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Dirk Mielenz
University of Erlangen-Nuremberg Division of Molecular Immunology
B cell activation and long-lived plasma cells
ca. 1000 Children were given dried pustules of convalescent Pock patients (Variolation, „Variola“ from lat. varius = blurry, checkered)
ca. 1500 variolation in Harems
1717 Mary Wortley Montagu introduces from Turkey variolation in Europe
1760 Variolation of the famillies of Maria Theresia and George III. make variolation popular
1776 Washington variolates its continental army
Lady Mary Montagu, wife of the British ambassodour to Turkey
Generation of antibodies is the basis for the vast majority of succesful vaccination strategies
Nutt et al., Nat. Rev. Immunol., 2015 H.M. Jäck, Division of Molecular Immunology, Erlangen
How succesful vaccination reduced incidence of infectious diseases
Duration of humoral immunity to common viral and vaccine antigens
Latent infection, recurrent re-exposure, repetitive infections, memory cell numbers or bystander memory is not necessarily an indicator for antibody titer longevity; it is likely the nature of the antigen
Viral antigens Very long halflife Protein antigens Shorter half-life
Amanna et al., NEJM, 2007
Relationship between long-term immunity and long term protection
M.K. Slifka, Vaccine, 2014
Are there different kinds of antibodies? What are the cells that secrete antibodies? Is there only one type of B cells? Is a B cell completely autonomous? How does a B cell develop into a plasma cell? How does a B cell sense an antigen – and where? Does a normal B cell make „good“ antibodies?
Questions – principles of antibody formation
Antibodies
Antibodies are bifunctional molecules secreted by differentiated B cells
Ig-Classes IgM (penta/hexameric) - blood
IgG blood
[IgD] IgA (dimer) - gut
IgE - mucosa
H-chain L-chain
VL
VH
CL
CH
CH
CH
CH
Effector function - constant “Bullet Part”
Pathogen/antigen recognition - variable
(„magic Part“)
IgM
H.M. Jäck
IgM - Antigen receptor on immature and naive/IgM memory B cells - First line defense during immune reaction - Very good agglutination - anti-inflammatory (artherosclerosis)
IgD - Antigen receptor on mature B cells
IgG - Main antibody in blood → Internal defense - Passes placenta
IgA - Main Ab on mucosal and gut surfaces (lung, gut, urogenital tract) → External defense - In tears, sputum, mother milk (passive immunity important for newbornes)
IgE - activates effector cells (mast cells, eosinophils) during worm infection - allergy
Antibodies
• Agglutination of bacteria (IgM)
• Neutralisation (IgG, IgA)
• Phagocytosis and Opsonization (IgM und IgG)
• Killing (antibody dependent cytotoxicity, ADCC; antibody dependent respiratory burst, ADRB)
• Mast cell activation (IgE) – inflammation
Antibody effector functions
• Immune regulation via activating and inhibitory Fc receptors
Hematopoietic stem cell
B cell Monocyte Erythocyte
Bone marrow, fetal liver (mouse, human)
CD4+ T cell
Thymus
CD8+ T cell
Lymphocyte Maturation and Migration
Bursa fabricii (chicken)
Primary lymphatic
organs
H.M. Jäck
Antibodies
Antibodies are bifunctional molecules secreted by differentiated B cells
Ig-Classes IgM (penta/hexameric) - blood
IgG blood
[IgD] IgA (dimer) - gut
IgE - mucosa
H-chain L-chain
VL
VH
CL
CH
CH
CH
CH
Effector function - constant “Bullet Part”
Pathogen/antigen recognition - variable
(„magic Part“)
IgM
H.M. Jäck
µH chain N C
B cell
B cell
VH Cµ
Transcription Translation
VH Exon
V(D)J recombination generates antibody diversity
Recombination
stem cell
J segments Cµ
V segments D segments
ca. 2.5 Mb (mouse) HC locus
κL chain N C
VL Exon
J V Cκ
LC locus
Generation of primary antibody diversity (repertoire)
S. Tonegawa Nobel Price 1987
Basel Institute of Immunology
H.M. Jäck
antigen
B cell repertoire (~ 1011 specificies)
B cell receptor
Clonal selection & expansion by proliferation B cell clone
Short-lived plasma cell
Differentiation
Memory B cell
Long-lived plasma cell
+TH - TH
Burnet Jerne Talmage
CLONAL Selection Theories (1956-58)
H.M. Jäck
The IgM BCR is a signaling machine and an endocytotic receptor
Activation Apoptosis
plasma membrane
Cytosol
L
lyn Syk
BLNK Btk PLCγ2
PKCβ
NF-AT NF-κB
IP3
Ca++
cdc42 rac rho
WASP
G/F Actin
Igα/β µ
antigen Costimulation (CD40, IL-4R, TLR)
µ
Antigen presentation
MHCII
Nucleus
,,lipid raft“
MHCII loading compartment
Integration of adaptive and innate signals in B cells – relevance for antiviral immunity and autoimmunity
Hobeika et al., J. Mol. Med., 2015
IgM BCR signaling
The BCR provides a „tonic“ survival signal The BCR provides signals for B cell activation by ultimatively triggering transcription factors The BCR organizes B cell signaling and its machinery integrates signals from CD40 and TLRs The BCR is an endocytotic receptor which delivers antigen super-efficiently into the MHCII loading compartment
B cell receptor signaling, CTMI, 2016, Kurosaki, Wienands (eds.)
Naive B cells
Plasmablast
1 µm
1 µm
anti
CD
40 /
IL4
1 µm LP
S
Grötsch B et. al, JEM 2014
antibody factory
naive B cell
?
Morphological and functional diversification of B cells
Morphological and functional diversification of B cells
Mature B cells are resting and have a low metabolic activity, consuming mainly fatty acids Activated B cells proliferate and have a high metabolic activity Plasma cells are quiescent but have an enormous energy turnover and a high, very specific metabolic activity Plasma cells need a good redox balance
Caro-Maldonado et al., J. Immunol., 2014 Anelli et al., Free Rad. Biol. Med., 2015 Lam et al., Immunity, 2016
103 IgM molecules / s x 102 disulfide bonds/IgM = 105 disulfide bonds / s / cell!! 105 sugar molecules / s / cell!
• Rapid T independent response against polysaccharides (encapsulated bacteria) (TLR)
• Natural IgM
• Very rapid differentiation into plasma cells
MZ
• Shuttle between MZ and follicle; transport antigen
• Prone to interact with T cells
• Give rise to class switched (IgG, IgA, IgE) antibodies + memory
Naive B cell subsets: B1 and B2 (marginal zone, MZ, and follicular, FO)
Cyster et al., Nat. Immunol., 2000
B1 (pleura) and MZ (spleen)
FO
IgG
IgG Log
Cab
in S
erum
primary secondary
3 2 1
0 4 8
IgM 10Ag 20Ag
IgM
0 4 8 t(d)
Primary and secondary antibody responses
• Normally not T cell help
• Bacterial components (PAMPs) (i.e., LPS=Lipopolysaccharide)
TLR3/7/9 (mouse)
• Highly repetitive antigens
• B1- and MZ B cells
→ No memory
Thymus-independent (TI) and dependent (TD) activation – The nature of the antigen matters!
• T cell help
• Antigen needs to contain protein in a digestable and presentable form
• Mainly FO B cells
• High affine IgG
• → Memory
(Thymus- Dependent)
(Thymus- Independent)
Klaus D. Elgert: „Immunology“, 2nd ed., WILEY-Blackwell, Hoboken, NJ, 2009
B cells at the crossroads of innate and adaptive immunity
Primary follicle Secondary
follicle +
Germinal center
CXCR5
IgM
Antigen
+/-TH
Expansion IgM
Naive B cell B cell
focus
Short-lived Plasma
cell
Long-lived plasma
cells
Memory B cell
IgG IgA IgE
IgG IgA IgE
Anatomy of the B Cell Response
IgD - B cells PNA - GC B cells CD3 - T cells
Spleen section - 7 days SRBC
B
T HEV
GC
T C
ell Z
one
B C
ell Z
one
+TH
White pulp
Fates of activated B cells
Muramatsu et al., Cell, 2016 Victora & Nussenzweig, Ann. Rev. Immunology, 2012
Weisel et al., Immunity, 2016
high affine antibodies class switched late
lower affinity class switched early
competition for T cells
AID
AID: activation induced deaminase
Molecular Changes at the Ig locus (pre GC/dark zone)
1. Somatic hypermutation
2. IgH class switch
IgM IgG, IgA, IgE
VH
CH
VL
CL
AID
Anti HIV broadly neutralizing Ab (bnAb) are highly mutated and have long CDR3
Escolano et al., Cell, 2016 Tian et al., Cell, 2016
Fates of activated B cells
Victora & Nussenzweig, Ann. Rev. Immunology, 2012 Weisel et al., Immunity, 2016
high affine antibodies class switched late
lower affinity class switched early
competition for T cells
M.K. Slifka, Vaccine, 2014
Characteristics of a vaccine antigen determine subsequent levels and duration of immunity – or: the
antigen has to go THROUGH the B cell in a BCR specific manner
A principle of plasma cell differentiation is proliferation, increasing the probability for plasma cell differentiation
Nutt et al., Sem. Immunology, 2011
The gene network controlling terminal plasma cell differentiation
Nutt et al.,Sem. Immunology, 2011 Ochiai et al., Immunity, 2013
IgG1 IgG3 IgG2a IgG2b IgA IgM0
200
400
600
800
1000
controlFra1tg
*** *** *
***
Ig (µ
g/m
l)
0 10 21 310.0
0.5
1.0
1.5
2.0controlFra1tg
*** *** ***
day post-immunization
NP-
spec
ific
IgG
11/
1600
0 (A
490)
0 100.0
0.1
0.2
0.3
controlFra1tg
*
day post-immunizationNP-
spe
cific
IgM
1/4
000
(A49
0)
0 10 21 310.0
0.2
0.4
0.6
0.8controlFra1tg
*** *** ***
day post-immunizationNP
-spe
cific
IgG
1/6
000
(A49
0)
Fra1(FosL1) tg mice do not respond well to immunization
Grötsch et al., JEM, 2014 H2-Fra1-LTRtg
Fra1 inhibits the formation of RER containing plasmablasts in vitro
1 µm 5 µm 5 µm
10 µm 2 µm 2 µm 5 µm
Fra1
tgcontrol
Uns'mulatedLPSCD40/BCR/IL-4
020406080
100
LPSunst. CD40/BCR/IL-4
Fra1
tg c
ell n
umbe
r
020406080
100
cells with prominent RERcells without prominent RER
unst. LPS CD40/BCR/IL-4
control c
ell n
umbe
r
Grötsch et al.,JEM, 2014
Ficoll purified cells
0 2 50.000
0.005
0.010
0.015
control
*Fra1ΔB cell
day post-stimulation
Blimp1
(rel
. exp
ress
ion)
0 2 50.000
0.002
0.004
0.006
0.008
0.010
controlFra1tg
*
day post-stimulation
Blimp1
(rel
. exp
ress
ion)
Blimp1IRF4
µmµs
wttgwttgwttgFra1
d0d2d3LPS
9745
kDa
66
+/+-/-+/+-/-+/+-/-Fra197
45
66
kDa
Blimp1
IRF4
µmµs
d0d2d3LPS
Fra1 controls Blimp1 and µs expression in vitro
Grötsch et al., JEM, 2014
Nutt et al., Nat. Rev. Immunol., 2015
Different kinds of plasma cells
Nutt et al., Nat. Rev. Immunol., 2015
Plasma cells need a niche
Nutt et al., Nat. Rev. Immunol., 2015
Generation of long lived plasma cells - overview
IgG
IgM
Naive Cell
Long-lived plasma cells
(IgG, IgA)
Long-lived plasma cells
(IgG, IgA)
Memory B cell (IgG, IgA) GC reaction
IgG Log
Cab
in
Ser
um
primary secondary
3 2 1
0 4 8
IgM 10Ag 20Ag
IgM
0 4 8 t(d)
Long-lived plasma cells
(IgG, IgA)
Memory B cell (IgG, IgA)
GC reaction
Short-lived plasma cell
(IgM) Ag+TH
IgM Short-lived plasma cell
(IgM) Naive B-Cell
Ag+TH
Summary
Duration of humoral immunity to common viral and vaccine antigens
Latent infection, recurrent re-exposure, repetitive infections, memory cell numbers or bystander memory is not necessarily an indicator for antibody titer longevity; it is likely the nature of the antigen
Viral antigens Very long halflife Protein antigens Shorter half-life
Amanna et al., NEJM, 2007