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
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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
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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
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
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)
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
Total strength of binding much greater than affinity of single antigen-antibody bond (avidity of
interaction)
Antibodies produced against one antigen may bind other, structurally similar antigens (cross-reaction)
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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
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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
Maturing lymphocytes selected at multiple steps during maturation to preserve useful specificities; selection
based on expression of intact antigen receptor components and what they recognize
o Prelymphocytes that fail to express antigen receptors die by apoptosis
o Immature T cells selected to recognize self MHC molecules in thymus (positive selection); after they
mature and enter peripheral tissues, they need to recognize same MHC molecules to be activated
 Antigen receptors on developing lymphocytes recognize MHC molecules in thymus and deliver
signals for survival and proliferation of cells, ensuring that cells with correct (self MHCrestricted) antigen receptors complete maturation process
o Immature B and T lymphocytes selected against high-affinity recognition of self antigens present in bone
marrow and thymus (negative selection), which eliminates potentially dangerous lymphocytes that may
be capable of reacting against self antigens present throughout body
Expression of B and T lymphocyte antigen receptors initiated by somatic recombination of gene segments that
code for variable regions of receptors, and diversity generated during this process
o Hematopoietic stem cells in bone marrow as well as early lymphoid progenitors contain Ig and TCR
genes in their inherited (germline) configuration; Ig H chain and L chain loci and TCR α chain and β chain
loci each contain multiple V region genes and one or a few C region genes
o Between V and C genes are joining (J) and diversity (D) gene segments
 All antigen receptor gene loci contain V, J, and C genes, but only Ig H chain and TCR β loci also
contain D gene segments
o Commitment of lymphocyte progenitor to become B lymphocyte associated with recombination of one
Ig VH gene segment with one D and one J segment (segments selected randomly)
 Committed but still developing B cells now has recombined V-D-J gene in H chain locus
 Gene transcribed, and in primary RNA, VDJ complex spliced onto first C region RNA, which
encodes µ chain, to form complete µ mRNA, which is translated to produce µ H chain, which is
first Ig protein synthesized during B cell maturation
 Similar sequence of DNA recombination and RNA splicing leads to production of L chain in B cells
and TCR chains in T lymphocytes
o Somatic recombination of V and J or V, D, and J gene segments mediated by group of enzymes
collectively called VDJ recombinase
 Lymphoid-specific component of VDJ recombinase composed of recombinase-activating gene
(RAG)-1 and RAG-2 proteins
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Recognizes DNA sequences that flank all antigen receptor V, D, and J gene segments; as
a result, recombinase brings V, D, and J segments close together and cleaves DNA at
specific sites
 DNA breaks repaired by ligases, producing full-length recombined V-J or V-D-J gene without
intervening DNA segments
 Lymphoid-specific component of VDJ recombinase expressed only in immature B and T
lymphocytes
 Same enzymes can mediate recombination of all Ig and TCR genes
 Intact Ig H and L chain genes expressed only in B cells
 TCR α and β genes expressed only in T cells
Diversity of antigen receptors produced by use of different combinations of V, D, and J gene segments in
different clones of lymphocytes (called combinatorial diversity) and even more by changes in nucleotide
sequences introduced at junctions of V, D, and J gene segments (junctional diversity)
o Combinatorial diversity limited by number of available V, D, and J gene segments, but junctional
diversity almost unlimited
o Junctional diversity produced by 3 types of sequence changes, each of which generates more sequences
than are present in germline genes
 Exonucleases may remove nucleotides from V, D, and J gene segments at time of recombination,
and if resulting combined sequences don’t contain stop or nonsense codons, many different and
new sequences may be produced
 Lymphocyte-specific enzyme (terminal deoxyribonucleotidyl transferase or TdT) catalyzes
random addition of nucleotides that aren’t parts of germline genes to sites of V(D)J
recombination, forming so-called N regions
 During intermediate stage in process of V(D)J recombination, before breaks in DNA repaired,
overhanging DNA sequences may be generated that are then filled in by P-nucleotides,
introducing even more variability at sites of recombination
o Nucleotide sequence at site of V(D)J recombination in antibody or TCR molecules made by one clone of
lymphocytes differs from sequence at V(D)J site of antibody or TCR molecules made by every other
clone
o Junctional sequences encode amino acids of CDR3 loop; thus, junctional diversity maximizes variability
in antigen-binding regions of antibodies and TCRs
o In process of creating junctional diversity, many genes produced that can’t code for proteins and are
therefore useless; risk of producing nonfunctional genes is why process of lymphocyte maturation
contains several checkpoints at which only cells with useful receptors selected to survive
Maturation of B lymphocytes occurs mainly in bone marrow; progenitors committed to B cell lineage proliferate,
giving rise to large number of precursors (pro-B cells)
o In next stage of maturation (making pre-B cells), Ig genes in H chain locus of one chromosome
recombine and give rise to µ H chain protein; most of this protein remains in cytoplasm (cytoplasmic µ is
hallmark of pre-B cells)
o Some of µ protein expressed on cell surface in association with 2 other, invariant, proteins that together
form surrogate light chain
 µ chain and surrogate light chain associated with Igα and Igβ signaling molecules to form preBCR complex (assembly of components of complex delivers signals that promote survival and
proliferation of cells on which pre-BCR expressed)
 This is first checkpoint in B cell development; selects and expands all pre-B cells that express
functional µ H chain; if µ chain protein not produced (e.g., faulty recombination of µ gene), cell
can’t be selected and it dies by apoptosis
o µ protein and pre-BCR complex signal 2 other processes
 Allelic exclusion – shuts off recombination of Ig H chain genes on second chromosome, so each
B cell can express Ig from only one of the parental alleles; helps ensure that each cell can
express receptors of single specificity
 Second signal triggers recombination at Ig L chain locus, first κ and then λ
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Whichever functional L chain produced associates with µ chain to form complete
membrane-associated IgM antigen receptor, which delivers signals that promote
survival and proliferation, preserving and expanding cells that express complete antigen
receptors (2nd checkpoint during maturation)
o Signals from antigen receptor shut off production of recombinase enzyme and further recombination at
unrecombined L chain loci; as result, each B cell produces either one κ or λ L chain from one of the
inherited parental alleles
 Presence of 2 sets of L chain genes increases chance of completing successful gene
recombination and receptor expression
o IgM-expressing B lymphocyte is immature B cell; further maturation may occur in bone marrow or
spleen
o Final maturation step involves coexpression of IgD with IgM, which occurs because recombined VDJ H
chain RNA may be spliced onto Cµ RNA or Cδ RNA, giving rise to µ or δ mRNA
o Ability of B cells to respond to antigens develops together with coexpression of IgM and IgD
 IgM+IgD+ cell is mature B cell; able to respond to antigen in peripheral lymphoid tissues
o B cell repertoire shaped by negative selection – if immature B cell binds antigen in bone marrow with
high affinity, further maturation stopped (either dies by apoptosis or may reactivate V(D)J recombinase
enyme, generate second L chain, and change specificity of antigen receptor (receptor editing))
 Antigens most commonly found in bone marrow self antigens that are abundantly expressed,
such as blood proteins, and membrane molecules common to all cells
 Negative selection eliminates potentially dangerous cells that can recognize and react against
ubiquitous self antigens
o Process of Ig gene recombination is random and can’t be inherently biased toward recognition of
microbes, but receptors produced able to recognize antigens of large number and variety of microbes
that immune system must defend against
 Repertoire of B lymphocytes generated randomly, selected positively for expression of intact
receptors, and selected negatively against strong recognition of self antigens
 What is left is collection of mature B cells able to recognize all microbial antigens that may be
encountered
o Most mature B cells are follicular cells
o Marginal zone B cells develop from same progenitors (pro-B cells) as follicular B cells
o B-1 cells may develop earlier and from different precursors
Process of T lymphocyte maturation has unique features largely related to specificity of different subsets of T
cells for peptides displayed by different classes of MHC molecules
o T cell progenitors migrate from bone marrow to thymus, where entire process of maturation occurs
o Most immature progenitors are pro-T cells (double-negative T cells), which expand in number, mainly
under influence of IL-7 produced in thymus
o Some progeny of double-negative cells undergo TCR β gene recombination, mediated by V(D)J
recombinase (γδ T cells undergo similar recombination involving TCR γ and δ loci, but are distinct
lineage)
o If successful VDJ recombination takes place on one chromosome and β chain protein synthesized, it is
expressed on surface in association with invariant protein (pre-Tα) to form pre-TCR complex of pre-T
cells; if complete β chain not produced in pro-T cell, that cell dies
o Pre-TCR complex delivers intracellular signals in response to assembly or recognition of some unknown
ligand; signals promote survival, proliferation, and TCR α gene recombination, and inhibit VDJ
recombination in 2nd TCR β chain locus (allelic exclusion)
o Failure to express αchain and complete TCR results in death of cell
o Surviving cells express both CD4 and CD8 coreceptors (double-positive T cells or double-positive
thymocytes)
o Different clones of double-positive T cells express different αβ TCRs; if TCR of T cell recognizes MHC
molecule in thymus, which has to be self MHC molecule displaying self peptide, that T cell selected to
survive; T cells that don’t recognize MHC molecule in thymus die by apoptosis (positive selection)
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During positive selection, T cells whose TCRs recognize class I MHC-peptide complexes preserve
expression of CD8 and lose expression of CD4; if T cell recognizes class II MHC-peptide complexes, that
cell maintains expression of CD4 and loses expression of CD8 (these are single-positive T cells)
During positive selection, T cells become functionally segregated: CD8+ T cells capable of becoming CTLs
on activation, and CD4+ cells are helper cells
Immature, double-positive T cells whose receptors strongly recognize MHC-peptide complexes in
thymus undergo apoptosis (negative selection); serves to eliminate T lymphocytes that could react in
harmful way against self proteins expressed in thymus
Both positive selection and negative selection mediated by recognition of same set of self MHC-self
peptide complexes in thymus (thymus can only contain self MHC molecules and self peptides; microbial
peptides concentrated in peripheral lymphoid tissues and tend not to enter thymus)
 If antigen receptor of T cell recognizes self MHC-self peptide complex with low avidity, result is
positive selection, whereas high-avidity recognition leads to negative selection
 High-avidity recognition happens if self peptide present in thymus and if T cell expresses TCR
that has high affinity for that self peptide; leads to autoimmunity
 Low-avidity recognition of self unlikely to be harmful
Ability to recognize foreign antigens relies on chance: T cells that weakly recognize self antigens in
thymus may strongly recognize and respond to foreign microbial antigens in periphery