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(review)
Phases of the humoral immune response
ISOTYPE SWITCH
Antibody isotype switching
Throughout the immune response the specificity of an antibody will
be essentially the same (notwithstanding affinity maturation)
The effector function of antibodies throughout a response needs to
change drastically as the response progresses.
Antibodies are able to retain Variable regions whilst exchanging
Constant regions that contain the structures that interact with cells.
Organisation of the functional human heavy chain C region genes
J regions
Cm
Cd
Cg3
Cg1 Ca1 Cg2
Cg4
Ce
Ca2
embrionális
Embryonal
L1 V1 L2 V2 Ln Vn D1 - 12
DNS
DNA
5'
szomatikus
rekombináció
Somatic
recombination
D–
D-J kapcsolódás
átrendeződött
Rearranged
DNA
DNS
L1
V1
Ln
Vn
Cm Cδ Cg3
CM CD CG3
J1-4
J
CA1Cε2
Ca1
CG1
CE2Cg1
CE1 Ca2
CG
2 CG
4 Cε1
CA2
C
g1
Cg4
D1D2 J1J2-4 Cm
CM Cδ
CD
5'
3'
szomatikus rekombináció
Somatic
recombination
V
-D-J kapcsolódás
CM Cδ
CD
D2J1 J2-4 Cm
3'
L1 V1
5'
Primerprimer
RNA transcript
RNS-átirat
mRNA
mRNS
3’
Transcription
transzkripció
5'
L1 V1D2 J1 J2-4 Cm
CM
Cδ
CD
IgM
Cγ1
IgG
Cγ2
IgG
Cγ3
IgG
Modification Cγ4
IgG
AAAA
transzláció
Translation
L V DJ Cm
C
polipeptid
Ig ISOTYPES
Cµ
3'
Processing
átalakítás
Cm
L1 V1 D2 J1 CM
naszcens
Nascent
polypeptide
módosítás
V
C
V–D–J
NEHÉZL
ÁNC (M
)
Heavy
chain
IIgM
Cα
IgA
Cε
IgE
Switch regions
Cm
Sm
Cd
Cg3
Sg3
Cg1
Sg1
Ca1
Sa1
Cg2
Sg2
Cg4
Sg4
Ce
Se
Ca2
Sa2
• Upstream of C regions are repetitive regions of DNA called
switch regions. (The exception is the Cd region that has no
switch region).
• The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n
is between 3 and 7.
• Switching is mechanistically similar in may ways to V(D)J
recombination.
• Isotype switching does not take place in the bone marrow, however,
and it will only occur after B cell activation by antigen and
interactions with T cells.
Switch recombination
Cm
Cd
Cg3
Cg1
Ca1
Cg2
Cg4
Cd
Ce
Cd
Ca2
Sg3
Cg3
Cg3
Cm
Sg1
Cm
Cg1
V23D5J4
Cg3
V23D5J4
Ca1
V23D5J4
Ca1
V23D5J4
Cg3
V23D5J4
Ca1
V23D5J4
Ca1
IgG3 produced.
Switch from IgM
IgA1 produced.
Switch from IgG3
IgA1 produced.
Switch from IgM
At each recombination constant regions are deleted from the genome
An IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1
Cm
L VDJ
Cδ
Rearranged DNA in
Cg2 Cg4 Ce Ca
3'
5'
S
S
S
S
IgM-producing cell
S
Switch regions
Cm
Cδ, Cg2, Cg4
ISOTYPE SWITCH
L VDJ
3'
5'
Ce
Ca
L VDJ
5'
3'
Rearranged DNA in
IgE-producing cell
L
VDJ
Primary RNA
3'
transcript
5'
AAAA
e-Heavy chain
Ce mRNA
All isotype switch
recombination is productive
Different recombination
signal sequences and
enzymes from VDJ
rearrangement
Happens after antigenic
stimulation
Regulated by external
signals, not random
Hyper IgM syndrome Type 2.
Activation Induced Cytidine Deaminase
RNS editing enzyme
NO HYPERMUTATION AND ISOTYPE SWITCH
SOMATIC HYPERMUTATION
MONOCLONAL ANTIBODIES
Monoclonal antibodies
- product of one B-lymphocyte clone
- homogeneous in antigenspecificity, affinity, and isotype
- can be found in pathologic condition in humans
(the product of a malignant cell clone)
- advantages against polyclonal antibodies: antibodies of a given
specificity and isotype can be produced in high quantity and
assured quality.
-therapeutic usage of monoclonals:
anti-TNF-α therapy in rheumatology,
tumor therapy
Possible use of monoclonal antibodies
- Identifying cell types
Immunohistochemistry
Characterization of lymphomas with CD (cluster of differentiation) markers
- Isolation of cells
Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral blood!)
- Blood group determination (with anti-A, anti-B, and anti-D monoclonals)
- Identification of cell surface and intracellular antigens
Cell activation state
- Targeted chemotherapy
CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma
Prevention of organ rejection after transplantation
Monoclonal antibodies as drugs?
Mouse monoclonal antibodies may elicit
an immune response upon administration
in human subjects.
(see immunogenicity-determining factors!)
How can we solve this problem?
VL
J2 gene product
V35 gene product
CDR1
CDR2
CDR3
Complementary Determining Region = hypervariable region
STRUCTURE OF THE VARIABLE
REGION
• Hypervariable (HVR) or complimentarity
determining regions (CDR)
Variability Index
HVR3
150
100
HVR2
HVR1
50
FR2
FR1
0
25
FR3
75
50
Amino acid residue
• Framework regions (FR)
FR4
100
SOMATIC HYPERMUTATION
Day 0.
Ag
Day7 7nap
CDR1 CDR2 CDR3
CDR1 CDR2
IgM
PRIMARY
immune response
Day
14 14
nap
IgM/IgG
Day 14.
Ag
Day
21 21
nap
IgG
SECONDARY
Immune response
Hypervariable regions
Plasma cell
clones
CDR3
1
2
3
4
5
6
7
8
AFFINITY
MATURATION
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ANTIBODY MEDIATED EFFECTOR FUNCTIONS
• Neutralization – binding of the antibody inhibits
the binding of the pathogen to the cell surface,
entry to the cell or multiplication
• Opsonization – binding of the antibody triggers
complement activation and binding to the cell
surface by complement (CR1) and IgG (FcgR)
receptors
• Cytophylic property - antibody isotypes have
distinct complement activating and FcR binding
activity
SECRETED ANTIBODIES BIND TO THE ANTIGEN
COMPLEMENT ACTIVATION
IMMUNE COMPLEX
OPSONIZATION
Ig Fc region
Conformational change?
Association?
FcR
CR
PHAGOCYTOSIS
Macrophage
COMPLEMENT ACTIVATION – classical
pathway
BINDING TO CELLS – cytophilic property
DEGRADATION
ISOTYPE DEPENDENT
IgG1 and IgG3 >> IgG2 és IgG4
ANTIBODY MEDIATED EFFECTOR FUNCTIONS
SPECIFIC ANTIBODY
Bacterial toxin
Bacterium in the
interstitium
Bacterium in the
plasma
Toxin
receptor
Neutralization
Opsonization
Complement activation
COMPLEMENT
Internalization
Phagocytosis
Phagocytosis and lysis
EFFECTOR FUNCTIONS OF ANTIBODIES
INHIBITION
Binding of bacteria to
epithelial cells
Binding of viruses to
receptor
Binding of bacterial
toxins to target cells
NEUTRALIZATION
Small proportion of
antibodies
OPSONIZATION
COMPLEMENT
ACTIVATION
Binding of antibody
increases phagocytosis
PLAZMA CELL
FcgR
Opsonization by C3b
Complement
C3b
FcgR
FcgR CR1
PHAGOCYTES
ENGULFMENT, DEGRADATION
T – CELLS PROMOTE B – CELL DIFFERENTIATION
T-CELL
ANTIGEN
CYTOKINES
B -CELL
PLASMA CELL
ISOTYPE SWITCH AND AFFINITY MATURATION OCCURS IN
COLLABORATION WITH T – CELLS ONLY
HOW T – CELLS RECOGNIZE ANTIGENS?
T CELL ACTIVATION
B- AND T-CELL RECEPTORS SHARE BASIC
STRUCTURE
mIg H
mIg L
a
TCR
T cell receptor
TCR
V
TCRa
C
TCR = a + 
The variable region of the -chain is generated by
gene rearrangements of the V – D – J gene segments
analogous to the generation of IgH diversity
The variable region of the a-chain is generated by the
recombination of V and J analogous to IgL
T-CELL
Single binding
C site
No somatic
mutation
The VARIABLE REGIONS OF a- AND -CHAINS ARE
GENERATED BY SOMATIC RECOMBINATION
Recombination of Va and Ja genes can occur
after multiple unsuccessful recombination
not functional
T-CELL
Antigen receptor
TCR
a/
next funcional
V
C
further funkcional (no allelic exclusion)
mRNS
CHARACTERISTICS OF T-CELL ANTIGEN RECOGNITION
1. The TCR is not able to interact directly with soluble or cell-bound antigen
2. T-cell activation can be induced by antigen in the presence of acessory
cells, only
3. T-cells recognize virus-infected cells
ACCESSORY CELL
ANTIGEN BINDING
NO INTERACTION
V
T-CELL
ACTIVATION
C
Antigen receptor
B-CELL
a/
T-CELL
T CELL RECEPTOR MEDIATED SIGNALING
Multisubunit Immune
Recognition Receptors
APC
MIRR
MHC
Antigen
Antigen
TCR
BCR
αβ
s
V s
s
sV
s
C s
s
sC
ss
P
P
D/E X7 D/E X2 YXXL/I X7 YXXL/I
ITAM
Immunoreceptor Tyrosine-based
Activation Motif
ACTIVATION
CD3
CD3
εδ
s s
s s
εγ
s s
s s
ζζ
s s
(review)
Phases of T cell response
(review)
BCR signaling
(review)
TCR signaling
Intracellular (cytoplasmatic) Ca2+ concentration increase
The intracytoplasmatic Ca2+ increase can be monitored by
fluorescent indicator dye. The intensity or the specificity of
fluorescence increase paralelly with the Ca2+ concentration.
Kinetic measurements connected to
signaling (1.)
Homogenous population – single cell property
measured one after the other - timing
Detected in
Detected in single cell
homogenous
population –
measured cell by
cell
Indicator – detection of intracellular Ca2+ signal
fluorescent Ca2+ indicators Fluo-3 or Indo-1
Kinetic measurements connected to
signaling (2.)
Fluo-3 AM – excitable by blue light
Indo-1 AM – excitable by UV light
These indicator dyes connected with apolar groups (e.g. acetoxy-methylester:
AM) – go across the cell membrane. In the cell, estherases cleveage these
groups - the fluorochrome became polar – catched in the cell
for example – ic Ca2+ signal in a single cell
antigen presentation by B cell to T cell
(click)
THE IMMUNOLOGICAL SYNAPSE
APC
T
APC
T CELL
THE INTERACTION OF T CELLS AND ANTIGEN
PRESENTING CELLS
interaction
recognition
1
2
3
4
5
6
stabilization
7
8
separation
Negulescu P.A. et. al. Immunity 4: 421-430, 1996
THE IMMUNOLOGICAL SYNAPSE
ANTIGEN PRESENTING CELL
ICAM-1
B7
CD48
CD4
CD2
CD28
adaptor
LFA-1
SIGNALING COMPLEX
T CELL
ICAM – Intercellular Adhesion Molecule
ACTIVATED
T CELL
RECOGNITION
EFFECTOR CELL
Plasma cell
B-lymphocyte
BCR + antigen
cytokines
Cytotoxic Tlimfocyte (Tc)
Antibody production
Cell killing
TCR + peptide + MHC-I
Effector cell retains specific
receptor
cytokines
Helper Tlymphocyte (Th)
TCR + peptide + MHC-II
Effector cells secrete cytokines
Macrophage activation
Lymphocyte activation
Inflammation
RECOGNITION OF EXOGENOUS AND ENDOGENOUS
ANTIGENES BY T-LYMPHOCYTES
Tc
Th
Exogenous Ag
Endogenous
Ag
Peptides of endogenous proteins
(virus, tumor) bind to class I MHC
molecules
Peptides of exogenous proteins (toxin,
bacteria, allergen) bind to class II MHC
molecules
Antigen presentation - T cells are co-stimulated
Signal 1 antigen & antigen
receptor
Th
APC
ACTIVATION
Signal 2
B7 family members (CD80 & CD86)
CD28
Costimulatory molecules are expressed by professional APC including dendritic cells,
monocytes, macrophages, and B cells, but not by cells that have no
immunoregulatory functions such as muscle, nerves, hepatocytes, epithelial cells etc.
THE ADHESION AND CO-STIMULATORY MOLECULES CD4 AND CD8
APC
MARKERS OF T CELL
SUBPOPULATIONS
MHCII
+
peptid
ADHESION MOLECULE
TCR
CD4
APC
MHCI
+
peptid
TCR
BINDS TO MHC
p56lck
p56lck
SIGNALING MOLECULE
+
CD4 T-sejt
CD8
CD8+ T-sejt
TARGET CELL
PROFESSIONAL APC
CD8
a1
s
s
a2
V4
a2
s
s
s
s
α β
Lck
Helper T-cell
SIGNAL
Lck
a b T-cell
Cytotoxic
a1
V3
2m
s
s
a3
V2
CD4
s
s
a3
s
s
2m
V1
2
1
a2
a1
ROLE OF CO-STIMULATION IN THE ACTIVATION OF
HELPER T CELLS
Th
Th
Th
CD40L
CD28
CD40
B7
B7
APC
APC
APC
NORMAL TISSUE CELLS DO NOT EXPRESS CD40 OR B7 CO-STIMULATORY
MOLECULES
Mechanism of co-stimulation in T cells
Antigen
1
IL-2
IL-2
IL-2Ra
IL-2Ra
Resting T cells
Express a low affinity
IL-2 receptor and g chains and
produce no IL-2
Signal 1
NFAT binds to the promoter of of the
a chain gene of the IL-2 receptor.
The a chain converts the IL-2R
to a high affinity form
Mechanism of co-stimulation in T cells
Costimulation
Antigen
Signal 2
1
2
Activates AP-1 and NFk-B to increase IL2 gene transcription by 3 fold
Stabilises and thus increases the half-life
of IL-2 mRNA by 20-30 fold
IL-2
IL-2Ra
IL-2 production increased by 100 fold
overall
Immunosuppressive drugs illustrate the importance of IL-2 in immune responses
Cyclosporin & FK506 inhibit IL-2 by disrupting TcR signalling
Rapamycin inhibits IL-2R signalling
THE HIGH AFFINITY IL-2 RECEPTOR
IL-2
Ligand binding
No signaling
α
β
γ
JAK
Janus kinase
CYTOSKELETON
Gene transcription
Proliferation
STAT
Signal Transducer and
Activatior of Transcription
INITIATION OF T CELL PROLIFERATION
IL-2R
adhesion
IL-2
recognition
IL-2Rα
costimulation
IL-1R
IL-1
IL-2R
G0
IL-2R
low affinity
G1
IL-2
M
G2
IL-2R
high affinity
S
transferrin
IL-2
insulin
PROLIFERATION
AUTOCRINE GROTH
FACTOR
CO-STIMULATION IS ESSENTIAL FOR PRIMING OF
NAIVE T LYMPHOCYTES
The antigen-specific and the co-stimulatory signals have to be
induced in concert to induce T lymphocyte activation
The antigen-specific and co-stimulatory signals can be
delivered simultaneously by professional antigen presenting
cells, only
The antigen-specific and the co-stimulatory singnals has to be
delivered by the same professional antigen presenting cell
T CELLS REQUIRE TWO SIGNALS TO GET ACTIVATED
Activated APC
Resting APC
B7
CD28
APC not presenting
antigen
B7
CD4
2 1
T-cell activation
CD4
CD28
1
T-cell anergy
CD4
CD28
2
No effect
ANTIGEN SPECIFIC ACTIVATION, ANERGY OR NEGLECTION
Anergy
Antigen
Naïve
T cell
1
Signal 1 only
IL-2
IL-2Ra
Epithelial
cell
Self peptide epitopes presented
by a non-classical APC e.g. an
epithelial cell
The T cell is unable to produce IL-2 and
therefore is unable to proliferate or be clonally
selected.
Unlike immunosupressive drugs that inhibit ALL
specificities of T cell, Signal 1 in the absence of
signal 2 causes T cell unresponsiveness to a
specific antigen
PROFESSIONAL ANTIGEN PRESENTING
CELLS
Express MHC class I and class II molecules
Express co-stimulatory molecules (B7, CD40)
Take up extracellular antigens
B cells
– soluble proteins, toxins (ADAPTIVE)
Macrophages
– extracellular pathogens (bacteria, yeast)
INNATE
– particles
Dendritic cells
– viruses, apoptotic cells
CHARACTERISTICS OF PROFESSIONAL ANTIGEN PRESENTING CELLS
Macrophage
Ag uptake
phagocytosis +++
Dendritic cell
phagocytosis +++
virus infection ++++
B - lymphocyte
Ag-specific mIg
++++
MHC expression induced +/+++
bacteria, cytokine
constitutive ++++
constitutive +++
immature/mature +++/++++ activation ++++
Pesented Ag
particulate Ag
intra/extracellular
pathogens
protein
virus protein, allergen
apoptotic cell
soluble protein
toxin
Co-stimulation
induced +/++
constitutive ++++
éretlen/érett +++/++++
induced +/+++
Localization
lymphoid tissue
connective tissue
body cavities
evenly
lymphoid tissue
connective tissue
epithelium
immature – tissue
mature – T cell area
Lymph node
lymphoid tissue
peripheral blood
follicles
ORIGIN AND DIFFERENTIATION OF DENDRITIC CELLS
BONE MARROW
MYELOID
CMP
CD34+
CLA+
LYMPHOID ?
CD34+
STEM
CELL
CLP
CD34+
CLABLOOD
CD11c+
CD1a+
CD11c+
CD1a-
CD14+
CD4+
IFNα
Monocyte/preDC1
Langerhans/LC Interstitialis Macrophage DC1
TISSUE
preDC2
DC2/plasmocitoid
CHANGES IN TISSUE ENVIRONMENT RESULTS IN
MACROPHAGE AND DENDRITIC CELL ACTIVATION
Phagocytosis of bacteria, degradation (LPS,
TLR)
DANGER SIGNAL
Macrophage
Monocyte
Dendritic cell
Activated macrophage
Activated dendritic cell
Virus, extracellular pathogenes, inflammatory cytokines (LPS,
TLR)
DANGER SIGNAL
BLOOD
TISSUE
LYMPH
FUNCTIONS OF DENDRITIC CELLS
Peptides
MH
C
Protein En
zym MHC I
es
MHC II
MHC II
Naíve
T cell
HIV-1
DC-SIGN
FcgRII
D
Naíve
T cell
Exo
som
es
Measles
CD91
HSP+peptide
CD14 Toll
aVb5
Toll
aVb3 PS-R
CD36
CR
N
B7
aVb3
aVb5
ICAM-1
CD1a
Glycolipid
FcgRII
AKTIVÁCIÓ
IDC
TISSUE
Antigen uptake
C
G
SI
MR
CD46
MR
ABCA1
I
MHC II
PS-R
CD40
MDR
CCR7
SLAM
MDC
LYMPHATIC TISSUES
Antigen processing and
presentation
MHC expression
Costimulatory molecules
ACTIVATION AND MIGRATION OF DENDRITIC CELLS
TISSUE
LYMPH NODE
LYMPH
Activated DC
TISSUE
Effector and memory T
cells
Inflammation
Pathogen
Naive T cells
ANTIGEN
Tissue DC
INTERACTION OG
DC AND T CELL
T CELL
ACTIVATION
BLOOD
DENDRITIC CELL T LYMPHOCITE
INTERACTION IN THE
LYMPHOID ORGANS
Activated dendritic cells in the
lymphatic tissues act as antigen
presenting cells
Tight contact with specific T cells
The danger hypothesis & co-stimulation
Full expression of T cell function and self tolerance
depends upon when and where co-stimulatory molecules are expressed.
Cell containing only
self antigens
No danger
Apoptotic cell death.
A natural, often useful
cell death.
No danger
APC
APC
Innocuous challenge to the immune system fails to activate APC and fails
to activate the immune system
Fuchs & Matzinger 1995
The danger hypothesis
Necrotic cell death
e.g. tissue damage,
virus infection etc
APC
DANGER
Pathogens recognised
by microbial patterns
APC
APC that detect ‘danger’ signals express costimulatory
molecules, activate T cells and the immune response
Some implications of the danger hypothesis
• There is no window for tolerance induction in neonates
• Neonatal T cells are not intrinsically tolerisable but the neonatal environment
predisposes to tolerance
• Antigens induce tolerance or immunity depending upon the ability of the
immune system to sense them as ‘dangererous’, and not by sensing whether
they are self or ‘non-self’.
• Apoptosis, the ‘non-dangerous’ death of self cells may prevent autoimmunity
when old or surplus cells are disposed of.
• Suggests that tolerance is the default pathway of the immune system on
encountering antigens.
• Explains why immunisations require adjuvants to stimulate cues of danger
such as cytokines or costimulatory molecule expression.
Doesn’t exclude self-nonself discrimination, but is very hard to
enequivocally disprove experimentally