCC).

.

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1) Neutralisation (Viruses, bacteria, toxins): IgG and IgA

Antibody classes - function I

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2) Opsonisation (supports phagocytosis): IgG1 and IgG3

Antibody classes - function II

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3) Complement activation (classic way): IgG and IgM

Antibody classes - function III

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The distribution of the antibody receptors on the different cells of the immune system/the tissue determines the site of activity of the antibody.

Function IgM IgD IgG IgA IgE

Neutralisation + - ++ ++ -

Opsonisation - - ++ + -

Mast cell sensitization - - - - +++

Complement activation +++ - + + -

Transport via epithelia + - - +++ -

Transport via placenta - - +++ - -

Diffusion into the tissue +/- - +++ ++ +

Serum level [mg/ml] 1.5 0.04 13.5 2.1 0.005

Antibody classes - function IV

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• IgG can be transported to the embryo by the placenta

• IgG and IgM are the dominant Ab- classes in the plasma

• IgA is secreted in all mucous membranes ( protective barrier)

• IgA can be transported to the infant by the breast milk

• IgE can be find on the mast cells in the skin and the mucous membranes

Antibody classes - function V

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Transient immune defect (immunoglobulin deficiency) after birth high susceptibility to infection of newborn babies

Antibody classes - function VI

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Antibody receptors I

The receptor function determines also the antibody effect

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Antibody receptors II

The receptor function determines also the antibody effect

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Antibody receptors III

The receptor function determines also the antibody effect

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B-cells • Development • Activation

Topics

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• B-cells develop constantly in the marrow (bone marrow) of lymphatic progenitor cells

B-cell development I

• The environment (stromal cells of the marrow) delivers the necessary milieu (surface molecules and cytokines) for the development

• The immunoglobulin genes get rearranged; this process takes place independent of the antigens

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• After the successful expression of the heavy chain the large pre-B-cell actively divides, before the rearrangement of the light chain starts

• The developed heavy chain is expressed with a surrogate L-chain partly on the surface of the large pre-B-cell (pre-B-cell-receptor).

B-cell development II

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• Beginning of the V-J-rearrangement at the κ-locus, on demand follows later the λ-Locus

• The development to a mature B-cell takes place beyond the bone marrow

B-cell development III

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In the bone marrow: Selection on self-tolerance

•Contact to antigens in the environment.

• Immature B-cells, with high binding affinity to the self-antigens, die (apoptosis); if they just bind weakly to the self-antigen, they get inactivated (anergy).

B-cell development IV

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• Self-tolerant, naive B-cells leave the bone marrow.

• Naive B-cells circulate through the blood in the secondary lymphatic organs.

• If the B-cells in the secondary lymphatic organs ’meet’ their suitable antigen, they get activated (transformation to a antibody-secreting plasma cell).

• Without antigen contact they circulate back with the lymph in the bloodstream.

• Lifetime of a non-activated B-cell: ca. 3 days.

B-cell development

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B-cells • Development • Activation

Topics

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B-cell activation I

The second signal can be submitted on two ways:

1) Thymus-dependent (via already activated T-helper- cells) = TD-antigens (thymus dependent)

2) Thymus-independent (just activated by antigen) = TI- antigen (thymus independent)

Naive B-cells need 2 signals for activation:

The first signal is the binding of a suitable antigen to the B-cell receptor (BCR) = Surface-Immunoglobulin

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TI-B-cell activation I

TI-1-antigens activate beyond the BCR a further receptor on the surface, e.g. CD14 against LPS or a complement receptor = co-stimulatory signal

TI-2-antigens activate the B-cell by simultaneous cross-linking of the BCR with many similar antigens, no co-stimulatory

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TI (1+2)-activation : B-cell Plasma cell

no germinal centre

no memory cells

no affinity maturation

switch till IgG3, IgG2 (how?)

TI-B-cell activation I

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TD-B-cell activation I

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Linked recognition = T-cell and B-cell recognise the same antigen

T-cell receptor

MHC II Peptide

CD4

TD-B-cell activation II

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T-cell receptor

MHC II Peptide

CD4

A T-cell expresses the co-stimulatory signals not before she is specific activated by a professional antigen-presenting cell (APC) (dendritic cells, macrophages).

TD-B-cell activation II

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TD-B-cell activation II

Linked recognition = T-cell and B-cell recognise the same antigen

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Summary: TD-B-cell activation Signals for the B-cell activation:

1) Binding of an antigen via BCR (B-cell receptor = membranous immunoglobulin)

2) Signals of the T-cell, which recognise the MHC-II-presenting peptide via TCR and co-receptor CD4 :

• Stimulation of CD40 (B-cell) by CD40-ligand (T-cell)

• Cytokines • Further co-stimulatory signals

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Cytokine of the Th-cell effect • Proliferation of the B-cell (most of all IL-5, IL-6) • Isotype-switch (e.g. IFN-γ to the IgG-switch, IL-4)

B-cells become after the activation to • B-memory cells • Antibody-secreting plasma cells

TD-B-cell activation III

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The primary activation of the naive B-cell by an activated Th-cell occurs also in the secondary lymph organs.

B-cells immigrate over venules with high epithelia (HEV) in the lymph organs and leave them, if they don´t ‘meet’ their specific antigens.

Primary humoral immune response II

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The B-cells immigrate partly of the primary focus in the medulla strands Plasma cells (antibody production, Isotype-switch).

Primary humoral immune response III

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Some B- and T-cells leave the primary foci and move in a primary lymph-follicle, where they build germinal centres. The B-cells proliferate quick (centroblasts) and pass through the somatic hypermutation.

Secondary humoral immune response I

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Summary: Secondary lymph organs I

Lymph follicle, Germinal centre

6 days

Hypermutation, Affinitätsreifung

Apoptosis

Rezirkulation

Activation of the B-cell by the T-cell

Plasma - zellen

Ab- release

Gedächtnis - zellen

Bone marrow

Langlebige Plasma - zellen

release, switch

Foci 3 days

Plasmazellen

Medulla strands

Hyper mutation, Affinity maturation

Rezirkulation recirculation

Plasma - zellen

Plasma cells

Gedächtnis - zellen

Sec. lymphatic organs

Memory cells

Langlebige Plasma - zellen

long-lasting plasma

cells

switch Ab-

switch

Plasmazellen

Primary

Plasma cells

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Memory cells

B-memory cells (Memory B-cells) circulate. They need antigens for antibody production and divide steadily in low frequency.

long-lasting Plasma cells

Long-lasting plasma cells occur first of all after a second antigen contact They move into the bone marrow (niche, that guarantees the survive). They need no antigen for antibody production. They will maybe survive for years/decades.

Summary: Secondary lymph organs II

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B-cells need at the affinity maturation absolutely 2 positive signals (otherwise apoptosis)

Signal 1: Antigen recognised

Signal 2: T-cell interaction

Summary: Secondary lymph organs III