Topic 4: Antigen Recognition in Adaptive Immunity

γδ TCR

-5% to 10% of T cells in the body -may recognize a variety of protein and nonprotein antigens, usually not displayed by classical MHC molecules -abundant in epithelia

antibody binding

-Antibodies are capable of binding a wide variety of antigens, including macromolecules and small chemicals. - The reason for this is that the antigen-binding CDR loops of antibody molecules can either come together to form clefts capable of accommodating small molecules or form more extended surfaces capable of accommodating many larger molecules, including portions of proteins. -Antibodies bind to antigens by reversible, noncovalent interactions, including hydrogen bonds, hydrophobic interactions, and charge-based interactions. - The parts of antigens that are recognized by antibodies are called epitopes, or determinants -Different epitopes of protein antigens may be recognized based on the sequence of a stretch of amino acids (linear epitopes) or on the shape (conformational epitopes).

combinatorial diversity

-Diversity of antigen receptors is produced by the use of different combinations of V, D, and J gene segments in different clones of lymphocytes -limited by the number of available V, D, and J gene segments

genes that encode B and T lymphocyte antigen receptors

-Hematopoietic stem cells and early lymphoid progenitors contain Ig and TCR genes in their inherited, or germline, configuration. In this configuration, Ig heavy-chain and light-chain loci and the TCR α-chain and β-chain loci each contain multiple variable region (V) gene segments, numbering about 30-45, and one or a few constant region (C) genes. Between the V and C genes are groups of several short coding sequences called diversity (D) and joining (J) gene segments. All antigen receptor gene loci contain V, J, and C genes, but only the Ig heavy-chain and TCR β-chain loci also contain D gene segments.

immature B cell

-IgM-expressing B lymphocyte -further maturation may occur in the bone marrow or after it leaves the bone marrow and enters the spleen

heavy chain class (or isotope) switching

-The antigen receptors of naive B lymphocytes, which are mature B cells that have not encountered antigen, are membrane-bound IgM and IgD. -After stimulation by antigen and helper T lymphocytes, the antigen- specific B lymphocyte clone may expand and differentiate into progeny that secrete antibodies. -Some of the progeny of IgM and IgD expressing B cells may secrete IgM, and other progeny of the same B cells may produce antibodies of other heavy-chain classes.

Lymphocyte Development

-The development of lymphocytes from bone marrow stem cells involves commitment of hematopoietic progenitors to the B or T cell lineage, the proliferation of these progenitors, the rearrangement and expression of antigen receptor genes, and selection events to preserve and expand cells that express potentially useful antigen receptors

Maturation of Selection of B Lymphocytes

-The maturation of B cells occurs mainly in the bone marrow. Progenitors committed to the B cell lineage proliferate (divide many times), giving rise to a large number of precursors of B cells, call pro- B cells. Subsequent maturation involves antigen receptor gene expression and selection.

allelic exclusion--> B cells

-The pre-BCR complex signals to shut off recombination of Ig heavy-chain genes on the second chromosome, because of which each B cell can express an Ig heavy chain from only one of the two inherited parental alleles. -helps ensure that each cell can only express a receptor of a single specificity.

How is the enormous diversity of TCR and BCR antigen receptors generated?

-The process of lymphocyte maturation first generates a very large number of cells each with a different antigen receptor and then preserves the cells with useful receptors. -Once these antigen receptors are expressed on developing lymphocytes, selection processes come into play that promote the survival of cells with receptors that can recognize antigens, such as microbial antigens, and eliminate cells that cannot recognize antigens in the individual or that have the potential to cause harm.

Mechanisms of V(D)J Recombination

-The somatic recombination of V and J, or of V, D, and J, gene segments is mediated by a lymphoid- specific enzyme, the VDJ recombinasend additional enzymes, most of which are not lymphocyte specific and are involved in repair of double-stranded DNA breaks introduced by the recombinase.

affinity

-The strength with which one antigen-binding surface of an antibody binds to one epitope of an antigen -Affinity often is expressed as the dissociation constant (Kd), which 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.

pro-T cells or double-negative T cells

-least developed progenitors in the thymus -do not express CD4 or CD8

NK-T cells

-less than 5% of all T cells -express αβ TCRs with limited diversity, and they recognize lipid antigens displayed by nonpolymorphic class I MHC-like molecules -functions of NK-T cells also are not well understood.

CDR3 hypervariable

-loop crucial for antigen recognition -centrally located in the TCR, positioned to interact directly with peptide antigen in the center of a MHC molecule. -randomly generated and NOT encoded in the genome

forms of antibodies

-membrane-bound antigen receptors on B cells or as secreted proteins -Secreted antibodies are present in the blood and mucosal secretions, where they function to defend against microbes

double-positive T cells

-surviving cells express the complete αβ TCR and both the CD4 and CD8 coreceptors -Immature, double-positive T cells whose receptors strongly recognize MHC-peptide complexes in the thymus undergo apoptosis.

mature B cell

-the IgM+IgD+ cell -it is able to respond to antigen in peripheral lymphoid tissues.

allelic exclusion-->T cells

The pre-TCR complex delivers intracellular signals once it is assembled, similar to the signals from the pre- BCR complex in developing B cells. These signals promote survival, proliferation, and TCR α gene recombination and inhibit VDJ recombination at the second TCR β-chain locus - Failure to express the α chain and the complete TCR again results in death of the cell.

Fab and Fc

- The fragment of an antibody that contains a whole light chain (with its single V and C domains) attached to the V and first C domains of a heavy chain contains the portion of the antibody required for antigen recognition and is therefore called Fab (fragment, antigen-binding). -The remaining heavy-chain C domains make up the Fc (fragment, crystalline) region; this fragment tends to crystallize in solution. -In each Ig molecule, there are two identical Fab regions that bind antigen and one Fc region that is responsible for most of the biologic activity and effector functions of the antibodies.

how are both positive selection and negative selection mediated by recognition of the same set of self MHC-self peptide complexes in the thymus?

- The likely explanation for these distinct outcomes is that if the antigen receptor of a T cell recognizes a self MHC-self peptide complex with low avidity, the result is positive selection, whereas high-avidity recognition leads to negative selection -If such a T cell were allowed to mature, antigen recognition could lead to harmful immune responses against the self antigen in the periphery, so the T cell must be eliminated.

Ipilimumab

- an antibody that binds to an inhibitory receptor (CTLA4) on tumor-specific T cells. Its Fc portion is engineered NOT to be recognized, and thus does not lead to killing of the T cells. Instead, by blocking the inhibitory receptor, Ipilimumab boosts the function of tumor-specific T cells and promotes tumor cell killing by T cell-mediated immunity.

junctional diversity

- changes in nucleotide sequences introduced at the junctions of the recombining V, D, and J gene segments -almost unlimited -produced by three mechanisms

T cell Receptors for Antigens

The TCR, which re cognizes pe ptide antigens dis playe d by MHC molecules, is a me mbrane -bound hete rodime ric protein composed of an α chain and a β chain, each chain containing one variable (V) region and one constant (C) region

Rituximab

- use the Fab portion to recognize and bind to a B cell-specific molecule (CD20) expressed by all B cells including B cell lymphoma and leukemia tumor cells. The Fc portion of Rituximab then directs killing via Fc-receptor bearing cells like natural killer (NK) cells and macrophages.

Antigen Receptors

-Antigen receptor chains are associated with membrane proteins whose function is to deliver intracellular signals following antigen recognition. -These signals, which are transmitted to the cytosol and the nucleus, may cause a lymphocyte to divide, to differentiate, or in certain circumstances to die. -The set of associated plasma membrane antigen receptor and signaling molecules in B lymphocytes is called the B cell receptor (BCR) complex, and in T lymphocytes it is called the T cell receptor (TCR) complex.

Selection of Mature B Cells

-Developing B cells are positively selected based mainly on expression of complete antigen receptors, and not on the recognition specificity of these cells. (This is fundamentally different in maturing T cells) -The B cell repertoire is further shaped by negative selection -->eliminates potentially dangerous cells that can recognize and react against ubiquitous self antigens. -Some B cells that encounter antigens in the bone marrow may die by apoptosis, also known as deletion -selected positively for expression of intact receptors and selected negatively against strong recognition of self antigens

avidity

-Each IgG, IgD, and IgE antibody molecule has 2 antigen-binding sites. -Secreted IgA is a dimer and therefore has 4 antigen-binding sites, and secreted IgM is a pentamer, with 10 antigen-binding sites. -Therefore, each antibody molecule can bind 2 to 10 epitopes of an antigen, or epitopes on 2 or more neighboring antigens. -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.

somatic recombination

-Essentially the same sequence of DNA recombination and RNA splicing leads to production of a light chain in B cells, except that the light-chain loci lack D segments, so a V region exon recombines directly with a J segment. The rearrangement of TCR α-chain and β-chain genes in T lymphocytes is similar to that of Ig L and H chains, respectively.

Early Steps in B Cell Maturation

-Ig heavy-chain locus rearranges first, and only cells that are able to make an Ig μheavy-chain protein are selected to survive and become pre-B cells. -Pre-B cells are defined by the presence of the Ig μ heavy- chain protein, mainly in the cytoplasm. -Some of the μ protein is expressed on the cell surface in association with two other, invariant proteins, called surrogate light chains because they resemble light chains and associate with the μ heavy chain. The complex of μ chain and surrogate light chains associates with the Igα and Igβ signaling molecules to form the pre-B cell receptor (pre-BCR) complex.

light chains

-The two types of light chains, called κ and λ, differ in their C regions. -Each B cell expresses either κ or λ but not both. -Each type of light chain may complex with any type of heavy chain in an antibody molecule, but unlike the heavy chains, the two types of light chains have no functional differences.

receptor editing.

-The λ light chain is produced only if the rearranged κ chain locus fails to express a functional protein or if the κ chain generates a potentially harmful self-reactive receptor and has to be eliminated

Isotopes of Antibodies

-There are five types of heavy chains, called μ, δ, γ, ε, and α, which differ in their C regions; in humans, there are four subtypes of γ chain and two of the α chain. -Antibodies that contain different heavy chains belong to different classes, or isotypes, and are named according to their heavy chains (IgM, IgD, IgG, IgE, and IgA). -Each isotype has distinct physical and biologic properties and effector functions.

Ig domain

-an Ig domain consists of two layers of a β-pleated sheet held together by a disulfide bridge. The adjacent strands of each β-sheet are connected by short, protruding loops; in the V regions of Ig molecules, these loops make up the three CDR loops responsible for antigen recognition

Ig gene recombination

-andom and cannot be inherently biased toward recognition of microbes -However, the receptors produced are able to recognize the antigens of many, varied microbes that the immune system must defend against.

Antibodies

-composed of four polypeptide chains—two identical heavy (H) chains and two identical light (L) chains—with each chain containing a variable region and a constant region. -The four chains are assembled to form a Y-shaped molecule. Each light chain is attached to one heavy chain, and the two heavy chains are attached to each other, all by disulfide bonds. -A light chain is made up of one V and one C domain, and a heavy chain has one V and three or four C domains. Each domain folds into a characteristic three-dimensional shape, called the immunoglobulin (Ig)domain

antigen-binding site

-composed of the V regions of both the heavy chain and the light chain, and the core antibody structure contains two identical antigen binding sites. -Each variable region of the heavy chain (called VH) or of the light chain (called VL) contains three hypervariable regions, or CDRs. - Of these three, the greatest variability is in CDR3, which is located at the junction of the V and C regions. --> As may be predicted from this variability, CDR3 is also the portion of the Ig molecule that contributes most to antigen binding.

VDJ recombinase

-composed of the recombination-activating gene 1 and 2 (RAG-1 and RAG- 2) proteins. It recognizes DNA sequences that flank all antigen receptor V, D, and J gene segments. As a result of this recognition, the recombinase brings two Ig or TCR gene segments close together and cleaves the DNA at specific sites. The DNA breaks are then repaired by ligases, producing a full- length recombined VJ or VDJ exon without the intervening DNA segments.

Lymphocytes

-derived from bone marrow resident pluripotent hematopoietic stem cells (HSC) - HSC differentiate into lymphoid and myeloid progenitor cells. -Lymphoid progenitors become one of three types of lymphocytes B, T, or NK cells. -B and T cells distinguish themselves by expression of highly specific antigen receptors. -B and T lymphocytes express different receptors that recognize antigens: membrane- bound antibodies on B cells and T cell receptors (TCRs) on T lymphocytes. -The principal function of cellular receptors in the immune system is to detect external stimuli and trigger responses of the cells on which the receptors are expressed. -Antigen receptors are clonally distributed, meaning that each lymphocyte clone is specific for a distinct antigen and has a unique receptor, different from the receptors of all other clones. -The total number of distinct lymphocyte clones is very large, and this entire collection makes up the immune repertoire.

negative selection

-eliminate potentially dangerous lymphocytes such as antigen receptors may recognize peptides of self proteins and prevent the development of autoimmune responses

TCRs

-exist only as membrane receptors on T cells.

receptor editing

-if an immature B cell binds an antigen in the bone marrow with high affinity, it may re-express the VDJ recombinase enzyme, undergo additional light-chain V-J recombination, generate a different light chain, and thus change the specificity of the antigen receptor, a process called

positive selection

-immature T cells are selected to survive only if they recognize MHC molecules in the thymus -ensures that cells that complete maturation will be capable of recognizing antigens displayed by the same MHC molecules on APCs

antibodies are also known as?

-immunoglobulins

monoclonal antibodies

-one of the most important technical advances in immunology, with far- reaching implications for clinical medicine and research. To produce monoclonal antibodies, B cells, which have a short life span in vitro, are obtained from an animal immunized with an antigen and fused with myeloma cells (tumors of plasma cells), which can be propagated indefinitely in tissue culture.

three mechanisms of junctional diversity

1. Exonucleases may remove nucleotides from V, D, and J gene segments at the sites of recombination. 2. A lymphocyte-specific enzyme called terminal deoxyribonucleotidyl transferase (TdT) catalyzes the random addition of nucleotides that are not part of germline genestothejunctions betweenVandD segments and D and J segments, forming so-called N regions. 3.During an intermediate stage in the process of V(D)J recombination, before breaks in the DNA are repaired, overhanging DNA sequences may be generated that are then filled in, forming P-nucleotides, introducing even more variability at the sites of recombination.

BCRs vs TCRs

-Membrane-bound antibodies, which serve as the antigen receptors of B lymphocytes, can recognize many types of chemical structures, while most T cell antigen receptors recognize only peptides bound to major histocompatibility complex (MHC) molecules. -B cell antigen receptors (BCR) and the antibodies that B cells secrete are able to recognize the shapes, or conformations, of macromolecules, including proteins, lipids, carbohydrates, and nucleic acids, as well as simpler, smaller chemical moieties. This broad specificity of B cells for structurally different types of molecules enables antibodies to recognize diverse microbes and toxins in their native form. -In striking contrast, most T cells see only peptides displayed on antigen-presenting cells (APCs) bound to MHC molecules. This specificity of T cells restricts their recognition to only cell-associated microbes

recent monoclonal antibody techniques

-More recently, monoclonal antibodies have been generated by using recombinant DNA technology to clone the DNA encoding human antibodies of desired specificity. Another approach is to replace the Ig genes of mice with human antibody genes and then immunize these mice with an antigen to produce specific human antibodies. Monoclonal antibodies are now in widespread use as therapeutic agents for many diseases in humans.

affinity maturation

-Most antibodies produced in a primary immune response have a Kd in the range of 10−6 to 10−9 M, but with repeated stimulation (e.g., in a secondary immune response), the affinity increases to a Kd of 10−8 to 10−11 M

how are monoclonal antibodies made ?

-Most monoclonal antibodies are made by fusing cells from immunized mice with mouse myelomas. Such mouse monoclonal antibodies cannot be injected repeatedly into human subjects, because the human immune system sees the mouse Ig as foreign and mounts an immune response against the injected antibodies. This problem has been partially overcome by genetic engineering approaches that retain the antigen-binding V regions of the mouse monoclonal antibody and replace the rest of the Ig with human Ig; such humanized antibodies are suitable for administration to people (although with prolonged use, even some humanized monoclonal may elicit anti-Ig antibody responses in treated individuals).

Lymphocyte Selection

-Selection is based on the expression of intact antigen receptor components and what they recognize. -Many attempts to generate antigen receptors fail because of errors during the gene recombination process. -Therefore, checkpoints are needed at which only cells with intact, functional antigen receptors are selected to survive and proliferate. -Prelymphocytes and immature lymphocytes that fail to express antigen receptors die by apoptosis. -The gene rearrangements in the developing lymphocytes randomly generate antigen receptors with highly diverse specificities. Some of these may be incapable of recognizing antigens in the individual.

Pre-BCR Complex in B Cell Maturation

-Signals from the pre-BCR complex promote the survival and proliferation of B lineage cells that have made a productive rearrangement at the Ig H-chain locus. This is the first checkpoint in B cell development, and it selects and expands the pre-B cells that express a functional μ heavy chain -Pre-B c ells that make out-of-frame (nonproductive) rearrangements at the heavy-chain locus fail to make the μ protein, cannot express a pre-BCR or receive pre-BCR signals, and die by programmed cell death (apoptosis). -allelic exclusion -triggers recombination at the Ig κ light-chain locus leading to production of the κ protein. -receptor editing -Whichever functional light chain is produced associates with the μ heavy chain to form the complete membrane-associated IgM antigen receptor -This receptor again delivers signals that promote survival, thus preserving cells that express complete antigen receptors, the second checkpoint during maturation. Signals from the antigen receptor also shut off production of the recombinase enzyme and further recombination at light chain loci.

single-positive T cells

-T cells whose TCRs recognize class I MHC-peptide complexes preserve the expression of CD8, the coreceptor that binds to class I MHC, and lose expression of CD4, the coreceptor specific for class II MHC molecules. Conversely, if a T cell recognizes class II MHC-peptide complexes, this cell maintains expression of CD4 and loses expression of CD8 -either CD8+ class I MHC restricted or CD4+ class II MHC restricted.

Selection of Mature T Cells

-TCR β gene recombination, mediated by the VDJ recombinase, occurs in some of these double-negative cells -If VDJ recombination is successful in one of the two inherited loci and a TCR β-chain protein is synthesized, it is expressed on the cell surface in association with an invariant protein called pre-Tα, to form the pre-TCR complex. If the recombination in one of the two inherited loci is not successful, recombination will take place on the other locus. If that too fails and a complete TCR β chain is not produced in a pro-T cell, the cell dies. -Different clones of double-positive T cells express different αβ TCRs. If the TCR of a T cell recognizes an MHC molecule in the thymus, which must be a self MHC molecule displaying a self peptide, and if the interaction is of low or moderate affinity, this T cell is selected to survive.

Antigen recognition by B and T lymphocytes

-TCRs only recognize peptide-MHC complexes and bind these with relatively low affinity, which may be why the binding of T cells to APCs has to be strengthened by additional cell surface adhesion molecules - The three-dimensional structure of the TCR is similar to that of the Fab region of an Ig molecule. -Unlike in antibodies, both TCR chains are anchored in the plasma membrane; TCRs are not produced in a secreted form and do not undergo isotype switching or affinity maturation during the life of a T cell.

CD3 and ζ role in TCR recognition

-The TCR recognizes antigen, but as with membrane Ig on B cells, it is incapable of transmitting signals to the T cell on its own. -Associated with the TCR is a complex of proteins, called the CD3 and ζ proteins, which together with the TCR make up the TCR complex -The CD3 and ζ chains transmit some of the signals that are initiated when the TCR recognizes antigen. In addition, T cell activation requires engagement of the coreceptor molecule CD4 or CD8, which recognize nonpolymorphic portions of MHC molecules and are also involved in signal transduction.

V and C regions

-The V and C regions are homologous to immunoglobulin V and C regions. In the V region of each TCR chain, there are three hypervariable, or complementarit y-determining, regions, each corresponding to a loop in the V domain. As in antibodies, CDR3 is the most variable among different TCRs.

alpha and beta chain of TCR

Both the α chain and the β chain of the TCR participate in specific recognition of both the MHC molecule and bound peptides simultaneously. -each TCR recognizes as few as one to three residues of the MHC-associated peptide