Antigen Recognition in Adaptive Immune System
Antigen Recognition in Adaptive Immune System
· Recognition of antigen is initiating event in lymphocyte responses; specific antigen recognition is task of membrane-bound antibodies on B cells and TCRs on T lymphocytes
· Principal function of cellular receptors in immune system is to detect external stimuli (antigens) and trigger responses of cells on which receptors are expressed
o To recognize large number and variety of antigens, antigen receptors must be able to bind to and distinguish between many, often closely related, chemical structures
· Antigen receptors clonally distributed (each clone of lymphocytes with particular specificity has unique receptor)
· Although each clone of B lymphocytes or T lymphocytes recognizes different antigen, antigen receptors transmit biochemical signals fundamentally same in all lymphocytes and unrelated to specificity
Antigen Receptors of Lymphocytes
· Antigen receptors of B and T lymphocytes recognize chemically different structures
o B lymphocyte antigen receptors (membrane-bound antibodies) and antibodies that B cells secrete able to recognize shapes (conformations) of native macromolecules, including proteins, lipids, carbs, and nucleic acids, as well as simple small chemical groups and parts of macromolecules
§ Broad specificity of B cells enables antibodies to recognize diverse microbes and toxins in their native form
o Most T cells see only peptides, and only when they are displayed on APCs bound to membrane proteins encoded in MHC genetic locus; allows T cells to recognize cell-associated microbes
· Antigen receptor molecules consist of regions (domains) involved in antigen recognition and therefore vary between clones of lymphocytes, and other regions that are required for structural integrity and for effector functions and are relatively conserved among all clones
o Antigen-recognizing portions of receptors are variable (V ) regions, and conserved portions are constant (C) regions
o Even within V regions, much of sequence variability concentrated within short stretches (hypervariable regions or complementarity-determining regions or CDRs) because they form parts of receptor that bind antigens
o By concentrating sequence variation in small regions of receptor, it’s possible to maximize variability while retaining basic structures of receptors
· Antigen receptors noncovalently attached to other invariant molecules whose function is to deliver activation signals triggered by antigen recognition to inside of cell
o Fact that specific antigen recognition and signal transduction mediated by different polypeptides allows variability to be segregated in one set of molecules (receptors themselves) while leaving conserved function of signal transduction in other (invariant) proteins
o Collection of antigen receptors and signaling molecules in B lymphocytes called B cell receptor (BCR) complex, and in T lymphocytes called TCR complex
o When adjacent antigen receptors of lymphocytes bind to 2+ antigen molecules, receptors pulled together into aggregate (cross-linking); brings associated signaling proteins of receptor complexes into close proximity
§ Enzymes attached to cytoplasmic portions of signaling proteins catalyze phosphorylation of other proteins
§ Phosphorylation triggers complex signaling cascades that culminate in transcriptional activation of many genes and production of numerous proteins that mediate responses of lymphocytes
· Immunoglobulins – called so because they are immunity-conferring proteins with characteristic electrophoretic mobility of plasma globulins
o Antibodies recognize microbial antigens and toxins by variable domains
o Constant regions of some secreted antibodies have ability to bind to other molecules that participate in elimination of antigens; include receptors on phagocytes and proteins of complement system
o B cell membrane-bound antibodies recognize antigens to initiate responses, and secreted antibodies neutralize and eliminate microbes and their toxins in effector phase of humoral immunity
o In cell-mediated immunity, effector function of microbe elimination performed by T lymphocytes
o Antigen receptors of T cells involved only in antigen recognition and T cell activation, and these proteins don’t mediate effector functions and aren’t secreted
· Antibody molecule – composed of 4 polypeptide chains (2 identical heavy (H) chains and 2 identical light (L) chains), with each chain containing one variable region and one constant region
o Each L chain attached to one H chain, and the H chains attached to each other, all by disulfide bonds
o L chain made of one V and one C domain, and each H chain made up of one V and 3-4 C domains
§ Each domain folds into characteristic 3D shape (Ig domain)
§ Ig domain consists of 2 layers of β-pleated sheet held together by disulfide bridge
§ Adjacent strands of each β-sheet connected by short loops, and these loops are sites of antigen recognition
§ Ig domains present in many other proteins in immune system as well as outside immune system, and most of these proteins involved in sensing signals from environment and from other cells (Ig superfamily)
o Each V region of H chains (VH) or L chains (VL) contains 3 hypervariable regions (CDRs)
§ Greatest variability in CDR3, located at junction of V and C regions; also portion of Ig molecule that contributes most to antigen binding
o Regions of antibody molecules named based on properties of proteolytic fragments of Igs
§ Fragment of antibody that contains whole L chain (single V and C domains) attached to V and first C domains of H chain contains portion of that antibody required for antigen recognition and is therefore called Fab (fragment antigen binding)
§ Remaining CH domains make up Fc region (fragment crystalline)
o In each Ig molecule, there are 2 identical Fab regions that bind antigen and one Fc region responsible for most of biologic activity and effector functions of antibodies
§ Some antibodies exist as multimers of 2 or 5 antibody molecules attached to one another
o Between Fab and Fc regions of most antibody molecules is flexible portion (hinge region); hinge allows 2 antigen-binding Fab regions of each antibody molecule to move independently of each other, enabling them to simultaneously bind antigen epitopes separated from one another by varying distances
§ C-terminal end of H chain may be anchored in PM (as in BCRs) or may terminate in tail piece that lacks membrane anchor so it is secreted
§ L chains not attached to PMs
o 2 types of L chains (κ and λ) that differ in C regions but not in function
§ Each B cell expresses either κ or λ, but not both
o 5 types of H chains (µ, δ, γ, ε, and α)
o Each type of L chain may complex with any type of H chain in antibody molecule
o Antibodies that contain different H chains belong to different isotypes (classes) and named according to H chains (IgM, IgD, IgG, IgE, and IgA) regardless of L chain class
§ Each isotype has distinct physical and biological properties and effector functions
§ Antigen receptors of naïve B cells (mature B cells that haven’t encountered antigen) are membrane-bound IgM and IgD
§ After stimulation by antigen and helper T lymphocyte, antigen-specific clone of B lymphocytes may expand and differentiate into progeny that secrete antibodies
· Some of progeny of IgM-expressing and IgD-expressing B cells may secrete IgM, and other progeny of same B cells may produce antibodies of other H chain classes (isotype switching or H chain class switching)
§ Although CH regions may switch during humoral immune responses, each clone of B cells maintains specificity because V regions don’t change
· L chain class also remains fixed throughout life of each B cell clone
o Antibodies capable of binding wide variety of antigens, including macromolecules and small chemicals because antigen-binding regions form flat surfaces capable of accommodating many different shapes
§ Antibodies bind to antigens by reversible, noncovalent interactions, including hydrogen bonds and charge interactions
§ Parts of antigens recognized by antibodies are epitopes (determinants)
§ Different antigenic determinants may be recognized based on sequence (linear epitopes) or shape (conformational epitopes); some epitopes hidden within antigen molecules and are exposed as result of physicochemical change
o Affinity often expressed as Kd (molar concentration of antigen required to occupy half the available antibody molecules in solution); lower Kd has higher affinity
§ With repeated stimulation (e.g., in secondary immune response) affinity increases (affinity maturation)
o Each antibody molecule can bind 2-10 epitopes of antigen as long as identical epitopes present sufficiently close together (e.g., on cell surface) in aggregated antigen or in some lipids, polysaccharides, and nucleic acids that contain multiple repeated epitopes
o Total strength of binding much greater than affinity of single antigen-antibody bond (avidity of interaction)
o Antibodies produced against one antigen may bind other, structurally similar antigens (cross-reaction)
o In B lymphocytes, Ig molecules noncovalently attached to 2 other proteins (Igα and Igβ) that make up BCR complex; when Ig receptor recognizes antigen, Igα and Igβ transmit signals to interior of B cell that initiate process of B cell activation
· Monoclonal antibodies – one of most important technical advances in immunology; B cells from animal immunized with antigen (short lifespan in vitro) fused with myeloma cells (tumors of plasma cells), which can be propagated indefinitely in tissue culture
o Myeloma cell line mutated to lack enzyme so it doesn’t grow in presence of certain toxic drug, whereas fused cells grow because normal B cells provide enzyme
o By fusing the 2 cell populations and selecting them by culture with drug, it’s possible to grow out fused cells derived from B cells and myeloma (hybridomas)
o Possible to select and clone continuously growing cells that secrete antibody of desired specificity from hybridoma (these are monoclonal antibodies)
o Most antibodies made by fusing cells from immunized mice with mouse myelomas; can’t be injected repeatedly into humans because human immune system sees mouse Ig as foreign and makes immune response against it
§ Problem overcome by retaining antigen-binding V regions of mouse monoclonal antibody and replacing rest of Ig with human Ig; humanized antibodies suitable for administration to people
o More recently, monoclonal antibodies synthesized by using recombinant DNA technology to clone DNA encoding human antibodies and selecting antibodies of desired specificity
o Also replace Ig genes of mice with human antibody genes and then immunize mice with antigen to produce specific human antibodies
· TCR for peptide antigen displayed by MHC molecules is membrane-bound heterodimer composed of α chain and β chain, each containing one V region and one C region
o Have CDRs just like antibodies (including TCR3 is most variable)
o 3D structure of TCR very similar to that of Fab region of Ig molecule
o Both TCR chains anchored in PM, and TCRs not produced in secreted form
o TCRs don’t undergo class switching or affinity maturation during life of T cell clone
o Both α chain and β chain participate in specific recognition of MHC molecules and bound peptides
§ Each TCR recognizes as few as 1-3 residues of MHC-associated peptide
§ Only a few peptides of even complex microbes (immunodominant epitopes) actually recognized by immune system
§ T cells can tell difference between complex microbes on basis of very few amino acid differences between immunodominant epitopes of microbes
o From 5-10% of T cells in body express receptors composed of γ and δ chains; structurally similar to αβ TCR but have very different specificities
§ γδ TCR may recognize variety of protein and non-protein antigens, usually not displayed by classical MHC molecules
§ T cells expressing γδ TCRs abundant in epithelia; γδ T cells recognize microbes commonly encountered at epithelial surfaces (we don’t know what they do)
o Less than 5% of all T cells express markers of NK cells (NK-T cells); express αβ TCRs, but recognize lipid antigens displayed by non-polymorphic class I MHC-like molecules
o TCR recognizes antigen, but incapable of transmitting signals to T cell
§ Associated with TCR is complex of proteins (CD3 and ζ proteins) that make up TCR complex
§ CD3 and ζ chains transmit some of signals initiated when TCR recognizes antigen
o T cell activation requires engagement of coreceptor molecules (CD4 or CD8) that recognize nonpolymorphic portions of MHC molecules and transmit activating signals
· Antibodies bind greatest variety of antigens with highest affinities (why antibodies can bind to and neutralize many different microbes and toxins present at low concentrations in circulation)
· Affinity of TCRs low (why binding of T cells to APCs has to be strengthened by additional cell surface adhesion molecules
Development of Immune Repertoires
· There are many clones of lymphocytes with distinct specificities, perhaps as many as 109, these clones arise before encounter with antigen
· Generation of diverse receptors intimately linked to process of lymphocyte maturation
· Maturation of lymphocytes from bone marrow stem cells consists of 3 types of processes: proliferation of immature cells, expression of antigen receptor genes, and selection of lymphocytes that express useful antigen receptors; events common to B and T lymphocytes, even though B lymphocytes mature in bone marrow and T lymphocytes mature in thymus
· Immature lymphocytes undergo tremendous proliferation at several stages during maturation
o Generation of useful antigen receptor genes fails more often than not in developing lymphocytes
o Proliferation of developing lymphocytes necessary to ensure that adequate number of cells will ultimately express useful antigen receptors and mature into functionally competent lymphocytes
o Survival and proliferation of earliest lymphocyte precursors stimulated mainly be growth factor IL-7 (produced by stromal cells in bone marrow and thymus)
§ IL-7 maintains and expands number of lymphocyte progenitors (mainly T cell progenitors) before they express antigen receptors, thus generating large pool of cells in which diverse antigen receptors may be produced
§ After antigen receptor proteins expressed, these receptors take over function of delivering signals for proliferation, ensuring only clones with intact receptors selected to expand
· Antigen receptors encoded by several gene segments (separate from one another in germline) that recombine during lymphocyte maturation
o Diversity generated mainly by varying nucleotide sequences at site of recombination
o Expression of diverse antigen receptors is central event in lymphocyte maturation