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©2015 Osaka University. All rights reserved.
Wataru Ise
WPI Immunology Frontier Research Center (IFReC)Osaka University
Acquired Immunity 2
- Vaccines & Immunological Memory -
Outline
1. What is vaccine (vaccination)?
2. What is immunological memory?
3. What are the issues in the researchfor immunological memory?
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©2015 Osaka University. All rights reserved.
Vaccine
“If you are prepared, you don’t have to worry”“Prevention is better than cure”
Vaccine=a biological preparation to prevent infectious diseases
Vaccination stimulates immune systemto recognize viruses and be ready to combat them
=cure diseases
=prevent diseases
Normal medicine
Vaccine
Types of vaccines
Vaccination
Virus infection
Protection!
1. Attenuated vaccines (measles, rubella, and mumps)created by reducing the virulence of a pathogen, but still keeping it viable
2. Inactivated vaccines (Influenza, Cholera, and Polio)created by killing the disease-causing microbewith chemicals, heat or radiation.
3. Toxoid (tetanus and diphtheria)bacterial toxin whose toxicity has been inactivated either by chemical (formalin) or heat treatment, while other properties, typically immunogenicity, are maintained
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©2015 Osaka University. All rights reserved.
Invention of the vaccine
“People who had already had cowpox would neverhave the often-fatal disease small pox”
HypothesisInfection with cowpox gives protection to smallpox
TestTook pus from the hand of a milkmaid with cowpox and scratched it into the arm of an 8-year old boyand six weeks later inoculated the boy with smallpox.
ResultsThe boy did not catch smallpox!
Edward Jenner(1749-1823)
Public domain | Wikimedia Commons
What happens if you get vaccinated?
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©2015 Osaka University. All rights reserved.
NK cell
Pathogen
Immune responses against invading pathogens
Neutrophil
Dendritic cell
Sensing of pathogens First defense agaist pathogens
Macrophage
Innate Immunity
Migration to lymph node
attack Antigenic peptide
Adaptive Immunity
CytokineB cell
B cell
Cytotoxic T cell(Cellular Immunity)
Antibody(humoural immunity)
Pathogens-specific attack
interaction
Co-stimulatory molecules
T cells or B cells express antigen receptors
Innate immune cells (macrophage or dendritic cells)uptake any kind of antigens and initiate immune responses(no antigen-specificity).
Cells in adaptive immune system (T cells or B cells) express antigen receptorson their surface and respond only to the specific antigens.
T cells or B cells have an almost infinite range of repertoireby rearrangement of antigen receptor genes.
B cells respond todifferent antigens
B cells undergo somatic hypermutation in germinal center and thus affinity or specificity of B cell receptors can be changed.
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©2015 Osaka University. All rights reserved.
Vaccination induces virus-specific antibody, T cells, and B cells
Influenza virus-specific T cell
Dendritic cell
Influenza virus-specific B cell
Influenza virus-neutralizing antibody
Signal
Stimulation
Vaccination againstinfluenza virus
Vaccination establishes “immunological memory”
No vaccination
Infection
Vaccination(immunization with Virus components)
Induction of virus-specificAb, T-cell, and B-cell
Protection!
NO Protection
Slow and weak immune response
Robust virus proliferation
Immune system “remembers” the virus and is ready to respond=Immunological Memory
Infection
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©2015 Osaka University. All rights reserved.
What is immunological memory?
A large amount of antibodiesare produced quickly
Immunological memory: Quick & Robust
Affinity of antibodiesbecome higher
Co
nce
ntr
atio
n o
f an
tib
od
ies
Aff
init
y o
f an
tib
od
ies
Pathogen
Secondary immunization
Primary immunization
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©2015 Osaka University. All rights reserved.
Immune memory cells
1,000
100,000
10,000
100
1,000,000
Memory B cell
Virus infection
30 days 60 daysNu
mb
er o
f V
iru
s-sp
ecif
ic B
cel
ls
Memory B cells
1,000
100,000
10,000
100
1,000,000
Memory B cell
Virus infection30 days 60 days
Nu
mb
er o
f V
iru
s-sp
ecif
icB
cel
ls
Most of the virus-specific B cells die.
However, small fraction of virus-specific B cells survive.
Virus-specific B cells expandand neutralize virus
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©2015 Osaka University. All rights reserved.
Memory B cells respond quickly and robustly
Naïve B cells
Memory B cell
Plasma cell
Pathogen
~7 days
~3 days More plasma cells
Higher affinity
QuickerPathogen
Does vaccination/ immunological memory work perfectly?
Need of flu vaccine every year
-Type of influenza viruses often differs year by year.
-Influenza viruses can mutate.
-Levels of protective antibody start to decline over time.
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©2015 Osaka University. All rights reserved.
Research for immunological memory
-What we know and what we don’t know about memory B cells-
Questions
Virus infection
Nu
mb
er o
f vi
rus
spec
ific
lym
ph
oc
ytes
Immune memory cells
Time (days)
1. Where are memory B cells in our body?
2. How do memory B cells respond to re-infection?
3. Why can a small fraction of cells surviveas memory B cells? How are memory B cells generated?
4. Are memory B cells really long-lived?
5. Are there any ways to induce memory B cellsefficiently?
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©2015 Osaka University. All rights reserved.
Question-1
Where are memory B cells present in our body?
B cells reside in lymph nodes and circulate in lymph or blood
Lymph node
Infection focus
Naïve lymphocytes enterlymph nodes from blood
Lymphocytes return toBlood via the thoracic duct
Heart
Antigens from sites of infectionreach lymph nodes via lymphatics
efferentlymphatic vessel
afferentlymphatic vessel
Lymph node
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©2015 Osaka University. All rights reserved.
Immune responses are elicited in lymph nodes
Germinal center
1. Antigens reach lymph nodes
Afferent lymphatic vessel
Efferent lymphatic vessel
2. Lymphocyte activation& Germinal center formation 3. Egress of activated lymphocytes
Activated T cells or B cells egress fromlymph nodes to periphery
Antigens
Efferent lymphatic vessel
Afferent lymphatic vessel
T cellZone
B cellZone
Pathogen
IgM/IgDCD38hi
CD138-
IgG/IgA/IgECD38hi
CD138-
Ig-CD38hi
CD138+
CD38lo
CD138-GL7hi
Fashi
Memory B cells can be distinguished from other type of B cells
Germinal center
T cell
FDC
Germinal centerB cell
Naïve B cell
Memory B cell
Plasma cell
Marker of memory B cell
-Switched Ig(=not naïve B cell)
-CD38hi(=resting)
-CD138 negative(=not plasma cell)
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©2015 Osaka University. All rights reserved.
Immunity. 2001 Feb;14(2):181-92.Takahashi Y, Ohta H, Takemori T.
NP
-bin
din
g
IgG
CD
38
IgG
CD
38
IgG
NP
-bin
din
g
IgG
GC B cells
Memory B ells
30 days
60 days
How to detect memory B cells
Memory B cells (IgG+, CD38hi)
GC B cells (IgG+, CD38lo)
Spleen of NP-CGG immunized mice
B cells reside in lymph nodes and circulate in lymph or blood
Lymph node
Infection focus
Naïve lymphocytes enterlymph nodes from blood
Lymphocytes return toBlood via the thoracic duct
Heart
Antigens from sites of infectionreach lymph nodes via lymphatics
efferentlymphatic vessel
afferentlymphatic vessel
Lymph node
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©2015 Osaka University. All rights reserved.
IgG RFP
CD38
Where do memory B cells localize in the secondary lymphoid tissues?
Germinal Center
Germinal Center
Follicle
The experiments in which memory B cells are labeled with fluorescence RFP
Memory B cells reside close to germinal centerAiba et al. PNAS (2010)
Day 60 after immunization
CD4+ T cells reside close to IgG+ memory B cells
High magnification
B cell follicleT cell area
IgG1+ cells
CD38CD4IgG1
IgG1 CD4 CD38, CD4, IgG1
Memory B cells can get help from CD4+ T cells efficiently upon re-infectionAiba et al. PNAS (2010)
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©2015 Osaka University. All rights reserved.
Question-2
How are memory B cells generated?
PathogenGerminal center
T cell
FDC
Germinal centerB cell
Naïve B cell
Memory B cell
Plasma cell
Transcription factors that regulate B cell development
Bcl6, Bach2
Blimp1, IRF4
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Identification of genes specifically expressed in memory B cells
Memory B cell Plasma cell
Gene chip data
GC B cellNaïve B cell
Kaji et al. J. Exp. Med. (2012)
Comparison of gene expression pattern
Identification and functional analysis of the candidate genes
Generation of genetically-modified mice(1)
A B X Y
A B X Y
A B X YGFP
Wild-type mice
Gene “X” Knock-out mice
(Function of gene X can be analyzed)
Gene “X” GFP Knock-in mice
(Expression pattern of gene X can be analyzed)
Fluorescent Protein
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©2015 Osaka University. All rights reserved.
Generation of conditional knock-out mice
X
ERT2-cre mice(Tamoxifen treatment can induce Cre-recombinase expression)
A B X Y
A B X Y
A B X Y
CreTamoxifen
Gene X is deleted
loxp loxp
Breeding
Generation of genetically-modified mice(2)
Function of gene X at specific time point can be analyzed
1,000
100,000
day30
10,000
100
1,000,000
Delete Gene X
Memory B cell
Research of memory B cells with gene targeting mice
day60
1,000
100,000
10,000
100
1,000,000
Delete Gene Y
Memory B cell
day60day30
Nu
mb
er o
f B
cel
l
If this is the case, this data suggests thatGene X is required for memory B cell generation
If this is the case, this data suggests thatGene Y is required for memory B cell maintenance
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©2015 Osaka University. All rights reserved.
Question-3 (1)
What is molecular basis for long-term survival of memory B cells?
Identification of signaling molecules that are essentialfor memory B cell survival
B cell receptor
Syk BtkBLNK
PLC-2
P3K
IP3
DAG
+
PKCER
Ca2+
Ca2+
IP3
receptor
Ca2+
Ca2+
CRACPIP2
PIP2 PIP3PIP3PIP3
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©2015 Osaka University. All rights reserved.
PLC-2 is required for maintenance of memory B cells
1,000
100,000
10,000
100
1,000,000
Number of memory B cells was decreased
Memory B cell
Deletion of PLC-2
PLC-2 deletion after memory B cell generation
# o
f T
cel
l#o
f m
emo
ry B
cel
l
day60day30
Hikida et al. J. Exp. Med. (2009)
Question-3 (2)
Are memory B cells really long-lived?
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©2015 Osaka University. All rights reserved.
Can memory B cells survive for a long time?
Immunization of protein Ag (PE)
Number of Ag (PE)-specificMemory B cells
Memory B cells appear to survive long-period timewithout antigen re-stimulation
Fre
qu
ency
of
PE
-bin
din
g
sple
nic
B-c
ells
(x1
0-4)
Days after priming
2.0
1.5
1.0
0.5
7 35 70 105 140 175 196Schittek et al. Nature (1990)
PathogenT cellFDC
Germinal centerB cell
Naïve B cell
IgM+ memory B cell
(IgM+)
IgG+ memory B cellIgM+ memory B cell
・Germinal center-independent・Low affinity
・Germinal center-dependent・High affinity
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IgM+ memory B cells are long-lived
Pape et al. Science (2011)
Long-term survival of IgM vs switched (IgG+IgA+IgE) memory B cells
IgM+ memory B cells are long-lived, whereas IgG+ memory B cells are short-lived
Heterogeneity in longevity or function among memory B cell subsets
Question-4
What kind of memory B cells should be inducedfor efficient vaccination?
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©2015 Osaka University. All rights reserved.
Important factors
1)High affinityAffinity of antibodies are increased as a result ofsomatic hypermutation in GC. Thus, a vaccine that sustainsGC response is desirable.
2)LongevityIf long-term immunological memory is established, repeatedvaccination is not needed.
3)Cross-reactivityInfluenza virus has many subtypes and is frequently mutated.Pre-existing antibodies may not be protective. Thus, induction of cross-reactive antibodies or memory B cells is the key for the efficient protection.
Antibody to Influenza HA
Head
Stem
HA:Hemagglutinin, a glycoprotein found on the surface of the influenza viruses
Antibodies to HA Head are easily induced
Mutations are frequently induced in HA Head
→Antibodies to HA Head are not efficaciousto different influenza subtypes
Antibodies to HA Stem are not easily induced
HA Stem is not mutated and highly conserved
→Antibodies to HA Stem are highly efficaciousto different influenza subtypes
How to generate HA-Stem specific memory B cellsis an important issues and extensively studied in the filed
Influenza Hemagglutinin
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