stage i stage ii stage iii stage iv - mit opencourseware i stage ii stage iii stage iv receptor gene...
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Stage I Stage II Stage III Stage IV
Receptor gene Elimination ofRearrangement self-reactive cells
Responders Effectors
Harvard-MIT Division of Health Sciences and TechnologyHST.176: Cellular and Molecular ImmunologyCourse Director: Dr. Shiv Pillai
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Y YY Immature
BSmall pre-BLarge pre-BEarly
pro-B
Ψ
Y
Y Y
Intermediate pro-B
Late pro-B
1. pre-BCR MEDIATED POSITIVE SELECTION
Mature B
A B C D E FC'
IgM IgD pre-BCR
2. BCR MEDIATED EMIGRATION
3. BCR MEDIATED MAINTENANCE
FOLLICLE
BONE MARROW
SPLEEN
DH to JH rearrangement VH to DJH VL to JL rearrangement
rearrangement
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Negative selection Positive selection I and Receptor editing
Pre-B receptor dependent Positive
A B C' Ψ
C Y
EDY FY Y
XLA selection II Syk KO
µKO
PERIPHERY Pre-pro B Pro-B I Pro-B2/preB
Large pre-B Small Pre-B Immature B Mature B
V to DJ out of frame
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HSC
IL-7 IL-7 SDF-1 SDF-1
progenitor lymphoid Common
pro-B B
D-JH
VDJ-VDJ-
VDJ+ In-frame
Large pre-B C'
V-DJH
Pre-B receptor expressing cells proliferate and allelically exclude
C
IL-7
pro/pre-B
VDJ+ In-frame
Large pre-B C'
VDJ+ In-frame
Large pre-B C'
VDJ+ In-frame
Large pre-B C'
pre-pro-B A
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Commitment Positive selection I Allelic exclusion
Positive selection II Negative selection
T
B
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Transcriptional regulation ofearly lymphoid development
CLP
DN DP SP
ProB PreB B
Tcf-1-/-
Sox-4-/-
Pax-5-/-
Gata-3-/-
E2a-/-
Ebf-/-
Ikaros-/-
PU.1-/-
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Entry versus commitment
• Commitment implies irreversibility and in wild type B cells has occurred when Ig H-chain gene rearrangement is initiated
• Certain transcription factors such as EBF and E2A are required to turn on genes required early in B cell development
• In the absence of Pax-5 cells “enter” the B lineage but remain highly plastic
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P r e - B r e c e p t o r B c e l l r e c e p t o rP r e - B r e c e p t o r B c e l l r e c e p t o r
Ig αIg β
ITAM
ι/vpreB
ω/λ5
Ig αIg β
ITAMSyk
Blk/Lyn/Fyn
Syk
Blk/Lyn/Fyn/Fgr (Src family kinases)
Btk (Bruton's tyrosine kinase)
(Src family kinases)Btk
Other signaling pathwaysOther Defective in X-linked agammaglobulinemia)
signaling pathways
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Ig αIg β
ITAM
ι/vpreB
ω/λ5
Btk
Syk
Blk/Lyn/Fyn
1)Survival 2) Proliferation 3) Allelic exclusion 4) Induction of κ
rearrangement 5) Shut off of surrogate
light chain expression
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B cell tolerance
See Immunobiology, by Janeway,C., Travers, P., Walport, M. and Capra, J., Garland Publishing, 5th edition, 2001 & Cellular and Molecular Immunology by Abbas, A., Pober, J., and Lichtman, A., W B Saunders; 4th edition.
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Multivalent
OR
Paucivalent
ANERGY
Chronic crosslinking modelCa++ influx seen but not sustainedNFAT and ERK activated normallyNFκB and JNK NOT activated
RECEPTOR EDITING
Rag gene reexpressionDeletion of old V κ-Jκ rearrangementNew κ or λ lightchain
CLONAL DELETION
BCR signals induce caspase activationApoptotic death
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BBT
Red Pulp
White pulp
marginal zoneand marginal sinus
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Poly A sitesδPoly A sitesµVDJ
AAAA
AAAA
Unspliced IgD message
mRNA for IgD heavy chain
AAAA mRNA for IgM heavy chain
L
Cap site ATG
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NO SIGNAL SIGNAL TRANSDUCTION
monosaccharide
antigen receptor
polysaccharide
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Fetal Liver HSC
HSC pro-B/pre-B
No TdTLimited diversity
YY
Y
Y
YB cells withbias towardsmultivalentTI-1 antigens
B-1 cells are self-renewingand express CD5
Microbial antigens
Bacterial LPSMicrobial antigens
B
B-1
B-1
B-1
?
IgM IgD
The B-1/CD5 B "lineage"
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Y CD5
B-1B CELLs
IgM
PERITONEUM /MUCOSAL SITES
YY
FOLLICULARB CELLS
CD1
CD21HIGH Y
MARGINAL ZONEB CELLS
IgM
IgM IgDCD21
SPLEEN
THREE DISTINCT TYPES OF PERIPHERAL B LYMPHOCYTES
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MZ and Follicular B cellsSee Immunobiology, by Janeway,C., Travers, P., Walport, M. and Capra, J., Garland Publishing, 5th edition, 2001 & Cellular and Molecular Immunology by Abbas, A., Pober, J., and Lichtman, A., W B Saunders; 4th edition.
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Are only “chosen” B cells selected by endogenousantigens?
OR
Do all B cells get tickled via the antigen receptor ?
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BCR
Btk
PLCγ2
PKCβ
Otherpathways
Otherpathways
Otherpathways
"No BCR signals"
MZ FO B-1
- - -
"Weak BCR signals"
" Weak andIntermediate BCRsignals"
"Weak, Intermediate,and Strong BCR signals"
Signal StrengthMutation
BCR
Btk
PKCβ
None
+ - -
+ + -
+ + +
PLCγ2 "Weak BCR signals" +* - -
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LONG-LIVED PERIPHERAL B CELL POPULATIONS
Strong BCR Signals …….B-1 cells
Intermediate strength BCR signals…….Follicular B cells
Relatively weak BCR signals …..MZ B cells
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Y YImmature B
Small pre-BLarge pre-BEarlypro-B
Ψ
Y
Y Y
Intermediate pro-B
Late pro-B
pre-BCR MEDIATEDPOSITIVE SELECTION
Mature B
A B C D E FC'
IgM IgDpre-BCR
DH to JH rearrangement VH to DJHrearrangement
VL to JL rearrangement
BCR MEDIATEDEMIGRATION
FOLLICLE
BONE MARROW
SPLEEN
CD1
CD21
MZB CELLS
?
IgM IgD
NewlyformedB cells
Y YIgD
FIII NF
FII
FI
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Y
Y
MHC class II
(loaded)
TCR
T
B
CD4
CD40
gp 39/CD40L
BCR
activated
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Cortex
Medulla
afferent lymphatics
efferent lymphatic
follicle (B cell zone)
1.Dendritic cells (interdigitating) in T cell zones2. Follicular dendritic cells in B cell areas3. Macrophages everywhere
artery
vein
T cell areas
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B
Y
Ag
X T
Focal aggregate
I
2
YY 3
4
Red pulp
White pulp
Germinal center
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X
low affinity
Follicular Dendritic cell
BT
high affinityT cell
somatic mutation
isotype switching
Memory B cells
Plasma cells
?Receptordiversification and rescue
T
APC
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Y
Y
MHCclass II
(loaded)
TCR
T
B
CD4
CD40
gp 39/CD40L
BCR
activated
?Signals to T cellgp 39/CD40L
Required for T-dependentimmune responses-proliferation-class switching-germinal center formation-somatic mutation
Activation of APCs via CD40to release IL-12 and drivea TH1 typeresponse
CD40
CD40L mutations lead toX-linked hyper-IgM syndrome
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Dark zoneLight zone
PALS
Follicle ( B cell area)
}Germinal center
(T cell area)
Centroblasts Centrocytes
memory B cel ls
plasma cells
apoptosis
V gene hypermutation
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Cµ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε Cα2JH
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VDJ
Sµ Sα Cα
Cγ1
Sγ1
Cγ3 Sγ3
Cδ
Cµ Looping out anddeletion
VDJ Sµ Cµ Cδ Sγ3 Cγ3 Sγ1 Cγ1 Sα Cα
VDJ Sα Cα Switched to IgA
γ2b, γ2a and ε
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VDJ
iENHSµ Sγ3
µ δ γ3 αIµ
C
LCR
Iγ3 SαIα
Cα3'E HS3B
HS43'αE
µ
µ
unspliced pre-MRNA
spliced Iµ transcript
γ2b, γ2a and ε C regions
Iµ Sµ
Iµ
Switch regions and I-regionpromoters
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Class Switching (Murine)
1. IL-4 promotes switching to IgG1 and IgE
2. TGF- β promotes switching to IgA
3. γ-IFN promotes switching to IgG2a
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Somatic mutation-I
1. Point substitutions. Non-templated singlebase changes in rearranged H- and L-chain Vregion genes
2. Requires T cell help, occurs in centrocytes
3. 10-4 to 10-3 base pairs/generation
4. Bell shaped curve of mutations starts inleader intron and ends about 1.5 kb downstream
5. Hotspot motifs
6. Transitions more common than transversions
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SOMATIC MUTATION -II
7. Requires enhancer
8. Mechanism:AID DEPENDENT DNADEAMINATION
a. Cytosines converted to uracils
b. Replication or error prone repair generates mutations
9. Accessibility? ? Need for transcription??
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AID required for both classswitching and somatic
mutation• AID is a novel Activation induced
cytidine deaminase
• Related to a protein involved in RNAediting
• Required for class switching and alsofor somatic mutation
• Aid-/- mice have large germinalcenters
• Humans lacking AID present withhyper IgM syndrome
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Y YYImmature B
Small pre-BLarge pre-BEarlypro-B
Ψ
Y
Y Y
Intermediate pro-B
Late pro-B
1. pre-BCR MEDIATEDPOSITIVE SELECTION
Mature B
A B C D E FC'
IgM IgDpre-BCR
DH to JH rearrangement VH to DJHrearrangement
VL to JL rearrangement
2. BCR MEDIATEDEMIGRATION
3. BCRMEDIATEDMAINTENANCE
FOLLICLE
BONE MARROW
SPLEEN
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For more information and examples, see Immunobiology, by Janeway,C., Travers, P.,
Walport, M. and Capra, J., Garland Publishing, 5th edition, 2001 & Cellular and MolecularImmunology by Abbas, A., Pober, J., and Lichtman, A., W B Saunders; 4th edition.