Administrative issues:
Recommended text: Goldsby/Kuby Immunology, 6th edition
(Note that Innate Immunity is not adequately covered in the 5th edition.)
Discussion sections start next week. The journal article Akira et al, and
the first problem set will be covered. Both are available on the website.
Office Hours: Questions about the lecture material are best addressed
during office hours (Tues 11-12). I will be holding extra office hours (date
and time TBA) before the first midterm.
Email: Please use email only for VERY simple yes/no questions or simple
administrative matters.
Great questions, keep them coming!
Innate Immunity
Innate immune effector mechanisms
Physical and biochemical barriers (defensins)
Phagocytosis and reactive oxygen species
Cell autonomous defenses
Apoptosis
Interferons and PKR
Innate immune recognition
discovery of the Toll-like receptors
mammalian TLRs and their ligands
non-TLR recognition of PAMPs
Connections between adaptive and innate immunity
Some microbes hijack cellular machinery to replicate
and spread. Intracellular pathogens include viruses
(influenza, HIV) and intracellular bacteria (listeria)
and intracellular parasites (malaria, toxoplasma).
cell-autonomous defense:
cell produces an immune
response that acts on itself
Apoptosis: Cellular Suicide
•Nuclear fragmentation
•Proteolysis
•Blebbing
•Death
Remnants
undergo
phagocytosis
Apoptosis versus Necrosis
•Tidy: contents of cells
degraded from within,
producing small cellular
“blebs”
•Programmed from inside the
cell
•Messy: contents of cell
released.
•Induced by external insult
Cell death by necrosis is more likely to produce inflammation.
Cells can avoid being hijacked by viruses by activating the Protein
Kinase R (PKR) pathway. PKR is triggered by dsRNA and interferon.
Interferons are cytokines that are
produced in response to viral infection.
Produce an “anti-viral state” in target
cells.
Acts on cell that produces it, as well as
neighboring cells.
Together with dsRNA, act to triggering
the Protein Kinase R
(PKR) pathway.
Shuts down protein synthesis machinery
of cells, thus preventing viral replication.
Protein Kinase R:
Interfering with Infection
eIF2a
dsRNA
or
interferon
Kinase
domain
RNAbinding
domain
PKR
Phosphorylated
PKR
(active)
Phosphorylated
eIF2a
(inactive)
Innate Immunity (finishing up)
Innate immune effector mechanisms
Physical and biochemical barriers (defensins)
Phagocytosis and reactive oxygen
Cell autonomous defenses
Apoptosis
Interferons and PKR
Innate immune recognition
discovery of the Toll-like receptors
mammalian TLRs and their ligands
non-TLR recognition of PAMPs
Connections between adaptive and innate immunity
Cell types of innate immunity
Components of the bacterial cell wall, such as
lipopolysaccaride (LPS), peptidoglycan, lipoprotein can
trigger the innate immune system.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Cross section of the cell
wall of a gram-negative
bacteria.
Comparison of the adaptive and innate immune responses
innate
adaptive
Response time
hours
days
Response to
repeat infection
identical to primary
(no memory)
stronger response upon
second exposure (memory)
Receptors that
Mediate pathogen
Recognition
pattern recognition receptors
e.g. Toll-like receptors (TLR)
limited diversity,
fixed in germline
antibodies and T cell antigen receptors (TCR)
Ligands
Pathogen associated molecular
patterns (PAMPs)
virtually any component of pathogen
unlimited diversity
generated by V(D)J recombination
What triggers innate immune responses?
( How do phagocytes know what to eat?)
• Long-known that bacterial cell wall components activate
phagocytes
• Hypothesis-- microbes contains pathogen associated
molecular patterns (PAMPs) which are recognized by
pattern recognition receptors (PRRs)
• A series of disparate observations in transcriptional
regulation and drosophila development lead to the
discovery of PRRs
Genetic analysis of
early embryonic
development in the
fruitfly, Drosophila
(Nusslein-Volhard and
colleagues)
The Dorsal Signaling Pathway controls
dorsal/ventral polarity in the Drosophila early
embryo
Dorsal
Toll (a receptor)
Cactus
Ventral
“Toll” is German slang for “weird”
Dorsal
(a transcription
factor)
NF-kB: a transcription factor which
binds to antibody genes (Baltimore and
colleagues).
Transcription
factor
Ig k gene
enhancer
Nuclear factor Igk locus B (as in A, B, C, etc.)
NF-kB: A critical transcription factor for
innate immunity
p65
p65
IkB
p50
Inactive,
cytoplasmic
(cactus)
p50
(dorsal)
Active,
nuclear
Mutant mice lacking NF-kB subunits have defects in innate immunity.
What is the mammalian receptor that leads to NF-kB activation in
response to infection?
Discovery of the mammalian Toll-like receptors
(TLR):
1997: Janeway and Medzhitov discovered a human protein
with structural similarity to drosophila Toll that could activate
immune response genes in human cells (TLR4).
1998: Beutler discovered that a mouse strain with an altered
response to bacterial lipopolysaccharide (called LPS or
endotoxin) was due to a mutation in the TLR4 gene.
There are 11 TLR family members in human and 12 in mice.
Each responds to a distinct set of microbial products.
Different mammalian Toll-like receptors (TLRs) are specific
for different classes of microbial products
Insert Fig 3-11
Different mammalian Toll-like receptors (TLRs) are specific
for different classes of microbial products
Toll-like receptors
(TLRs)
link microbial
products (PAMPs) to
transcription
factor activation
in a signaling
pathway that is
conserved between
mammals and insects
Toll-mutant drosophila are
susceptible to fungal infections
A more detailed look at the signaling pathway down-stream of
Toll-like Receptors (TLRs)
A more detailed look at the signaling pathway down-stream of
Toll-like Receptors (TLRs)
Pathogen recognition receptors (PRR) in the innate
immune system: TLRs are not the whole story
scavenger receptors
NOD proteins (cytoplasmic PRR)
etc, etc
The benefits and hazards of TLR signaling
Septic shock can be caused by systemic
infection with gram negative bacteria
(Salmonella).
Shock is caused by overwhelming
production of cytokines and is induced by
presence of LPS in blood stream.
Mice lacking TLR4 are resistant to LPSinduced shock.
Mice lacking TLR4 are also more sensitive
to infection with gram negative bacteria.
(local vs. systemic effects of TLR
signaling?)
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Triggering of PRRs on macrophage or dendritic cells can induce a
LARGE variety of events including:
Increased phagocytosis
Production of cytokines and inflammatory mediators:
Interferons to induce anti-viral state
Chemokines to attract migrating cells
Etc, etc.
Increased cell migration
Changes in expression of molecules involved in T cell
antigen presenting cell function.
Innate Immunity
Innate immune effector mechanisms
Physical and biochemical barriers (defensins)
Phagocytosis and reactive oxygen
Cell autonomous defenses
Apoptosis
Interferons and PKR
Innate immune recognition
discovery of the Toll-like receptors
TLRs and their ligands
non-TLR recognition of PAMPs
Connections between adaptive and innate immunity
When innate immune signaling is insufficient to clear a pathogen, the
adaptive immune system kicks in.
Innate immune signaling turns on the adaptive immune response.
Macrophage and dendritic cells serve as antigen-presenting cells for adaptive
immune cells (T cells).
T cell interacting with a macrophage
(antigen presenting cell)
TLR-signaling activates innate immune cells (macrophage and dendritic cells).
Innate immune cells that have been activated by TLR-signaling are much more
effective antigen presenting cells than resting immune cells.
TLR signaling within phagosomes determines fate of
that phagosome (destruction vs antigen presentation).
Pathogen
(non-self, TLR signaling)
Material in phagosome
enters antigen
presentation pathwaypresentation to T cells
Dying infected cell
(self, no TLR signaling)
Material in
phagosome disposed
of inside cell-no
presentation to T cells
Blander and Medzhitov 2006 Nature v440 p808
Comparison of innate and adaptive immune recognition
Receptors that mediate
innate immune recognition: Tolllike receptors (TLR)
Receptors that mediate adaptive immune recognition:
Antibody and the T cell receptor (TCR)
The genes encoding the antigen receptors of T and B cells are
assembled by DNA rearrangement as these cells develop. As a result
The genes encoding the T cell antigen receptor (TCR) are assembled
of V(D)Jby
recombination,
every
and T
cells expresses
a unique
DNA rearrangement
asB
T cells
develop
in the thymus
version of the antigen receptor.
V segments
D
J
TCR  locus:
structure in germline
DNA rearrangement
(rag1, rag2)
TCR  locus:
structure in T cells
transcription
RNA splicing
translation


T cell
C
A a result of V(D)J recombination every mature B cell expresses a unique antibody.
Encounter with an antigen leads to clonal expansion of B cells with a particular
specificity.
What’s coming up in the next couple of weeks
(Innate Immunity)
Antibodies and antigens I (emphasis on antibody structure)
Antibodies and antigens II
(emphasis on
antigen-antibody
binding interactions)
Techniques based on
antibodies
V(D)J recombination
B cell development and
function
Antigens & Antibodies I
Discovery of antibodies
Basic Antibody Structure
brief review of protein structure
disulfide linked tetramer: 2 heavy and 2 light chains
myeloma proteins and the primary structure of antibody
crystal structure of antibody: the Ig domain
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgE
The advantages of multivalency
effector functions of antibody isotypes
Early Observations of Acquired (adaptive)
Immunity
•Thucydides, historian: 430 BC noted that those who had
survived plague could nurse the sick and not become sick
again.
•1600s, Turks and Chinese practiced “variolation”:
intentional exposure to material from smallpox lesions to
provide protection against smallpox.
• Edward Jenner: ~1800 noted that exposure to vaccinia
virus (cowpox) protects against smallpox. Tested first
vaccine using cowpox.
•Louis Pasteur: ~1880
•finds that exposure
to attenuated bacteria
(chicken cholera)
protects against live
bacteria.
•First vaccine against
rabies
Von Behring & Kitasato: ~1890 discovered
“passive immunization”
•Immunize rabbits with tetanus bacteria.
•Isolate serum (non-cellular portion of blood) from immunized
rabbits, inject “immune serum” into naïve rabbit
•Challenge treated and untreated rabbits with lethal dose of live
tetanus bacteria: only treated mice survived.
•Passive immunization with pooled human immunoglobulin is
currently used to treat immunodeficiency and to provide
temporary resistance to infection (hepatitis, rabies, measles,
tetanus).
•Tiselius & Kabat: 1930s. Discovered that the g-globulin fraction of
serum contains antibodies.
Immunize rabbits with chicken ovalbumin.
Here a purified foreign protein (chicken ovalbumin), rather than a
pathogen, is the “antigen”, or substance that induces an antibody response
response. (“model antigen”)
A serum sample from an immunized rabbit contains a substance with the capacity
to bind to ovalbumin, called “antibody”.
Serum contains many different proteins. Which serum fraction contains the
antibody?
•Tiselius & Kabat: 1930s. Discovered that the g-globulin fraction of
serum contains antibodies.
Immunize rabbits with chicken ovalbumin. Isolate serum, divide into 2 parts.
Separate one serum sample into different protein fractions (based on size, charge) using
electrophoresis.
Mix another serum sample with ovalbumin and remove the insoluble immune complexes
(containing ovalbumin plus serum proteins that bind ovalbumin).
Re-analyze depleted serum by electrophoresis and compare to the starting serum sample.
Blue: serum
from immune rabbit
Black: serum with specific
binding activity depleted
The g-globulin fraction of
serum contains most antibody:
Termed “immunoglobulin”
Antigens & Antibodies I
Discovery of antibodies
Basic Antibody Structure
brief review of protein structure
disulfide linked tetramer: 2 heavy and 2 light chains
myeloma proteins and the primary structure of antibody
crystal structure of antibody: the Ig domain
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgE
The advantages of multivalency
effector functions of antibody isotypes
Levels of Protein Structure
Non-covalent forces that hold
antigens and antibodies together
Energy of chemical interactions
involved in stability of protein
structure
Covalent
Ionic
Hydrogen
Van der Waals
Hydrophobic
Bond Length
0.15 nm
0.25 nm
0.30 nm
0.35 nm
0.35 nm
Energy
90 kcal/mol
3 kcal/mol
1 kcal/mol
0.1 kcal/mol
0.1 kcal/mol
The Alpha-Helix
The Beta-Sheet
Ribbon Diagrams
Antigens & Antibodies I
Discovery of antibodies
Basic Antibody Structure
brief review of protein structure
disulfide linked tetramer: 2 heavy and 2 light chains
myeloma proteins, Ig domains, and hypervariable regions
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgE
The advantages of multivalency
effector functions of antibody isotypes
Basic Immunoglobulin (antibody) structure
Biochemical methods to
characterize IgG structure
(Porter and Edelman 1960s)
1972 Nobel Prize for medicine:
partial proteolysis
chromatography
Fab: fragment antigen binding
Fc: fragment crystalizes
cleavage of disulfide bonds
chromatography
Heavy chain ~50 Kda
Light chain ~25 Kda
Basic Immunoglobulin (antibody) structure
2 identical
antigen binding
sites/molecule
Each antibody molecules consists of two
identical heavy chains (50 kDa) and two
identical light chains (25 kDa).
Chains are held together by inter-chain
disulfide bonds.
Structure made up of repeating structural
units of ~110 amino acids called Ig
domain (2/light chain and 4/heavy chain).
N-terminal Ig domain is variable (antigen
binding domain). The C-terminal Ig
domains are constant (effector functions).
Basic Immunoglobulin (antibody) structure
Antibodies are bivalent (2 identical antigen binding sites).
Membrane-associated vs.
secreted immunoglobulin
Distinct carboxy-termini
Basic Immunoglobulin (antibody) structure
2 identical
antigen binding
sites/molecule
Each antibody molecules consists of two
identical heavy chains (50 kDa) and two
identical light chains (25 kDa).
Chains are held together by inter-chain
disufide bonds.
Structure made up of repeating structural
units of ~110 amino acids called Ig
domain (2/light chain and 4/heavy chain).
N-terminal Ig domain is variable (antigen
binding domain). The C-terminal Ig
domains are constant (effector functions).
Variations in the heavy chain lead to
different antibody isotypes, and membrane
vs secreted forms of antibody.
Antigens & Antibodies I
Discovery of antibodies
Basic Antibody Structure
brief review of protein structure
disulfide linked tetramer: 2 heavy and 2 light chains
myeloma proteins, Ig domains, and hypervariable regions
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgE
The advantages of multivalency
effector functions of antibody isotypes
In a normal individual, antibodies are extremely heterogeneous.
Myeloma protein: key to determining Ig
structure
• Heterogeneity of antibodies makes sequencing
impossible (each B cell clone produces a unique version
of antibody).
• Multiple myeloma: cancer derived from an antibody
producing cells (plasma B cell).
• Myeloma patients have large amounts of one particular
Ig molecule in their serum (and urine)
• Many patients produce a large amount of one light chain,
known as “Bence-Jones” proteins.
When the amino acid sequences of several different
Bence-Jones proteins were compared, they were found to
consist of two repeating units of ~110 amino acids: one
variable and one constant.
Antibody
molecules are
composed of
repeats of a single
structural unit
known as the
“immunoglobulin
domain”
Protein homology
• Identity or similarity between domains in two or more
proteins
• Most easy to see at the level of primary amino acid
sequence (computer programs find it)
• Sometimes no obvious primary sequence homology but
striking structural homology
• Homology can sometimes predict structure and function
All Ig domains have a
similar 3D structure
known as an
“Immunoglobulin Fold”.
2 -pleated sheets come
together to form a
sandwich, held together
by disulfide bond and
hydrophobic interactions.
3 flexible loops at end:
correspond to
hypervariable regions of
primary sequence (HV).
The Immunoglobulin
Fold is a very commonly
used structural motif
amongst cell surface proteins
Ig domain: Genome Project Champion!
The variability of antibodies occurs within 3 discrete regions
of the primary sequence: hypervariable regions HV1-3
The hypervariable regions (HV1-3) are separated in primary structure,
but come together in the tertiary structure where they form the antigen
binding site. Alias Complementary Determining Regions or CDR1-3.
The HV regions form
loops at the end of the Ig
domain.
The intervening framework regions (FR1-4)
make up the rest of the
structure.
The quaternary structure of immunoglobulin
Associations between Ig domains.
Interchain disulfide bonds
Hinge region allows flexible movement
of Fc regions
6 CDR (3 from HC, 3 from LC) combine
to make up antigen binding site
Because the CDR are highly variable, each antibody molecule
has a unique antigen binding site with its own dimensions and
complementarity.
Antibodies that
bind to large
proteins antigens
Antibodies that
bind to small
molecules