B CELLS

THE TWO ARMS OF THE IMMUNE SYSTEMTHE TWO ARMS OF THE IMMUNE SYSTEM

Monocytes, Macrophages, Dendritic cells, Granulocytes, NK

cells and Complement componentsB and T cells

Monocytes, Macrophages, Dendritic cells, Granulocytes, NK

cells and Complement components

Overview of B cell–mediated immunity

B CELLS

Structure of antibody

Antigen binding/hypervariable regions

Clonal proliferation

B cell differentiation, memory cells, plasma cells

Antibody-mediated effector functions

Izotypes

B cell mediated antigen presentation

IMMUNOGLOBULINSIMMUNOGLOBULINS

structure and function

STRUCTURE OF ANTIBODY STRUCTURE OF ANTIBODY

SS

Light chain (L)

Heavy chain (H)

VL

CL

VH

CH

Symmetric core structure 2 identical heavy chain, 2 identical light chain

Variable regions antigen bindingConstant regions

STRUCTURESTRUCTURE

• heavy and light chains

• disulfide bonds– inter-chain– intra-chain

hinge region

carbohydrate

disulfide bond

CH1

VL

CL

VH

CH2 CH3

• variable and constant regions

• hinge region

• domains– VL & CL

– VH & CH1 - CH3 (or CH4)

• oligosaccharides

hinge region

carbohydrate

disulfide bond

CH1

CL

VH

CH2 CH3

VL

immunoglobulin domen

STRUCTURESTRUCTURE

s

s

s

s

s

s

s

s

s ss s

CH2

CH3

s

s

s

s

s

s

s

s

ss

VL

VH

CL

CH1 ss

ss

ss

ss

ss

effektor funkciók

konstans domének

antigénkötés

variábilis domének

ANTIBODY DOMAINS AND THEIR FUNCTIONSANTIBODY DOMAINS AND THEIR FUNCTIONS

Constant domain

Effector functions

Antigen recognition

Variable domain

Two identical binding siteHeavy chain and light chain compose the antigen binding surface

THE ROLE OF THE HINGE REGIONTHE ROLE OF THE HINGE REGION

RIBBON STRUCTURE OF IGGRIBBON STRUCTURE OF IGG

B cells

Structure of antibody

Antigen binding/hypervariable regions

Clonal proliferation

B cell differentiation, memory cells, plasma cells

Antibody-mediated effector functions

Izotypes

B cell mediated antigen presentation

VLVLVHVH

ANTIGEN BINDINGANTIGEN BINDING

Variable domens responsible for antigen binding

DIFFERENT VARIABLE REGIONS DIFFERENT VARIABLE REGIONS DIFFERENT DIFFERENT ANTIGEN-BINDING SITES ANTIGEN-BINDING SITES DIFFERENT SPECIFICITIES DIFFERENT SPECIFICITIES

3 CDR regions in a V domain

VH & VL domains 3+3 CDR

HYPERVARIABLE REGION –HYPERVARIABLE REGION – COMPLEMENTARY DETERMINING REGION (CDR)COMPLEMENTARY DETERMINING REGION (CDR)

HYPERVARIABLE REGION – HYPERVARIABLE REGION – COMPLEMENTARY DETERMINING COMPLEMENTARY DETERMINING

REGION (CDR)REGION (CDR)

COMPLEMENTARY DETERMINING REGION (CDR)COMPLEMENTARY DETERMINING REGION (CDR)

B cells

Structure of antibody

Antigen binding/hypervariable regions

Clonal proliferation

B cell differentiation, memory cells, plasma cells

Antibody-mediated effector functions

Izotypes

B cell mediated antigen presentation

Cc. (minimum) 10 mCc. (minimum) 10 milliillion various (10on various (1077) B lymphocyte clones with ) B lymphocyte clones with different different aantigntigeen-recognizing receptorsn-recognizing receptors

CCc. (minimum) 10 – 1000 mc. (minimum) 10 – 1000 milliillion various (10on various (1077 - - 99) ) TT lymphocyte lymphocyte clones with different clones with different aantigntigeen-recognizing receptorsn-recognizing receptors

DIVERSITY OF LYMPHOCYTESDIVERSITY OF LYMPHOCYTES

AAll lymphocytes have a different receptor ll lymphocytes have a different receptor

10101212 l lyymphocytes in our body ( B and T lymphocytesmphocytes in our body ( B and T lymphocytes))

Several antibodies are expressed on B cells, (arround 100.000) but all of them with the same specificity

Antigen

ActivationClonal expansion

B cell

Antigen receptor, BCR

Ag

Clonal antigen receptors are expressed exclusively on T- and B lymphfocyties.

Antigen

Antigen

Antigen

ANTIGEN RECOGNITION BY SPECIFIC BCR INDUCES ANTIGEN RECOGNITION BY SPECIFIC BCR INDUCES CLONAL EXPANSION OF THE SPECIFIC B CELLS.CLONAL EXPANSION OF THE SPECIFIC B CELLS.

ANTIGEN RECOGNITION BY SPECIFIC BCR INDUCES ANTIGEN RECOGNITION BY SPECIFIC BCR INDUCES CLONAL EXPANSION AND CLONAL EXPANSION AND DIFFERENTIATIONDIFFERENTIATION OF OF

THE SEPCIFIC B CELLS.THE SEPCIFIC B CELLS.

ANTIGEN RECOGNITION BY SPECIFIC BCR INDUCES ANTIGEN RECOGNITION BY SPECIFIC BCR INDUCES CLONAL EXPANSION AND DIFFERENTIATION OF CLONAL EXPANSION AND DIFFERENTIATION OF

THE SEPCIFIC B CELLS.THE SEPCIFIC B CELLS.

Primary lymphoid organs:- Bone marrow- Thymus

Secondary lymphoid organs:- Spleen- Lymphatic vessels- Lymph nodes- Adenoids and tonsils- MALT (Mucosal Associated Lymphoid Tissue) GALT (Gut Associated Lymphoid Tissue) BALT (Bronchus Associated Lymphoid Tissue) SALT (Skin Associated Lymphoid Tissue)

NALT (Nasal Associated Lymphoid Tissue)

LYMPHOID ORGANSLYMPHOID ORGANS

LYMPHOCYTES REACTING WITH SELF ANTIGEN LYMPHOCYTES REACTING WITH SELF ANTIGEN DURING THEIR DEVELOPMENT IN THE PRIMARY DURING THEIR DEVELOPMENT IN THE PRIMARY LYMPHOID ORGANS, BECOME INACTIVATED OR LYMPHOID ORGANS, BECOME INACTIVATED OR

DIE BY APOPTOSIS. DIE BY APOPTOSIS.

Antibodies are natural products that appear on the cell surface as receptors and selectively react with the antigen.

Lymphocyte receptors are variable and carry various antigen-recognizing receptors.

‘Non-self’ antigens/pathogens encounter the existing lymphocyte pool (repertoire).

Antigens select their matching receptors from the available lymphocyte pool, induce clonal proliferation of specific clones and these clones differentiate to antibody secreting plasma cells.

The clonally distributed antigen-recognizing receptors represent about ~107 – 109 distinct antigenic specificities.

MACFARLANE BURNET (1956 - 1960)MACFARLANE BURNET (1956 - 1960) CCLLOON SELEN SELECTION HYPOTHESISCTION HYPOTHESIS

MACFARLANE BURNET (1956 - 1960)MACFARLANE BURNET (1956 - 1960) CCLLOON SELEN SELECTION HYPOTHESISCTION HYPOTHESIS

-Lymphocytes are monospecific cells

-Antigen engagemnt result in the activation of lymphocytes

-Activated lymphocytes differentiate and proliferate but keep their antigen specificity

-Lymphocytes reacting with self antigen during their development in the primary lymphoid organs, become inactivated or die by apoptosis.

1. BCR (cell surface antibody) recognize the antigen

2. Clonal proliferation of specific B cells3. Differenciation of activated B cells to plasma cell (antibody production) or memory

cell

4. To distinguish self-nonself is with the selection and killing of self dangerous clones

Activated B cells Plasma cells

Ag

B cells

Structure of antibody

Antigen binding/hypervariable regions

Clonal proliferation

Antibody-mediated effector functions

Izotypes

B cell mediated antigen presentation

B cell: Antibody on the cell surface (BCR) function: cell activation

Plasma cell: production of antibody antibod y-mediated effector functions

TWO FORMS OF ANTIBODYTWO FORMS OF ANTIBODY-cell surface (BCR)-soluble (on the surface of plasma cells antibody is not expressed) Cell surface and soluble antibodies recognize the same antigen

ANTIBODYANTIBODYBCR (BCR (B cell receptorB cell receptor))

MEMBRANE BOUND!

Associated chains for signaling

Transmembrane domain

Cytoplasmic domain

Antigen recognition and B cell activation

SOLUBLE (freely circulating)

Antigen recognition and effector functions.

Produced by plasma cells

B cell

B CELL ACTIVATIONB CELL ACTIVATION

BCR oligomerization results in B cell activation, proliferation and differentiation

!

VLVLVHVH

ANTIGEN BINDINGANTIGEN BINDING

Variable domens responsible for antigen binding

•detect antigen

•precipitate antigen

•block the active sites of toxins or pathogen-associated molecules

•block interactions between host and pathogen-associated molecules

The variable domain can:

•detect antigen

•precipitate antigen

•block the active sites of toxins or pathogen-associated molecules

•block interactions between host and pathogen-associated molecules

The variable domain can:

Complement proteins

•detect antigen

•precipitate antigen

•block the active sites of toxins or pathogen-associated molecules

•block interactions between host and pathogen-associated molecules

The variable domain can:

B cells

Structure of antibody

Antigen binding/hypervariable regions

Clonal proliferation

Antibody-mediated effector functions

Izotypes

B cell mediated antigen presentation

ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:

1. Neutralization (variable domen)

Fc part:

2. Complement activation

Via opsonization:

3. Phagocytosis

4. ADCC (antibody dependent celular cytotoxicity)

5. (mast cell degranulation)

NEUTRALIZATIONNEUTRALIZATION

Covering of the pathogen’s surface prevents replication and growth

WHY DO ANTIBODIES NEED AN FC REGION?WHY DO ANTIBODIES NEED AN FC REGION?

•inflammatory and effector functions associated with cells

•inflammatory and effector functions of complement

•the trafficking of antigens into the antigen processing pathways

Fc region can activate

IMMUNOGLOBULIN FRAGMENTS: IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPSSTRUCTURE/FUNCTION RELATIONSHIPS

antigen binding

complement binding site

placental transfer

binding to Fc receptors

FC RECEPTORSFC RECEPTORSRECOGNIZE THE CONSTANT (FC) PART OF ANTIBODIESRECOGNIZE THE CONSTANT (FC) PART OF ANTIBODIES

!

Expression of Fc receptors on the cell surface is constitutive (relativelly)

Fc receptors are not activated by free/lonely antibody but by immunocomplexes

FC RECEPTORSFC RECEPTORS

FcR Affinity for Immunoglobulin Cell Distribution Function

FcγRI (CD64) High (Kd < 10-9 M); binds IgG1 and IgG3, can bind monomeric IgG

Macrophages, neutrophils; also eosinophils

Phagocytosis; activation of phagocytes

FcγRIIA (CD32) Low (Kd > 10-7 M) Macrophages, neutrophils; eosinophils, platelets

Phagocytosis; cell activation (inefficient)

FcγRIIB (CD32) Low (Kd > 10-7 M) B lymphocytes Feedback inhibition of B cells

FcγRIIC (CD32) Low (Kd > 10-7 M) Macrophages, neutrophils, NK cells

Phagocytosis, cell activation

FcγRIIIA (CD16) Low (Kd > 10-6 M) NK cells Antibody-dependent cell-mediated cytotoxicity

FcγRIIIB (CD16) Low (Kd > 10-6 M); GPI-linked protein

Neutrophils Phagocytosis (inefficient)

FcΕRI High (Kd > 10-10 M); binds monomeric IgE

Mast cells, basophils, eosinophils

Cell activation (degranulation)

FcΕRII (CD23) Low (Kd > 10-7 M) B lymphocytes, eosinophils, Langerhans cells

Unknown

FcαR (CD89) Low (Kd > 10-6 M) Neutrophils, eosinophils, monocytes

Cell activation?

Complement proteins

IMMUNCOMPLEX IMMUNCOMPLEX complexe of (1)antigens-(2)antibodies (3)complement components complex

ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:

1. Neutralization (variable domen)

Fc part:

2. Complement activation

Via opsonization:

3. Phagocytosis

4. ADCC (antibody dependent celular cytotoxicity)

5. (mast cell degranulation)

COMPLEMENT ACTIVATIONCOMPLEMENT ACTIVATION

GENERATES INFLAMMATION

OPSONIZATION

KlLLING of PATHOGEN

REMOVE IMMUNKOPLEXES

ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:

1. Neutralization (variable domen)

Fc part:

2. Complement activation

Via opsonization:

3. Phagocytosis

4. ADCC (antibody dependent celular cytotoxicity)

5. (mast cell degranulation)

OPSONIZATIONOPSONIZATIONFlagging a pathogen

Antigen binding portion (Fab) binds the pathogen, the Fc region

binds phagocytic cells Fc-receptors speeding up the

process of phagocytosis

Opsonins:ANTIBODY

Complement componentsAcute phase proteins

ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:

1. Neutralization (variable domen)

Fc part:

2. Complement activation

Via opsonization:

3. Phagocytosis

4. ADCC (antibody dependent celular cytotoxicity)

5. (mast cell degranulation)

DEGRANULATION OF NK CELLSDEGRANULATION OF NK CELLS

ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY (ADCC)ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY (ADCC)

ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:ANTIBODY-MEDIATED EFFECTOR FUNCTIONS:

1. Neutralization (variable domen)

Fc part:

2. Complement activation

Via opsonization:

3. Phagocytosis

4. ADCC (antibody dependent celular cytotoxicity)

5. (mast cell degranulation)

INNATE IMMUNITY INNATE IMMUNITY

Killing:

•Phagocytosis

•Soluble mediators

•Complement system

•NK cells

Antibody-mediated effector functions accelerates and facitlitates the effector functions of innate immune system

B cells

Structure of antibody

Antigen binding/hypervariable regions

Clonal proliferation

Antibody-mediated effector functions

Izotypes

B cell mediated antigen presentation

s

s

s

s

s

s

s

s

s ss s

CH2

CH3

s

s

s

s

s

s

s

s

ss

VL

VH

CL

CH1 ss

ss

ss

ss

ss

effektor funkciók

konstans domének

antigénkötés

variábilis domének

ANTIBODY DOMAINS AND THEIR FUNCTIONSANTIBODY DOMAINS AND THEIR FUNCTIONS

Constant domain

Effector functions

Antigen recognition

Variable domain

HUMAN IMMUNOGLOBULIN CLASSESHUMAN IMMUNOGLOBULIN CLASSESencoded by different structural gene segments (isotypes)

• 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

light chain types

• kappa (κ)• lambda (λ)

Expression of Fc receptors on the cell surface is constitutive (relativelly)

Different cells express various Fc receptors

Antibodies with diferent izotype activates distinct cells, effector functions

Fc receptors are not activated by free/lonely antibody but by immunocomplexes

FC RECEPTORSFC RECEPTORS

IZOTYPE SWITCHIZOTYPE SWITCH

Ig isotype Serum concentration

Characteristics, functions

12-14 mg/ml

Major isotype of secondary (memory) immune response

Complexed with antigen activates effector functions (Fc-receptor binding, complement activation

Trace

amounts

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

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

MAIN CHARACTERISTICS OF ANTIBODY ISOTYPESMAIN CHARACTERISTICS OF ANTIBODY ISOTYPES

free IgM pentamer (star shape)

Antigen bound IgM (crab shape)

valence

2

2

2

2-4-6

2-8-10-12

FcR Affinity for Immunoglobulin Cell Distribution Function

FcγRI (CD64) High (Kd < 10-9 M); binds IgG1 and IgG3, can bind monomeric IgG

Macrophages, neutrophils; also eosinophils

Phagocytosis; activation of phagocytes

FcγRIIA (CD32) Low (Kd > 10-7 M) Macrophages, neutrophils; eosinophils, platelets

Phagocytosis; cell activation (inefficient)

FcγRIIB (CD32) Low (Kd > 10-7 M) B lymphocytes Feedback inhibition of B cells

FcγRIIC (CD32) Low (Kd > 10-7 M) Macrophages, neutrophils, NK cells

Phagocytosis, cell activation

FcγRIIIA (CD16) Low (Kd > 10-6 M) NK cells Antibody-dependent cell-mediated cytotoxicity

FcγRIIIB (CD16) Low (Kd > 10-6 M); GPI-linked protein

Neutrophils Phagocytosis (inefficient)

FcΕRI High (Kd > 10-10 M); binds monomeric IgE

Mast cells, basophils, eosinophils

Cell activation (degranulation)

FcΕRII (CD23) Low (Kd > 10-7 M) B lymphocytes, eosinophils, Langerhans cells

Unknown

FcαR (CD89) Low (Kd > 10-6 M) Neutrophils, eosinophils, monocytes

Cell activation?

(Classes/subclasses)

Sequence variability of H/L-chain constant regions

Sequence variability of H and L-chain variable regions (individual, clone- specific)

Allelic variants

VARIABILITY IN DIFFERENT REGIONS OF THE IG VARIABILITY IN DIFFERENT REGIONS OF THE IG DETERMINES IG CLASSES OR SPECIFICITYDETERMINES IG CLASSES OR SPECIFICITY

isotype idiotypeallotype

B cells

Structure of antibody

Antigen binding/hypervariable regions

Clonal proliferation

Antibody-mediated effector functions

Izotypes

B cell mediated antigen presentation

ANTIGEN PRESENTATION OF B CELLSANTIGEN PRESENTATION OF B CELLS

B-se jt

c itokinek

C D4TC R

MHC II+ p ep tid

T-se jt

2

1

+++

B CELL-MEDIATED ANTIGEN PRESENTATIONB CELL-MEDIATED ANTIGEN PRESENTATION

•cell surface antigen receptor on B cells (BCR)

allows B cells to sense their antigenic environment

connects extracellular space with intracellular signalling

machinery

•secreted antibody

neutralization

opsonization

complement fixation

NK cell –mediated killing

IMMUNOGLOBULIN STRUCTURE-FUNCTION RELATIONSHIPIMMUNOGLOBULIN STRUCTURE-FUNCTION RELATIONSHIP

ANTIBODY-MEDIATED FUNCTIONS:ANTIBODY-MEDIATED FUNCTIONS:

Cell surface (BCR):-antigen recognition-B cell activation-(antigen presentation)

Soluble:effekctor functions1. Neutralization (variable domen)

Fc part:

2. complement activation

Via opsonization:

3. Phagocytosis

4. ADCC