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Fundamental Immunology
Module 1:
Innate and Adaptive
Immune Responses
Danielle PEREZ-BERCOFF
[email protected]
BioTech 3
2019-2020
Introduction
3 Modules:
•
Innate immune response
Initiation of adaptive immune response
CD4 T cells
Danielle Perez Bercoff
•
T and B ontogeny
Humoral response (B cells) (adaptive)
Danielle Perez Bercoff
•
Cytotoxic responses
- cellular response : CD8 T cells (adaptive)
- NK cells (innate)
??
Module 1: Course outline
• Introduction to Immunology
• Innate Immunity
• The Complement System
• Innate response: Cellular effectors
- Cellular effectors
- Immune sensors
- Induction of co-stimulatory molecules
• HLA and antigen presentation
- Antigen presentation by HLA
- Antigen processing
• Activation of T lymphocytes
Danielle Perez-Bercoff
Biotech3:2019-2020
Module 1: Course outline
• Introduction to Immunology
• Innate Immunity
• The Complement System
• Innate response: Cellular effectors
- Cellular effectors
- Immune sensors
- Induction of co-stimulatory molecules
• HLA and antigen presentation
- Antigen presentation by HLA
- Antigen processing
• Activation of T lymphocytes
Danielle Perez-Bercoff
Biotech3:2019-2020
Introduction
Immunology and vaccinology history
Edward Jenner
Late of 18th century: Edward Jenner  beginning of immunology
Vaccinia (mild disease of cowpox) seemed to confer protection
against smallpox (often fatal disease)
1796: inoculation with cowpox protected the recipient against smallpox
 concept of vaccination
1979: WHO announces the eradication of smallpox
Immunology and vaccinology history
Introduction
Robert Koch
Paul Ehrlich
Nobel Prize 1905 Nobel Prize1908
Robert Koch :
Founder of bacteriology.
“infectious diseases are caused by
specific microorganisms"
Identified infectious nature of
Tuberculosis, Malaria & Anthrax
Tuberculosis is caused by a
bacterium (Koch’s bacillus): first to
prove it by staining the pathogen,
using Ehrlich’s staining methods
Louis Pasteur
In the 1880s, Louis Pasteur
developed a vaccine against
cholera in chicken (attenuation)
Then developed a
rabies vaccine 
spectacular
success upon its
first trial in a boy
bitten by an
infected dog
Introduction
Basic principles of Immunology
Bacteria
Viruses
Fungi
Induction of
defense
mechanisms
1
Breach in natural
barrier
Innate Response =
Immediate and non specific
Local
=> Inflammatory reaction
• primary effectors
• cellular effectors
2
Adaptive Response =
Specific, acquired
Need for time
= latency
Introduction
2 axes of immune response: overview of immune effectors
Immediate elimination of
pathogens
Non specific response
Activation of adaptive IR
Not adaptable
• Monocytes/M
• Dendritic cells
• Neutrophils, basophils,
eosinophils…
• Platelets
• NK, NKT,
• T-cells
Specific response
Immune memory
•
•
•
•
Complement
Collectins
Cytokines
Chemokines
• T Lymphocytes
• B Lymphocytes
• Antibodies
• Cytokines
• Chemokines
Humoral = soluble
Innate Immune Response
3 phases in mammalian immune response
The response to an initial contamination occurs in three phases:
1
2
• Anatomi barrier
• antimicrobial enzymes & peptides
• Complement system
Cells (innate IR):
PAMP  PRR
3
Adaptive IR
• B + T lymphocytes
• Expansion of Ag-specific lymphocytes
• memory
Introduction
Basic principles of Immunology: effector cells
Introduction
Basic Principles in Immunology: Immune System Network
Innate Immunity
Adaptive Immunity
monocytes
macrophages
CD4 T lymphocytes
basophils
eosinophils
neutrophils
CD8 T lymphocytes
Dendritic cells
Induction of co-stimulatory
molecules
platelets
B lymphocytes
NK , iNKT cells
Soluble Factors
Cellular interactions
Introduction
Lymphopoïesis
Basic Principles in Immunology: Immune cell precursors
Adaptive IR
Innate IR
Introduction
Basic Principles in Immunology: Ilymphoid organs
Compartmentalized immune system
Primary lymphoid organs (central):
• Bone marrow: hematopoiesis &
B cell ontogeny
• Thymus: T-cell ontogeny
Secondary lymphoid organs
(or peripheral):
Initiation of Adaptive Immune Response
lymph nodes (axillary, inguinal,
mesenteric)
spleen
MALT (Peyer‘s patches)
+ Mucous membrane:
skin, intestine, rectum, respiratory
system …
Trigger of innate response + effector
adaptive response
Introduction
innate Immunity
Innate Immunity
Becomes an Antigen
Presenting Cell (APC)
& initiates adaptive
Immune Response
Epithelial cells
Neutrophils
Natural Killer
Macrophages
Pro-inflammatory cytokines & chemokines
Dendritic cell
Introduction
Basic Principles of Immunology: adaptive immunity
Adaptive Immunity
Active
Natural
Infection
Induced/artificial
Vaccination
Passive
Natural
Tansfer of maternal
antibodies
Induced/artificial
Ig
Introduction
Basic Principles of Immunology: adaptive immunity & memory
Immune memory
- Immune memory is mediated by T and B cells
Primary response
≈ 1st infection, vaccination
Secondary response
≈ 2nd infection, vaccination boost
Faster
more intense
longer
more diversified
Clonal
expansion
Clonal
contraction
Latency
1st injection
2nd injection
Time
Introduction
Definitions
Antigen (Ag): Substance (toxin, protein) that stimulates an immune response (IR) in
the body
It is able to specifically react with an antibody or a cellular receptor and able to
stimulate antibody production or a cellular reaction
Antigen capacity to induce an immune response = immunogenicity
Antigen capacity to specifically combine with Ab or T cell receptors = antigenicity
Antigen specificity: antigen property to react specifically with a particular antibody or
T receptor recognition of a particular antigen structure, the epitope – the site on the
surface of the antigen to which an antibody attaches
Antibody (Ab): Substance produced in response to antigen administration and
able to specifically bind with this antigen
After a second infection or injection, the immune response is faster and more intense.
It is the immunological memory
CELLS of the immune system: All the cells of the immune system are leucocytes
originating from the bone marrow from a common precursor, the pluripotent stem cells.
Some cells are mature when they leave the bone marrow (BM). Other cells complete
their differentiation outside the bone marrow.
Introduction
Immune system consequences
BENEFICIAL
HARMFUL
- Elimination of infectious pathogens
- Elimination of tumoral cells
- Immune surveillance
- Hypersensitivity (allergy)
- Inflammation (chronic diseases)
- Auto-immune diseases
Module 1: Course outline
• Introduction to Immunology
• Innate Immunity
• The Complement System
• Innate respone: Cellular effectors
- Cellular effectors
- Immune sensors
- Induction of co-stimulatory molecules
• HLA and antigen presentation
- Antigen presentation by HLA
- Antigen processing
• Activation of T lymphocytes
Danielle Perez-Bercoff
Biotech3:2019-2020
Innate Immune Response
The agents that cause disease
Five groups of pathogen: viruses, bacteria, fungi, protozoa and helminths (worms)
Two major compartments: extracellular and intracellular
Cell damage or killing
Innate Immune Response
The agents that cause disease
Different ways for pathogens to damage tissues
Non-secreted constituents of
bacterial structure
Lipopolysaccharide (LPS) of
the outer cell membrane of
gram-neg bacteria  many of
the symptoms of infection
(fever, pain, rash, hemorrage,
septic shock) are due largely to
LPS
2
Innate Immune Response
Natural barriers: epithelial surfaces
Epithelia = physical barrier
• Skin
• mucosal epithelia : respiratory, urogenital and gastrointestinal tracts
• Tight junctions: seal against external environment
• Cilia, flow and movement (peristalsis in the gut)
2
Innate Immune Response
Natural barriers: epithelial surfaces
Epithelia = physical barrier
+ microbiological barrier
gut commensal flora (= 1014 bacteria)
skin, mouth commensal flora
produces antimicrobial substances
strengthens the barrier functions of epithelia
2
Innate Immune Response
Natural barriers: epithelial surfaces
Epithelia = physical barrier
+ microbiological barrier
+ chemical barrier:
Mucus (prevents microorganism adhesion)
pH (skin, stomach)
Digestive enzymes and fatty acids
Soluble factors (antibacterial enzymes and peptides)
Innate Immune Response
Epithelial damage
Pathogens overcome innate host defenses to establish infection
Spread countered by
inflammatory response
& blood clotting
Introduction
Basic principles of Immunology
Humoral = soluble
Innate Immune Response
Primary effectors : antibacterial enzymes
Antimicrobial proteins:
- secreted by epithelial cells and phagocytes
- mucosal tissue protection
Lysozyme
(tears, saliva, phagocytes, paneth cells)
Enzyme: glycosidase
 cleavage of peptidoglycan component of
the bacterial cell wall
 more effective on Gram (+) bacteria than
Gram (-) because of LPS layer
Secretory phospholipase A2
(paneth cells)
Highly basic enzyme
Insertion in bacterial cell wall
 phospholipid degradation
 Death of bacteria
PLA2
Innate Immune Response
Primary effectors : antimicrobial peptides
Antimicrobial peptides: - ancient, evolutionnary conserved peptides
- generated by proteolytic processing from inactive propeptides
α and β-Defensins (amphipatic structure)
(epithelial cells, phagocytes)
Disrupt the cell membrane of bacteria and form
a pore within minutes
Cathelicidins
Reg III “Lecticidins” (C-type lectins)
Innate Immune Response
Primary effectors
Platelets:
- coagulation, wound healing
- production of several mediators:
* vaso-active:
histamines – serotonine
dilation → œdema
constriction → pain
* inflammatory:
prostaglandin - leucotriene – chemoattractants
Collectins : Surfactant protein A & D (SP-A and SP-D), MBL
- scavenger receptor
- present in fluid that bathes epithelial surface of the lung
- Pathogen opsonization  neutralization + phagocytosis
- Activation of complement cascade
Opsonization: process of coating the surface of pathogens, making them more susceptible
to phagocytosis by macrophages
Module 1: Course outline
• Introduction to Immunology
• Innate Immunity
• The Complement System
• Innate respone: Cellular effectors
- Cellular effectors
- Immune sensors
- Induction of co-stimulatory molecules
• HLA and antigen presentation
- Antigen presentation by HLA
- Antigen processing
• Activation of T lymphocytes
Danielle Perez-Bercoff
Biotech3:2019-2020
Innate Immune Response
Complement system - Introduction
• Complement system :
• Enhances (complements) the ability of
antibodies and phagocytes to clear
microbes and damaged cells
• Discovered in the 1890s by Jules
Bordet as a heat-labile substance
(loss of ability to neutralize cholera
when serum is heated)
• 30 soluble proteins
(liver production, plasma circulation)
• Enzymatic cascade
the majority of proteins are proteases
• highly regulated system
Jules Bordet
Nobel Prize 1919
• Serum from Guinea pigs
surviving immunization with
cholera kills the bacterium
in vitro.
• Heat removes this ability
• Protective activity
maintained when heated
serum injected to new Gp
2 components:
Heat-labile
+ heat-resistant
Believed complement was
aspecific.
Paul Ehrlich
Nobel Prize1908
• Introduced the term
complement
• His theory: immunization
induces specific receptors
(Ab) on cells, which are
shed
• Named amboceptors b/c
bind Ag + complement
(heat-labile component)
• Believed each Ag had its
own specific complement.
Believed complement was
specific.
Complement can act in combination
with Ab or on its own
Innate Immune Response
Complement system - Nomenclature
Nomenclature :
• Small molecules circulating in blood, produced mainly by hepatocyes or M
• Synthesized as inactive proteins which are activated by proteolytic cleavage
and activate one-another = zymogens (pro-enzymes)
• Inactive complement molecules : Cn factor for classical pathway
or letter for alternative pathway
• Active complement molecules with suffix ‘a’ (small fragment) or 'b’ (large fragment)
• Ex: C4 is cleaved into
C4a (small fragment) &
C4b (large fragment with catalytic activity)
• Exceptions:
historically C2a was the large fragment with
catalytic activity, but now called C2b.
C1q, C1r and C1s  3 different components
making up C1
• Complement receptor: CR
Innate Immune Response
Complement system – Complement cascade
Inactive zymogens
infection
Activation of the cascade by serial proteolytic cleavage
Amplification of the signal
Stimulation of
phagocytes to
clear Ag
via
opsonzation
Inflammation
to attract
additional
phagoytes &
neutrophils
Cell or
pathogen lysis
by Membrane
Attack Complex
(MAC)
Large
fragment
Small
fragment
Large
fragment
C3b
C3a, C4a, C5a
C5b
Innate Immune Response
Complement system - Introduction
• 3 biochemical systems
activate the complement
cascade:
1. Lectin (MBL & ficolins)
2. Classical (C1q – Ab)
3. Alternative (spontaneous
hydrolysis)
• C3 convertase:
- Cleaves C3  C3a + C3b
- Lectin & Classical pathways: C4bC2b
- Alternative pathway: C3b(2)Bb
• C3b:
- main central molecule
(coating of bacteria)
- Influences adaptive immunity:
 Opsonization  enhances
pathogen presentation
(Ag) to T cells by APCs
 B cells express
complement Rec (CR)
Inflammation
Phagocytosis
Lysis
Chap I. Innate Immune Response
Complement system - Classical pathway
Classical pathway
• Initiated by C1 complex: large subunit C1q (pathogen sensor)
+ 2 serine proteases (C1r and C1s)
= C1qr2s2
• C1q: Collectin
N-terminal globular domain
C-terminal collagen-like domain is a hexamer of trimers
• Main function of C1q is to bind to the constant region of
antibodies (Fc portion) attached to pathogens via their Agbinding-site (Fab)
 links complement to recognition provided by adaptive
immunity (natural Ab = Ab produced without infection)
• Mainly binds pentameric IgM and IgG
(ideally 6 IgG complexed to Ag)
• C1q can bind to the surface of pathogens
• Acts as link between innate & adaptive immunity
Chap I. Innate Immune Response
Complement system - Classical pathway
Classical pathway
• Binding of C1q globular heads to pathogen  conformational change in (C1r:C1s)2
 Activation of the C1r (serine protease)  autocleavage
 C1r cleaves the 2 C1s (serine protease)
 C1s cleaves:
C4  C4a + C4b
C2  C2a + C2b
Binding of C4b + C2b = C4b2b
= classical C3 convertase
C3
C3b
C3a
N.B: C2b is
the active
component
of C3 & C5
convertases
Innate Immune Response
• Similar to Classical pathway
Instead of C1q
•
Complement system - Lectin pathway
Lectin pathway
Initiated by:
- Mannan-Binding Lectin (MBL)
a collectin synthesized in the liver
N-terminal collagen-like domain
C-terminal C-type lectin domain
Assembled in trimers
High Avidity for repetitive carbohydrate
structures present on microbial surface
- Ficolins (L-, M- and H- ficolin)
Synthesized in liver, lung and blood cells
N-terminal collagen-like domain
C-terminal fibrinogen-like domain
Specificity for acetylated sugars
•
Associated with serine protease:
MBL-associated serine protease (MASP-1 & -2)
instead of C1r & C1s
•
Binding of MBL to mannose activates MASP-1
(~C1r)  Activation of MASP-2 (~C1s)
Chap I. Innate Immune Response
Complement system - Classical pathway
Lectin pathway
• Binding of MBL or Ficolin to pathogen surface  conformational change in MBL
 Activation of MASP-1 (serine protease)
 MASP-1 cleaves MASP-2 (serine protease)

MASP-2 cleaves:
C4  C4a + C4b
C2  C2a + C2b
Binding of C4b + C2b = C4b2b
= C3 convertase
C3
C3b
C4b2b
C4b2b
C4b2b
C2b
C4b2b
C4b2b
C3a
Innate Immune Response
Complement system - Alternative pathway
Alternative pathway : amplification of C3b
• Alternative pathway C3 convertase : C3bBb
• Special place because it can generate more of itself  once some C3b has been formed
by whichever pathway, the alternative pathway acts as an amplification loop   C3b
• Activation by two different ways: * classical or lectin pathway :
C3b linked to surface of pathogen alters the conformation of factor B
 factor D (a protease) cleaves Factor B into Ba + Bb = C3 convertase (C3bBb)
* spontaneous hydrolysis
Innate Immune Response
Complement system - Alternative pathway
Alternative pathway: Spontaneous hydrolysis
Spontaneous hydrolysis (C3H2O) due to breakdown of internal thioester bond
If no pathogen
C3b rapidly
inactivated by
Factor H & Factor I
If Pathogen ++++
Hydrolyzed C3b reacts with –OH or -NH3+ group on
surface of pathogen
 No lysis by factor H
 Binding of factor B
 Factor B is cleaved by Factor D (protease)
 Alternative C3 convertase C3bBb or C3(H2O)Bb
 C3bBb is stabilized by properdin (factor P)
Innate Immune Response
Complement system - Alternative pathway
Amplification of C3b lysis
•
Stabilized C3 convertase C3bBbP cleaves more C3
 C3b binds B, D & P
 (+) feedback loop & signal amplification
Alternative pathway acts as amplifcation system
Role of C3 convertase : decorate Bacteria with C3b
•
C3bBb accumulates on non-self surface & binds a 2nd C3b
 C3bBbC3bP = C5 convertase
•
C3 signal on cell surface is limited by endogenous
regulatory proteins : CD35, CD46, CD55 (DAF), CD59
 Compete with Bb for binding C3b (Factor H, CR1, DAF)
or cleave it (Factor I)  no self-destruction
•
Pathogens lack complement inhibitors
 Distinction between self and non-self
•
N.B: some pathogens evade complement response by
expressing complement regulatory proteins
Innate Immune Response
Complement system - C3b & C5 convertase
• C3 & C5 convertase activity:
C4b2b
• Different C3 convertases cleave C3 to form C3b + C3a
 C5 convertase =
- Classical pathway = C4b-C2b-C3b
- Alternative pathway = C3b2-Bb
C3a intiates local
inflammation
• opsonizes
pathogen to
facilitate
phagocytosis
C4b2b3b
C4b2b3b
C3b
• C4b2b3b &
= C5
convertase
C3bBb3b
• C5 convertases bind C5
 cleave C5
• N B: C3 is the most abundant complement protein
in plasma (1.2 mg/mL)  up to 1000 molecules of C3b
can bind in the vicinity of a single active C3 convertase
C4b2b3b
C2b
C4b2b3b
Innate Immune Response
Complement system
C3 convertase:
-
Classical pathway = C4bC2b
Alternative pathway = C3bBb
C5 Convertase:
-
Classical pathway = C4bC2bC3b
Alternative pathway =C3b2Bb
Innate Immune Response
Complement system - opsonization and phagocytose
First function of complement system: opsonization
•
Facilitate the uptake and destruction of
pathogens by phagocytic cells
•
Occurs through the specific recognition
of bound complement by complement
receptors (CRs)
* CR1 (CD35) = C3b Rec with the help of
C5a (Rec for C5a is G-prot)
* CR2 (CD21), CR3 (CD11b), CR4
(CD11c) and CRIg bind C3b or its
inactive forms
• Phagocytosis by M:
 pathogen destruction
Phagocytosis by DCs:
 pathogen destruction + Ag presentation
•
deficiency in C3  susceptibility to
infection by a wide range of extracellular
bacteria (Streptococcus pneumonia)
Cellular distribution of CR
CR1 (CD35) : mono, PMN, B cells, fDC
CR2 (CD21) : B cells, fDC
CR3 (CD11b/CD18) : mono, fMN, DC
CR4 (CD11c/CD18) : mono, PMN, DC
Innate Immune Response
Complement system - Local inflammatory response
Second function of complement system: local inflammatory response
•
The small complement fragments C3a &
C5a act on Chemokine (CC) Receptors on
endothelial cells and Mast cells to produce
inflammatory response
•
N.B: If produced in a large amounts, C5a &
C3a could induce anaphylactic shock
 referred to as anaphylatoxins
(C5a > C3a > C4a)
• Increase vascular permeability
• Induce contraction of smooth muscles
•
Induce synthesis of adhesion molecules
by endothelial cells
•
Induce release of inflammatory molecules
(TNF-) by Mast cells
•
Recruit Ab, complement and phagocytic
cells out of blood to the site of infection
•
Increased fluid hastens the movement of
APC to the LN  initiation of adaptive IR
Chap I. Innate Immune Response
Complement system - Membrane-Attack Complex (MAC)
Third function of complement system: MAC
•
First step: C5 convertase cleaves C5
 release of C5b
•
C5b triggers assembly of a complex of
one molecule of each C6, C7 & C8
•
Conformational change of C7
 hydrophobic site inserted in the
membrane
•
C8 binds to C5b
 hydrophobic domain of C8-
inserts in mb & hydrophilic domain
of C8- induces polymerization
•
Up to 16 molecules are added to the
assembly  generate a channel
 disrupt the bacterial cell membrane
and kill the bacteria
•
N.B: these components seem to be quite limited in host defenses  opsonizing and inflammatory
actions of the complement system are more important for host defense against infection
Chap I. Innate Immune Response
Complement system - Regulation
Regulation of complement activation
• Activation events must remain confined to site of infection
/bacterium where it is initiated
• Cleavage of C4b exposes a highly reactive thioester bond
(thioester bond: S-C=O)
 C4b can bind covalently carbohydrates on pathogen or
adjacent proteins
 If no bond, thioester bond is cleaved by H2O
 inactivation
• C2 susceptible to cleavage by C1s only when bound to C4b
 C2b active only when bound to C4b i.e in form of C3
convertase C4b2b
Innate Immune Response
Complement system - Regulatory proteins
27
Regulatory proteins of complement system
(on cell surface: avoid cleavage of cells)
• C1 inhibitor (C1 INH, serpin) = a serine
protease inhibitor
C4b2b
b
C2b
C4b2b
• DAF(CD55), C4BP and CR1 displace C2b
from C4b + cleave C4b to an inactive form
C4b2b
• Factor I (protease) cleaves C3b to
iC3b (with help of co-factors)
• CR1 (CD35) and Factor H displace C3b
and act as cofacteurs in the cleavage of
C3b by Factor I
• CD59 prevents final assembly of MAC at
the C8 to C9 stage
C4b2b3b
C4b2b
Innate Immune Response
Complement system - Regulatory proteins
Summary of Regulatory proteins
b
b
Regulation of all three pathways of complement activation to protect the host
from destructive effects
Innate Immune Response
Pathogens produce several types of
proteins that can inhibit complement
activation:
• Express proteins that bind to soluble
regulatory proteins (C4BP or Factor H)
Ex: Factor H binding protein (fHbp) & PorA
Neisseria meningitidis inactivation of C3b
• Secrete proteins that directly inhibit
components of complement
Ex: Staphylococcus aureus produces:
- Staphylococcal protein A (Spa) binds to
Ab and interferes with C1 recruitment
- Staphylokinase (SAK) cleaves Ig
 Inhibition of complement activation
+ avoid phagocytosis
- Staphylococcal component inhibitor
(SCIN) binds C3 convertase
 Inhibition from classical & alternative
complement pathways
Inhibition of complement by pathogen
Innate Immune Response
Complement system – summary Complement cascade
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