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THE IMMUNE SYSTEM

Chapter One
Elements of the Immune System and their Roles in Defense
1.0

1.1.

1.2.

1.3.

Elements of the immune system and their roles in defense


Immunology
o study of the physiological mechanisms that humans use to defend their bodies from
invasion by all sorts of other organisms
Immune system
o cells collectively dedicated to the defense of the human body
o crucial to human survival
o takes time to build up its strongest response to an invasion, time during which the invader
can multiply and cause disease
Immunity
o gives future protection from the disease
o the immune system must first battle the invader before immunity is provided
Immunization
o also vaccination
o a procedure where a disease may be prevented by prior exposure to the infectious agent
in a form that cannot cause disease
o provides opportunity for immune system to gain experience for protective response with
little risk to health
Numerous commensal microorganisms inhabit healthy human bodies
Main purpose of immune system: protect body from infectious disease
o Mostly from microorganisms smaller than a human cell
Human body is a vast resource-rich environment in which microbes can live, feed and reproduce
~1000 microbial species live in a healthy adult human gut
o commensals
o community is called microbiota
Animals have evolved with commensal microbes
o becoming more tolerant and dependent upon them
in humans, commensals enhance nutrition by processing digested food and
making several vitamins
also protect against disease since they prevent colonization of other dangerous
microbes
Escherichia coli produces antibacterial proteins called colicins
Pathogens are infectious organisms that cause disease
Pathogen
o any organism with potential to cause disease
o four kinds: bacteria viruses, fungi and internal parasites
o opportunistic pathogens
may not cause disease at first, but can cause one if the immune system is
weakened or if it reaches a suitable place for infection
Over time, relationship between pathogen and its human host changes, affecting severity
o evolution of special adaptations of the pathogens to invade, replicate and transmit
o human populations evolve a degree of built-in genetic resistance to common diseasecausing organisms and lifetime immunity to endemic diseases
The skin and mucosal surfaces form barriers against infection
Skin
o bodys first defense against infection
o forms tough, impenetrable barrier of epithelium protected by layers of keratinized cells
o epithelium
layers of cells that line outer surface and inner cavities of the body
o may be breached by physical damage that exposes soft tissues

1.4.

Mucosal surfaces
o also mucosae
o internal surfaces with tissues that are specialized for communication with their
environment
o more vulnerable to microbial invasion
o bathed in mucus
contains glycoproteins, proteoglycans and enzymes
protects epithelial cells from damage
limits infection
Antimicrobial substances
o secreted by all epithelial surfaces
o sebum by sebaceous glands
fatty acids and lactic acids that inhibit bacterial growth on skin
o antimicrobial peptides by all epithelia
perturbs membranes of microbes
o tears and saliva
lysozyme degrades cell walls
o acidic environments in stomach, vagina and skin
The innate immune response causes inflammation at sites of infection
Innate immune response
o controls the infections that are highly localized
o two parts
recognition of the presence of the pathogen
involves soluble proteins and cell-surface receptors
o binds to pathogen and its products or to human cells
o also binds to serum proteins that become altered in its presence
recruitment of destructive effector mechanisms
kill and eliminate pathogen
o provided by effector cells of various types
engulf bacteria, kill virus-infected cells or attack
protozoan parasites
o also provided by a battery of serum proteins called complement
helps effector cells by marking pathogens with molecular
flags
also attacks pathogens
o innate: qualities a person is born with
genetically programmed set of responses
o receptor proteins contribute to recognition of pathogens
ligands: peptides, proteins, glycoproteins, proteoglycans, peptidoglycan,
carbohydrates, glycolipids, phospholipids and nucleic acids
o infection
cells in damaged tissues send out soluble proteins called cytokines that interact
with other proteins to trigger innate immune response
cytokines induce local dilation of blood capillaries
induces gaps between cells of endothelium and makes it permeable for
blood plasma leakage onto connective tissue
expansion of fluid volume causes edema
swelling that puts pressure on nerve endings and causing pain
cytokines change adhesive properties of vascular endothelium
inviting white blood cells to attach to it
o inflammation
induced by the innate immune response in the infected tissue

1.5.

The adaptive immune response adds to an ongoing innate immune response


Some infections outrun the innate immune response
o calls on white blood cells called lymphocytes
Adaptive immune response
o organized around an ongoing infection
o adapts to nuances of the infecting pathogen
o adaptive immunity
provides a highly specialized defense
o effector mechanisms
differences from innate: the way in which lymphocytes recognize pathogens
innate: recognizes features shared by groups of pathogens
adaptive: recognize pathogens by using cell-surface receptors of just one
molecular type
o can be made specific for a particular pathogen by using only
those lymphocyte receptors that bind to the infecting pathogen
o such receptors are encoded by genes that are modified greatly
during lymphocyte development
o each lymphocyte is programmed to make one variant of the
basic receptor type
only lymphocytes bearing receptors that recognize the infecting pathogen
participate in the adaptive response effector cells specific for pathogen
clonal selection (proliferation) and clonal expansion (differentiation)
o some lymphocytes selected during an adaptive immune response persist in the body to
provide long-term immunological memory
memory cells allow subsequent encounters to elicit a stronger and faster
adaptive immune response
adaptive immunity provided by this is called acquired immunity or protective
immunity
o primary immune response
first time an adaptive immune response is made to a given pathogen

1.6.
1.7.

secondary immune response


second and subsequent times an adaptive immune response is made and when
immunological memory applies

Adaptive immunity is better understood than innate immunity


Immune system cells with different functions all derive from hematopoietic stem cells
Leukocytes
o white blood cells
o principal cells of the immune system
hematopoiesis
o developmental process that continually generates blood cells
o pluripotent hematopoietic stem cell
common progenitor wherein leukocytes, erythrocytes ad megakaryocytes derive
from
o hematopoietic cells
blood cell types + their progenitor cells
o anatomical sites
early embryo: yolk sac, fetal liver
third to seventh month of fetal life: spleen
birth: bone marrow
adults: specifically in the bone marrow of the skull, ribs, sternum, vertebral
column, pelvis,and femurs
o active throughout life
o self renewal
in which hematopoietic stem cells divide to give further hematopoietic stem cells
o mature stem cells commit to one of three cell lineages: erythroid, myeloid and
lymphoid
erythroid progenitor erythroid lineage of blood cells
oxygen-carrying erythrocytes
platelet-producing megakaryocytes
o giant cells that arise from fusion of multiple precursor cells
o with nuclei containing multiple sets of chromosomes
o permanent residents of bone marrow
o platelets
small packets of membrane-enclosed cytoplasm that
break off
initiate and participate in clotting reactions
myeloid progenitor myeloid lineage of cells
granulocytes
o also polymorphonuclear leukocytes
o have prominent cytoplasmic granules containing reactive
substances against microbes
o naming based on staining of cytoplasmic granules with
commonly used histological stains

neutrophil
captures, engulfs and kills microorganism
phagocytes
effector cells of innate immunity
can work in anaerobic conditions
short-lived and die at site of infection, forming pus
most abundant
o eosinophil
second most abundant
defends against helminth worms and other intestinal
parasites
granules bind with acidic stain eosin
o basophil
least abundant
regulates immune response to parasites
granules are acidic and binds with basic stains like
hematoxylin
monocytes
o leukocytes that circulate the blood
o bigger than granulocyte, with distinctive indented nucleus
o mobile progenitors of macrophages
travel in blood to tissues where they mature into
macrophages
macrophages
o large phagocyte
o large, irregularly shaped cells characterized by an extensive
cytoplasm
o general scavenger cells of the body
phagocytoses and disposes of dead cells and cell debris
o long-lived commanders that provide warning to other cells
o orchestrate local response to infection
o secrets cytokines to recruit neutrophils
dendritic cells
o resident in bodys tissues with distinctive star-shaped
morphology
o acts as cellular messengers that are sent to call up an adaptive
immune response when needed
mast cells
o resident in all connective tissues
o granules like basophil
o still no known progenitor
lymphoid progenitor lymphoid lineage of white blood cells
large granular lymphocytes
o also natural killer or NK cells
o effector cells of innate immunity
o important in the defense against viral infections
o enters infected tissues and kills virus-infected cells
o also releases cytokines
small lymphocytes
o responsible for adaptive immune response
o circulates in quiescent and immature form that is functionally
inactive

1.8.

1.9.

1.10.

Immunoglobulins and T-cell receptors are the diverse lymphocyte receptors of adaptive immunity
small lymphocytes comprise several sub-lineages distinguished by cell-surface receptors and
functions
o B lymphocytes
also B cells
cell-surface receptors are immunoglobulins, or Igs
plasma cells
effector B cells
secretes antibodies
o soluble forms of Igs
o T lymphocytes
also T cells
cell-surface receptors are T-cell receptors
o receptors are structurally similar and are products of genes that are cut, spliced and
modified during lymphocyte development
o each B cell expresses a single type of Ig and each T cell expresses a single type of T-cell
receptor
o antigen
any molecule, macromolecule, virus particle or cell that contains a structure
recognized and bound by an Ig or a T-cell receptor
On encountering their specific antigen, B cells and T cells differentiate into effector cells
B cells only differentiate into antibody-producing plasma cells upon encountering its antigen
Antigen-activated effector T-cells
o cytotoxic T cells
kill cells infected with viruses or bacteria
similar to NK cells (which is for innate)
o helper T cells
secretes cytokines
helps other cells of immune system become fully activated effector cells
regulatory T cells
controls activities of cytotoxic and other types of T cells
Antibodies bind to pathogens and cause their inactivation or destruction
antibodies
o secreted by plasma cells
o circulates in blood and can enter infected tissues

o
o

1.11.

1.12.

humoral immunity
reduces infection
binds tightly to a site on a pathogen so as to inhibit growth, replication or
interaction with human cells (neutralization)
o facilitates engulfment and destruction of extracullar microorganisms and toxins
(opsonization)
covers pathogen surfaces which only has a few different molecules at high
density
neutrophils and macrophages bind to antibody molecule thru cell-surface
receptors
Most lymphocytes are present in specialized lymphoid tissues
lymphoid tissues and organs
o where vast majority of lymphocytes are found
o includes: bone marrow, thymus, spleen, adenoids, tonsil, appendix, lymph nodes and
Peyers patches
less organized tissue found in lining of extensive mucosal surfaces of respiratory,
gastrointestinal and urogenital tracts
o types
primary or central lymphoid tissues
where lymphocytes develop and mature to stage
bone marrow, thymus
o B and T lymphocytes originate in bone marrow, B cells complete
maturation in bone marrow, T cells migrate to thymus for
maturation
secondary or peripheral lymphoid tissues
where mature lymphocytes become stimulated to respond to invading
pathogens
o lymph nodes
lie at junctions of an anastomosing network of lymphatic vessels called
lymphatics
originates in connective tissues throughout body and collects plasma that
leaks out of blood vessels
lymph is returned to blood through thoracic duct
empties to left subclavian vein in neck
mature B and T cells move through the body in both blood and lymph
lymphocyte recirculation
o pattern of movement between blood and lyph
o allows lymphocyte population to continually survey secondary lymphoid organs for
infection
Adaptive immunity is initiated in secondary lymphoid tissues
large initial dose of pathogens is usually necessary to cause disease
o must colonize tissue in sufficient numbers to overwhelm cells and molecules of innate
immunity
pathogens are carried from sites of infection to nearest lymph node by lymphatics
o draining lymph node
receives fluid collected at an infected site
o anatomy of lymph node provides meeting places for lymphocytes from blood to encounter
pathogens from infections
T cells to T-cell areas and B cells to B-cell areas known as lymphoid follicles
pathogens and pathogen-laden dendritic cells arrive thru afferent lymphatic
vessels
several unite at nodes and leave as a single efferent lymphatic vessel
o during infection, pathogen-specific B cells proliferate to form germinal center in each
follicle

1.13.

1.14.

small fraction of B and T cells bearing receptors that bind to pathogen are stimulated to
divide and differentiate into effector cells
dendritic cells active T cells
some T cells move to associated lymphoid follicle to activate B cells
o effector T cells and antibodies go to infected tissues where they work with innate
immunity counterparts to subdue infection
recovery involves clearance of infectious organisms from body and repair of damage caused by
infection and immune response

The spleen provides adaptive immunity to blood infections


spleen
o serves as filter for the blood
o removes damaged or senescent
red blood cells
o secondary lymphoid organ that
defends body against blood-borne
pathogens
pathogens in blood are
taken up by splenic
macrophages and
dendritic cells stimulate
B and T cells that arrive
from blood
o red pulp
where red blood cells are
monitored and removed
o white pulp
where white blood cells
gather to provide adaptive
immunity
o organization similar to lymph node
asplenia
o an immunodeficiency disease
in which inheritance of mutant gene/s leads to defects of immune systems
functions
Most secondary lymphoid tissue is associated with the gut
mucosal lymphoid tissues
o gut-associated lymphoid tissues (GALT)
include tonsils, adenoids, appendix, Peyers patches
o bronchial-associated lymphoid tissue
o mucosa-associated lymphoid tissue

o
o
o

pathogens arrive by direct delivery across mucosa, mediated by M cells


lymphocytes enter from the blood and leave via lymphatics if not activated
activated lymphocytes stay within mucosal system

Chapter Two
Innate Immunity: The Immediate Response to Infection
2.0

2.1

2.2

2.3

Innate immunity: the immediate response to infection


microorganisms are always with us
o almost all microbes we come into contact with are prevented from ever causing infection
three lines of defense
o innate immunity
o innate immune mechanisms that are mobilized once cells of the immune system have
detected presence of infection
o adaptive immunity
Physical barriers colonized by commensal microorganisms protect against infection by pathogens
outside surface of the body
o skin
o mucosal epithelia
lines digestive, respiratory and urogenital tracts
o provides effective physical and chemical barriers
commensal microorganisms
o colonizes skin and mucosal surfaces of healthy individuals
o deters pathogen invasion
o pathogen must compete successfully for nutrients and space in order to become an
infection
before birth, no commensal microbes
o at birth and contact with mothers vagina, commensals start to populate, which are from
family members, friends and pats
mucosal surfaces are better
o 1000 species of bacteris in human gut
o many commensals have developed symbiotic relationships with their hosts
microbiota
o population of commensal microorganisms
o its acquisition after birth influences and shapes immune system
Intracellular and extracellular pathogens require different types of immune response
extracellular infections
o caused by pathogens that live and replicate in spaces between human cells
accessible to soluble, secreted molecules of the immune systems
intracellular infections
o caused by pathogens that replicate inside human cells
host cells are attacked by the immune system
interferes with pathogens life cycle
exposes pathogens to soluble molecules of immune system
many pathogens have life cycles with both extracellular and intracellular forms
Complement is a system of plasma proteins that mark pathogens for destruction
defense mechanisms come to play when pathogen penetrates epithelial barrier
o complement system
system of soluble proteins made constitutively by the liver
coats surface of bacteria and extracellular virus particles to make the more easily
phagocytosed
most are proteolytic enzymes or proteases
inactive: zymogen
infection triggers complement activation
series or cascade of enzymatic reactions
each enzyme is highly specific for the complement component it cleaves
belong to large family of serine proteases
made up of >30 proteins
o complement component 3 (C3)

2.4

most important
people lacking C3 are prone to successive severe infections
activation cleavage of C3 small C3a fragment + large C3b fragment
C3b fragments
covalently bonds to pathogens surface
o complement fixation
C3b becomes firmly fixed to pathogen
tags the pathogen for destruction by phagocytes
organizes foration of protein complexes that damage pathogens
membrane
C3a fragment
acts as chemoattractant to recruit effector cells from blood to site of
infections
unusual feature due to high-energy thioester bond within glycoprotein
C3 enters bloodstream in inactive form
o thioester is sequestered and stabilized within hydrophobic
interior
when cleaved, bond is exposed and becomes subject to nucleophilic
attack by water molecules, or by amino and hydroxyl groups of proteins
and carbohydrates on pathogen surfaces
three pathways of complement activation
o all lead to C3 activation, deposition of C3b on pathogens surface and recruitment of
similar effector mechanisms
o alternative pathway of complement activation
start of infection
o lectin pathway of complement activation
part of innate immunity
induced by infection
requires some time before it gains strength
o classical pathway of complement activation
innate and adaptive immunity
requires binding of antibody or C-reactive protein to pathogens surface
At the start of an infection, complement activation proceeds by the alternative pathway

C3 is made in liver and secreted into blood in a conformation that sequesters the thioester bond
in an inactive form within the hydrophobic interior of protein
C3 spontaneously changes its conformation and exposes the bond, at a slow rate and without
being cleaved
thioester bond becomes active and makes a covalent bond that attaches C3 to another molecule
with an amino or hydroxyl bond
o usually involves a water molecule iC3 or C3(H2o)
o hydrolysis is first step in alternative pathway
environment near surface of certain pathogens ncreases rate at which C3 is hydrolyzed to give
iC3
o iC3 binds to inactive complement factor B cleavage by protease factor D small
fragment Ba (released) + large fragment Bb (has protease activity, remains bound to iC3)
o iC3bBb complex is a protease that specifically cleaves C3 into C3a and C3b fragments
C3 convertases
o proteases that cleave and activate C3
o e.g. iC3Bb
C3b also binds factor B and facilitates cleavage of factor B by factor D Ba + C3bBb complex
on microbial surface
C3bBb
o also a potent C3 convertase
o alternative C3 convertase

o
o

2.5

works right at the surface of pathogen


also binds to C3 and cleaves it into C3a and C3b
large proportion of C3b fragments it produces becomes fixed to pathogen
positive feedback process = progressive amplification of C3 cleavage

Regulatory proteins determine the extent and site of C3b deposition

complement control proteins


o regulates C3bBb
o two broad categories
one comprises plasma proteins that interact with C3b attach to human and
microbial cell surfaces
properdin (factor P) increases speed and power of complement
activation

2.6

2.7

binds to C3 convertase C3bBb on microbial surfaces and


preventing its degradation by proteases
factor H counteracts effect of properdin
o binds to C3b and facilitates its further cleavage to a form called
iC3b by factor I
o fragment iC3b cannot assemble a C3 convertase
o factor H + factor I = decreased number of C3 convertase on
pathogen surface
o has binding site for sialic acid
some bacteria have sialic acid to mimic human cells
other includes membrane proteins on human cells that prevent complement
fixation at the cell surface
decay-accelerating factor (DAF)
o binds to C3b component of the alternative C3 convertase
(C3bBb) dissociation and inactivation
membrane cofactor protein (MCP)
o binds to C3b component of C3bBb susceptible to cleavage
and inactivation by factor I (similar to factor H)
complement control protein (CCP) modules
o structurally similar modules in which complement control protein modules are built from
o ~60 amino acids, two -pleated sheets, stabilized by disulfide bonds
o proteins made up of CCP modules: regulators of complement activation
Phagocytosis by macrophages provides a first line of cellular defense against invading
microorganisms
macrophages
o first effector cells
o mature forms of circulating monocytes that have taken residence in tissues
o in liver: Kupffer cells
o long-lived phagocytic cells
o both innate and adaptive
o may be nonspecific; but receptors on its surface binds to specific ligands on microbial
surface, like the C3b fragments
complement receptor 1 (CR1) in macrophage
also serves to protect surface of cells on which it is expressed, like MCP
and factor H
makes C3b susceptible to cleavage by factor I
complement receptor 3 (CR3) and complement receptor 4 (CR4)
bind to iC3b fragments on icrobial surfaces
o iC3b has no convertase activity, but facilitates phagocytosis as
ligand for CR3 and CR4
members of surface glycoproteins, the integrins
o opsonization
coating of a pathogen with protein that facilitates phagocytosis
The terminal complement proteins lyse pathogens by forming membrane pores
C3b binds to alternative C3 convertase to produce an enzyme that acts on C5 component of
complement
o alternative C5 convertase
Bb + 2 C3b C3b2Bb
complement component C5
o structurally similar to C3; lacks thioeaster bond
o cleaved by C5 convertase smaller C5a fragment + larger C5b fragment
o C5b initiates formation of membrane-attack complex
makes holes in membranes of bacterial pathogens and eukaryotic cells
C6 and C7 bind to C5b
o exposes hydrophobic site in C7

2.8

2.9

inserts into lipid bilayer


C8 binds to C5b
o hydrophobic site in C8 is exposed
o inserts into membrane initiates polymerization of C9
C9
o forms transmembrane pores

regulated by soluble and surface-associated proteins


o soluble proteins prevent soluble complex of C5b with C6 and C7 from associating with
cell membranes
e.g. S protein, clusterin, factor J
o surface-associated proteins prevent recruitment of C9 by complex of C5b, C6, C7 and C8
e.g. homologous restriction factor (HRF) and CD59 (protectin)
with DAF, linked to plasma membrane by glycosylphosphatidylinositol
lipid tails
o impaired synthesis of tail paroxysmal nocturnal
hemoglobinuria
epsodes of complement-mediated lysis of RBC that lack
these proteins
Small peptides released during complement activation induce local inflammation
soluble C3a and C5a fragments are also active
o increases inflammation at the site of complement activation thru binding to receptors on
several cell types
o inflammation: major consequence of innate immune response to infection
o may induce anaphylactic shock; thus, anaphylatoxins
acute inflammatory reaction that occurs simultaneously throughout the body
contraction of smooth muscle and degranulation of mast cells and basophils
release of histamine increase capillary permeability
o C5a is more stable and potent than C3a
o phagocytes, endothelial cells, and mast cells have receptors for C5a and C3a
o C5a acts directly on neutrophils and monocytes
increase adherence to blood vessel walls
chemoattactant to direct their migration towards sites of complement fixation
increases phagocytic capacity ofcells
increased expression of CR1 and CR3
Several classes of plasma protein limit the spread of infection
coagulation system
o activated by damage to blood vessels
o cascade of plasma enzymes that forms blood clots
immobilizes microbes
decreases loss of blood an fluid
o platelets

2.10

releases variety of highly active substances from storage granules


i.e. prostaglandins, hydrolytic enzymes, growth factors
contributes to antimicrobial defense, wound healing, inflammation

kinin system
o includes vasoactive peptide bradykinin
causes vasodilation
increases supply of soluble and cellular materials of innate immunity to infected
site
o second enzymatic cascade of plasma proteins
o triggered by tissue damage
Streptococcus pyogenes
o acquires human protease plasmin on its surface
response: protease inhibitors (2-macroglobulins)
Antimicrobial peptides kill pathogens by perturbing their membranes
defensins
o 35-40 amino acids, rich in arginine residues
o two classes
-defensins
HD5 and HD6; also cryptdins
o secreted by Paneth cells
o specialized epithelial cells of small intestine at base of intervillial
crypts
at least six
-defensins
secreted by broad range of epithelial cells, esp. skin, respiratory tract,
and urogenital tract
at least four
HBD1, HBD2, HBD3, etc.

o
o
o

amphiphatic in character
allows them to penetrate microbial membranes and disrupt their integrity
secreted at mucosal surfaces
also produced by neutrophils
packaged in their granules
synthesized in inactive polypeptides that are cleaved to release active fragment

2.11

Pentraxins are plasma proteins of innate immunity that bind microorganisms and target them to
phagocytes
pentraxins
o family of cyclic multimeric proteins
o binds to surfaces of various pathogens and binds them for destruction
o 200-residue pentraxin domain at carboxy-terminal end of polypeptide
o two subfamilies
short pentraxins
by hepatocytes in liver
represented by serum amyloid P component (SAP)
long pentraxins
made by myeloid, endothelial, and epithelial cells
represented by PTX3
o function as bridging molecules
binds pathogens with one binding site on their molecular surface and use a
second to bind to human cell-surface receptors, like CD89 on phagocytes
when cell-surface receptors are cross-linked by pentraxin-coated pathogen, the
phagocyte is signaled to engulf and destroy to pathogen
o similar work to antibodies in adaptive immunity

Chapter Three
Innate Immunity: the Induced Response to Infection
3.0

3.1

3.2

Innate immunity: the induced response to infection


human body has several lines of defense
if immediate cellular and molecular defenses are successful in repelling the pathogen, it may not
even be perceived by the human
next lines of defense have to be induced by the infection
o takes about 4 days to be ordered up, marshaled and brought into service
o involves soluble and cellular receptors that detect presence of infection
o recruits leukocytes to do something about infection
o results to damaged integrity of blood vessels, flooding of the infected tissue with plasma
and rising temperatures
Cellular receptors of innate immunity distinguish non-self from self
innate immune system has an extensive array of receptors to cope with numerous microbial
species
o expressed by macrophages, NK cells, and other cells of innate immunity
o recognize structural features that distinguish microbial carbohydrates, lipids, proteins and
nucleic acids from mammalian counterparts
able to distinguish between self and non-self
enables induced innate immune response to be directed with precision at
invading organism
o each receptor can recognize multiple pathogenic species that share a particular cellsurface structure
o >100 different innate immune receptors
each immune cell can only express a subset
Tissue macropaghes carry a battery of phagocytic and signaling receptors
all tissues of the body contain resident macrophages
o ready to attack any encroaching microorganism
o array of receptors in macrophage surface, i.e. CR1 and CR2, that bind with invaders and
trigger phagocytosis
ligands are called phagocytic receptors (bacterial carbohydrates and lipids)
lectins
o cell-surface receptors and plasma proteins that recognize carbohydrates
o C-type lectins
subset of lectins that share carbohydrate recognition domain in which a calcium
ion coordinates the interaction
on macrophages: mannose receptor and dectin-1
ligand-binding domain known as C-type lectin domain (CTLD)
different C-type lectins differ in number of CTLDs (dectin-1 has 1,
mannose receptor has 8)
o R-type lectins
binds sulfated galactosamine residues
has structural similarity to ricin, thus R-type
o scavenger receptors
first observed to scavenge damaged molecules of low-density lipoprotein from
the blood
SR-A
recognizes lipopolysaccharide of Gram-negative bacteria, teichoic acids
of Gram-positive bacteria, and CpG-rich bacterial DNA
SR-B
recognizes lipopeptides
macrophage receptor with collagenous structure (MARCO)
recognizes Gram-negative and Gram-positive bacteria

3.3

has triple helices that form rigid rods and serve as binding sites for
microbial ligands
o allow-carbohydrate-recognition domains and other ligand binding
sites to be held at a distance from the cell surface

CR3 and CR4


recognizes a range of lipids on microbial surfaces
members of a protein family called integrins, which contributes to adhesive
interactions between cells
many ligands recognized are arrayed at high density on microbial surface
o enables numerous receptor molecules to bind ligands in a cooperative fashion
o secure capturing the pathogen receptor-mediated endocytosis
receptor-bound pathogen is surrounded by macrophage membrane and
internalized into a membrane-bound vesicle (endosome or phagosome)
phagosomes + lysosomes = phagolysosomes
acidic vesicles that are loaded with degradative enzymes and toxic
substances for destroying the pathogens
signaling receptors
o complements phagocytic eceptors
o recognizes the pathogen and instructs macrophage to recruit additional innate immunity
to the infected tissue
o e.g. Toll-like receptors
recognizes a variety of microbial ligands
TLR4 is expressed by macrophages and associates with CD14 to recognize LPS
Recognition of LPS by TLR4 induces changes in macrophage gene expression
Toll-like receptor proteins
o family of 10 genes encodes Toll-like receptor proteins TLR1-TLR10
o with variable extracellular domain for recognizing pathogens and a conserved
cytoplasmic signaling domain for conveying this information to the inside of cell
signaling domain: Toll inteleukin-1 receptor (TIR)
observed first in Toll-like receptors and receptors for interleukin-1
pathogen-recognition domain: leucine-rich repeat region (LRR)
variation in its number gives Toll-like receptors their specificities
o functional Toll-like receptors consist of two Toll-like receptor proteins (heterodimers or
homodimers)
TLR4 protein only associates with itself = homodimeric TLR4 receptor
when infected with Gram-negative bacteria, macrophages bind the bacteria with phagocytic
receptors
o LPS on macrophage binds to CD14 on macrophage surface, a co-receptor to TLR4
o LPS can also be picked by soluble LPS-binding protein in plasma and delivered to CD14
on macrophage surface
TLR4 dimer associates with a protein called MD2 and form complex with CD14
and LPS
causes cytoplasmic TIR domain of TLR4 to bind to a similar TIR domain of
MyD88 protein
adaptor protein that brings together two signaling components
a second domain of MyD88 binds to next member of the pathway, IRAK4
o causes IRAK4 to phosphorylate itself, dissociate from the
complex, and phosphorylate another adaptor protein, TRAF6
o additional factors lead to the activation of a kinase complex, inhibitor of B kinase (IKK)
activates transcription factor called nuclear factor B (NFB)
major roles in innate and adaptive immune responses
held in cytoplasm in an inactive complex with inhibitor of B (IB)
translocates to the nucleus after activation
initiates transcription of genes for cytokines, adhesion moecules and
other proteins necessary to establish inflammation

phosphorylates IB
releases NFB from inhibition

X-linked hypohidrotic ectodermal dysplasia and immunodeficiency or NEMO deficiency


o lacks one of the subunit of IKK; thus, impaired activation of NFB
o susceptible to bacterial infections
low capacity to activate macrophages
o gene for IKK or NFB essential modulator (NEMO) is on X-chromosome
thus, it is more common in boys than girls
o NFB also has functions in development
abnormalities in development of tissues derived from embryonic ectoderm
Activation of resident macrophages induces a state of inflammation at sites of infection

cytokines
o small soluble proteins used for communication between cells
o in case of infection, a cell may secrete a cytokine that binds to a specific cytokine
receptor on surface of another type of cell
induces intracellular signals that change behavior of second cell
o short-lived molecules that exert their influence over a short distance
when infection has been detected in tissue, resident macrophages become activated to recruit
other cells by secreting several cytokines
o inflammatory cytokines
creates a state of inflammation in infected tissue
causes infected tissue to swell and become red, painful and hot
e.g. interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), CXCL8 and
tumor necrosis factor- (TNF-)

3.4

monocytes and NK cells are also recruited to the infected tissue


NK cells proliferate and secrete cytokines and sustain macrophage
activation
monocytes recruited from blood mature into macrophages in the infected
tissue

3.5

for phagocytosis and disposal of immense number of neutrophils


that die in containing the infection
increase in temperature caused by IL-6
acts on local muscle and fat cells, causing them to adjust metabolism
and generate more heat

NOD-like receptors recognize bacterial degradation products in the cytoplasm


NOD-like receptors (NLRs)
o family of receptors in the cytoplasm that detects products derived from the intracellular
degredation of phagocytosed pathogens
o e.g. NOD1 and NOD2
recognize components of the bacterial cell wall
o NOD = nucleotide-binding oligomerization domain (NOD)
central domain
o carboxy-terminal side of the NOD domain
pathogen-recognition domain made up of LRR
NOD1
recognizes -glytamyl diaminopimelic acid
o degradation product of peptidoglycan of Gram-negative bacteria
NOD2
recognizes muramyl dipeptide
o degradation product derived from peptidoglycan of most bacteria
o amino-terminal side of NOD domain
caspase-recruitment domain (CARD)
structurally similar to a domain on some signaling proteins that is used to
recruit proteases called caspases
recruit signaling proteins that have the same type of CARD domain as
the NOD receptors

3.6

when NOD receptors recognizes its ligand, the CARD


domain of the receptor dimerizes with CARD domain of
a serine-threonine kinase called RIPK2
causes RIPK2 to phosphorylate and activate
kinase TAKI
phosphorylates and activates IKK
o IKK activates NFB, leading to
circular activation
Inflammasomes amplify the innate immune response by
increasing the production of IL-1
Toll-like receptor and IL-1 receptor share the same TIR
signaling domain
signaling through the IL-1 receptor is central to innate immunity
and inflammation and IL-1 is the most involved cytokines in
these processes
IL-1 is a family of 11 related cytokines
o IL-1
most studied because of its major contribution
to chronic inflammatory diseases
destructive potential
production and secretion are strictly regulated
synthesized on ribosomes in the cytoplasm
and kept as an inactive precursor protein of 35
kDa called proIL-1
generated by cleavage by protease caspase 1
supply of caspase 1 is highly regulated
synthesized as precursor procaspase
1
o needs to undergo proteolytic
cleavage to become active
enzymatic activity of caspase depends
on a cysteine residue in the active site
and their site of cleavage is next to an
aspartic acid residue
active form is 17 kDa
once a macrophage has been activated by infection, it can
accelerate the production of IL-1 by a feedback mechanism
o secreted IL-1 binds to IL-1 receptors on the
macrophage
increases the transcription and translation of
proIL-1
other signals cause the supply of caspase 1 to increase
release of ATP by the macrophage causes the ion channels to activate and lower
the intracellular concentration of potassium ions
ionc change assembly of inflammasome through oligomerization of
NOD-like receptor called NLRP3 or cryopyrin
o no CARD domain
o function of CARD provided by an adaptor protein that links
procaspase 1 to NLRP3
o procaspase 1 is bound by CARD of the adaptor protein and
becomes oligomerized as inflammasome assembles
o creates a high local concentration of procaspase 1 in
inflammasome autoproteolysis of procaspase 1 and
generation of active caspase 1
o caspase 1 cleaves proIL-1

maturation of IL-1 can occur in the cytoplasm or in specialized secretory granules


release: shedding of microvesicles, exocytosis of secretory lysosomes and by activity of
specific membrane transporters
Neutrophils are dedicated phagocytes and the first effector cells recruited to sites of infection
phagocytic cells are the principal means by which the immune system destroys invading
pathogens
neutrophils
o short-lived dedicated killers that circulate in the blood, awaiting a call from a macrophage
to center infected tissue
o first population of effector cells to be recruited
o type of granulocyte or polymorphonuclear leukocytes
o aka microphage
o most abundant white blood cells
o mature cells are kept in bone marrow for about 5 days
o arrival of neutrophils is the first in the inflammatory response
o specialized for working under anaerobic conditions
o forms creamy pus after death
pyogenic pus-forming
Inflammatory cytokines recruit neutrophils from the blood to the infected tissue
CXLCL8
o inflammatory cytokine made by pathogen-activated macrophages
o one of the family of about 40 chemoattractant cytokines called chemokines
serve to direct flow of leukocyte traffic throughout the body
o recruits neutrophils that are circulating in the blood and set them to work at site of
infection
adhesion molecules
interactions between these determine leukocyte movement between
blood and tissue
grouped according to four structural types
without infection neutrophils travel rapidly across capillaries and do not interact with endothelium
o with infection and inflammatory cytokines, blood vessels dilate and endothelial cells
express adhesion molecules called selectins
o blood flow decreases, and neutrophils make contact with vascular endothelium in postcapillary venules
x
interactions between selectin and sialyl-Lewis carbohydrate groups of
glycoproteins on neutrophil surface
surface rolling
TNF- induces vascular endothelium expression of ICAM-1 and ICAM-2
o adhesion molecules that are ligads for CR3 and LFA-1 on neutrophils
o interactions are initially weak, but strengthen when CXCL8 binds to CXCR1 and CXCR2
chemokine receptors on neutrophil
contain seven membrane-spanning helices that develop intracellular signals
through an associated GTP-binding protein
leads to conformation changes in CR3 and LFA-1 strengthens hold on
ICAM-1 and ICAM-2
immobilize neutrophil on vascular endothelium
extravasation
o neutrophil leaves blood, squeezing through gaps between neighboring endothelial
o involves LFA-1 and CR3, and CD31
o neutrophils pattern of gene expression changes to make it more active in phagocytosis
and killing pathogens; also secretes CXCL8 for more neutrophil recruitment
neutrophil reaches membrane by secreting elastase and other enzymes that degrade laminins
and collagens of basement membrane
o direction: gradient of CXCL8; neutrophils move up the gradient toward source of
chemokine
o
o

3.7

3.8

3.9

3.10

Neutrophils are potent killers of pathogens and are themselves programmed to die
neutrophils have a range of phagocytic receptors that recognize microbial products
o complement receptors that facilitate the phagocytosis of pathogens opsonized by
complement fixation
o range is greater than macrophages; more diverse microbicidal substances in granules
o devote more resources to storage and delivery of antimicrobial weaponry
neutrophil engulfs pathogen into a phagosome
o degradative enzymes are released upon pathogen, which are rapidly killed
o phagosomes have three types of preformed neutrophil granule
primary or azurophilic granules
marked by presence of enzyme myeloperoxidase
first made
packed with proteins and peptides that disrupt and digest microbes
o lysosome, defensins, myeloperoxidase, neutral proteases like
cathepsin G, elastase and proteinase 3
o bactericidal permeability-increasing protein that binds LPS and
kills Gram-negative batera
binded together by negatively charged matrix of sulfated proteoglycans
secondary or specific granules
marked by protein lactoferrin
o competes with pathogens for metal ions
also contain lysozyme and several membrane proteins, including
components of NADPH oxidase
o essential enzyme assembled in phagosome after fusion with
three types of granules
o produces superoxide radicals converted into hydrogen
peroxidase by superoxide dismutase
consumes hydrogen ions
raises pH to 7.8-8.0
activates antimicrobial peptides and proteins
after, pH becomes normal
neutrophils lysosomes fuse with phagosome to
form phagolysosome
tertiary or gelatinase granules
marked by enzyme gelatinase
o metal-containing protease that restricts growth of bacteria by
sequestering iron
lysosomes fuse with phagosomes
o lysosomes contribute acid hydrolases
respiratory burst
o transient increase in oxygen consumption
o produces several toxic oxygen species that diffuse out of cell and damage other host
cells
o to limit damage: accompanied by synthesis of enzyme that inactivate these molecules,
like catalase (hydrogen peroxide water + oxygen
mature neutrophil dies after it uses up its granule contents
o through apoptosis; then phagocytosed by macrophage
o through netosis
produces neutrophil extracellular traps (NETs)
traps and kills pathogens
nucleus swell and bursts chromatin dissolves
continues to trap and kill microbes even after death
through bactericidal defensins, several proteases and calprotectin
Inflammatory cytokines raise body temperature and activate the liver to make the acute-phase
response

3.11

fever
o

systemic effect of cytokines IL-1, IL-6 and TNF-


acts on temperature-control sites in hypothalamus and on muscle and fat cells
called pyrogens
o some pathogen products also raise bodys temperature
raised body temperature
o helps immune system fight infections
o adaptive immunity becomes more potent
o human cells become more resitant to effects of TNF-
acute-phase response
o another systemic effect of IL-6 (and IL-1 and TNF-)
o a change in the spectrum of soluble plasma proteins that are made and secreted by the
hepatocytes of the liver
o acute-phase proteins
proteins that change their concentration by at least 25%
e.g. C-reactive protein, and serum amyloid A protein
concentrations rise several hundred fold
CRP is used to diagnose for infection, inflammation and tissue damage
C-reactive protein (CRP)
o member of pentraxin family of proteins
o five identical subunits slab-like pentameric molecule with a hole in the middle
o binds to bacteria
target: phosphorylcholine
component of lipopolysaccharides in bacterial cell walls
o also binds to fungi, yeasts and parasites
o acts as opsonin
triggers classical pathway of complement fixation
o also binds to surface of phagocytes
can deliver pathogens to these cells
serum amyloid A protein
o small protein; 100 amino acids
o associates with high-density lipoprotein protein particles
o can interact with cell-surface receptors, like Toll-like receptors and CD36 scavenger
receptors activate cells produce inflammatory cytokines
The lectin pathway of complement activation is initiated by the mannose-binding lectin
mannose-binding lectin (MBL)
o C-type lectin
o binds to mannose-containing carbohydrates of bacteria, fungi, protozoa and viruses
o an acute-phase protein
o structure
resembles a bouquet of flowers in which each stalk is a triple helix made form
three identical polypeptides carbohydrate-recognition domain
15 to 18 potential sites for attachment
o triggers lectin pathway of complement activation
o serves as opsonin that facilitates uptake of bacteria by monocytes
o collectin
combine properties of collagen and lectin
lectin pathway of complement activation
o mannose-binding lectin as a complex with MBL-associated serine protease (MASP) 1
and 2
two molecules each of MASP-1 and MASP-2 associate with main stalk of MBL
o MBL complex bnds to mannose-containing macromolecule at pathogen surface 1
MASP-2 becomes active and cuts itself activated MASP-2 cuts second MASP-2
o substrates of activated MASP-2 proteases are C4 and C2 complement components
C4 is similar to C3 in structure

o
o
o

o
o

3.12

3.13

C2 is a serine protease zymogen similar to factor B


C4 reacts with activated MASP-2 large C4b fragment and small C4a fragment
cleavage exposes thioester bond of C4b C4b fixation to pathogen surface
C4a is an anaphylotoxin
C2 reacts with activated MASP-2 large C2a fragment and small inactive fragment
(C2b)
C2a binds to pathogen-bonded C4b
C4b + C2a = C4bC2a
classical C3 convertase
also a component of classical pathway
where lectin and classical pathway converges
C4bC2a binds and cleaves C3 C3b fragments that attach to pathogen surface
C3b + factor B C3bBb
alternative convertase
where lectin and classical pathway converge with alternative pathway

C-reactive protein triggers the classical pathway of complement activation


once C-reactive protein has bound to a bacterium it can interact with C1
C1 molecule
o bouquet of six flowers
18 C1q polypeptides + 2 C1r + 2 C1s
each stalk = triple helix of C1q molecules
C-reactive protein binds to C1q stalks 1 C1r to cut itself, the other C1r and the two C1s C1
becomes an active protease
active C1 cleaves C4 covalent attachment of C4b to pathogen surface
active C1 cleaves C2 C2a + C4b C4bC2a (classic C3 convertase)
complement activation
o at start of infection, mainly alternative pathway
o as inflammatory response develops and acute-phase proteins are produced lectin and
classical pathways provide increased complement activation
Toll-like receptors sense the presence of the four main groups of pathogenic microorganisms
other members of the family of Toll-like receptors rrecognize presence of viral, fungal and
parasitic infections

3.14

3.15

Genetic variation in Toll-like receptors is associated with resistance and susceptibility to disease
variation of TLRs is increased by the different alleles of these genes
o allotypes
proteins encoded by different alleles of the same gene
o genetic polymorphism
can influence function of a TLR
septic shock
o life-threatening condition
o occurs when a bacterial infection spreads to blood and becomes systemic
o macrophages throughout the body release TNF- widespread dilation of blood
vessels; massive leakage of fluid blood supply is perturbed and organ failure may
occur
Internal detection of viral infection induces cells to make an interferon response
human cells have sensor proteins in cytoplasm that can detect viral nucleic acids
response: production of cytokines
o type 1 interferons
interferes with propagation of infection
immediate effects
interfere with viral replication in infected cell
signal neighboring uninfected cells to resist viral infection
further effects
alert cells of immune system about the infection
make infected cells vulnerable to attack by killer lymphocytes
all cells can make this
RIG-1-like receptors (RLRs)
o detects viral RNAs in the cytoplasm
o e.g. retinoic-acid-inducible gene 1 (RIG-1), melanoma differentiation-associated protein 5
(MDA-5)
consists of an RNA helicase-like domain that recognizes RNA + two CARD
domains that interact with mitochondrial antiviral signaling protein (MAVS)
dimerization of RIG-like helicases, initiation of signaling pathway
phosphorylation of transcription factor interferon-response factor 3 (IRF3) IRF3

3.16

3.17

3.18

dimerizes and enters nucleus intiate transcription of IFN- genes (requires


NFB and AP-1)
family of genes on chromosome 9 encode type I interferons
IFN-, multiple forms of IFN-, and several additional types (IFN-, IFN, IFN-, IFN- and IFN-)
o IFN- acts in autocrine and paracrine fashion
interferon response
o when interferon binds to receptor, intracellular Jak1 and Tyk2 kinases associated with
receptor initiate reactions that change expression of a variety of human genes

Plasmacytoid dendritic cells are factories for making large quantities of type I interferons
plasmacytoid dendritic cells
o secrete up to 1000-fold more interferon than other cells
o professional interferon-producing cells
o combine phenotypic features of lymphocytes and myeloid dendritic cells
o present in blood and lymphoid tissues
o detects presence of viral infection by TLR7 and TLR9 signal through MyD88
activation of IRF7 translocation to nucleus mass production of type I inferferon
Natural killer cells are the main circulating lymphocytes that contribute to the innate immune
response
natural killer cells (NK cells)
o one of the main populations of lymphocytes that circulate the blood
o large and active lymphocytes that are more speedily induced to respond to infection,
cancer or other forms of stress
o two distinctive functions
kill cells infected virus
maintain or increase state of inflammation in the infected tissue
o like T cells in adaptive immunity
similar functions
many of the effector and cell-surface molecules of NK cells are also found in T
cells
o lymphocyte that expresses CD5 and lacks CD3
Two subpopulations of NK cells are differentially distributed in blood and tissues
NK cells are abundant in blood
o 5 25% of lymphocytes
o present in most other tissues
two subpopulations on the basis of abundance of CD56 glycoprotein
dim
o CD56 NK cells
more than 90% of blood NK cells
fewer CD56 molecules
greater capacity for killing cells

3.19

bright

CD56
NK cells
~80% of NK cells in tissues other than blood
more CD56 molecules
o uterine NK cells (uNK)
most abundant lymphocyte in womans uterus
fluctuates with menstrual cycle
essential to forming the placenta
cooperates with fetal trophoblast cells in enlarging maternal blood vessels
bright
mostly CD56
type
poor at killing cells
good at secreting growth factors and non-inflammatory cytokines
NK-cell cytotoxicity is activated at sites of virus infection
NK cells can only discharge their weapons at most intimate
contact with a target cells
o limits killing at one target cell at a time
decision to kill or not to kill is made by the sum of interactions
between many different types of NK cell receptors and their
cognate ligands on target cell
ground state for interaction of an NK cell with a target cell is
one of active inhibition based on inhibitory receptors
o inhibitory interaction must first be overcome
IFN- and IFN-
o first cytokines NK cells are exposed to
o effects:
induce NK-cell mitosis and proliferation
induce NK-cell differentiation into cytotoxic
effector cells that kill virus infected cells
in infected cell, NK cell makes initial transient contacts with
any tissue cell it encounters
o if cell is healthy, it moves on
o if cell is infected, firmer adhesion, with CR3 and lFA-1
adhesion molecule, occurs
o interacting sets of receptors and ligands concentrate
at localized regions at surfaces of the two cells
immunological synapse (NK-cell synapse)
o diverse activating and inhibitory receptors are
recruited into NK-cell synapse
if inhibitory signals predominate, target cell is
unharmed
if activating signals predominate, execution
proceeds
once committed to killing target cell, NK cells strengthens the adhesive interactions at synapse
and undergoes intracellular reorganization that facilitates a quick, precise, and clean delivery of
toxic cargo
various enzymes, proteins and proteoglycans disrupt plasma membrane
o selective proteolytic attack on target cell programmed cell death or apoptosis
after NK cell has detached, target cells DNA starts to fragment by own nucleases disrupted
nucleus and loss of membrane integrity and normal cell morphology cell shrinking by shedding
of vesicles
o remains are disposed by macrophage
NK cells express 3 of 10 Toll-like receptors
o TLR3 (double-stranded viral RNA) and TLR7 + TLR8 (single stranded RNA)
can recognize their ligands + further activated by signaling coming through TLR3,
TLR7 and TLR8 through a unique MyD88-dependent pathway IRF7
transcription factor production of IFN- and IFN-

3.20

3.21

pathway uses adaptor molecule, Toll-receptor associated activator of interferon


(TRIF)
NK cells and macrophages activate each other at sites of infection
IL-12 directs activation and recruitment of NK cells
o formerly NK-cell stimulatory factor
o surface receptor for IL-12
consists of two polypeptides, IL-12R1 and IL-12R2
in the presence of IL-12, they are brought together generation of intracellular
signals that lead to NK cell activation
IL-15 is critical for activation, proliferation and survival of NK cells
o made by macrophages
o dependent upon contact between NK cell and macrophage
o disfavors differentiation of NK cells into cytotoxic effector cells
o favors differentiation into effector NK cells
produce cytokines and act on macrophages and other cells heightened state
of inflammation
interferon- (IFN-)
o type II interferon
o only one form
o NK cells are a principal source
o targets: macrophages
o interaction of IFN- with receptor on macrophage signals that further activate
macrophage more efficient in phagocytosis, destruction of pathogens and secreting
inflammatory cytokines
Interaction between dendritic cells and NK cells influence the immune response
myeloid dendritic cells are developmentally related to macrophages
o similarly found as resident cells in all tissues of body
o sentinels that sample environment to detect infection and use that information to
stimulate responses of lymphocytes against pathogen
o takes up pathogens and products and be infected with viruses and other intracellular
pathogens changes in proteins on surfaces monitored by array of cell receptors on
NK-cell surfaces
when NK cell detects pathogen-induced changes formation of synapse
between them expression of IL-15
when NK cells outnumber dendritic cells, NK cells become cytotoxic cells and kill dendritic cells
o when dendritic cells outnumber NK cells, the NK cells secrete cytokines that induce
dendritic cells to differentiate into a form that leaves the infected tissue and migrates in
lymph

Chapter Four
Antibody Structure and the Generation of B-Cell Diversity
4.0

Antibody structure and the generation of B-cell diversity


antibodies
o clears the body of extracellular pathogens and their toxins in adaptive immune response
o secreted for of B-cell receptor for antigen
o produced by effector B lymphocytes (or plasma cells)
o circulate as major part of plasma; are always present at mucosal surfaces
o can recognize all types of biological macromolecule
o binding to bacterium/virus particle can disable pathogen
o very variable proteins; specific
can only bind to only one antigen
o diverse in their antigen-binding specificities
o antibody repertoire
total number of different specific antibodies that can be made by an individual
immunoglobulins
o general term for cell-surface B-cell antigen receptors and secreted antibodies
o each immature B cell becomes committed to producing one specific immunoglobulin
o when antigen binds to receptor, B cell is stimulatd to proliferate into plasma cells
secretes large amounts of antibodies with same specificity
example fo clonal selection
agammaglobulinemia
o inability to make immunoglobulins
o renders people highly susceptible to certain types of infection
The structural basis of antibody diversity
antibodies
o function: to recognize and bind its corresponding antigen to deliver bound antigen to
other components of immune system
o binding and interaction are carried out by different parts of antibody molecule
one part is highly variable
contains the site of antigen binding and confers specificity on the
antibody
constant part
interacts with other immune-system componenets
o five isotypes
IgG, IgM, IgD, IgA, IgE
distinguished on the basis of structural differences in the constant part of the
molecule
confers distinctive effector functions on isotypes
4.1
Antibodies are composed of polypeptides with variable and constant regions
antibodies are glycoproteins that are built from a basic unit of four polypeptide chains
o consists of two identical heavy chains (H chains) and two identical, smaller light chains (L
chains)
o assembles into a structure that looks like letter Y
each arm of Y is made up of a complete light chain paired with the amino
terminal part of a heavy chain
linked by a disulfide bond
stem of Y consists of the paired carboxy-terminal portions of the two heavy
chains
o vary greatly in amino acid sequences
variable region or V region
variability is the basis for the great diversity of antigen-binding
specificities that antibodies site
antigen-binding site

4.2

o pair of variable regions


o one from a heavy and one from a light chain
o each Y-shaped antibody has two, one at the end of each arm
constant regions or C regions
limited variation in amino acid sequences between different antibodies
a relatively unstructured portion in the middle of the heavy chain forms a flexible hinge region
o can be cleaved by proteases to produce two types of antibody fragents
Fab
fragment antigen binding
binds antigen
Fc
fragment crystallizable
o it readily crystallizes
mediates effector functions of antibody molecule by binding to serum
proteins and cell-surface receptors
o hinge allows the two Fabs to adopt many different spatial orientations
differences in heavy-chan C regions define five many isotypes or classes of immunoglobulins
IgG, IgM, IgD, IgA, IgE
heavy chains: , , , ,
light chains: or
o each antibody contains either one, but not both at the same time

Immunoglobulin chains are folded into compact stale protein domains


its structure helps antibodies withstand relatively harsh conditions
o heavy and light chains each consist of a series of similar sequence motifs
100-110 amino acids long
folds into a compact and stable protein called an immunoglobulin domain
o variable domain (V domain)
VH in heavy chain and VL in the light chain
forms an antigen-binding site
o constant domains (C domains)
light chain: single C1 domain
heavy chain: depends on isotype
heavy chains of IgG, IgD, and IgA
o CH1, CH2, CH3
IgM and IgE have fourth C domain
o C H4
structure of single immunoglobulin domain
o two sheets held together by strong hydrophobic interactions between constituent amino
acid side chains
o stabilized by disulfide bonds between the two sheets
o adjacent strands within sheets are connected by loops of polypeptide chain
immunoglobulin-like domains
o domains similar to immunoglobulins found in many other proteins

o
o

4.3

4.4

common in cell surface and secreted proteins of the immune system


immunoglobulin superfamily

An antigen-binding site is formed from the hypervariable regions of heavy-chain V domain and a
light-chain V domain
difference in amino acid sequences are concentrated in hypervariable regions (HV)
o three hypervariable regions are found in each V domain
three of the loops exposed at the end of the domain farthest from constant region
also known as complementarity determining regions (CDR1, CDR2, CDR3)
provides a binding surface that is complementary to that of antigen
o flanked by much less variable framework regions
corresponds to strands and remaining loops
pairing of a heavy and a light chain in an antibody molecule brings together the hpervariable
loops from each V domain composite hypervariable surface
o forms antigen-binding site at tup of each Fab arm
Antigen-binding sites vary in shape and physical properties
antibodies are most effective against infection when they bind to exposed and accessible
molecules making up surface of a pathogen
o epitope
also antigenic determinant
part of antigen to which an antibody binds
usually either carbohydrate or protein
complex macromolecules will usually have several different epitopes
typically composed of a cluster of amino acids or a small part of a polysaccharide
chain
multivalent
when an antigen contains more than one epitope or has more than once
copy of same epitope
o antigen-binding sites are versatile
can accommodate epitopes with different range of structures
o epitopes of protein antigens two broad roups
linear epitopes
epitopes composed of several amino acids in a protein sequence
formed by accessible loops of antigen
discontinuous epitopes
formed by two or more parts of protein antigen that are separated in the
amino acid sequence
dependent upon 3D structure
o binding is based solely on noncovalent forces
antigen-binding sites of antibodies are rich in aromatic amino acids
can participate in many van der Waals and hydrophobic interactions
complemented by electrostatic interactions and hydrogen bonds
better general fit stronger bonds formed

small differences in shapes and chemical properties


effective antibodies are those that bind tightly to an antigen and do not let go
catalytic antibodies
some catalyze chemical reactions involving the antigen they bind
Monoclonal antibodies are produced from a clone of antibodyproducing cells
antibodies are useful reagents for detecting and quantifying a
particular antigen
o traditional method for making antibodies of desired
specificity is to immunize animals with antigen and prepare
anti-sera from blood
specificity and quality dependent upon purity of the
immunizing antigen preparation
o more modern method: B cells isolated from immunized
animals and immortalized with a tumor cells to form
hybridoma produces antibodies indefinitely
cells making antibodies of desired specificity are
identified and selected for further preparation
monoclonal antibodies
antibodies made from hybridoma cell line
are identical
replaced antisera is reagents in many
clinical assays since 1970s
used to identify numerous previously
unknown surface proteins of cells of the
immune system
flow cytometry
measured reactions of monoclonal
antibodies with different types of cells
used to analyze cell populations
inperipheral blood and detect perturbations
caused by disease
intravenous antibodies
Monoclonal antibodies are used as treatments for a variety of diseases
monoclonal antibodies are used in therapy and diagnosis
o first successful therapeutic application: mouse monoclonal antibody specific for CD3
antigen on human T cell
prevents rejection of transplanted kidneys showing early symptoms of T-cell
mediated graft rejection
could only be used once for each patient
patient makes antibodies against C regions of the mouse antibody
problem is reduced by using chimeric monoclonal antibodies
combines mouse V regions with human C regions
e.g. rituximab
o treat several diseases like non-Hodgkin B cell lymphoma
o specific for CD20, which is present on normal and malignant
human B cells and influences and influences B-cell proliferation
o targets normal and malignant B cells for destruction by NK cells
o despite temporary loss of normal B cells, treated patients
continue to make antibodies because plasma cells lack CD20
and are unaffected by rituximab
o normal populations are reconstituted within a year after
treatment
o humanizing mouse antibodies by genetic engineering
o

4.5

4.6

sequences encoding CDR loops are used re place corresponding CDR


sequences in human immunoglobulin gene
e.g. omalizumab
specific for human IgE used to treat cases of severe allergic asthma
prevents recruitment of mast cells, eosinophils and basophils to allergic
response
o fully human monoclonal antibodies
antibodies made by mice whose mouse Ig genes have been replaced wth human
counterparts
e.g. adalimumab
specific for inflammatory TNF-
used for treatment of rheumatoid arthritis, a conditions in which chronic
inflammation of the joints is perpetuated by TNF-
Generation of immunoglobulin diversity in B cells before encounter with antigen
immunoglobulin genes are organized differently from other genes
o number of different antibodies produced is virtually limitless
o genes are in fragmented form that cannot be expressed
o heavy chain and light-chain loci consists of families of gene segments, sequentially
arrayed along chromosome
with each set containing alternative versions of parts of the immunoglobulin V
region
o inherited through the germline germline form or germline configuration
o for expression: gene segments must be rearranged (only in developing B cells) heavy
and light chains are produced Ig appears at B-cell surface
4.7
The DNA sequence encoding a V region is assembled from two or three gene segments
immunoglobulin genes are found at three chromosomal locations
o heavy-chain locus on chromosome 14, light-chain locus on chromosome 2 and lightchain locus on chromosome 22
o different gene segments encode the leader peptide (L), the V region (V), and the constant
region (C) of heavy and light chains
C genes gene segments encoding C regions
o gene segments encoding leader peptides and C regions consist of exons and introns and
are ready to be transcribed
o V regions are encoded by two (VL) or three (VH) gene segments that must be selected
from arrays of similar gene segments
two types of gene segments that encode light-chain V region
variable (V) gene segment
joining (J) gene segment
additional heavy-chain locus
diversity (D) gene segment)
o lies between arrays of V and J gene segments
V region of a light chain is encoded by combination of one V and one J
segments, whereas C region is encoded by single C gene segment
difference between two segments are in sequences that encode CDR1
and CDR2
CD3 is determined by sequence diversity introduced at the junction
between V and J segments
either or light-chain loci gives rise to functional gene
V region of a heavy chain is encoded by the combination of one V, one D and
one J segment
diversity in CDR1 and CDR2 is determined by differences in sequences
of heavy-chain V gene segments
CD3 is determined by differences in the D gene segments and junctions
they make with the V and J gene segments

4.8

Random recombination of gene segments produces diversity in the antigen-binding sites of


immunoglobulins
somatic recombination
o during development of B cells, the arrays of V, D, and J segments are cut and re-splced
by DNA recombination
o brings together gene segent of each type for form DNA sequence encoding the V region
o for light chain, single recombination occurs between VL and JL segment
o for heavy chain, two recombinations are needed
D + JH segments DJ + VH segment
segments joined together are selected at random
o carried out by enzymes that cut and paste DNA and it exploits some mechanisms more
universally used by cells for DNA recombination and repair
o directed by recombination signal sequences (RSS)
flanks 3 side of V segment, both sides of D segment and 5 side of the J
segment
two types
one comprises defined heptamer and a defined nonamer separated by
12-bpspacer
other comprises a heptamer and nonamer separated by a 23-bp spacer
ensures gene segments ar ejoined in correct order
12/23 rule
recombination cannot happen when both segments have the same type
of RSS

V(D)J recombinase
set of enzymes needed to recombine V, D, and Jsegments
two of its components made only in lymphocytes
specified by recombination-activating genes (RAG1 and RAG2)
RAG-1 + RAG-2 + high mobility group of proteins RAG complex
Recombination
RAG complex binds to one of the RSSs flanking the sequences and
recruits the other RSS to the complex
Cleaves DNA to generate clean break at ends of two heptamer
sequences
RSSs are aligned and held in place by RAG coplex while broken ends
are rejoined (non-homologous end-joining)
bringing together ends of two gene segments to form coding joint in the
chromosome while joining ends of the removed DNA in a signal joint

4.9

4.10

light chain diversity


o for light chain, 35 V gene segments and 5 J segments can be recombined in 175
different ways
o for light chain, 30 V gene segments and 4 J segments can be recombined in 120
different ways
heavy chain diversity
o 40 VH segments, 23 D segments and 6 JH segments can produce 5520 combinations
Recombination enzymes produce additional diversity in the antigen-binding site
recombination process introduces changes in nucleotide
sequence and adds nucleotides at random points at coding
joints
o initial cleavage of DNA within RAG complex generates
single stranded structures, called DNA hairpins, at
ends of cleaved gene segments
o DNA repair enzymes open hairpins single-stranded
end with bases that are complementary in two DNA
strands now on same strand new nucleotide
sequence, palindromic in double-stranded DNA
o additional nucleotides are called P nucleotides
(palindromic nucleotides)
o ends of opened hairpins can be modified
by exonucleases
remove germline nucleotide
by enzyme terminal deoxynucleotidyl
transferase (TdT)
adds nucleotides randomly
N nucleotides (not encoded in
germline DNA)
o junctional diversity
contribution of P and N nucleotides to coding
joints
increases diversity in resulting amino-acid
sequence of CDR3s of immunoglobulin
chains
in light chains, CDR3s determined by junction between V and J segment
in heavy chains, CDR3s are formed by Da segment and its two junctions with V
and J segments
important source of variability
7
increases diversity by 3 x 10
Developing and nave B cells use alternative mRNA splicing to make both IgM and IgD
nave B cells
o circulating B cells that have yet to encounter antigen
o expresses both IgM and IgD
simultaneous expression of both and chains is accomplished by differential
splicing of same primary RNA transcript

o
o

4.11

rearrangement of V, D, and J segments brings gene promoter and enhancer into closer
juxtaposition transcription of rearranged gene
resulting RNA is spliced and processed and mRNA is translated to give a heavychain protein
rearranged heavy-chain locus, exons encoding leader peptide and V region on 5
side (upstream) of DNA encoding nine different C regions
closest is gene. followed by gene
in each C gene, separate exons encode each immunoglobulin domain
in mature B cells, transcription starts upstream exons encoding leader peptide and the V
region, continues through and C genes and terminates downstream of gene,
before 3 C gene
long RNA transcript is spliced and processed in two different ways
one yields mRNA for heavy chain
another yields mRNA for heavy chains

Each B cell produces immunoglobulin of a single antigen specificity


allelic exclusion
o process of Ig-gene rearrangement is tightly controlled so that only one heavy chain and
one light chain are finally expressed
o ensures B cell produces IgM and IgD of single antigen specificity
o only one heavy-chain locus and one light-chain locus are rearranged
in population of B cells, same functional light-chain gene rearrangement is found associated with
different heavy-chain gene rearrangement
o the converse is also true
random combinatorial association of heavy and light chains makes important contribution to the
overall diversity of immunoglobulins
an encounter with given pathogen engages subset of B cells that will make antibodies that bind
only to the pathogen
4.12
Immunoglobulin is first made in a membrane-bound form that is
present on the B-cell surface
when B cell first makes IgM and IgD, heavy chains have a
hydrophobic sequence near carboxy terminus by which Ig
associate with cell membranes
immunoglobulin molecules enter endoplasmic reticulum as soon
as they are synthesized
o they must associate with additional transmembrane
proteins called Ig and Ig
o they travel to B-cell surface in a complex with
immunoglobulin molecule
o this complex of IgM, Ig and Ig forms receptor for
antigen on nave B cells
longer tails of Ig and Ig components of B-cell receptor provide function of signal transduction
Diversification of antibodies after B cells encounter antigen
binding of antigen to surface immunoglobulin of mature nave B cell cell proliferation and
differentiation secretion of antibodies
4.13
Secreted antibodies are produced by an alternative pattern of heavy-chain RNA processing
after an encounter with antigen, isotypes are produced as secreted antibodies
o IgM antibodies are produced n large aounts and are important in protective immunity
against range of infections
o IgD antibodies are produced mainly within respiratory tract
all classes of Ig are made in two forms
o one bound to cell membrane that serves as B-cell receptor for antigen
o another, the antibody, secreted and soluble effector molecule

4.14

4.15

during differentiation, B cells go from making only membrane-bound form to making only soluble,
secreted form
o difference between two forms lies at carboxy terminus of heavy chain
membrane-associated Ig has hydrophobic anchor sequence
encoded by two small, separate exons downstream of hydrophilic
sequence
antibody has hydrophilic sequence
encoded at 3 end of exon encoding the fourth C-region domain
difference determined by differential RNA splicing and processing of same
primary transcript
Rearranged V-region sequences are further diversified by somatic hypermutation
diversity generated during gene rearrangement is concentrated on CDR3s of V H and VL domains
somatic hypermutation
o further diversification of whole of V-domain coding sequence
o introduces point mutations almost randomly and at a high rate throughout rearranged V
regions
o constant regions are not affected
o rate: one mutation per V region sequence per cell division
o dependent on activation-induced cytidine deaminatase (AID)
only made by proliferating B cells
converts cytosine in single-stranded DNA to uracil
acts during transcription when two DNA strands become temporarily separated
other enzymes convert the uracil to any one of the four bases potentially
causing a mutation
o some mutants have substitutions in antigen-binding site that increases affinity for antigen
cells bearing such mutants compete most effectively for binding to antigen and
are preferentially selected to mature into antibody-secreting plasma cells
changes are concentrated at CDR loops
affinity maturation
as adaptive immune response proceeds, antibodies of progressively
higher affinity for the infecting pathogen are produced
Isotype switching produces immunoglobulins with different C regions but identical antigen
specificities
IgM is the first class of antibody made in a primary immune response
o surface IgM of B-cell receptor is monomeric
o secreted effector consists of circular pentamor of Y-shaped immunoglobulin monomers
has 10 antigen-binding sites
binds strongly to surface of pathogens with multiple repetitive epitopes
limitations; bulky, low-affinity binding site, restricted recruitment of effector
mechanisms
isotype switching or class switching
o produces antibodies with other effector functions
o further DNA recombination enables rearranged V-region coding sequence to be used
with other heavy-chain C genes
o dependent on AID
o accomplished by a recombination within the cluster of C genes that excises the
previously expressed C gene and brings a different one into juxtaposition with assembled
V-region sequence
o switch sequences or switch regions
flanking the 5 side of each C gene (except gene)
highly repetitive sequences that mediate recombination
o mechanism
initiation of transcription of C-region gene to which cell will be switch
AID targets cytosines in S1 and switch region of the class to replace IgM/IgD for
deamination to uracil

4.16

uracil is removed by uracil-DNA-glycosylase (UNG)


APE1 excises abasic nucleotide, leaving a nick in DNA strand
nicks on both strands of DNA in both switch sequences facilitate their
recombination
o hyper-IgM syndrome
immunoglobulin genes in B cells of patients lacking a functional AID gene do not
undergo somatic hypermutation or isotype switching only antibodies that these
patients make are low-affinity IgM produced in greater amounts
Antibodies with different C regions have different effector functions
five classes of human immunoglobulin: IgG, IgM, IgD, IgA and IgE
o IgG has four subclasses: IgG1, IgG2, IgG3, IgG4
numbered according to their relative abundance in plasma
heavy chains designated as 1, 2, 3 and 4
o IgA has two subclasses: IgA1 and IgA2
heavy chains designated as 1 and 2
o , , and heavy-chain C regions are made up of three C domains
o and heavy chains have four C domains
o each C domain is encoded by separate exon in relevant C gene
o additional exons are used to encode the hinge region
antibodies can clear pathogens from the body in various ways
o neutralizing antibodies
directly inactivate a pathogen or a toxin and prevent it from interacting with
human cells
o opsonins
coats pathogens with proteins that facilitates its elimination
opsonized pathogens are more efficiently ingested by phagocytes, which have
receptors for Fc receptors of some antibodies
at beginning of immune response, antibodies bind antigen with low affinity
o avidity
overall strength of binding at multiple sites
monomeric IgA
o made by plasma cells in lymph nodes, spleen and bone marrow and is secreted into the
bloodstream
o can also be made as a dimer
two monomers being joined by a J chain identical to pentameric IgM
made principally in lymphoid tissues underlying mucosal surfaces
secreted nto lumen of gut
main antibody in other secretons
o more IgA is made than any other isotype
IgE is highly specialized toward recruiting effector functions of mast cells in epithelium, activated
eosinophils present at mucosal surfaces and basophils in blood
o carries high-affinity receptor that binds IgE in absence of antigen
o presence of antigen triggers strong physical anf inflammatory reactions that can expel
and kill infecting parasites
IgD is made by a cryptic switch region between and genes
o B cells that make IgD concentrated in upper airways of the bronchial tract, particularly
tonsils
makes antibodies that interact with commensal and pathogenic respiratory
bacteria
o basophils have high-affinity with IgD in the absence of IgE
when antigen is present, it triggers it to orchestrate a local immune response that
eliminates bacteria
IgG is most abundant antibody in internal body fluids
o made principally in lymph nodes, spleen and bone marrow
o circulates in lymph and blood

4.17

conformational flexibility
allows antigen-binding sites in the two Fabs and effector-binding sites in Fc to
move in a partly independent manner and assumes wide range of different
positions with respect to each other

The four subclasses of IgG have different and complementary functions

differences between four subclasses are in constant region of heavy chain and most are within
the hinge
o IgG1 is the most abundant and versatile of four subclasses
Intermediate in flexibility, susceptibility to proteolysis and capacity to activate
complement
o IgG2 is the second most abundant subclasses
hinge is similar to IgG1 but contains more disulfide bonds that reduce flexibility,
susceptibility to proteolysis and capacity to activitate complement
preferentially made against the highly repetitive carbohydrate antigens of
microbial surfaces
infections with encapsulated bacteria are poorly controlled in IgG2-deficient
individuals
o IgG3 is best at activating complement

has much longer hinge region


greater flexibility in binding antigens and makes Fc more accessible for binding to
C1
more susceptible to cleavage
associated with recurrent infection leading to chronic lung disease
IgG4 is least abundant subclass of IgG
does not activate complement as it binds C1q poorly
can exchange a module composed of one heavy chain and one light chain with
that from another IgG4 molecule
most IgG4 molecules have two different heavy chains and two different light
chains
functionally monovalent and can only impede pathogens by mechanism of
neutralization
anti-inflammatory
blocks IgE when it binds to allergen and reducing severity of allergic reaction

Chapter Five
Antigen Recognition by T Lymphocytes
5.0

Antigen recognition by T lymphocytes


T lymphocytes recognize and bind antigen through highly variable antigen-specific receptors
o more diverse roles than B cells
involves interactions with other cells
T-cell receptor (TCR)
o similar to immunoglobulins
similar structure
produced as a result of gene rearrangement
highly variable
diverse in antigen specificity
o recognize and bind mainly to peptide antigens
derived from pathogens proteins
each clone of T cells expresses a single species of antigen receptor
effector functions of T-cells are carried out through antigen-specific interactions with other cells of
the body
o T-cell receptors recognize a composite structure on a human cell surface composed of a
peptide antigen bound by a MHC
o ligand = peptide + MHC on cell surface
major histocompatibility complex (MHC)
o genes that encode MHC molecules
o clustered in a chromosomal region
o some are highly polymorphic
T-cell receptor diversity
T-cell receptor
o membrane-bound glycoprotein
o resembles a single antigen-binding arm of immunoglobulin olecule
o composed of two different polypeptide chains and one antigen-binding site
o each chain has a variable and constant region
o gene rearrangement produces sequence variability
o no further change after T cell is stimulated with antigen
5.1
The T-cell receptor resembles a membrane-associated Fab
fragment of immunoglobulin
T-cell receptor
o consists of two different polypeptide chains
T-cell receptor chain (TCR) and T-cell
receptor chain (TCR)
genes have germline organization as
immunoglobulin heavy-chain and lightchain genes
o comprising sets of gene
segments that must be
rearranged to form a functional
gene
each mature T cell expresses only one
functional chain and chain
each chain has a variable region (V region) and
constant region (C region)
each chain consists of an amino-terminal V domain, followed by a C
domain and a membrane-anchoring domain
antigen-recognition site = V + V
amino acid sequences shows that sequence variation of the chains is clustered
into regions of hypervariability

5.2

5.3

corresponds to loops at end of domain farthest from T-cell membrane


o forms binding site for antigen complementarity-determining
regions (CDRs)
o three CDR loops: CDR1, CDR2, CDR3
o T-cells ony has one binding site for antigen
Only used as cell-surface receptors
T-cell receptor diversity is generated by gene rearrangement
human T-cell -chain locus is on chromosome 14 and -chain is on chromosome 7
o simplicity of T-cell receptor region only one C gene and two C genes (but two are
functionally the same)
o -chain is otherwise similar to an immunoglobulin light chain locus
containing only sets of V and J gene segments
o -chain locus is similar to an immunoglobulin heavy chain locus
containing D gene segments + V and J gene segments
T-cell receptor gene rearrangement occurs during T-cell development in the thymus
o in -chain gene, V gene segment + J gene segment (via somatic DNA recombination)
V-region sequence
o in -chain gene, D + J gene segment + V gene segment V-region sequence
o gene segments are flanked by recombination signal sequences
o RAG complex and same DNA-modifying enzymes are involved
o additional, nontemplated P and N nucleotides are inserted
severe combined immunodeficiency disease (SCID)
o one of causes: absence of RAG proteins
o functional B and T lymphocytes are absent
o Omenn syndrome
missense mutations that produce partially a ctive RAG proteins
after gene rearrangement, the genes consist of exons encoding leader peptide, the V region, the
C region and membrane-spanning region
o separated by introns
o primary RNA transcript is spliced to remove the introns mRNA
o translation of -chain and -chain mRNA produces -chain and -chain endoplasmic
reticulum : T-cell receptor
The RAG genes were key elements in the origin of adaptive immunity

V(D)J recombination
o mechanism of gene rearrangement found in both T and B cells
o generates clonal diversity in antigen receptors
o two subunits of RAG recombinase
key to the recombination
specific to adaptive immunity
RAG genes
o lack introns
o resembles the transposase gene of transposon

type of genetic element that can make and


move copies of itself to different chromosomal
locations
o found on chromosome 11
5.4
Expression of the T-cell receptor on the cell surface requires
association with additional proteins
heterodimers of and chains cannot leave the endoplasmic
reticulum
o : heterodimer must associate with four invariant
membrane proteins
CD3 complex
CD3, CD3, CD3
genes on chromosome 11
chain
gene on chromosome 1
o at surface, CD3 proteins and chain remain in stable
association with T-cell receptor T-cell receptor
complex
CD3 proteins and chain transmit signals to
cell interior after antigen has been recognized
5.5
A distinct population of T cells expresses a second class of T-cell receptor with and chains
second type of T-cell receptor
o formed by T-cell receptor chain (TCR) and T-cell receptor
o cannot be expressed by a T cell also expressing : receptor
o : T cells
o organization resembles and loci
major differences
gene segments are within -chain locus on chromosome 14, between
V and J gene segments
-chain locus is on chromosome 7
contains fewer V gene segments, thus less diverse
o partly compensated by junctional diversity, wherein two D
segments can be incorporated in final gene sequence for chain
Antigen processing and presentation
antigen receptors of nave T cells have antigen-binding sites that are structurally similar to Ig
o but do not recognize
broad range of
structures
o foreign antigen short
peptide produced by
intracellular degradation
of a pathogens proteins
o human protein binds
with peptide antigen
where it can be
recognized and
engaged by a T-cell
recptor
antigen processing
o production of peptide
antigens inside human
cells
antigen presentation
o display of peptide
antigens of surface of

5.6

5.7

human cells in the precise manner that can be bound by a T-cell receptor
T-cell receptors recognize peptide antigens bound to MHC molecules
antigen recognized by T-cell receptors are short peptides
o around 8-25 amino acids in legth
o generated by degradation of pathogen ad its proteins
o simplifies antigen recognition
concentrates on only protein
ignores complexity of three-dimensional structures of protein
o major source: uptake and killing of pathogens by macrophage and dendritic cells
o antigen processing
process by which peptide antigens are produced from pathogens and their
products
antigenic peptide must be brought to cell surface in order to be bound
o peptide binds to membrane-associated protein
o major histocompatibility complex (MHC) molecules
dedicated human glycoproteins that bind and transport peptide antigens
can bind to only a single peptide
o complex of MHC molecule and peptide antigen is recognized by T-cell receptor
o antigen presentation
process where MHC molecule presents the peptide antigen to T-cell receptor
o antigen-presenting cell (APC)
a cell that can present antigens

Two classes of MHC molecule present peptide antigens to two types of T cell
two types of MHC molecule are used for different molecules
o MHC class I
presents antigens from intracellular pathogens
pathogen cytosolic degradation peptides endoplasmic reticulum
binding with MHC class I cell surface
effector: cytotoxic T cells
recognizes MHC class I + antigen
defends against intracellular pathogens
cell-surface protein CD8
o MHC class II
presents antigens from extracellular pathogens
pathogen lysosomal degradation endosomal vesicles with MHC class II
cell surface
effector: helper T-cell
recognizes MHC class II + antigens
defends against extracellular infection
cell-surface protein CD4
T-cell co-receptors
o CD4 and CD8

5.8

5.9

CD4 and CD8 bind to a site topographically separate from the site bound by Tcell receptor
similar functions but different structures
CD4: single polypeptide, four extracellular Ig-like domains
CD8: heterodimer, each has one extracellular Ig-like domain
o cooperate with T-cell receptor in recognition of complexes of peptide antigens and MHC
molecules
cytotoxic CD8 T cells
o kill cells infected with virus, bacterium or some other intracellular pathogen
helper CD4 T cells
o facilitates macrophage activation, phagocytosis
improvement, secretion of cytokines and chemokines
o also helps B cells
human immunodeficiency virus (HIV)
o causes acquired immunodeficiency virus (AIDS)
o selectively infects CD4 T cells
o binds to CD4 and gains entry to cell where it
replicates
o number of CD4 T cells gradually declines
The two classes of MHC molecule have similar three dimensional structures
MHC class I molecule
o transmembrane heavy chain (-cain) + 2-microglobulin
heavy chain: three extracllular domains
1, 2
o forms peptide-binding site
o farthest from membrane
3
o supports the peptide-binding site
o Ig-like
encoded by gene in the MHC
2-microglobulin
supports peptide-binding site
Ig-like
MHC class II molecule
o two transmembrane chains: and
each contribute one domain to peptide-binding site and one Ig-like supporting
domain
both encoded by MHC genes
Ig-like domains of MHC class I and II
o also provide specific binding sites for the CD4 and CD8 co-receptors

MHC molecules bind a variety of peptides


peptide-binding sites

5.10

5.11

deep groove on surface of MHC molecule, within which a single peptide is held tightly by
noncovalent bonds
o constraints on length and amino acid sequence
dictated by structure of groove, differing between MHC class I and II molecules
promiscuous binding specificity or promiscuous specificity
o MHC class I
length is limited because two ends of peptide are grasped by pockets situated at
ends of peptide-binding groove
8-10 amino acids long, mostly nine
differences accommodated ny a kink in the extended conformation of the
bound peptide
hydrophobic or a basic residue at carboxy terminus
o MHC class II
two ends of peptide are not pinned down into pockets at each end of peptidebinding groove
peptides extend out from each end of groove longer and more variable in
length
13-25 amino acids long, some longer
MHC class I and MHC class II molecules function in different intracellular compartments
human cells consist of two topological compartments
o one compartment is sealed off from outside of the cell
comprises of nucleus and cytosol
connected by nuclear pores
o vesicular system
contiguous with outside of the cell
comprises the endoplasmic reticulum, Golgi apparatus, lysosomes and smaller
endocytic and exocytic vesicles
synthesis on cytosol
o inefficient incomplete and improperly folded proteins tagged for degradation and
recycling
o virus infection
synthesis of viral proteins subject to same scrunity
some peptides produced by protein degradation are transported into
endoplasmic reticulum bind to MHC class I if length and sequence is right
self proteins and self peptides
human proteins and peptides
non-self proteins and non-self peptides
pathogen proteins and peptides
vesicular system
o major functions
uptake by phagocytosis
endocytosis of nutrients, molecular signals and damaged molecular and cellular
components of the circulation
materials lysosomes degradation peptides
junction connecting endocytic and exocytic pathways binding to MC class I
olecules
Peptides generated in the cytosol are transported to the endoplasmic reticulum for binding to
MHC class I molecules
proteasome
o constitutive proteasome
o a large barrel-shaped protein complex that degrades damaged, poorly-folded or uses
proteins in the cytosol
o makes up 1% of cellular protein
o structure
core

5.12

four rings of seven polypeptide subunits of 20-30kDa


o 1, 2 and 5
has protease activities
19S regulatory caps
recognizes proteins destined for degradation and steer them into
catalytic chamber
o with infection, can be modified to favro production of peptids that bind to MHC class I
molecules
induced by IFN-
secreted by NK cells during innate immune response
induces production of alternative subunits that replace the subunits
o alters proteolytic activities increased cleavage after
hydrophobic residues and decreased cleavage after acidic
residues
induces production of PA28 and PA28 subunits 20S proteasomeactivator complex
o replaces 19S regulatory cap
o speeds release of peptides
o immunoproteasome
modified form of proteasome in cells exposed to IFN-
antigenic peptides are transported into endoplasmic reticulum
o transporter associated with antigen processing (TAP)
transports peptides across endoplasmic reticulum
heterodimer of TAP-1 and TAP-2
needs ATP
bare lymphocyte syndrome type 1
o also MHC class I deficiency
o TAP protein is nonfunctional
MHC class I molecules bind peptides as a part of peptide-loading complex
newly synthesized MHC class I heavy chains and 2-microglobulin are also translocated into ER
o calnexin
binds with MHC class I heavy chains when they first enter ER
retains proteins until folded and associated correctly
releases only when it has folded, formed its disulfide bonds and bonded
with 2-microglobulin
calcium-dependent lectin
o peptide-loading complex
incorporates heterodimer into it after being released from calnexin
central component: tapasin
bridging protein that brings the heterodimer of 2-microglobulin and class
I heavy chain into close proximity to TAP
o effect: empty peptide-binding groove of MHC class I is positioned
to receive peptides from TAP
Ig-like domains bind to 2 and 3 domains; interacts with TAP through
membrane-spanning regions
interaction with MHC class I heavy chain causes peptide-binding groove
to be in a more open conformation
o makes it more selective to peptides that bind tightly
calreticulin
soluble chaperone, related to calnexin
binds to MHC class I heavy chain
ERp77
thiol reductase linked to tapasin via disulfide bond
stabilizes peptide-binding loading complex by interacting with calreticulin
and protecting disulfide bond of 2 domain

5.13

5.14

peptide editing
process by which tapasin helps heterodimer of class I heavy chain and
2-microglobulin to try on a variety of peptides to find one that fits really
well
when a peptide binds strongly but is too long to fit snugly in groove
o endoplasmic reticulum aminopeptidase (ERAP) will remove
amino acids until the fit is good
when peptide falls out of groove during transport, calreticulin in vesicle
membrane will stabilize the empty MHC class I and recycle it back into
ER
arrival of peptide that binds tightly breaks the hold of tapasin leave peptide-loading
complex exit from ER in a vesicle Golgi stacks MHC class I glycosylation
plasma membrane

Peptides presented by MHC class II molecules are generated in acidified intracellular vesicles
proteins of extracellular bacteria, extracellularvirus particles, soluble protein antigens
o exploits mechanisms that cells use to take up nutrients, hormones, and molecular signals
from environment
o produce intracellular vesicles derived from plasma membrane and contains extracellular
material
o endosomes, phagosomes
membrane-enclosed vesicles
o become part of an interconnected vesicular system
carries materials to and from cell surfaces
o interiors become acidified by proton pumps + fuse with other vesicles like lysosomes
phagolysosomes with enzymes that degrade vesicle contents and produce peptides
derived from proteins
microbes in extracellular environment
o taken up by macrophages and dendritic cells by phagocytosis and degraded within
phagolysosomes
o B cells also bind via surface Ig and internalize antigens via endocytosis degradation
within vesicular system
peptides produced within phagolysosomes become bound to MHC class II molecules
o peptide: MHC class II complexes are carried to cell surface by outward-going vesicles
Invariant chain prevents MHC class II molecules from binding peptides in the endoplasmic
reticulum
newly synthesized MHC class II and chains in ribosomes membranes of ER
o chain and chain associate with invariant chain
prevents MHC class II molecules from binding peptides in ER

5.15

stabilizes conformation until itt binds peptide


delivers to specialized endocytic vesicles where they bind peptides
MHC class II compartment (MIIC)
o contains cathepsin S and other proteases that attack invariant chain series of
cleavages class II-associated invariant-chain peptide (CLIP)
covers up MHC class II peptide-binding site
o HLA-DM
facilitates removal of CLIP and binding of another peptide
specialized form of MHC class II molecule that cannot bind peptides
concentrated on MIIC
analogous to tapasin
induces conformational changes that opens up peptide-binding groove
once peptide binds, MHC class II molecule releases HLA-DM and leaves MIIC
compartment in vesicles that deliver them to plasma membrane
o HLA-DO
specialized MHC class II molecule that antagonizes HLA-DM
when HLA-DO binds to HLA-DM, HLA-DM cannot release CLIP
o balance between HLA-DM and HLA-DO concentrations is controlled by IFN-
increases expression of HLA-DM

Cross-presentation enables extracellular antigens to be


presented by MHC class I
people affected with hepatitis C virus make a cytotoxic
CD8 T-cell response that kills hepatocytes infected with
virus
o nave T cells are presented with virus-derived
peptides on MHC class I molecules from
dendritic cells or macrophages
o Kupffer cells are not affected
only way Kupffer cells can acquire viral
proteins is through phagocytosis or
endocytosis of dead, dying or
disintegrating infected hepatocytes
pathway for MHC class II molecule
presentation
o cross-presentation
o cross-priming
when an immune response is initiated
by cross-presentation
o possible mechanisms
phagocytosis allows complexes of
MHC class I molecules and viral
peptide to be transferred from dead

hepatocyte to membranes of intracellular vesicles inside Kupffer cell transport


to cell surface
viral components are delivered from endocytic vesicles into the cytosol for
degradation by proteasome and presentation by usual class I pathway
5.16
MHC class I molecules are expressed by most cell types, MHC class II molecules are expressed
by few cell types
professional antigen-presenting cells
o B cells, macrophages and dendritic cells
5.17
The T-cell receptor specifically recognizes both peptide and MHC molecule
T-cell receptor contacts both peptide and surrounding surface of MHC molecule
o binds with peptide:MHC class I complex with long axis of its binding site oriented
diagonally across the peptide-binding groove of MHC class molecule
o binds with peptide: MHC class II complex in similar orientation
o CD3 loops form central part of binding site and grasp side chain of one of the amino
acids in idle of peptide
o CDR1 and CDR2 loops form periphery of binding site and contact the helices of MHC
molecule
The major histocompatibility complex
major histocompatibility complex
o chromosome 6 in humans
o where genes for MHC molecules and other proteins involved in antigen processing and
presentation are encoded
o first recognized as site of genes that rejects tissues transplanted from unrelated donors
o magnitude of diversity is smaller than Ig or T-cell receptors
o region of the genome most strongly correlated with human disease
5.18
The diversity of MHC molecules in the human population is due to multigene families and genetic
polymorphism
human MHC human leukocyte antigen complex (HLA complex)
o antibodies used to identify MHC molecules react with white cells of blood but not red cells
o MHC class I = HLA class I; MHC class II = HLA class II
MHC class I and II
o encoded by stable genes that neither rearrange nor undergo any other developmental or
somatic process of structural change
o diversity from two sources
gene families
multiple similar genes encoding
genetic polymorphism
presence of multiple alleles
o terms
isotype
protein producets of the different genes in a MHCc class I or class II
family
alleles
different forms of any given gene
allotypes
encoded proteins of alleles
isoform
denotes any particular MHC protein
o there are six MHC class I isotypes
HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G
highly polymorphic: A, B, C
function: present antigens to CD8 T cells and form ligands for receptors
on NK cells
polymorphism is property of the heavy chain, as 2-microglobulins are
monomorphic

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5.20

oligomorphic: E and G
function: forms ligands for NK-cell receptors
monomorphic:
function: acts as a chapterone that retrieves other HLA class I molecules
that lose their peptides
o there are five MHC class II isotypes
HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR
highly polymorphic: HLA-DP, HLA-DQ, HLA-DR
function: present peptide antigens CD4 T-cells
polymorphism is contributed by and/or cells
o HLA-DR: is highly polymorphic
o HLA-DP and HLA-DQ: both chains are polymorphic
oligomorphic: HLA-DM and HLA-DO
supervise peptide loading of HLA-DP, HLA-DQ and HLA-DR
The HLA class I and class II genes occupy different regions of the HLA complex
human leukocyte antigen (HLA) complex
o human MHC
o consists of 4M base pairs of DNA on short arm of chromosome 6
o divided into three regions
class I region
farthest from the centromere
contains six expressed HLA class I genes
also has several nonfunctional class I genes and gene fragments
class II region
other end of the complex
contains all class II genes and several nonfunctional class II genes
class III region
also central MHC
separates class I and class II genes
around 1M base pairs
invariant 2-microglobulin is located on human chromosome 15
o for every HLA class II isotype
genes encoding and are called A and B respectively (i.e. HLA-DMA and HLADMB)
when there are more than one gene, a number in series is added (i.e. HLA-DQA1
and HLA-DQA2)
o haplotype
the particular combination of HLA alleles found on a given chromosome 6
o meiotic recombination at only about 2%
homozygous individuals are rare but are usually healthy
can only express three class I (HLA-A, HLA-B and HLAC) and three
class II (DP, DQ and DR) isoforms
heterozygous individuals can express up to six class I and sixteen class II
isoforms
maximum is when each haplotype contains two functional DRB genes +
different allele for all polymorphic HLA class I and class II genes
Other proteins involved in antigen processing and presentation are encoded in the HLA class Ii
region
other genes in HLA complex embrace a variety of functions
o HLA complex has more than 200 genes
o class II region is almost dedicated to genes involving to processing and presentation of
antigens
encodes two polypeptides of TAP peptide transporter, gene for tapasin and
genes encoding for two of three proteolytic subunits (LMP2 and LMP7) specific to
immunoproteosome

coordinated by cytokines IFN-, -, and


produced at sites of infection at early stages of immune response
stimulates cells in vicinity to increase expression of HLA class I heavy chains, 2microglobulin, TAP and the LMP2 and LMP7 subunits
IFN coordinates expression of HLA-DM, HLA-DP, HLA-DQ, HLA-DR, and
invariant chain genes
turned on by MHC class I transactivator (CIITA)
o also induced by IFN
o impairment leads to MHC class II deficiency
o majority of genes of class I region are not involved in immune system
genes encoding class I molecules and related class I-like molecules are found on
several different chromosomes
HLA class I molecules and class I-like molecules encompass a broader range of
functions
uptake of IgG in gut, regulation of iron metabolism and regulation of NKcell function in the innate immune response
o MHC class I is older form of MHC molecule, and MHC class II evolved from MHC class I
MHC polymorphism affects the binding of peptide antigens and their presentation to T cells
alleles of highly polymorphic MHC genes encode proteins that differ by 1-50 amino acid
substitutions
o mainly locaed in domains that bind peptide and interact with T-cell receptor
1 and 2 domains of MHC class I
1 and 1 domains of MHC class II
HLA-DR: 1 is invariant
HLA-DP and HLA-DQ
variation in the peptide-contact residues on the floor and sides of peptide-binding groove
determines the types of peptides that each isoform binds
o anchor residues
most peptides that bind an MHC isoform have the same amino acid or chemically
similar
arises because side chains of amino acids are bound by complementary pockets
within the groove
anchors peptide to MHC molecule
less defined in MHC class II
inaccessible to T-cell receptor when in a complex of peptide bound to an MHC
molecule
other peptides are more diverse and are available for contact with T-cell
receptors
o peptide-binding motif
anchor residues + particular MHC isoform
number is limited so MHC allotypes differ by only a few amino acids binds
overlapping populations of peptides
o MHC restriction
any given T-cellr receptor is therefore specific for the complex of a particular
peptide bound to a particular MHC molecule
antigen-specific T-cell response is restricted to a particular MHC type
MHC diversity results from selection by infectious disease
MHC diversity is due to natural selection
o likely sources of selection are infections caused by pathogens
advantage of having multiple MHC genes or having polymorphic MHC genes
o contributes different-peptide-binding specificities greater number of pathogen-derived
peptides improved strength of immune response
o for heterozygotes, two different peptide-binding specificities can be brought into play
o high degree of polymorphism ensures that most members of the populations are
heterozygous
o

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5.22

balancing selection
selective processes that act to maintain a variety of MHC isoforms
o directional selection
a different mode of selection that favors certain MHC alleles or combinations or
alleles at expense of others
imposed by specific, epidemic disease
disrupts the balance
evidence: numerous HLA differences between human populations of different
ethnicities and geographical origins
recently formed MHC alleles will confer advantage on host and be selected during epidemics
o pathogens adapt to MHC
o variants arise through point mutations and several recombinations
o favored: new HLA alleles in which a small segment of one allele has been replaced by
homologous section of another + addition of several acid substitutions that change
contact residues
interallelic conversion or segmental exchange
MHC polymorphism triggers T-cell reactions that can reject transplanted organs
any cells having T-cell receptors that respond to self peptides and MHC molecules at healthy cell
surfaces are eliminated
o prevents persons T cells from attacking own tissues and causing disease
encompasses only the MHC isoforms expressed by that person
o self MHC
autologous
others: allogenic
o alloreactive T cells
T cells that can respond against the cells of another individual
o alloreaction
a powerful T-cellr esponse that attacks a graft
cause: alloreactive T cells are activated by allogenic HLA molecules in
graft
solution: donors should be selected to have combinaitons of HLA alleles
that are similar to the patient
o HLA type
combination of HLA alleles that a person has
o alloantibody
any antibody raised in one member of a species against an allotypic protein from
another member of the sme species
mother and fetus
when HLA molecules of fetus derive from father but not expressed by
mother
preexisting alloantibodies can react with allogenic MHC class I molecules
of graft almost untreatable graft rejection
o

5.23