Antigen Capture and Presentation to Lymphocytes
 Adaptive immune responses initiated when antigen receptors of lymphocytes recognize antigens
 Antigen receptors of B lymphocytes (membrane-bound antibodies) recognize wide variety of macromolecules
(proteins, polysaccharides, lipids, and nucleic acids) as well as small chemicals in soluble or cell surfaceassociated form
 Most T lymphocytes can see only peptide fragments of protein antigens, and can do so only when peptides
presented by specialized peptide display molecules on host cells
o May be generated only against protein antigens of microbes associated with host cells
 Immune responses by antigens must overcome several barriers
o Low frequency of naïve lymphocytes specific for any one antigen (may be less than 1 in 105 lymphocytes)
o Different kinds of microbes need to be combated by different types of adaptive immune responses;
immune system has to react in different ways to the same microbe in different stages of its life
 B cells capable of recognizing many more different types of molecules than T cells without need for specialized
processing and often not on surface of host cells
Antigens Recognized by T Lymphocytes
 Majority of T lymphocytes recognize peptide antigens bound to and displayed by MHC molecules of APCs
o MHC is genetic locus whose principal products function as peptide display molecules of immune system
o Requirement of T cells to see peptides only on MHC molecules called MHC restriction
 Each T cell has dual specificity: TCR recognizes some residues of peptide antigen and recognizes residues of MHC
molecule displaying that peptide
o Relatively small subpopulations of T cells that recognize lipid and other non-peptide antigens displayed
by non-polymorphic class I MHC-like molecules
 Naïve T lymphocytes need to see protein antigens presented by dendritic cells (most effective “professional”
APC) to initiate clonal expansion and effector cell differentiation
o Differentiated effector T cells need to see antigens, presented by various APCs , to activate effector
functions of T cells in humoral and cell-mediated immune responses
Capture of Protein Antigens by Antigen-Presenting Cells
 Protein antigens of microbes that enter body captured mainly by dendritic cells and concentrated in peripheral
lymphoid organs, where immune responses initiated
 Epithelial and subepithelial tissues contain network of dendritic cells; same cells present in T-cell-rich areas of
peripheral lymphoid organs and, in smaller numbers, in most other organs
o In skin, epidermal dendritic cells called Langerhans cells; immature because they are inefficient at
stimulating T lymphocytes; express membrane receptors that bind microbes, such as receptors for
terminal mannose residues on glycoproteins
 Dendritic cells use receptors to capture and endocytose microbial antigens
 Some soluble microbial antigens enter dendritic cells by pinocytosis
 Microbes stimulate innate immune reactions by binding to TLRs and other sensors of microbes in dendritic cells,
as well as in epithelial cells and resident macrophages in tissue
o Results in production of inflammatory cytokines such as TNF and IL-1
o Combination of TLR signaling and cytokines activates dendritic cells
 Activated dendritic cells lose adhesiveness for epithelia and express surface receptor CCR7 (specific for
chemokines) produced in T cell zones of lymph nodes
o Chemokines direct dendritic cells to exit epithelium and migrate through lymphatic vessels to lymph
nodes draining epithelium
o During process of migration, dendritic cells mature from cells designed to capture antigens into APCs
capable of stimulating T lymphocytes; maturation reflected in increased synthesis and stable expression
of MHC molecules, which display antigen to T cells, and of other molecules (costimulators) required for
full T cell responses
 Soluble antigens in lymph picked up by dendritic cells that reside in lymph nodes, and blood-borne antigens
handled in essentially same way by dendritic cells in spleen
 Net results is protein antigens of microbes that enter body transported to and concentrated in regions of lymph
nodes where antigens most likely to encounter T lymphocytes
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Naïve T lymphocytes continuously recirculate through lymph nodes and express CCR7 (promotes entry
into T cell zones of lymph nodes)
 Dendritic cells bearing captured antigen and naïve T cells poised to recognize antigens come together in lymph
nodes
 Very efficient – antigens introduced anywhere in body get T cell response within 12-18 hours
 Dendritic cells are principal inducers of responses because they are most potent APCs for activating naïve T
lymphocytes; can also influence nature of response
o Various subsets of dendritic cells can direct differentiation of naïve CD4+ T cells into distinct populations
that function in defense against different types of microbes
 Macrophages phagocytose microbes and display antigens of microbes to effector T cells, which activate
macropahges to kill the microbes
 B lymphocytes ingest protein antigens and display them to helper T cells within lymphoid tissues; important for
development of humoral immune responses
 All nucleated cells can present antigens derived from microbes in cytoplasm to CTLs
 Dendritic cells involved in initiating responses of CD8+ T lymphocytes to antigens of intracellular microbes
o Dendritic cells ingest infected cells and display antigens present in infected cells for recognition by CD8+
T lymphocytes (cross-presentation or cross-priming)
o Dendritic cells that ingest infected cells may also present microbial antigens to CD4+ helper T
lymphocytes
o Both classes of T lymphocytes specific for same microbe activated close to one another; important for
antigen-stimulated differentiation of naïve CD8+ T cells to effector CTLs (often requires help of CD4+)
o Once CD8+ T cells differentiate into CTLs, they kill infected host cells without need for dendritic cells or
signals other than recognition of antigen
Structure and Function of Major Histocompatability Complex Molecules
 MHC molecules – membrane proteins on APCs that display peptide antigens for recognition by T lymphocytes
o MHC is principal determinant of acceptance or rejection of tissue grafts exchanged between individuals
o Physiologic function of MHC molecules is to display peptides derived from protein antigens to antigenspecific T lymphocytes
 Human MHC proteins called human leukocyte antigens (HLAs)
 In all species, MHC locus contains 2 sets of highly polymorphic genes (class I and class II MHC genes)
o MHC locus also contains many non-polymorphic genes; some of which code for proteins involved in
antigen presentation, and others code for proteins whose function not known
 Class I and class II MHC molecules are membrane proteins that each contain peptide-binding cleft at aminoterminal end; classes differ in subunit composition, but are very similar in overall structure
o Each class I molecule consists of α chain noncovalently attached to protein (β2-microglobulin encoded by
gene outside MHC)
 Amino-terminal α1 and α2 domains form peptide-binding cleft (groove)
 Floor of peptide-binding cleft is region that binds peptides for display to T lymphocytes, and
sides and tops of cleft are regions contacted by T cell receptor (which contacts part of displayed
peptide as well)
 Polymorphic residues of class I molecules (amino acids that differ among different individuals’
MHC molecules) located in α1 and α2 domains of α chain
 Some polymorphic residues contribute to variations in floor of peptide-binding cleft and
thus in ability of different MHC molecules to bind peptides
 Others contribute to variations in tops of clefts and thus influence recognition by T cells
 α3 domain invariant and contains binding site for T cell co-receptor CD8
 T cell activation requires recognition of MHC-associated peptide antigens by TCR and
simultaneous recognition of MHC molecule by co-receptor
 CD8+ T cells can only respond to peptides displayed by class I MHC molecules
o Each class II MHC molecule consists of α chain and β chain
 Amino-terminal regions of both chains (α1 and β1 domains) contain polymorphic residues and
form cleft
 Non-polymorphic β2 domain contains binding site for T cell co-receptor CD4
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Because CD4 binds to class II MHC molecules, CD4+ T cells can only respond to peptides
presented by class II MHC molecules
MHC genes codominantly expressed
o 3 polymorphic class I genes (HLA-A, HLA-B, and HLA-C) and each person inherits one of these from each
parent; any cell can express 6 different class I molecules
o In class II locus, every individual inherits one pair of HLA-DP genes (DPA1 and DPB1, encoding α and β
chains), one pair of HLA-DQ genes (DQA1 and DQB1, encoding α and β chains), one HLA-DRα gene
(DRA1), and 1-2 HLA-DRβ genes (DRB1 and DRB3, -4 or -5)
 Heterozygous individual can inherit 6 or 8 class II MHC alleles, 3 or 4 from each parent (one set
each of DP and DQ, and 1-2 of DR)
 Because of extra DRβ genes, and because some DQα molecules encoded on one chromosome
can associate with DQβ molecules encoded from other chromosome, total number of expressed
class II molecules may be considerably more than 6
o Set of MHC alleles present on each chromosome is MHC haplotype
o Each HLA allele has numerical designation (HLA-A2, HLA-B5, HLA-DR3 for example could be one person’s
HLA haplotype)
o All heterozygous individuals have 2 HLA haplotypes, one from each chromosome
MHC genes highly polymorphic; many different alleles present among different individuals in population
o Because polymorphic residues determine which peptides presented by which MHC molecules, existence
of multiple alleles ensures that there are always some members of population that will be able to
present any particular microbial protein antigen
o Evolution of MHC polymorphism ensures a population will be able to deal with diversity of microbes and
won’t succumb to newly encountered or mutated microbe because at least some individuals will be able
to mount effective immune responses to peptide antigens of these microbes
o Variations in MHC molecules (accounting for polymorphism) result from inheritance of distinct DNA
sequences and not induced by gene recombination
Class I molecules expressed on all nucleated cells, but class II molecules expressed mainly on dendritic cells,
macrophages, and B lymphocytes
o Class II molecules also expressed on thymic epithelial cells and endothelial cells and can be induced on
other cell types by cytokine interferon-γ
Peptide-binding clefts of MHC molecules bind peptides derived from protein antigens and display peptides for
recognition by T cells
o Pockets in floors of peptide-binding clefts of most MHC molecules; side chains of amino acids in peptide
antigens fit into MHC pockets and anchor peptides in cleft of MHC molecule
o Peptides anchored in cleft by side chains (anchor residues) contain some residues that bow upward and
are recognized by antigen receptors of T cells
Each MHC molecule can present only one peptide at a time because there is only one cleft, but each MHC
molecule capable of presenting many different peptides
o So long as pockets of MHC molecule can accommodate anchor residues of peptide, that peptide can be
displayed by MHC molecule; only one or two residues in peptide have to fit into MHC molecule’s cleft
o MHC molecules have broad specificity for peptide binding; essential for antigen display function of MHC
molecules because each individual has only a few different MHC molecules that must be able to present
vast number and variety of antigens
MHC molecules bind only peptides and not other types of antigens; this is why MHC-restricted CD4+ and CD8+
cells can recognize and respond to only protein antigens
MHC molecules acquire peptide cargo during biosynthesis and assembly inside cells
o MHC molecules display peptides derived from microbes inside host cells, and this is why MHC-restricted
T cells recognize cell-associated microbes and are mediators of immunity to intracellular microbes
o Class I molecules acquire peptides from cytosolic proteins, and class II molecules from proteins in
intracellular vesicles
o Only peptide-loaded MHC molecules stably expressed on cell surfaces because MHC molecules must
assemble both their chains and bound peptides to achieve stable structure, and empty molecules
degraded inside cells
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Requirement for peptide binding ensures that only useful MHC molecules expressed on cell
surfaces for recognition by T cells
o Once peptides bind to MHC molecules and are displayed on cell surface, they stay bound for long time
(up to days); slow off-rate ensures that after MHC molecule acquired on peptide, it will display peptide
long enough to maximize chance particular T cell will find peptide it can recognize and initiate response
 In each individual, MHC molecules can display peptides derived from foreign (microbial) proteins as well as
peptides from that individual’s own proteins
o New MHC molecules constantly being synthesized, ready to accept peptides, and are adept at capturing
any peptides present in cells
o Single T cell may need to see peptide displayed by only as few as 0.1%-1% of 105 MHC molecules on
surface of APC, so even rare MHC molecules displaying peptide enough to initiate immune response
o T cells specific for self antigens either killed or inactivated
o MHC molecules presenting self peptides is key to normal surveillance function of T cells; T cells
constantly patrolling body looking at MHC-associated peptides, not reacting to peptides derived from
self proteins but able to respond to rare microbial peptides
 MHC molecules capable of displaying peptides but not intact microbial protein antigens
Processing and Presentation of Protein Antigens
 Extracellular proteins internalized by specialized APCs (dendritic cells, macrophages, and B cells) processed in
vesicles and displayed by class II MHC molecules, whereas proteins in cytosol of any nucleated cell processed in
cytoplasm and displayed by class I MHC molecules
o 2 pathways of antigen processing involve different cellular organelles and proteins
o Designed to sample all proteins present in extracellular and intracellular environments
o Segregation of antigen-processing pathways also ensures different classes of T lymphocytes recognize
antigens from different compartments
 APCs may internalize extracellular microbes or microbial proteins by binding to surface receptors specific for
microbial products or to receptors that recognize antibodies or products of complement activation that are
attached to microbes
o B lymphocytes internalize proteins that specifically bind to cells’ antigen receptors
o Some APCs may phagocytose microbes or pinocytose proteins without any specific recognition event
o After internalization into APCs by any pathway, microbial proteins enter acidic endosomes
(phagosomes), which may fuse with lysosomes, where the proteins are broken down by proteolytic
enzymes, generating many peptides of varying lengths and sequences
o APCs constantly synthesize class II MHC molecules in ER; each newly synthesized class II molecule carries
attached protein (invariant chain) which contains sequence (class II invariant chain peptide or CLIP) that
binds tightly to peptide-binding cleft of class II molecule
 Thus, cleft of newly synthesized class II molecule is occupied, and inaccessible class II molecule
begins transport to cell surface in exocytic vesicle, which then fuses with endosomal vesicle
containing peptides derived from ingested extracellular proteins
 Same endosomal vesicle contains class II-like protein (DM) that functions to remove CLIP from
class II MHC molecule; after removal of CLIP, cleft of class II molecule becomes available to
accept peptides
 If class II MHC molecule able to bind one of peptides generated from ingested proteins, complex
becomes stable and is delivered to cell surface
 If MHC molecule doesn’t find peptide it can bind, empty molecule is unstable and is degraded by
proteases in endosomes
o One protein antigen may give rise to many peptides, only a few of which (1-2) may bind to MHC
molecules present in cells; only these peptides derived from intact antigen stimulate immune responses
in individual; such peptides are immunodominant epitopes of antigen
 Antigenic proteins may be produced in cytoplasm from viruses living inside infected cells, from some
phagocytosed microbes that may leak from, or be transported out of, vesicles into cytoplasm, and from mutated
or altered host genes (as in tumors)
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Proteins targeted for destruction by proteolysis; proteins unfolded, covalently tagged with multiple
copies of ubiquitin, and threaded through proteolytic organelle (proteasome), where unfolded proteins
degraded by enzymes
o Some classes of proteasomes efficiently cleave cytosolic proteins into peptides with size and sequence
properties typical of class I MHC-binding peptides
o Transporter associated with antigen processing (TAP) located in ER membrane; binds peptides from
cytoplasm and actively pumps them across ER membrane into interior of ER (reverse of normal direction
of protein traffic)
o Newly synthesized class I MHC molecules loosely attached to interior face of TAP molecule; thus as
peptides enter ER, they can be captured by class I molecules
o If class I molecule finds peptide with right fit, complex is stabilized and transported to cell surface
o During transport, class I MHC-peptide complex may intersect endosome, but it isn’t available to bind
peptides, and being stable, it’s able to resist proteolysis by endosomal proteases
o If class I MHC molecule doesn’t find peptide in ER, molecule becomes unstable and is degraded by
proteases
Viruses can remove newly synthesized MHC molecules from ER, inhibiting transcription of MHC genes, and block
peptide transport by TAP
o By inhibiting class I MHC pathway, viruses reduce presentation of their antigens to CD8+ T cells and are
thus able to evade adaptive immune system
o Viral evasion strategies partly counterbalanced by ability of NK cells of innate immune system to
recognize and kill virally infected cells that have lost class I MHC expression
Restriction of T cell recognition to MHC-associated peptides ensures T cells see and respond only to cellassociated antigens; partly because MHC molecules are cell membrane proteins and partly because peptide
loading and subsequent expression of MHC molecules dependent on intracellular biosynthetic and assembly
steps; MHC molecules can be loaded with peptides only inside cells, where antigens of phagocytosed and
intracellular pathogens present, so T lymphocytes can recognize antigens of only phagocytosed and intracellular
microbes
By segregating class I and class II pathways of antigen processing, immune system able to respond to
extracellular and intracellular microbes in different ways best able to combat these microbes
o Extracellular microbes captured by APCs, including B lymphocytes and macrophages, and are presented
by class II molecules
 Because of specificity of CD4 for class II, class II-associated peptides recognized by CD4+ T
lymphocytes, which function as helper cells
 Helper T cells help B lymphocytes produce antibodies and phagocytes destroy ingested
microbes, thereby activating 2 effector mechanisms best able to eliminate extracellular and
ingested microbes
o Cytosolic antigens processed and displayed by class I MHC molecules, which are expressed on all
nucleated cells because all nucleated cells can be infected with some viruses
 Class I-associated peptides recognized by CD8+ T lymphocytes, which differentiate into CTLs,
which kill the infected cells and eradicate the infection (most effective for cytoplasmic microbes)
Function of MHC-associated antigen processing pathway important because T cells themselves can’t distinguish
between extracellular and intracellular microbes
o During virus’s extracellular life, it is combated by antibodies and phagocytes activated by helper T cells,
but once virus in cytoplasm of cells, it can be eradicated only by CTL-mediated killing of infected cells
o Segregation of class I and class II antigen presentation pathways ensures correct, specialized immune
response against microbes in different locations
APCs also express second signals for T cell activation; antigen is necessary signal 1, and signal 2 provided by
microbes or APCs reacting to microbes
o Different types of microbial products and innate immune responses may activate APCs to express
molecules that are second signals for lymphocyte activation
o Many bacteria produce LPS (endotoxin); when bacteria captured by APCs for presentation of their
protein antigens, LPS acts on APCs via TLR and stimulates expression of costimulators and secretion of
cytokines
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Costimulators and cytokines act in concert with antigen recognition by T cell to stimulate proliferation
and differentiation of T cells
Antigens Recognized by B Cells and Other Lymphocytes
 B lymphocytes use membrane-bound antibodies to recognize wide variety of antigens, including proteins,
polysaccharides, lipids, and small chemicals
o Antigens may be expressed on microbial surfaces or in soluble form
 B cells differentiate in response to antigen and other signals into plasma cells
o Secreted antibodies enter circulation and mucosal fluids and bind to antigens, leading to their
neutralization and elimination
 Antigen receptors of B cells and antibodies secreted usually recognize antigens in native conformation, without
any requirement for antigen processing or display by specialized system
 Macrophages in lymphatic sinuses may capture antigens that enter lymph nodes and present antigens, in intact
(unprocessed) form, to B lymphocytes in follicles
 B cell-rich lymphoid follicles of lymph nodes and spleen contain population of cells called follicular dendritic cells
(FDCs) whose function is to display antigens to activated B cells
o Antigens displayed by FDCs coated with antibodies or by complement byproducts such as C3b and C3d
o FDCs use receptors for one end of antibody molecules (Fc receptors) to bind antigen-antibody
complexes, and receptors for complement proteins, to bind antigens with these proteins attached
o Antigens seen by specific B lymphocytes during humoral immune responses, and they function to select
B cells that bind antigens with high affinity
 Other smaller populations of T cells that recognize different types of antigens
o NK-T cells specific for lipids displayed by class I-like CD1 molecules
o γδ T cells recognize wide variety of molecules, some displayed by class I-like molecules and others
apparently requiring no specific processing or display