Immunology:

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Immunology the only science where you can
still get a Nobel prize with two test-tubes and
a mouse
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Immunology the science of why you dont
get measles twice
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Defense against Disease
Non-specific and specific strategies
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The Challenge

• Pathogens microorganisms that are capable of
  causing disease
• Viruses
• Bacteria
• Fungi

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The Viruses

• Modus Operandi
• Dock with receptors on target cell surface
• Insert viral DNA or RNA into host cell
• Use host cell machinery to replicate new
  viruses
• Lyse host cell and spread to nearby cells
• Lytic vs. Lysogenic life cycles
• Examples smallpox, chickenpox, polio, HIV

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Bacteria

• Modus operandi
• Set up shop in tissues but remain EXTERNAL
  to cells
• Reproduce rapidly
• Secrete exotoxins or contain endotoxins as
  part of cell wall
• Examples Escherichia coli, Clostridium
  botulinum, Salmonella

Figure from Holt Biosources
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Fungi

• Modus Operandi
• Similar to bacteria-
• reproduce rapidly
• Damage cells directly or indirectly
• by secreting enzymes
• Examples Athletes Foot,
• Pneumocystis carinii (fungal pneumonia)

http//www.ces.ncsu.edu/depts/pp/bluemold/
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• First line defenses Nonspecific anatomical
  barriers and secretions that prevent entry, such
  as skin, saliva, tears (lysozyme), mucus, stomach
  acid, fever
• Second line defenses Inflammation
• A nonspecific response triggered by histamine
  secreted by basophils when tissue is damaged

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If all else failsThe Immune Response

• A highly specific, long lasting response tailored
  to combat pathogens
• Vocabulary
• Antigen- a molecule (usually carried on the
  surface of a pathogen) that is capable of
  eliciting an immune response
• B-Lymphocytes- white blood cells that produce
  and secrete antibodies
• T-Lymphocytes- white blood cells that serve as
  part of the cell-mediated immune response

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Self- Nonself Recognition

• Critical to appropriate immune system function
• Tcells learn to distinguish self from non self
  as they mature in the thymus
• All nucleated self cells display unique Human
  Leukocyte Antigens (HLA)
• As T cells mature, they produce and display a
  variety of randomly-defined receptors
• Any T cell with receptors that bind to self
  MHC-HLA complexes will be deleted in the thymus
• Only T cells that do NOT bind to self cells with
  high affinity will emerge from the thymus and
  enter circulation

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Immune Response- Step by Step
1. Pathogen (carrying foreign antigens) enters
and survives the inflammatory response 2. Some
pathogens remain exposed in tissues where their
antigens may be recognized by circulating B
cells OR 3. Macrophages engulf pathogens and
display their antigens on MHC (major
histocompatibility complex) proteins. Macrophage
has now become an Antigen Presenting Cell (APC)
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In a specific immune response, only those T and B
cells that can bind to the antigens of the
pathogen are selected to participate in the
response.
Proliferation of cells with receptors capable of
binding epitopes of the antigen
Mixture of T and B cells with different
antigen specificities
An antigen with 2 epitopes - red epitope, blue
epitope
Clonal selection of lymphocytes during the
specific immune response
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Cell-Mediated ImmunityT cell Response

• Helper T cells (a.k.a. TH or CD-4 T cells)
  constantly interact with macrophages
• When TH cell finds a macrophage that is
  presenting antigen (APC) it becomes activated
• Activated TH cells secrete cytokines, proteins
  that stimulate both T and B cells
• Stimulated cytotoxic T cells (a.k.a. killer or
  CD-8 T cells) divide rapidly, bind directly to
  pathogen infected cells and secrete enzymes that
  lyse infected cells

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Central Role of Helper T Cells
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Humoral ImmunityB cell response

• If a circulating B cells receptors bind to
  foreign antigens, the B cell becomes activated
• Activated B cells divide into Memory B cells and
  Plasma B cells
• Plasma B cells rapidly produce and secrete
  antibodies (immunoglobulins)
• Clonal selection amplifies the production of
  cells that produce effective antibodies

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Immunity
1. specificity 2. tolerance 3. memory
Paradigms self vs
non-self protection vs kill
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Cells of the Immune System
ne
baso
T
white blood cells
MO
eos
B
NK
CD4
MAC
DC
MHC II
CXCR4
CD8
CCR5
MHC II
MHC I
TCR
MHC I
CD3
CD3
TCR
Helper T cell
Cytotoxic/ suppressor T cell
Derived from JM Austyn KJ Wood Principles of
Cellular and Molecular Immunology 1993
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The Immune System
LYMPH NODE
SKIN / MUCOSAL SURFACE
migration
migration
T cell area
Cytotoxic T cells
Th2
B
DC
B
B cell area
Th2
pathogen
B
B
B
Isotype switch affinity maturation
Cellular immunity
INFLAMMATION
IgM IgG IgA IgE
migration
B
Bone Marrow
B
Antibody response
AFC
memory
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Immunity
Innate Immunity
Adaptive Immunity
Barriers (skin) Secretions (lysozyme) Complement I
nflammation Granulocytes NK cells Macrophages Phag
ocytosis
B cells-- Antibody production T cells-- Cell
mediated immunity Immunoregulation
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Overview
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Generation of an adaptive immune response

• During an adaptive immune response,T cells which
  recognize specific antigen(s) are selected for
  differentiation into armed effector cells which
  undergo clonal expansion to produce a battery of
  antigen-specific cells.
• Clonal expansion refers to the process by which
  antigen-specific T cells or B cells are
  stimulated to reproduce clones of themselves to
  increase the systems repertoire of
  antigen-specific effectors.
• Activation of antigen-specific T cells (the
  initiation of the adaptive response) occurs in
  the secondary lymph tissues (lymph nodes and
  spleen).
• This activation depends upon antigen presentation
  by a professional antigen presenting cell (APC)
  along with simultaneous co-stimulation. (eg., B7
  on the APC, CD28 on the T cell).

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Antigen Presentation
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Antigen recognition by T and B cells T and B
cells exhibit fundamental differences in antigen
recognition.
B cells recognize antigen free in solution
(native antigen). T cells recognize antigen
after it has been phagocytosed, degraded and
small pieces of the antigen have been bound by
MHC molecules.
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Antigens and epitopes
Figure 1
Concept C.M. Gray, PhD
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"B-lymphocytes have sIg molecules that recognize
epitopes directly on antigens. T-lymphocytes have
TCR molecules that recognize epitope only after
it has been bound to MHC molecules."
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T cell epitopes
T cell epitopes always consist of sequential
amino acids
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Properties of T cell epitopes - linear peptide
fragments of the antigen consisting of 11-20
amino acids - must be able to bind
simultaneously to the T cell receptor and the MHC
molecule - often amphipathic (having both
hydrophobic and hydrophilic residues)
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The Immune system
Class I
Class II
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?
-N
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MHC structure

• MHC classes I and II have an almost identical 3-D
  structure.
• Both classes of MHC are polygenic (each cell has
  many MHC genes) and polymorphic (there are many
  alleles for each locus), but the MHC genes do not
  undergo recombination.
• Note Human MHC are called HLA (human leukocyte
  antigen).

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MHC / T cell interactions
Class II MHC
Class I MHC
target cell
Antigen presenting cell
CD8
CD4
CD4 T cell
CD8 T cell
TCR complex
TCR complex

• The MCH/peptide-TCR interaction is facilitated by
  the CD4 or CD8 co-receptor.

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ANTIGEN-PRESENTING CELLS
Present antigen to T cells in conjunction with
MHC molecules and also provide co-stimulatory
signals
STIMULATION
T cell receptor
ANTIGEN-PRESENTING CELL
T CELL
MHC molecule
Antigen peptide
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ANTIGEN-PRESENTING CELLS
1. MACROPHAGES Distributed throughout body,
versatile Not efficient presenters of antigen
until activated When activated (e.g. by IFN-?)
? increase expression of MHC molecules and
co-stimulatory molecules
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ANTIGEN-PRESENTING CELLS
2. DENDRITIC CELLS Most effective
antigen-presenters, distributed throughout
body Derived from three different sources, but
all have characteristic starfish shape Able
to activate virgin T cells (unlike other
antigen- presenting cells)
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2. DENDRITIC CELLS In resting state, express
only small amounts of MHC, but have large
internal reserves of MHC molecules
MHC II
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2. DENDRITIC CELLS Following encounter with
antigen, dendritic cell leaves tissues ?
migrates to lymph node ? loads foreign antigens
onto the reserve MHC molecules ? displays
MHCantigen peptide Production of
co-stimulatory molecules upregulated ? Virgin
helper T cells activated immediately
Foreign antigen
T cell
Travel to lymph node
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ANTIGEN-PRESENTING CELLS
3. ACTIVATED B CELLS B cells can present
antigen to T cells - must be activated first
(to increase expression of MHC II molecules)
B cells bind specific antigens (ie recognised by
their BCRs) ? ingest antigen ? process
antigen ? present Advantage of B cells over
other APCs antigen concentration -
if antigen in small amounts, B cell acts like
magnet to collect and concentrate it
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The antigen presenting cells, continued
Note this B cell is not a plasma cell -- a
plasma cell is shown above. Plasma cells do not
present antigen. They simply pump out antibody
for a few days then die.
Dendritic Cell
Macrophage
B cell
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MHCII
Macrophage (APC)
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1.
Phagocytosis
MHCII
Macrophage (APC)
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Destruction
2.
1.
Phagocytosis
MHCII
Macrophage (APC)
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Infection, Cytokines (e.g. TNF, IFN)
APC Activation
Destruction
2.
1.
Phagocytosis
MHCII
Macrophage (APC)
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Infection, Cytokines (e.g. TNF, IFN)
APC Activation
Destruction
2.
Altered processing
1.
Phagocytosis
MHCII
Peptides
3.
Macrophage (APC)
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Infection, Cytokines (e.g. TNF, IFN)
APC Activation
Destruction
2.
Altered processing
1.
Phagocytosis
MHCII
Peptides
3.
4.
Macrophage (APC)
Antigen Presentation
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Infection, Cytokines (e.g. TNF, IFN)
APC Activation
Destruction
2.
Altered processing
1.
CD4 T cell Activation
Phagocytosis
MHCII
TcR
Peptides
3.
4.
Macrophage (APC)
Antigen Presentation
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Different MHC present different epitopes
MHC TYPES HLA-A2 HLA-A3 HLA-B7
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Super-motifs A1 A2 A3 A24 B7 B27
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T-Cells
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TCR compared to Immunoglobulins

• Similarities
• Both have specific Antigen-binding region created
  by the variable regions of two polypeptide
  chains.
• Both display great potential for diversity via
  genetic recombination at the genome level
• Differences
• A TCR is monovalent (has one binding site). An
  Ig is bivalent (has two binding sites).
• The TCR has no secreted form. It is always
  membrane-bound.
• The TCR does not recognize free antigen. Antigen
  must be presented to a T cell on an MHC molecule
  (next week).
• There is no class switching for the TCR. Once
  made, the TCR does not change.

Epitope-binding site
? chain
? chain
Variable region
Constant region
Transmembrane region
Immunoglobulin
T cell Receptor
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The T cell Receptor, cont.

• The TCR only recognizes specific peptide/MHC
  complexes expressed on the surfaces of cells
• A TCR complex is composed of one heterodimeric
  TCR (ususally ?/?), plus a 5-polypeptide CD3
  complex which is involved in cell signalling for
  T cell activation.
• Each TCR is produced through genetic
  recombination and recognizes one small peptide
  epitope (about 8-13 amino acids).
• One T cell expresses only one specific type of
  TCR.

CD3 is the activation complex for the TCR
Binding of antigen/MHC to the TCR stimulates CD3.
CD3 then sends an activation signal to the
inside of the T cell.
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Clonal expansion of CD4 T cells
antigen presentation by dendritic cells to naïve
CD4 T cells In the lymph node
Priming and activation
dentritic cell
Clonal expansion
Clones of effector CD4 T cells expressing the
same TcR
Antigen removed and host protected
99 of expanded cells die by apoptosis
respond to recall antigens and mount a rapid
secondary response
Memory cells
Figure 2
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T helper Cell Differentiation

• Type 1 response
• immunity to mycobacteria
• inflammation
• rheumatoid arthritis, diabetes

IFNg TNFa

• Type 2 response
• IgE antibody responses
• Immunity to some parasites
• allergic diseases

IL4 IL13 IL10
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Interactions Regulating T helper Cell
Differentiation
IL2
g
IFN


IL2

pathogen
CD40
apoptosis through Fas engagement
-
g
IL4
IFN
THp
APC
-
B7
IL2 IFNg

local microenvironment

IL10
IL2 IL4
IL4/IL10

-
-
TGFb
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Th1 and Th2 Differentiation
IL4
antigen presenting cell
NK cell
IL4R
IL12R
IFNg
CD28
IFNaR

IFNgR



-
apc
-
STAT6

STAT4

STAT1




T-bet
GATA-3
-


T-bet ? Th1
GATA-3 ? Th2
T Cell
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B-Cells
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The Antibody Response
Secondary Response
IgG, IgA, IgE
Primary Response
T Cell Dependent
IgD/IgM
t
Ag
Ag
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Memory Primary Immune Response (10-17
days) Secondary Immune Response (2-7 days)
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Figure 19.6
Diversity During lymphocyte differentiation
Immunoglobulin genes undergo somatic
recombination Produce a coding sequence specific
to each cell (and cell lineage). Hundreds of
thousands of different possibilities within one
individual. Collectively can recognize millions
of different molecules
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ACTIVATION OF B CELLS

• B cell must be activated to produce antibodies
• - T cell dependent
• - T cell independent

BCR
B cell
ANTIBODY
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T CELL-DEPENDENT ACTIVATION
Two signals 1. Recognition of cognate antigen
by BCRs on the surface of the B cell ? clustering
of BCRs ? signal sent to nucleus
antigen
1
B cell
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T CELL-DEPENDENT ACTIVATION
Two signals 2. Helper T cell (Th cell) gives
co-stimulatory signal, especially from direct
contact (ligation of CD40 on B cell by CD40L on
Th cell)
2
1
T cell
CD40
CD40L
B cell
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T CELL-INDEPENDENT ACTIVATION
B cell activated with no help from T cell, in
response to antigens that can cross-link the
BCRs - antigens with repeated epitopes (e.g.
carbohydates on surface of bacteria) - mitogens
(bind to surface of B cells and cluster BCRs,
regardless of specificity of the BCR)
bacterium
mitogen
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B CELL MATURATION
Divided into 3 steps 1. Isotype switching 2.
Affinity maturation 3. Career choice
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1. ISOTYPE SWITCHING
Following gene rearrangement (to determine
specificity of antigen-binding site), the
immature B cell displays 2 types of BCR an IgM
type and an IgD type If the immature B cell
meets its cognate antigen, and if it is
activated, it will start to proliferate and to
produce antibodies (initially IgM). The B cell
can then change the class of antibody it produces
(ie, to IgG, IgA or IgE)
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1. ISOTYPE SWITCHING
To change antibody class, the constant region
of the heavy chains must be changed. (?, µ, a, e
and d) This is done by gene deletion, which
cuts out the µ and d gene segments, bringing the
next segment (e.g. ?) into the V/D/J exon (?
IgG would be produced)
?
?
C? C? C? C? C?
V/J/D
?
C? C? C?
V/J/D
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1. ISOTYPE SWITCHING
Note the V/D/J (variable) region remains the
same, therefore, specificity for antigen
remains the same
Selection of isotype not random influenced by
cytokine environment (which results from
interplay of various other cells) ? production
of most appropriate Ig isotype
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2. AFFINITY MATURATION
Results in increased affinity for the cognate
antigen (better fit) The V, J and D gene
segments have a higher rate of mutation than
other parts of the genome (somatic hypermutation)
?changes in the variable (antigen-binding)
region, which may - increase the affinity of
the antibody for its antigen - decrease the
affinity of the antibody for its antigen -
affinity of the antibody for its antigen remains
the same
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2. AFFINITY MATURATION
The B cells with increased affinity for the
cognate antigen are stimulated more often/more
strongly ? proliferate more than others The B
cells with decreased affinity for the cognate
antigen will not be stimulated, will not
proliferate, and undergo apoptosis End result
growing collection of B cells that produce
antibodies with the closest fit (the best match)
for the cognate antigen
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POOR FIT
apoptosis
BEST FIT
OK FIT
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Specificity Antigens Foreign molecule induces
antibody response Contains several
epitopes Antibodies Immunoglobulins secreted in
response to antigens Each B or T cell
produces one kind and recognize specific epitopes
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3. CAREER CHOICE
(i) Plasma cells - default option -
antibody factories - found in spleen, medulla of
lymph nodes, bone marrow - develop from
stimulated B cells
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3. CAREER CHOICE
(ii) Memory B cells - reserve of
antigen-sensitive cells - results from repeated
ligation of CD40 on B cell (ie, repeated
activation from Th cell) - most memory cells
have undergone somatic hypermutation (ie have
high affinity BCRs) - have lower requirements
for activation (ie are stimulated more easily
and more quickly) THEREFORE secondary immune
response is greater and faster and has high
affinity for antigen
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Virgin B cell
IgM
IgM
IgG
ANTIGEN
IgG
Memory B cells
ANTIGEN
Memory B cells
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