Preview of ImmunoglobulinsPreview of ImmunoglobulinsIg TypeIg Type StructureStructure FunctionFunction LocationLocation

IgGIgG

IgAIgA

IgMIgM

IgEIgE

Ch 12- Humoral ImmunityCh 12- Humoral Immunity The Humoral Immune Response =The Humoral Immune Response =antibody. antibody. B cells and plasma cells synthesize antibody B cells and plasma cells synthesize antibody

molecules in response to challenge by molecules in response to challenge by antigen. antigen.

Antibodies provide Antibodies provide protection from re-challenge by an infectious protection from re-challenge by an infectious

agent, agent, block spread of the agent in the bloodblock spread of the agent in the blood facilitate elimination of the infectious agent. facilitate elimination of the infectious agent.

So many types of antibodiesSo many types of antibodies Large repertoire of antibody molecules must be Large repertoire of antibody molecules must be

available to available to recognize the tremendous number of recognize the tremendous number of

infectious agentsinfectious agents molecules that challenge our bodies. molecules that challenge our bodies.

Must interact with host systems and cells (e.g., Must interact with host systems and cells (e.g., complement, macrophages)complement, macrophages) promote clearance of antigenpromote clearance of antigen activation of subsequent immune responses (activation of subsequent immune responses (Box 12-1Box 12-1). ).

Serve as the cell surface receptors Serve as the cell surface receptors stimulate the appropriate B cell antibody factories stimulate the appropriate B cell antibody factories

grow and produce more antibody grow and produce more antibody in response to antigenic challenge. in response to antigenic challenge.

Immunogens, Antigens, and EpitopesImmunogens, Antigens, and Epitopes Proteins and carbohydrates associated with an Proteins and carbohydrates associated with an

infectious agent, whether a bacterium, fungus, infectious agent, whether a bacterium, fungus, virus, or parasite, are considered foreign to the virus, or parasite, are considered foreign to the human host and have the potential to induce an human host and have the potential to induce an immune response. immune response.

A protein or carbohydrate that challenges the A protein or carbohydrate that challenges the immune system and can initiate an immune immune system and can initiate an immune response is called an response is called an immunogenimmunogen ( (Box 12-2Box 12-2). ).

. .

Immunogens continuedImmunogens continued Immunogens may contain more than one antigen Immunogens may contain more than one antigen

(e.g., bacteria). (e.g., bacteria). An An antigenantigen is a molecule that is recognized by specific is a molecule that is recognized by specific

antibody or T cells. antibody or T cells. An An epitope (antigenic determinant)epitope (antigenic determinant) is the actual is the actual

molecular structure that interacts with a single antibody molecular structure that interacts with a single antibody molecule. molecule.

Within a protein, an epitope may be formed by a specific Within a protein, an epitope may be formed by a specific sequence sequence (linear epitope)(linear epitope) or a three-dimensional or a three-dimensional structure structure (conformational epitope).(conformational epitope).

Antigens and immunogens usually contain several Antigens and immunogens usually contain several epitopes, each capable of binding to a different antibody epitopes, each capable of binding to a different antibody molecule. molecule.

As described later in this chapter, a As described later in this chapter, a monoclonal antibodymonoclonal antibody recognizes a single epitoperecognizes a single epitope

Not an Immunogen-Not an Immunogen-HAPTENSHAPTENS Not all molecules are immunogens. Not all molecules are immunogens. Proteins are the best immunogens, carbohydrates are Proteins are the best immunogens, carbohydrates are

weaker immunogens, and lipids and nucleic acids are weaker immunogens, and lipids and nucleic acids are poor immunogens.poor immunogens.

immunogens must be of sufficient size, and proteins immunogens must be of sufficient size, and proteins must be degradable by phagocytes so that they can must be degradable by phagocytes so that they can be presented to lymphocytes, to initiate an immune be presented to lymphocytes, to initiate an immune response. response.

Haptens (incomplete immunogens)Haptens (incomplete immunogens) are often too are often too small to immunize (i.e., initiate a response) an small to immunize (i.e., initiate a response) an individual but can be recognized by antibody. individual but can be recognized by antibody.

Haptens can be Haptens can be made immunogenicmade immunogenic by attachment to by attachment to a a carrier molecule,carrier molecule, such as a protein. For example, such as a protein. For example, dinitrophenol conjugated to bovine serum albumin is dinitrophenol conjugated to bovine serum albumin is an immunogen for the dinitrophenol hapten. an immunogen for the dinitrophenol hapten.

ADJUVANTSADJUVANTS During artificial immunization (e.g., vaccines), During artificial immunization (e.g., vaccines),

an an adjuvant adjuvant is used to enhance the response to is used to enhance the response to antigen. antigen.

TheyThey prolong the presence of antigen in the prolong the presence of antigen in the tissuetissue

Activate or promote uptake of the immunogen Activate or promote uptake of the immunogen by dendritic cells (DCs), macrophages, and by dendritic cells (DCs), macrophages, and lymphocytes. lymphocytes.

Activates responses that mimic a natural Activates responses that mimic a natural antigenic challengeantigenic challenge

ADJUVANTS contADJUVANTS cont Cells are stimulated and antigen is released slowly Cells are stimulated and antigen is released slowly

when emulsified in complete Freund's adjuvant when emulsified in complete Freund's adjuvant (consisting of heat-killed mycobacteria in mineral (consisting of heat-killed mycobacteria in mineral oil). oil).

Complete Freund's adjuvant is not for human use, Complete Freund's adjuvant is not for human use, but newer, less toxic adjuvants are being tested for but newer, less toxic adjuvants are being tested for use with human vaccines. use with human vaccines. liposomes (defined lipid complexes), liposomes (defined lipid complexes), bacterial cell wall components, bacterial cell wall components, molecular cages for antigen, molecular cages for antigen, polymeric surfactants.polymeric surfactants.

Cholera toxin and Cholera toxin and Escherichia coliEscherichia coli lymphotoxin are lymphotoxin are potent adjuvants for secretory antibody potent adjuvants for secretory antibody (immunoglobulin [Ig] A). (immunoglobulin [Ig] A).

No immune responseNo immune response Some molecules will not elicit an immune Some molecules will not elicit an immune

response in an individual. response in an individual. During growth of the fetus, the body develops During growth of the fetus, the body develops

immune toleranceimmune tolerance toward self-antigens and toward self-antigens and any foreign antigens that may be introduced any foreign antigens that may be introduced before maturation of the immune system. before maturation of the immune system.

Later in life, tolerance may develop under Later in life, tolerance may develop under special conditions; for example, ingestion of special conditions; for example, ingestion of high concentrations of bovine myelin can cause high concentrations of bovine myelin can cause an individual to develop tolerance to myelin. an individual to develop tolerance to myelin.

This has been proposed as a potential therapy This has been proposed as a potential therapy for the autoimmunopathogenesis that causes for the autoimmunopathogenesis that causes multiple sclerosis.. multiple sclerosis..

Structure denotes ResponseStructure denotes Response The type of immune response initiated by an The type of immune response initiated by an

immunogen depends on its molecular structure. immunogen depends on its molecular structure. A A primitive but rapid antibody responseprimitive but rapid antibody response can be can be

initiated toward initiated toward bacterial polysaccharides, bacterial polysaccharides, peptidoglycan, or flagellin.peptidoglycan, or flagellin.

Termed Termed T-independent antigensT-independent antigens,, these these molecules have a large, repetitive structure, molecules have a large, repetitive structure, which is sufficient to activate B cells directly to which is sufficient to activate B cells directly to make antibody without the participation of T-cell make antibody without the participation of T-cell help. help.

In these cases the response is limited to In these cases the response is limited to production of production of IgMIgM antibody antibody and fails to stimulate and fails to stimulate an anamnestic (booster) response. an anamnestic (booster) response.

Moving from non to specificMoving from non to specific The transition from an IgM response to an IgG, IgE, The transition from an IgM response to an IgG, IgE,

or IgA response is a big change in the B cell and is or IgA response is a big change in the B cell and is equivalent to differentiation of the cell. equivalent to differentiation of the cell.

This requires help, in the form of This requires help, in the form of cytokines, from T cytokines, from T cells. cells.

The The antigen must be recognized and stimulate both antigen must be recognized and stimulate both T and B cells.T and B cells.

T-dependent antigensT-dependent antigens are usually proteins; they are usually proteins; they stimulate all five classes of immunoglobulins and stimulate all five classes of immunoglobulins and can elicit an can elicit an anamnesticanamnestic (secondary-booster) (secondary-booster) response. response.

More on specific responsesMore on specific responses Determined by:Determined by: structure of the antigen,structure of the antigen, the amount, the amount, route of administration, route of administration, and other factors influence the type of immune and other factors influence the type of immune

response, response, including the types of antibody produced. including the types of antibody produced. For example, oral or nasal administration of a For example, oral or nasal administration of a

vaccine promotes production of a secretory form of vaccine promotes production of a secretory form of IgAIgA (sIgA) that would not be produced on (sIgA) that would not be produced on intramuscular challengeintramuscular challenge

Basic structureBasic structure

Immunoglobulin Types and StructuresImmunoglobulin Types and Structures Composed of at least two heavy chains and two light Composed of at least two heavy chains and two light

chains, a dimer of dimers. chains, a dimer of dimers. They are subdivided into classes and subclasses They are subdivided into classes and subclasses

based on the structure and antigenic distinction of based on the structure and antigenic distinction of their heavy chains. their heavy chains.

IgG, IgM, and IgA are the major antibody forms,IgG, IgM, and IgA are the major antibody forms, whereas IgD and IgE make up less than 1% of the whereas IgD and IgE make up less than 1% of the

total immunoglobulins. total immunoglobulins. The IgA and IgG classes of immunoglobulin are The IgA and IgG classes of immunoglobulin are

divided further into subclasses based on differences in divided further into subclasses based on differences in the Fc portion. the Fc portion. There are four subclasses of IgG, designated as IgG1 There are four subclasses of IgG, designated as IgG1

through IgG4, and two IgA subclasses (IgA1 and IgA2) (through IgG4, and two IgA subclasses (IgA1 and IgA2) (Figure 12-1Figure 12-1). ).

Antibody StructureAntibody Structure Y-shaped molecules with two major structural Y-shaped molecules with two major structural

regions that mediate the two major functions of regions that mediate the two major functions of the molecule (the molecule (Figure 12-1Figure 12-1 and and Table 12-1Table 12-1). ).

The The variable-region/antigen-combining sitevariable-region/antigen-combining site must be able to identify and specifically interact must be able to identify and specifically interact with an epitope on an antigen. with an epitope on an antigen.

A large number of different antibody molecules, A large number of different antibody molecules, each with a different variable region, are each with a different variable region, are produced in every individual to recognize the produced in every individual to recognize the seemingly infinite number of different antigens seemingly infinite number of different antigens in nature. in nature.

Antibody Structure IIAntibody Structure II The The Fc portionFc portion (stem of the antibody Y) interacts with host (stem of the antibody Y) interacts with host

systems and cells to promote clearance of antigen and systems and cells to promote clearance of antigen and activation of subsequent immune responses. activation of subsequent immune responses.

Responsible for Responsible for Fixation of complement Fixation of complement Binding of the molecule to cell surface immunoglobulin receptors Binding of the molecule to cell surface immunoglobulin receptors

(FcR)(FcR) on on macrophages, macrophages, natural killer cells, natural killer cells, T cells. T cells.

For For IgG and IgA,IgG and IgA, the Fc portion interacts with other proteins the Fc portion interacts with other proteins to promote to promote transfer across the placenta and the mucosatransfer across the placenta and the mucosa, , respectively (respectively (Table 12-2Table 12-2).).

In addition, each of the different types of antibody can be In addition, each of the different types of antibody can be synthesized with a synthesized with a membrane-spanning portionmembrane-spanning portion to make it to make it a a cell surface antigen receptorcell surface antigen receptor. .

More specific structure variationMore specific structure variation IgG and IgA have a flexible IgG and IgA have a flexible hinge regionhinge region

rich in proline and susceptible to cleavage rich in proline and susceptible to cleavage by proteolytic enzymes. by proteolytic enzymes.

Digestion of IgG molecules with Digestion of IgG molecules with papainpapain yields two yields two FabFab fragments and one fragments and one FcFc fragment (see fragment (see Figure 12-2Figure 12-2). ).

Each Fab fragment has one antigen-binding Each Fab fragment has one antigen-binding site. site.

PepsinPepsin cleaves the molecule, producing an cleaves the molecule, producing an F(ab')2F(ab')2 fragment with two antigen-binding fragment with two antigen-binding sites and a sites and a pFc'pFc' fragment. fragment.

Can you tell the differenceCan you tell the difference

IgG’s------------------------------------------

IgA’s------------------------------------------

TYPESTYPES The different types and parts of immunoglobulin can The different types and parts of immunoglobulin can

also be distinguished using antibodies directed also be distinguished using antibodies directed against different portions of the molecule.against different portions of the molecule.

Isotypes Isotypes (IgM, IgD, IgG, IgA, IgE)(IgM, IgD, IgG, IgA, IgE) are determined are determined by antibodies directed against the Fc portion of the by antibodies directed against the Fc portion of the molecule (molecule (isoiso meaning the same for all people.) meaning the same for all people.)

AllotypicAllotypic differences occur for antibody molecules differences occur for antibody molecules with the same isotype but contain protein sequences with the same isotype but contain protein sequences that differ from one person to another (in addition to that differ from one person to another (in addition to the antigen-binding region). the antigen-binding region). (Every one ["allo"] of (Every one ["allo"] of them cannot have the same IgG.)them cannot have the same IgG.)

The The idiotypeidiotype refers to the protein sequences in the refers to the protein sequences in the variable region that generate the large number of variable region that generate the large number of antigen-binding regions. antigen-binding regions. (There are many different (There are many different idiots.)idiots.)

ChainsChains Each antibody molecule is made up of heavy Each antibody molecule is made up of heavy

and light chains encoded by separate genes. and light chains encoded by separate genes. The basic immunoglobulin unit consists of The basic immunoglobulin unit consists of

two heavy (H)two heavy (H) and and two light (L) chains.two light (L) chains. IgM and IgA consist of multimers of this basic IgM and IgA consist of multimers of this basic

structure. structure. The heavy and light chains of immunoglobulin The heavy and light chains of immunoglobulin

are fastened together by are fastened together by interchain disulfide interchain disulfide bonds.bonds.

Light ChainsLight Chains

Two types of light chains-Two types of light chains-κ and λκ and λ-are -are present in all five immunoglobulin present in all five immunoglobulin classes, although only one type is classes, although only one type is present in an individual molecule. present in an individual molecule. Approximately 60% of human Approximately 60% of human immunoglobulin molecules have κ light immunoglobulin molecules have κ light chains, and 40% have λ light chains. chains, and 40% have λ light chains.

Heavy ChainsHeavy Chains There are There are five types of heavy chains,five types of heavy chains, one for each isotype one for each isotype

of antibody of antibody (IgM, μ; IgG, γ; IgD, δ; IgA, α; and IgE, ε).(IgM, μ; IgG, γ; IgD, δ; IgA, α; and IgE, ε). Intrachain disulfide bonds define molecular domains within Intrachain disulfide bonds define molecular domains within

each chain. Light chains have a variable and a constant each chain. Light chains have a variable and a constant domain. domain.

The heavy chains have a variable and three (IgG, IgA) or four The heavy chains have a variable and three (IgG, IgA) or four (IgM, IgE) constant domains. (IgM, IgE) constant domains.

Variable domains on the heavy and light chains Variable domains on the heavy and light chains interact to form the antigen-binding site. interact to form the antigen-binding site.

Constant domains Constant domains Are the molecular structure to the immunoglobulin Are the molecular structure to the immunoglobulin define the interaction of the antibody molecule with host systemsdefine the interaction of the antibody molecule with host systems

The heavy chain of the different antibody molecules can also be The heavy chain of the different antibody molecules can also be synthesized with a membrane-spanning region to make the antibody synthesized with a membrane-spanning region to make the antibody an antigen-specific cell surface receptor for the B cellan antigen-specific cell surface receptor for the B cell

Recognizing Ig ShapesRecognizing Ig Shapes

IMMUNOGLOBULIN DIMMUNOGLOBULIN D Molecular mass of 185 kDaMolecular mass of 185 kDa Accounts for less than 1% of serum Accounts for less than 1% of serum

immunoglobulins. immunoglobulins. Exists primarily as membrane IgDExists primarily as membrane IgD Serves with IgM as an antigen receptor on Serves with IgM as an antigen receptor on

early B-cell membranes early B-cell membranes Helps initiate antibody responses by Helps initiate antibody responses by

activating B cell growth.activating B cell growth. IgD and IgM are the only isotypes that can be IgD and IgM are the only isotypes that can be

expressed together by the same cell. expressed together by the same cell.

IMMUNOGLOBULIN MIMMUNOGLOBULIN M First antibody produced in response to antigenic First antibody produced in response to antigenic

challenge challenge Can be produced in a T-cell-independent manner. Can be produced in a T-cell-independent manner. IgM makes up 5% to 10% of the total IgM makes up 5% to 10% of the total

immunoglobulins in adults and has a half-life of 5 immunoglobulins in adults and has a half-life of 5 days.days.

Pentameric moleculePentameric molecule with five immunoglobulin with five immunoglobulin units joined by disulfide bonds and the units joined by disulfide bonds and the J chain,J chain,

Total molecular mass of 900 kDa. Total molecular mass of 900 kDa. Has 10 antigen-binding sites.Has 10 antigen-binding sites. The most efficient immunoglobulin for fixing The most efficient immunoglobulin for fixing

(binding) complement.(binding) complement. A single IgM pentamer can activate the classical A single IgM pentamer can activate the classical

complement pathway. complement pathway.

Classical Complement Classical Complement PathwayPathway

click me to seeclick me to see

IgM continuedIgM continued Monomeric IgM is found with IgD on the B-cell Monomeric IgM is found with IgD on the B-cell

surface, where it serves as the receptor for antigen. surface, where it serves as the receptor for antigen. Because IgM is relatively large, it cannot spread Because IgM is relatively large, it cannot spread

from the blood into tissue. from the blood into tissue. IgM is particularly important for immunity against IgM is particularly important for immunity against

polysaccharide antigens on the exterior of polysaccharide antigens on the exterior of pathogenic microorganisms.pathogenic microorganisms.

It also promotes phagocytosis and promotes It also promotes phagocytosis and promotes bacteriolysis by activating complement through its bacteriolysis by activating complement through its Fc portion. Fc portion.

IgM is also a major component of rheumatoid factors IgM is also a major component of rheumatoid factors (autoantibodies). (autoantibodies).

IMMUNOGLOBULIN GIMMUNOGLOBULIN G Comprises approximately 85% of the immunoglobulins in Comprises approximately 85% of the immunoglobulins in

adults. adults. Molecular mass of 154 kDa, based on two L chains of 22,000 Molecular mass of 154 kDa, based on two L chains of 22,000

Da each and two H chains of 55,000 Da each. Da each and two H chains of 55,000 Da each. The four subclasses of IgG differ in structure (see Figure 12-1), The four subclasses of IgG differ in structure (see Figure 12-1),

relative concentration, and function. relative concentration, and function. Production of IgG requires T-cell help. Production of IgG requires T-cell help. IgG, as a class of antibody molecules, has the longest half-life IgG, as a class of antibody molecules, has the longest half-life

(23 days) of the five immunoglobulin classes, (23 days) of the five immunoglobulin classes, Crosses the placenta, Crosses the placenta, Principal antibody in the Principal antibody in the anamnestic or booster response.anamnestic or booster response. IgG shows high avidity (binding capacity) for antigens, IgG shows high avidity (binding capacity) for antigens, fixes complement, fixes complement, stimulates chemotaxis, stimulates chemotaxis, Acts as an opsonin to facilitate phagocytosis. Acts as an opsonin to facilitate phagocytosis.

Anamnestic or Booster ResponseAnamnestic or Booster Response

A rapidly increased antibody level A rapidly increased antibody level following renewed contact with a specific following renewed contact with a specific antigen, even after several years. Also antigen, even after several years. Also known as booster responseknown as booster response

IMMUNOGLOBULIN AIMMUNOGLOBULIN A Comprises 5% to 15% of the serum Comprises 5% to 15% of the serum

immunoglobulins and has a half-life of 6 days. immunoglobulins and has a half-life of 6 days. Molecular mass of 160 kDa and a basic four-Molecular mass of 160 kDa and a basic four-

chain monomeric structure. chain monomeric structure. However, it can occur as monomers, dimers, However, it can occur as monomers, dimers,

trimers, and multimers combined by the J trimers, and multimers combined by the J chain (similar to IgM). chain (similar to IgM).

In addition to serum IgA, a In addition to serum IgA, a secretory IgAsecretory IgA appears in body secretions and provides appears in body secretions and provides localized immunity. localized immunity.

IgA production requires specialized T-cell help IgA production requires specialized T-cell help and mucosal stimulation. and mucosal stimulation.

Ig A continuedIg A continued Adjuvants, such as cholera toxin and attenuated Adjuvants, such as cholera toxin and attenuated

SalmonellaSalmonella bacteria, can promote an IgA response bacteria, can promote an IgA response IgA binds to a IgA binds to a poly-Ig receptorpoly-Ig receptor on epithelial cells for on epithelial cells for

transport across the cell. transport across the cell. The poly-Ig receptor remains bound to IgA and is then The poly-Ig receptor remains bound to IgA and is then

cleaved to become the cleaved to become the secretory componentsecretory component when when secretory IgA is secreted from the cell. secretory IgA is secreted from the cell.

An adult secretes approximately 2 g of IgA per day.An adult secretes approximately 2 g of IgA per day. Secretory IgA appears in colostrum, intestinal and Secretory IgA appears in colostrum, intestinal and

respiratory secretions, saliva, tears, and other respiratory secretions, saliva, tears, and other secretions. secretions.

IgA-deficient individuals have an increased incidence IgA-deficient individuals have an increased incidence of respiratory tract infections. of respiratory tract infections.

IMMUNOGLOBULIN E (ellergic)IMMUNOGLOBULIN E (ellergic) Accounts for less than 1% of the total Accounts for less than 1% of the total

immunoglobulins and has a half-life of approximately immunoglobulins and has a half-life of approximately 2.5 days. 2.5 days.

Most IgE is bound to Fc receptors on Most IgE is bound to Fc receptors on mast cells,mast cells, on on which it serves as a receptor for allergens and which it serves as a receptor for allergens and parasite antigens. parasite antigens.

When sufficient antigen binds to the IgE on the mast When sufficient antigen binds to the IgE on the mast cell, the mast cell releases histamine, prostaglandin, cell, the mast cell releases histamine, prostaglandin, platelet-activating factor, and cytokines. platelet-activating factor, and cytokines.

IgE is important for protection against parasitic IgE is important for protection against parasitic infection and is responsible for infection and is responsible for anaphylactic anaphylactic hypersensitivityhypersensitivity (type 1) (rapid allergic reactions). (type 1) (rapid allergic reactions).

ImmunogeneticsImmunogenetics The antibody response can recognize as The antibody response can recognize as

many as 108 structures but can still many as 108 structures but can still specifically amplify and focus a response specifically amplify and focus a response directed to a specific challenge. directed to a specific challenge.

The mechanisms for generating this antibody The mechanisms for generating this antibody repertoire and the different immunoglobulin repertoire and the different immunoglobulin subclasses are tied to the genetic events that subclasses are tied to the genetic events that accompany the development (differentiation) accompany the development (differentiation) of the B cell (Figures 12-3 and 12-4). of the B cell (Figures 12-3 and 12-4).

Immunogenetics cont.Immunogenetics cont. Human chromosomes 2, 22, and 14 contain Human chromosomes 2, 22, and 14 contain

immunoglobulin genes for κ, λ, and H chains, immunoglobulin genes for κ, λ, and H chains, respectively. respectively.

Finalizing antibodiesFinalizing antibodies Somatic mutationSomatic mutation of the immunoglobulin of the immunoglobulin

gene occurs later in gene occurs later in activated,activated, growing B cells growing B cells to add to the enormous number of possible to add to the enormous number of possible coding sequences for the variable region and coding sequences for the variable region and to fine-tune a specific immune response. to fine-tune a specific immune response.

Final B Cell differentiationFinal B Cell differentiation The final steps in B-cell differentiation to memory The final steps in B-cell differentiation to memory

cells or plasma cells do not change the antibody cells or plasma cells do not change the antibody gene. gene.

Memory cellsMemory cells are long-lived, antigen-responsive B are long-lived, antigen-responsive B cells expressing the CD45RO surface marker. cells expressing the CD45RO surface marker.

Memory cells can be activated in response to Memory cells can be activated in response to antigen later in life to divide and then produce its antigen later in life to divide and then produce its specific antibody. specific antibody.

Plasma cellsPlasma cells are terminally differentiated B cells are terminally differentiated B cells with a small nucleus but a large cytoplasm filled with with a small nucleus but a large cytoplasm filled with endoplasmic reticulum. endoplasmic reticulum.

Plasma cells are antibody factories. Plasma cells are antibody factories.

Can you explain in general terms- what is happening in this picture?

Antigen Binding SignalAntigen Binding Signal An initial repertoire of IgM and IgD immunoglobulins An initial repertoire of IgM and IgD immunoglobulins

is generated in pre-B cells by the genetic events is generated in pre-B cells by the genetic events previously described (Figure 12-6).previously described (Figure 12-6).

Expression of cell surface IgM and IgD accompany Expression of cell surface IgM and IgD accompany differentiation of the pre-B cell to the B cell. differentiation of the pre-B cell to the B cell.

Cell surface antibody is associated with signal Cell surface antibody is associated with signal transduction receptors, Ig-α (CD79a) and Ig-β transduction receptors, Ig-α (CD79a) and Ig-β (CD79b), in the membrane through which antigen (CD79b), in the membrane through which antigen binding initiates an activation signal. binding initiates an activation signal.

A cascade of protein tyrosine kinases, A cascade of protein tyrosine kinases, phospholipase C, and calcium fluxes that activate phospholipase C, and calcium fluxes that activate transcription and cell growth mediate the activation transcription and cell growth mediate the activation signal. signal.

Other surface molecules, including the CR2 (CD21) Other surface molecules, including the CR2 (CD21) complement (C3d) receptor, amplify the activation complement (C3d) receptor, amplify the activation signal. signal.

Antigen Specific B cellsAntigen Specific B cells T-independent antigens cross-link sufficient T-independent antigens cross-link sufficient

numbers of surface antibody to stimulate numbers of surface antibody to stimulate growth of the antigen-specific B cells.growth of the antigen-specific B cells.

In this manner, the B cells that best recognize In this manner, the B cells that best recognize the different epitopes of the antigen are the different epitopes of the antigen are selected to increase in number in a process selected to increase in number in a process termed termed clonal expansion.clonal expansion.

Production of antibody to T-dependent Production of antibody to T-dependent antigens requires interaction of the B cell with antigens requires interaction of the B cell with the helper T cell through CD40 (on the B cell), the helper T cell through CD40 (on the B cell), CD40L (T cell), and the action of cytokines CD40L (T cell), and the action of cytokines (interleukin-4 [IL-4], IL-5, IL-2, or interferon-γ) (interleukin-4 [IL-4], IL-5, IL-2, or interferon-γ) and the C3d component of complement. and the C3d component of complement.

Clonal expansionClonal expansion Of the antigen-specific B cells increases the number Of the antigen-specific B cells increases the number

of antibody factories making the relevant antibody, of antibody factories making the relevant antibody, increases the strength of the antibody response is increases the strength of the antibody response is

thus increased. thus increased. Activation of the B cells also promotes Activation of the B cells also promotes somatic somatic

mutationmutation of the variable region, increasing the of the variable region, increasing the diversity of antibody moleculesdiversity of antibody molecules directed at the directed at the specific antigen. specific antigen. WHY IS THIS IMPORTANT?WHY IS THIS IMPORTANT?

The B-cell clones that express antibody with the The B-cell clones that express antibody with the strongest antigen binding are preferentially strongest antigen binding are preferentially stimulated, selecting for a better antibody response. stimulated, selecting for a better antibody response.

More differentiation More differentiation techniquestechniques Different combinations of cytokines produced Different combinations of cytokines produced

by helper T cells induce class switching. by helper T cells induce class switching. TH1-helper responses (IL-2, interferon-γ) TH1-helper responses (IL-2, interferon-γ)

promote production of IgM and IgG. TH2-promote production of IgM and IgG. TH2-helper responses (IL-4, IL-5, IL-6, IL-10) helper responses (IL-4, IL-5, IL-6, IL-10) promote production of IgM, IgG, IgE, and promote production of IgM, IgG, IgE, and IgA. IgA production is especially promoted IgA. IgA production is especially promoted by IL-5 and transforming growth factor-β by IL-5 and transforming growth factor-β (TGF-β).(TGF-β).

Memory cells are developed with T-cell help.Memory cells are developed with T-cell help. Terminal differentiation produces the ultimate Terminal differentiation produces the ultimate

antibody factory, the plasma cell. antibody factory, the plasma cell.

Explain the graph

Primary Antibody responsePrimary Antibody response Characterized by the initial production of IgM. Characterized by the initial production of IgM. As the response matures, IgG antibodies As the response matures, IgG antibodies

rapidly increase in concentration (Figure 12-7). rapidly increase in concentration (Figure 12-7). IgM antibodies appear in the blood within 3 IgM antibodies appear in the blood within 3

days to 2 weeks after exposure to a novel days to 2 weeks after exposure to a novel immunogen. immunogen.

The first antibodies that are produced react with The first antibodies that are produced react with residual antigen and therefore are rapidly residual antigen and therefore are rapidly cleared. After the initial lag phase, however, the cleared. After the initial lag phase, however, the antibody titer increases logarithmically to reach antibody titer increases logarithmically to reach a plateau. a plateau.

Re-exposureRe-exposure Reexposure to an immunogen, a Reexposure to an immunogen, a secondary secondary

response,response, induces a heightened antibody induces a heightened antibody response (also termed response (also termed anamnestic anamnestic responseresponse). ).

The antibodies develop more rapidly, last The antibodies develop more rapidly, last longer, and reach a higher titer. longer, and reach a higher titer.

The antibodies in a secondary response are The antibodies in a secondary response are principally of the IgG class, although IgM principally of the IgG class, although IgM antibodies can also be detected in response antibodies can also be detected in response to some infections. to some infections.

During the Immune ResponseDuring the Immune Response Antibodies are made against different epitopes of Antibodies are made against different epitopes of

the foreign object, protein, or infectious agent.the foreign object, protein, or infectious agent. Specific antibody is a mixture of many different Specific antibody is a mixture of many different

immunoglobulin molecules made by many different immunoglobulin molecules made by many different B cellsB cells (polyclonal antibody),(polyclonal antibody),

Each immunoglobulin molecule differing in the Each immunoglobulin molecule differing in the epitope that it recognizes and the strength of the epitope that it recognizes and the strength of the interaction. interaction.

Different antibody molecules are made against Different antibody molecules are made against different epitopes on the antigen, and each binds different epitopes on the antigen, and each binds with different strengths (with different strengths (avidity,avidity, multivalent binding multivalent binding of antibody to antigen; of antibody to antigen; affinity,affinity, monovalent binding monovalent binding to an epitope) for the same antigen. to an epitope) for the same antigen.

Classic Complement PathwayClassic Complement Pathway

Monoclonal AntibodiesMonoclonal Antibodies Identical antibodies produced by a single clone of cells or by Identical antibodies produced by a single clone of cells or by

myelomas (cancers of plasma cells) or hybridomas.myelomas (cancers of plasma cells) or hybridomas. Hybridomas are cloned, laboratory-derived cells obtained by Hybridomas are cloned, laboratory-derived cells obtained by

the fusion of antibody-producing spleen cells and a myeloma the fusion of antibody-producing spleen cells and a myeloma cell. cell.

In 1975, Kohler and Millstein developed the technique for In 1975, Kohler and Millstein developed the technique for producing monoclonal antibodies from B-cell hybridomas.producing monoclonal antibodies from B-cell hybridomas.

The hybridoma is immortal and produces a single The hybridoma is immortal and produces a single (monoclonal) antibody. (monoclonal) antibody.

This technique has revolutionized the study of immunology This technique has revolutionized the study of immunology because it allows selection (cloning) of individual antibody-because it allows selection (cloning) of individual antibody-producing cells and their development into cellular factories producing cells and their development into cellular factories for production of large quantities of that antibody. for production of large quantities of that antibody.

Monoclonal antibodies have been commercially produced for Monoclonal antibodies have been commercially produced for both diagnostic reagents and therapeutic purposes. both diagnostic reagents and therapeutic purposes.

ELISAELISA

Enzyme-Linked ImmunoSorbent Assay. Enzyme-Linked ImmunoSorbent Assay. The test could be called an antibody The test could be called an antibody

detection test which would be much detection test which would be much clearer to the general public. But actually, clearer to the general public. But actually, the test can be used to test for a wide the test can be used to test for a wide range of substances. range of substances.

How does ELISA work?How does ELISA work? First, your blood is taken. First, your blood is taken. Only the serum part is needed; all blood cells are separated out so Only the serum part is needed; all blood cells are separated out so

they won't get in the way of the test. they won't get in the way of the test. Samples of your blood serum are put onto the bottom of plastic Samples of your blood serum are put onto the bottom of plastic

dishes. Your blood serum may or may not have the antigen dishes. Your blood serum may or may not have the antigen indicating a disease is present. indicating a disease is present.

An antibody is added that determines the presence of a disease. So, An antibody is added that determines the presence of a disease. So, if the laboratory has been asked to test for Lyme disease, for if the laboratory has been asked to test for Lyme disease, for example, antibodies to the bacteria that causes Lyme disease will be example, antibodies to the bacteria that causes Lyme disease will be added to the plastic dish. added to the plastic dish.

The antibody will stick to any antigen that it fits into, namely those The antibody will stick to any antigen that it fits into, namely those that indicate the presence of the disease for which you are being that indicate the presence of the disease for which you are being tested. For example, if you have antigens to Lyme disease in your tested. For example, if you have antigens to Lyme disease in your blood serum, those antibodies will stick to them. blood serum, those antibodies will stick to them.

In order to see the result, another compound is added that sticks to In order to see the result, another compound is added that sticks to the antibody added. This compound lights up, or fluoresces, or it the antibody added. This compound lights up, or fluoresces, or it changes color. changes color.

Finally, a machine reads exactly how much lighting up is happening, Finally, a machine reads exactly how much lighting up is happening, which is related to how much antigen is in your blood serum. which is related to how much antigen is in your blood serum.

False PositiveFalse Positive

What is the Cut-off Reading?What is the Cut-off Reading? Since some antibodies may stick to other Since some antibodies may stick to other

substances in your blood serum, there may be a tiny substances in your blood serum, there may be a tiny bit of material that lights up in a negative result. So bit of material that lights up in a negative result. So there is a cut-off point that indicates the result really there is a cut-off point that indicates the result really means the presence of the disease-causing antigen means the presence of the disease-causing antigen in the blood serum and not just some background in the blood serum and not just some background junk for which the enzyme is lighting up. Below that junk for which the enzyme is lighting up. Below that cut-off does not indicate a real positive result. Your cut-off does not indicate a real positive result. Your doctor may order the test be repeated if results are doctor may order the test be repeated if results are close to the cut-off point. close to the cut-off point.

Complement ContinuedComplement Continued The complement system is an alarm and a weapon against The complement system is an alarm and a weapon against

infection, especially bacterial infection. infection, especially bacterial infection. Activated directly by bacteria and bacterial products Activated directly by bacteria and bacterial products

(alternate or properdin pathway),(alternate or properdin pathway), by lectin binding to sugars by lectin binding to sugars on the bacterial cell surface on the bacterial cell surface (mannose-binding protein),(mannose-binding protein),

Or activated by complexes of antibody and antigen Or activated by complexes of antibody and antigen (classical (classical pathway)pathway) (Figure 12-8). (Figure 12-8).

Activation initiates a cascade of proteolytic events that Activation initiates a cascade of proteolytic events that produce chemotactic factors produce chemotactic factors to attract phagocytic and inflammatory cells to the site,to attract phagocytic and inflammatory cells to the site, increase vascular permeability to allow access to the site of increase vascular permeability to allow access to the site of

infection, infection, bind to the agent to promote their phagocytosis bind to the agent to promote their phagocytosis

(opsonization)(opsonization) and elimination, and elimination, and directly kill the infecting agent. and directly kill the infecting agent. The three activation pathways of complement coalesce at a The three activation pathways of complement coalesce at a

common junction point, the activation of the common junction point, the activation of the C3 component.C3 component.

Alternate C pathwayAlternate C pathway Activated directly by bacterial cell surfaces and their Activated directly by bacterial cell surfaces and their

components (e.g., endotoxin, microbial polysaccharides), as components (e.g., endotoxin, microbial polysaccharides), as well as other factors. well as other factors.

Activated before the establishment of an immune response to Activated before the establishment of an immune response to the infecting bacteria because it does not depend on antibody the infecting bacteria because it does not depend on antibody and does not involve the early complement components (C1, and does not involve the early complement components (C1, C2, and C4). C2, and C4).

Initial activation of the alternate pathway is mediated by Initial activation of the alternate pathway is mediated by properdin factor Bproperdin factor B binding to C3b and then with binding to C3b and then with properdin factor properdin factor D,D, which splits which splits factor Bfactor B in the complex to yield the in the complex to yield the Bb active Bb active fragmentfragment that remains linked to that remains linked to C3b (activation unit).C3b (activation unit).

The C3b sticks to the cell surface and anchors the complex. The C3b sticks to the cell surface and anchors the complex. Inactive Inactive BaBa is split from this complex, leading to cleavage and is split from this complex, leading to cleavage and activation of many C3 molecules (amplification). activation of many C3 molecules (amplification).

The complement cascade continues in a manner analogous to The complement cascade continues in a manner analogous to the classical pathway. the classical pathway.

CLASSICAL PATHWAYCLASSICAL PATHWAY

Cascade is initiated by Cascade is initiated by binding to the binding to the Fc Fc portion of antibody that is bound to cell portion of antibody that is bound to cell surface antigens, or in an immune complex surface antigens, or in an immune complex with soluble antigens.with soluble antigens.

AggregationAggregation of antibody of antibody (IgG or IgM, not IgA (IgG or IgM, not IgA or IgE)or IgE) changes the structure of the heavy changes the structure of the heavy chain to allow binding to complement (see chain to allow binding to complement (see Figure 12-8). Figure 12-8).

Complement Component Function

C1 *

C2

C3

C4

C5

C6-9

Classical Complement Pathway Classical Complement Pathway continued.continued.

The first complement component, designated The first complement component, designated C1,C1, consists consists of a complex of three separate proteins designated of a complex of three separate proteins designated C1q, C1q, C1r, and C1sC1r, and C1s (see Figure 12-8). (see Figure 12-8).

One molecule each of C1q and C1s with two molecules of One molecule each of C1q and C1s with two molecules of C1r comprises the C1 complex or C1r comprises the C1 complex or recognition unit.recognition unit.

C1q facilitates binding of the recognition unit to cell surface C1q facilitates binding of the recognition unit to cell surface antigen-antibody complexes. antigen-antibody complexes.

Activation of the classical complement cascade requires Activation of the classical complement cascade requires linkage of C1q to two IgG antibodies through their Fc linkage of C1q to two IgG antibodies through their Fc regions.regions.

In contrast, one pentameric IgM molecule attached to a In contrast, one pentameric IgM molecule attached to a cell surface may interact with C1q to initiate the classical cell surface may interact with C1q to initiate the classical pathway. pathway.

Binding of C1q activates C1r (referred to now as C1r*) and Binding of C1q activates C1r (referred to now as C1r*) and in turn C1s (C1s*). C1s* then cleaves in turn C1s (C1s*). C1s* then cleaves C4C4 to C4a and C4b, to C4a and C4b, and and C2C2 to C2a and C2b. to C2a and C2b.

ComplementComplement The ability of a single recognition unit to split numerous C2 and The ability of a single recognition unit to split numerous C2 and

C4 molecules represents an amplification mechanism in the C4 molecules represents an amplification mechanism in the complement cascade. complement cascade.

The union of The union of C4bC4b and and C2aC2a produces produces C4b2a,C4b2a, which is known as which is known as C3 convertase.C3 convertase.

This complex binds to the cell membrane and cleaves This complex binds to the cell membrane and cleaves C3C3 into into C3aC3a and and C3bC3b fragments. fragments.

The C3b protein has a unique thioester bond that will covalently The C3b protein has a unique thioester bond that will covalently attach C3b to a cell surface or be hydrolyzed. attach C3b to a cell surface or be hydrolyzed.

The C3 convertase amplifies the response by splitting many C3 The C3 convertase amplifies the response by splitting many C3 molecules. molecules.

The interaction of C3b with C4b2a bound to the cell membrane The interaction of C3b with C4b2a bound to the cell membrane produces produces C4b3b2a,C4b3b2a, which is termed which is termed C5 convertase.C5 convertase.

This activation unit splits This activation unit splits C5C5 into into C5aC5a and and C5bC5b fragments and fragments and represents yet another amplification step. represents yet another amplification step.

Lectin PathwayLectin Pathway Is also a bacterial and fungal defense mechanism. Is also a bacterial and fungal defense mechanism.

Mannose-binding proteinMannose-binding protein (previously known as (previously known as RaRF) is a large serum protein that binds to RaRF) is a large serum protein that binds to nonreduced mannose, fucose, and glucosamine on nonreduced mannose, fucose, and glucosamine on bacterial and other cell surfaces. bacterial and other cell surfaces.

Mannose-binding protein resembles and replaces Mannose-binding protein resembles and replaces the C1q component and on binding to bacterial the C1q component and on binding to bacterial surfaces, activates the cleavage of mannose-binding surfaces, activates the cleavage of mannose-binding protein-associated serine protease. protein-associated serine protease.

Mannose-binding protein-associated serine protease Mannose-binding protein-associated serine protease cleaves the C4 and C2 components to produce the cleaves the C4 and C2 components to produce the C3 convertase, the junction point of the complement C3 convertase, the junction point of the complement cascade. cascade.

BIOLOGIC ACTIVITIES OF BIOLOGIC ACTIVITIES OF COMPLEMENT COMPONENTSCOMPLEMENT COMPONENTS Cleavage of the C3 and C5 components produces important Cleavage of the C3 and C5 components produces important

factors that enhance clearance of the infectious agent by factors that enhance clearance of the infectious agent by promoting access to the infection site and by attracting the promoting access to the infection site and by attracting the cells that mediate protective inflammatory reactions. cells that mediate protective inflammatory reactions.

C3bC3b is an is an opsoninopsonin that promotes clearance of bacteria by that promotes clearance of bacteria by binding directly to the cell membrane to make the cell more binding directly to the cell membrane to make the cell more attractive to phagocytic cells such as neutrophils and attractive to phagocytic cells such as neutrophils and macrophages, which have receptors for C3b. C3b can be macrophages, which have receptors for C3b. C3b can be cleaved further to generate cleaved further to generate C3d,C3d, which is an activator of B which is an activator of B lymphocytes. lymphocytes.

Complement fragments Complement fragments C3aC3a and and C5aC5a serve as powerful serve as powerful anaphylatoxinsanaphylatoxins that stimulate mast cells to release that stimulate mast cells to release histamine, which histamine, which enhances vascular permeability and smooth enhances vascular permeability and smooth muscle contraction.muscle contraction.

C3aC3a and and C5aC5a also act as attractants also act as attractants (chemotactic factors)(chemotactic factors) for neutrophils and macrophages. for neutrophils and macrophages.

These cells also express receptors for C3b, are phagocytic, These cells also express receptors for C3b, are phagocytic, and promote inflammatory reactions. and promote inflammatory reactions.

What do you remember?What do you remember? What do C3 and C5 do?What do C3 and C5 do? Is the complement pathway over with the use Is the complement pathway over with the use

of C1-C5?of C1-C5? Can you guess what C6-C9 do?Can you guess what C6-C9 do?

Membrane attack complexMembrane attack complex The terminal stage of the classical pathway involves The terminal stage of the classical pathway involves

creation of the creation of the membrane attack complex,membrane attack complex, which is which is also called also called the lytic unitthe lytic unit (Figure 12-9). (Figure 12-9).

The five terminal complement proteins (C5 through The five terminal complement proteins (C5 through C9) associate into a membrane attack complex on C9) associate into a membrane attack complex on target cell membranes to mediate injury.target cell membranes to mediate injury.

Initiation of membrane attack complex assembly Initiation of membrane attack complex assembly begins with C5 cleavage into C5a and C5b begins with C5 cleavage into C5a and C5b fragments. fragments.

A (C5b,6,7,8)1(C9)n complex forms and drills a hole A (C5b,6,7,8)1(C9)n complex forms and drills a hole in the membrane, leading to the hypotonic lysis of in the membrane, leading to the hypotonic lysis of cells. cells.

The C9 component is similar to perforin, which is The C9 component is similar to perforin, which is produced by cytolytic T cells and natural killer cells. produced by cytolytic T cells and natural killer cells.

So do you even need T-So do you even need T-cells or macrophages?cells or macrophages?

If your complement works so well to kill If your complement works so well to kill invaders-why do you need Leukocytes invaders-why do you need Leukocytes and Lymphocytes?and Lymphocytes?

Is there such a thing as inappropriate Is there such a thing as inappropriate complement activation?complement activation?

If so-what would happen?If so-what would happen?

Innapropriate Innapropriate Complement ActivationComplement Activation

Humans have several mechanisms for preventing Humans have several mechanisms for preventing generation of the C3 convertase to protect against generation of the C3 convertase to protect against inappropriate complement activation. inappropriate complement activation.

These include C1 inhibitor, C4 binding protein, These include C1 inhibitor, C4 binding protein, Factor H, Factor I, and the cell surface proteins, Factor H, Factor I, and the cell surface proteins, which are decay-accelerating factor (DAF) and which are decay-accelerating factor (DAF) and membrane cofactor protein. membrane cofactor protein.

In addition, CD59 (protectin) prevents formation of In addition, CD59 (protectin) prevents formation of the membrane attack complex. the membrane attack complex.

Most infectious agents lack these protective Most infectious agents lack these protective mechanisms and remain susceptible to complement. mechanisms and remain susceptible to complement.

A genetic deficiency in these protection systems can A genetic deficiency in these protection systems can result in disease. result in disease.

Antigen Antibody Antigen Antibody ComplexesComplexes