stage i stage ii stage iii stage iv - mit opencourseware i stage ii stage iii stage iv receptor gene...

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

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

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

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

Commitment Positive selection I Allelic exclusion

Positive selection II Negative selection

T

B

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-/-

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

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

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

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.

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

BBT

Red Pulp

White pulp

marginal zoneand marginal sinus

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

NO SIGNAL SIGNAL TRANSDUCTION

monosaccharide

antigen receptor

polysaccharide

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"

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

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.

Are only “chosen” B cells selected by endogenousantigens?

OR

Do all B cells get tickled via the antigen receptor ?

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" +* - -

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

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

Y

Y

MHC class II

(loaded)

TCR

T

B

CD4

CD40

gp 39/CD40L

BCR

activated

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

B

Y

Ag

X T

Focal aggregate

I

2

YY 3

4

Red pulp

White pulp

Germinal center

X

low affinity

Follicular Dendritic cell

BT

high affinityT cell

somatic mutation

isotype switching

Memory B cells

Plasma cells

?Receptordiversification and rescue

T

APC

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

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

Cµ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε Cα2JH

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

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

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

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

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??

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

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

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.