B cell PowerPoint Presentation

B  cell PowerPoint Presentation

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development. Tony . DeFranco, . 10/. 29/14. 5 . Themes in B cell development. + . Ig. class switch and somatic mutation. Theme 1: Checkpoints . in B cell development: feedback from . Ig. gene rearrangements. ID: 625432

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Presentations text content in B cell

Slide1

B

cell

development

Tony

DeFranco,

10/

29/14

Slide2

5 Themes in B cell development+ Ig class switch and somatic mutation

Theme 1: Checkpoints

in B cell development: feedback from

Ig

gene rearrangements

Theme 2: Bone marrow microenvironment

Theme 3: Lineage

commitment: transcription factors

Theme 4: Central

and peripheral tolerance of B cells

Theme 5: 3

different types of mature B

cells

Molecular

mechanisms of class switch recombination and somatic

hypermutation

: Activation-induced

cytidine

deaminase

(AID)

Slide3

Lymphocyte Development

Lymphocyte development is designed

to generate functional lymphocytes

with

useful

antigen receptors

that are

not self-reactive

Much of what happens during lymphocyte

development is designed to improve the

efficiency

of adaptive immunity

Slide4

B cell Development: Relevance

Immunodeficiencies

that affect B cell development

B cell malignancies (pre-B ALL, etc.)

Alterations in B cell tolerance may underlie some autoimmune diseases

B cell development is an

especially well understood example of mammalian cell development

Slide5

Overview of B cell development

Slide6

Theme 1: Ig rearrangement checkpoints forB cell development

IgH unrearr DJ VDJ VDJ VDJ

IgL unrearr unrearr unrearr rearranging VJ

(surrogate L chain)

Slide7

B cell development:distinctive cell surface markers(mouse)

B220 - + ++ +++

CD43 (S7) + + - -CD25 - - + -c-kit + + - -

Human B cell precursors: see Blom & Spits 2006

chain expressed in cytoplasm

Slide8

Pro-B to pre-B checkpoint requires Pre-BCR signaling

Surrogate light chain (

VpreB

+

l

5):

Expressed only in

proB

/

preB

cells

Triggers signaling (self

aggregation?Ligand

present on stromal cells?)

Expression turned off by pre-BCR signaling

Slide9

Pre-BCR checkpoint regulates V(D)J recombination

Controlled by

chromatin accessibility

; epigenetic marks due to histone modifications match those associated with transcription

Slide10

Theme 2: the bone marrow microenvironment

Role of IL-7 in murine B cell development

Lack

of Notch ligands in the bone marrow favor B lineage

development (Notch ligands in thymus are important for commitment to the T lineage)

Slide11

Theme 2: the bone marrow microenvironment

Based on in vitro culture experiments, the bone

marrow microenvironment has several key properties:

Pro

-B cells (

-) grow

indefinitely in vitro

only in contact with stromal cell layer from bone marrow

Pre-B cells (

+) will

grow in vitro

for a short period in response to IL-7 and in the absence of stromal cell

contact (matches in vivo “large pre-B cell”)

Slide12

Theme 2: the bone marrow microenvironment

Hypothesis:

pro

-B cells fill up a niche of sites bound to the appropriate stromal

cells

cells that lose contact, stop dividing and can attempt V(D)J recombination of

IgH

locus

pre

-BCR

replaces the stromal signal, so combines with IL-7R to induce burst of pre-B proliferation

Slide13

IL-7 and proliferation of B cell precursors

Clark, et al. Nat. Rev.

Immunol

. 14: 69-80, 2014

Slide14

Pre-BCR and IL-7R: cooperation vs. antagonism?

Clark, et al. Nat. Rev.

Immunol

. 14: 69-80, 2014

Slide15

B-ALL have lost pre-BCR signaling through Blnk

Rickert Nat. Rev. Immunol. 13: 578-591, 2013

Slide16

Pax5-/-

E2A-/-

EBF-/-

Knockouts of several transcription factors block B cell development at discrete stages

Theme

3:

B lineage commitment: control by transcription factors

Slide17

A hierarchy of transcription factors specifies B cell fate

Slide18

Pax5 and commitment to the B cell lineage

E2A and EBF are needed to turn on B cell specific genes including Pax5, which turns on additional B cell-specific genes

Slide19

Pax5 and Commitment to B cell lineage

Culture Pax5-/- bone marrow in vitro to get pro-B cell culturesSee if the cells can differentiate into other hematopoietic lineages (add various growth factors)

Nutt et al. Nature 1999

Slide20

Pax5 and Commitment to B cell lineage

Nutt et al. Nature 1999

Slide21

Pax5 and commitment to the B cell lineage

Pax5

seems to act in two ways

It promotes progression down the B cell lineage (expression of

Ig

a

,

Blnk

)

It shuts off genes needed to go down other lineages (M-CSF receptor, pre-

T

a

,

Notch1) or associated with other lineages (myeloperoxidase,

perforin

, etc.)

Slide22

Repression of genes needed to become T cell or myeloid cell

Ikaros

Repression of genes needed to become myeloid cell

A

network

of transcription factors specifies B cell fate

PU.1

Slide23

Theme 4: Tolerance of B cells

Sorted based on phenotype and/or specificity, etc.

Test antibodies for properties, self reactivity, etc.

Method for studying the self-reactivity of human B cells

Slide24

The primary repertoire of B cells includes many self-reactive cells

Meffre

and

Wardemann

, COI 20:632, 2008

Slide25

pre-B

Mature

PlasmaCell

IgMμ

IgM

Antigen

Independent

Antigen Dependent

Immature

Periphery

Bone Marrow

IgM

IgM

IgD

T2

T1

autoreactive

deletion

or editing

autoreactive

deletion

or anergy

autoreactive

anergy

(w/o T cell help)

Negative Selection

Positive Selection

Pre BCR

antigen encounter &

proliferation

Theme

4:

Fate of self-reactive B cells

Slide26

Receptor Editing Mechanisms

1. Upstream V

can rearrange to downstream J

2. Upstream V

can rearrange to KDE (



deleting element) deleting C

; this would be followed by a rearrangement of another light chain allele

KDE

V

k

J

k

C

k

Slide27

Receptor editing vs. clonal deletion

Contact with antigen in bone marrow leads to maturational arrest (no exit from bone marrow) and

receptor editing

Contact with antigen in periphery leads to

deletion

Bone

marrow stromal cells promote survival to allow editing to

occur

Slide28

Clonal deletion vs. clonal anergy

Anti-lysozyme transgenic mice with high affinity

antibody:

-

presence

of soluble lysozyme either as transgenic or injected leads to

anergy

(

Goodnow

et al.)

-membrane

-bound form of lysozyme induces

deletion

Anti-DNA

transgenics

(

autoantigen

of lupus):

-

mIg

with high affinity for

dsDNA

results in strong

editing

and

deletion

-

mIg

with lower affinity leads to

anergy

(

Weigert

, Erikson)

Slide29

Characteristics of Anergic B cells

Anergic

B cells exhibit chronic low grade BCR

signaling

and attenuated response to further stimulation

Anergic

B cells localize to the edge of the T cell zone next to B cell follicles, same as acutely stimulated naïve B cells.

Anergic

B cells have decreased survival in

vivo

due to decreased ability to respond to the survival factor

BAFF (

BLyS

).

Anergy

in the presence of competent helper T cells is enforced by Fas killing.

NOTE: B cell

anergy

is best thought of as a range of phenotypes from deep

anergy

to light

anergy

Slide30

Theme 5: 3 types of mature B cells

B1, marginal zone, and follicular B cells

Slide31

Three types of mature B cells

Recirculating follicular B cells (aka

conventional B cells

, B2 cells): circulate between LN follicles and blood: size of population determined by BAFF levels

Marginal zone B cells: reside in marginal zone of spleen where they can respond to particulate antigen in blood (bacteria, etc.); also dependent on BAFF for

survival. Also dependent on Notch signaling

B1 B cells: prominent in peritoneal and pleural cavities, present in spleen, absent in lymph node. Produce

natural antibody

and also respond to T-independent antigens. (less dependent on BAFF)

Slide32

Biological roles of three types of B cells

B1

follicular

MZ + B1

MZ + FO

Slide33

BCR signaling and B cell fate

(maturation B1)

(follicular>MZ?)

Slide34

Changes to rearranged

Ig

genes during immune response

Slide35

Ig Heavy chain class (isotype) switching

Slide36

Affinity maturation and antibody responses

Slide37

from Longacre and Storb Cell 102: 541, 2000.

Ig mutations are localized near transcription start site

Slide38

Activation-induced cytidine deaminase (AID)

Discovered as an induced gene in a cell line with inducible class-switch recombination (subtractive hybridization)

Transfection into B cell lines induces class switch recombination

AID KO mice have strong defect in class switch recombination AND in somatic

hypermutation

Hyper-

IgM

syndrome type 2 (autosomal) is due to mutation in AID; very similar phenotype to mice (no

IgG

, IgA,

IgE

; very much reduced somatic mutation)

Slide39

AID: How does it work?

AID is highly related to APOBEC-1, a

cytidine

deaminase

that edits mRNA for

Apolipoprotein

B (via a targeting subunit

) and APOBEC-3, which mutates retroviral genomes

indirect action or direct action in class switch and

hypermutation

?

AID could edit mRNAs for factors that act in class switch and factors that act in class switch

OR

it could act directly in both processes

Slide40

AID as a mutator of DNA

AID is mutagenic in bacteria and mutations are increased by deficiency in Uracil-DNA glycosylase (enzyme that removes U from DNA and triggers DNA repair)

Class switch is inhibited and hypermutation perturbed in UNG-deficient mice

These results favor the hypothesis that AID directly acts on C residues in DNA to promote class switch and hypermutation

Slide41

In hypermutation: U in DNA could lead to direct mutations and secondary mutations via mismatch repair and/or error-prone DNA polymerasesIn class switch recombination:U in DNA could lead to nick formation by repair enzymes:nicks on both strands-->ds breaks-->recombination

Model for direct actions of AID in somatic mutation and class switch


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