acquired immunity 2 - vaccines & immunological memory

©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?

1


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

2


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?

3


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.

4


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

5


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

6


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

7


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.

8


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?

9


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

10


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)

11


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

12


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)

13


Question-2

How are memory B cells generated?

PathogenGerminal center

T cell

FDC


Naïve B cell

Memory B cell

Plasma cell

Transcription factors that regulate B cell development

Bcl6, Bach2

Blimp1, IRF4

14


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

15


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

16


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

17


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?

18


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


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

19


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?

20


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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acquired immunity 2 - vaccines & immunological memory

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