4_antigen recognition_3ed [compatibility mode]

ANTIGEN RECOGNITION IN THE ADAPTIVE IMMUNE SYSTEM

Sitti Wahyuni, MD, PhDDepartment of Parasitology,

Medical Faculty, Hasanuddin University

1S.Wahyuni/lecture/immunology/S15/13/2014

DURING LECTURE/DISCUSSION

S.Wahyuni/lecture/immunology/S1 25/13/2014

The principal function of cellular receptors is to detect external stimuli and trigger responses of the cells on which the receptors are expressed.

Antigen receptors of lymphocytes must be able to bind to and distinguish between many, often closely related, chemical structures.

Antigen receptors on lymphocyte B and T are clonally distributed: each clone having a particular specificity has a unique receptor

The total number (repertoire) of lymphocyte specificities is very large

Antigen receptors transmit biochemical signals that are fundamentally the same in all lymphocytes and are unrelated to specificity

5/13/2014 3S.Wahyuni/lecture/immunology/S1

Questions to be addressed

How do the antigen receptors of lymphocytes recognize extremely diverse antigens and transmit quite conserved activating signals to the cells?

How is the vast diversity of receptor structures generated in lymphocytes?

5/13/2014 S.Wahyuni/lecture/immunology/S1 4

Antigen Receptors of Lymphocytes

Features of antigen receptors of B and T lymphocytes The antigen receptors of B and T lymphocytes recognize

chemically different structures. B lymphocyte antigen receptors (membrane-bound

antibodies) are able to recognize the shapes, or conformations, of native macromolecules (proteins, lipids, carbohydrates, and nucleic acids and simple small chemical groups)

T cells see only peptides that are displayed on antigen-presenting cells (APCs) bound to MHC


Antigen receptor molecules consist The antigen-recognizing portions: variable (V)

regions The conserved portions: constant (C) regions. Within the V regions there are variability which

are called hypervariable regions, or complementarity-determining regions (CDRs)


Antigen receptors are noncovalently attached to other invariant molecules whose function is to deliver signal to the inside of the cell

The collection of antigen receptors and signaling molecules in B lymphocytes: B cell receptor (BCR) complex, in T lymphocytes: T cell receptor (TCR) complex.

When bind to antigen molecules, the receptors are pulled together into an aggregate (cross-linking)

Cross linking will catalyze enzymes that are attached to the cytoplasmic portions of the signaling proteins and leads the phosphorylation of other proteins.

Phosphorylation triggers complex signaling cascades that culminate in the transcriptional activation of many genes

5/13/2014S.Wahyuni/lecture/immunology/S1 7

5/13/2014 S.Wahyuni/lecture/immunology/S1 8Properties of antibodies and T cell antigen receptors (TCRs).

Antibodies

Antibody molecule is composed of 4 polypeptide chains=

2 identical heavy (H) chains & 2 identical light (L) chains Each chain containing variable region & constant region The four chains are assembled to form a Y-shaped molecule. Each light chain is attached to one heavy chain 2 heavy chains are attached to each other by disulfide bonds. A light chain is made up of one V and one C domain A heavy chain has one V and three or four C domains. Each domain folds into a characteristic three-dimensional

shape, which is called the immunoglobulin (Ig) domain. 5/13/2014 S.Wahyuni/lecture/immunology/S1 9


The structure of antibodies: immunoglobulins G (IgG) and M (IgM).

Variable region Regio of heavy & light chain that recognize & bind

to antigen In heavy chain (called VH) or of the light chain

(called VL) Contains three hypervariable regions (CDRs), the

greatest variability is in CDR3, which is located at the junction of the V and C regions

Constant region Region that is not attached to

antigen


Regions of antibody based on the properties of proteolytic fragments of immunoglobulins. Fab (fragment antigen binding)

Contains a whole light chain (with its single V and C domains) attached to the V and first C domains of a heavy chain

Required for antigen recognition Fc (fragment cystalline)

Contain the remaining heavy chain Hinge region

Between the Fab and Fc regions A flexible portion called the hinge region & allows the 2

antigen-binding Fab regions of each antibody molecule to move independently of each other


Light chain There are two types of light chains, called and ,

that differ in their C regions but do not differ in function.

Each B cell expresses either or , but not both Heavy chain

There are five types of heavy chains, called , , , , and , that also differ in their C regions

Antibodies that contain different heavy chains are said to belong to different isotypes, or classes, and are named according to their heavy chains (i.e., IgM, IgD, IgG, IgE, and IgA)



Features of the major isotypes (classes) of antibodies

Antibodies are capable of binding a wide variety of antigens, (macromolecules and small chemical) because antigen-binding regions of antibody molecules form flat surfaces capable of accommodating many different shapes

Antibodies bind to antigens by reversible, noncovalent interactions, including hydrogen bonds and charge interactions.

The parts of antigens that are recognized by antibodies are called epitopes, or determinants.



Binding of an antigen by an antibody

Affinity The strength with which one antigen-binding surface of an

antibody binds to one epitope of an antigen Is expressed as the dissociation constant (Kd) = is the molar

concentration of an antigen required to occupy half the available antibody molecules in a solution; the lower the Kd, the higher the affinity.

In a primary immune response the Kd between 10-6 to 10-9 M In a secondary immune response, the Kd between 10-8 to 10-

11 M Affinity maturation: the increase in antigen-binding strength


Antigen-binding site The site where antibody bind to epitope Each IgG, IgD, and IgE antibody molecule has two

antigen-binding sites IgA has four antigen-binding sites IgM has 10 antigen-binding sites

The total strength of binding is much greater than the affinity of a single antigen-antibody bond and is called the avidity of the interaction.

Antibodies produced against one antigen may bind other, structurally similar, antigens cross-reaction.


Monoclonal antibodies Antibody that has similar spesificity Has far-reaching implications for clinical medicine and research Methods

An animal is immunized with an antigen x B cell will produce plasma cells specific for x antigen Plasma cells is taken from animal spleen and fused with

myeloma cell line that had been treated with toxic drug to inhibit its grow

Fused cells grow and are called hybridomas From a population of hybridomas, it is possible to select and

clone the continuously growing cells that secrete the antibody x


Most of antibody monoclonal are made by fusing cells from immunized mice with mouse myelomas

These mouse monoclonal antibodies cannot be injected repeatedly into human subjects it will be regarded by immune system as foreign

The solution: the antigen-binding V regions is retained & the rest is replaced with human Ig "humanized" antibody


B cell receptors for antigens

Antigen receptor of B cell (B cell receptor-BCR) Is the antibody M or B that located

at the membrane of B cells Composed of

2 light chains and 2 heavy chain Each chain containing one variable (V) region constant region Only heavy chains are anchored in the plasma membrane Produced in a secreted form as effector Undergo class switching or affinity maturation during the life

of a B cell clone.


T cell receptors for antigens

Antigen receptor of T cell (T cell receptor-TCR) Displayed by MHC molecules A membrane-bound heterodimer Composed of an chain and a chain Each chain containing one variable (V) region and one

constant (C) region The V region of each TCR chain has 3 hypervariable, or complementarity-determining (CDR) and the CDR3 is the most variable among different TCRs.

Both TCR chains are anchored in the plasma membrane TCRs are not produced in a secreted form TCRs do not undergo class switching or affinity maturation

during the life of a T cell clone. 5/13/2014 S.Wahyuni/lecture/immunology/S1 23


4-5 The structure of the T cell antigen receptor (TCR)

and the chain of the TCR participate in specific recognition of MHC molecules and bound peptides

Each TCR recognizes 1-3 residues of peptide Note: only a few peptides of even complex microbes,

called the immunodominant epitopes, are actually recognized by the immune system.

This means that T cells can tell the difference between complex microbes on the basis of very few amino acid differences between the immunodominant epitopes of the microbes.



The recognition of a peptide-MHC complex by a T cell antigen receptor

TCR A lymphocyte T in innate immunity About 5% - 10% of total T cells in the body Structurally similar to the TCR but have very

different specificities May recognize a variety of protein and nonprotein

antigens, usually not displayed by classical MHC molecules.

Abundant in epithelia.


NK-T cells Comprising less than 5% of all T cells Express markers of natural killer (NK) cells and are

called NK-T cells. NK-T cells express TCRs, but they recognize

lipid antigens displayed by nonpolymorphic class I MHC-like molecules

The functions of NK-T cells also are not well understood.



Features of antigen recognition by immunoglobulins (Igs) and T cell antigen receptors (TCRs).

Development of Immune Repertoires

How the enormous diversity of these B and T cels receptors is produced?

There are many clones of lymphocytes with distinct specificities, perhaps as many as 109, and these clones arise before encounter with antigen.

There are not enough genes in the human genome for every possible receptor to be encoded by a different gene.

The immune system has developed mechanisms for generating extremely diverse repertoires of B and T lymphocytes

The generation of diverse receptors is intimately linked to the process of lymphocyte maturation.


Maturation of lymphocytes

The maturation of lymphocytes from bone marrow stem cells consists of three types of processes: proliferation of immature cells expression of antigen receptor genes selection of lymphocytes that express useful antigen

receptors .


Lymphocyte maturation

Involving random genetic recombination events Proliferation of lymphocyte stimulated mainly by interleukin-7

(IL-7) produced by stromal cells in the bone marrow and the thymus.

IL-7 maintains and expands the number of lymphocyte progenitors

After antigen receptor proteins are expressed, these receptors take over the function of delivering the signals for proliferation


5/13/2014 S.Wahyuni/lecture/immunology/S1 33Steps in the maturation of lymphocytes

Antigen receptors are encoded by several gene segments that are separate from one another in the germline and recombine during lymphocyte maturation.

Diversity is generated during this recombination process mainly by varying the nucleotide sequences at the site of recombination.

The expression of diverse antigen receptors is the central event in lymphocyte maturation


Maturing lymphocytes are selected at multiple steps during their maturation to preserve the useful specificities.

Selection is based on the expression of intact antigen receptor components and what they recognize. Positive selection: Only select lymphocytes that

express antigen receptor Negative selection: Only select lymphocytes that

has low affinity to self antigens present in the bone marrow and thymus


Production of diverse antigen receptors

The expression of B and T lymphocyte antigen receptors is initiated by somatic recombination of gene segments that code for the variable regions of the receptors, and diversity is generated during this process.

Hematopoietic stem cells in the bone marrow as well as early lymphoid progenitors contain Ig and TCR genes in their inherited, or germline, configuration.


Each loci of heavy & light chain of BCR and & chain of TCR contain Multiple variable region (V) genes One or a few constant region (C) genes Several small stretches of nucleotides, called joining (J) Diversity (D) gene segments.

All antigen receptor gene loci contain V, J, and C genes The Ig heavy chain and TCR loci also contain D gene segments Gene recombination is combination V, J, and C segment genes

(with or without D) the segments are selected randomly


5/13/2014 S.Wahyuni/lecture/immunology/S1 39Recombination and expression of immunoglobulin (Ig) genes

The somatic recombination of V and J, or V, D, and J, gene segments is mediated by a group of enzymes collectively, VDJ recombinase.

The lymphoid-specific component of the VDJ recombinase (recombinase-activating gene (RAG)-1 and RAG-2 proteins) recognizes DNA sequences that flank all antigen receptor V, D, and J gene segments.

The recombinase brings the V, D, and J segments close together and cleaves the DNA at specific sites.

The DNA breaks are then repaired by ligases, producing a full-length recombined V-J or V-D-J gene without the intervening DNA segments

The lymphoid-specific component of the VDJ recombinase is expressed only in immature B and T lymphocytes. 5/13/2014 S.Wahyuni/lecture/immunology/S1 40

Diversity of antigen receptors is produced by: the use of different combinations of V, D, and J gene

segments in different clones of lymphocytes (called combinatorial diversity)

by changes in nucleotide sequences introduced at the junctions of V, D, and J gene segments (called junctionaldiversity)

Combinatorial diversity is limited by the number of available V, D, and J gene segments, but junctional diversity is almost unlimited.

This junctional diversity is produced by three types of sequence changes, each of which generates more sequences than are present in the germline genes.



Mechanisms of diversity in antigen receptors

Maturation and selection of B lymphocytes


Steps in the maturation and selection of B lymphocytes (Occur in Bone marrow)

Maturation and selection of T lymphocytes

The process of T lymphocyte maturation are largely related to the specificity of different subsets of T cells for peptides displayed by different classes of MHC molecules.

The most immature progenitors are called pro-T cells or double-negative T cells because they do not express CD4 or CD8.

These cells expand in number mainly under the influence of IL-7 produced in the thymus.

Some of the progeny of double-negative cells undergo TCR gene recombination, mediated by the V(D)J recombinase.



Steps in the maturation and selection of major histocompatibility complex (MHC)-restricted T lymphocytes

SUMMARY In the adaptive immune system, the molecules responsible for

specific recognition of antigens are antibodies and T cell antigen receptors.

Antibodies (also called immunoglobulins) may be produced as membrane receptors of B lymphocytes and as proteins secreted by antigen-stimulated B cells that have differentiated into antibody-secreting plasma cells. Secreted antibodies are the effector molecules of humoral immunity, capable of neutralizing microbes and microbial toxins and eliminating them by activating various effector mechanisms.

TCRs are membrane receptors and are not secreted.


The core structure of antibodies consists of two heavy chains and two light chains forming a disulfide-linked complex. Each chain consists of a variable (V) region, which is the portion that recognizes antigen, and a constant (C) region, which provides structural stability and, in heavy chains, performs the effector functions of antibodies.

T cell receptors consist of an chain and a chain. Each chain contains one V region and one C region, and both chains participate in the recognition of antigens, which for most T cells are peptides displayed by MHC molecules.

The V regions of Ig and TCR molecules contain hypervariable segments, also called complementarity-determining regions, which are the regions of contact with antigens.


The genes that encode antigen receptors consist of multiple segments that are separate in the germline and are brought together during the maturation of lymphocytes. In B cells, the Ig gene segments undergo recombination as the cells mature in the bone marrow, and in T cells the TCR gene segments undergo recombination during maturation in the thymus.

Receptors of different specificities are generated in part by different combinations of V, D, and J gene segments. The process of recombination introduces variability in the nucleotide sequences at the sites of recombination by adding or removing nucleotides from the junctions. The result of this introduced variability is the development of a diverse repertoire of lymphocytes, in which clones of cells with different antigen specificities express receptors that differ in sequence and recognition, and most of the differences are concentrated at the regions of gene recombination.


During their maturation, lymphocytes undergo alternating cycles of proliferation and antigen receptor expression and traverse several checkpoints at which they are selected such that only cells with complete functional antigen receptors are preserved and expanded. In addition, T lymphocytes are positively selected to recognize peptide antigens displayed by self MHC molecules.

Immature lymphocytes that strongly recognize self antigens are negatively selected and prevented from completing their maturation, thus eliminating cells with the potential of reacting in harmful ways against self tissues.


Review questions

What are the functionally distinct domains (regions) of antibody and TCR molecules? What features of the amino acid sequences in these regions are important for their functions?

What are the differences in the types of antigens recognized by antibodies and TCRs?

What mechanisms contribute to the diversity of antibody and TCR molecules? Which of these mechanisms contributes the most to the diversity?

What are some of the checkpoints during lymphocyte maturation that ensure survival of the useful cells?

What is the phenomenon of negative selection, and what is its importance?


4_antigen recognition_3ed [compatibility mode]

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