Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

(1)

in the Teaching Material of

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(2)

B-CELL RECEPTOR SIGNALING

Tímea Berki and Ferenc Boldizsár Signal transduction

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(3)

B-cell development

• Haematopoietic development is a highly regulated multistep process in which pluripotent HSC

differentiate through intermediate progenitors to mature cells in the blood

• These processes are regulated by transcription factors and signaling pathways

• The generationof lymphoid progenitors depends on:

c-Kit, Flt3, and IL-7R

(4)

Early lymphopoiesis depends on:

• PU.1: crucial for myeloid and lymphoid progenitors

• IKAROS: controls development of lymphoid progenitors

• Bcl11a: zinc finger transcrition factor, in its absence development is blocked in CLP

• E2A: helix loop helix protein, B-cell fate determinant, turns on B-cell specific genes

• EBF: early B-cell factor, B-cell fate determinant, turns on B- cell specific genes

• Pax5: in its absence cells are blocked at pro-B stage, self

renew, broad developmental potential. Pax5 represses

non-B genes

(5)

The stage specific crucial events

involved in B lymphoid commitment

LMPP: lymphoid primed multipotent progenitor, CLP: common lymphoid progenitor,

BCP: B lineage committed progenitor LMPP

Lymphoid priming

E2A/IKAROS/PU1

RAG1/2, TdT, IgH, IL-7Rα, Notch-1, Ebf1

BCP

B-lineage commitment

EBF1/PAX5

λ5, VpreB1, mb1, B29, OcaB, Pax5 CLP

B-lineage priming

E2A/EBF1

CD19, Tcf4, Aiolos, Irf4, Irf8, CD55

(6)

Genetic control of lineage

commitment in early lymphopoiesis

Low level of PU.1 → IL7Ra expression → lymphoid lineages (IL7Ra+)

PU.1 low

Myeloid prog.

HSC CLP

Pro-T

Pro-B Pre-

NK Pre-

DC

Ikaros

EA2 EBF Pax5

Id2

Notch PU.1 high

IL-7Ra+

IL-7Ra-

(7)

Early B-cell development in the bone marrow

A schematic diagram of early B lymphopoiesis, showing the successive differentiation stages and the rearrangement status of IgH and IgL genes

PU.1

Ikaros c-Kit

Fit3

Bcl11a Il-7R

E2A EBF

Pax5 Foxp1

(D_H► J_H) D_H► J_H D_H► J_H ^VH ► D_HJ_H Pre-BCR⁺ ^V^L^► J_L BCR⁺

Myeloid

Erythroid T NK

HSC CLP Pre-

pro-B

ELP Pro-B Large

pre-B

Small pre-B

Imm.

B

(8)

The Ig gene rearrangement

• DH–JH rearrangements are initiated in the earliest lymphocyte progenitors (ELPs) at a low level and are completed as the cell

progresses to the CLP and pre-pro-B cell (also referred to as CLP2) stage.

• VH–DJH recombination takes place in pro-B cells, and successful

rearrangement leads to expression of the Igm protein as part of the pre- BCR in large pre-B cells.

• Subsequent rearrangement of the IgL locus in small pre-B cells results in the expression of the BCR (consisting of m heavy and k or l light chains) on immature B cells (Imm. B).

• The approximate points of the developmental arrest in mice that have defective transcription factors PU.1, Ikaros, Bcl11a, E2A, EBF, Pax5 and Foxp1 (black) or signalling components involved in signalling through c- Kit, Flt3 or IL-7R (grey) are indicated above.

• E2A- and EBF-deficient progenitors in the bone marrow resemble pre- pro-B cells but lack all Ig rearrangements.

(9)

Expression pattern of

transcription factors involved in B cell commitment and differentiation

Progenitor CMLP, CLP

Commitment to B lineage

Bone marrow PreB → Imm.B

Peripheral

maturation Germinal center Plasma cells

PU.1 E2A EBF Ikaros Pax5 LEF-1 NF-kB Aiolos Bcl-6 Blimp-1 XBP-1

(10)

Immunreceptor Tyrosin-based Activation Motif

Activating receptors

Immunreceptor Tyrosin-based Ativation Motif (ITAM):

D/E-x2- YxxL/I-x6-9Yx2L/I

Inhibiting receptors

Immunreceptor Tyrosin-based Inhibition Motif (ITIM):

L/V/S-YxxL/V

ACTIVATION

ITAM ITAM

INHIBITION

ITAM ITIM

(11)

Acute antigen signaling

LYN LYN SYK SYK

BCR

P P

Iga Igβ

P P

PLC2 BTK

BLNK VAV GRB2 SOS RAS ERK

DAG PKC

IP3 Ca²⁺ Transcription factors Proliferation, activation and antibody secretion Antigen

PlP₃ PlP₂

PI3K

(12)

SYK P P BCR

ITAM

PLC2

PI3K Iga Igβ

PIP₃ PIP₂

BTK BLNK

GRB2

AKT1

RAS

RAF DAG

MEK

ERK PKCβ

CARMA1 MALTI BCL10

p50 p65 IKKa

IKK

IKKβ

TRAF3

NIK

p100 RELB IP₃

Ca²⁺

p52 RELB NFAT

NF-B1 Cytoplasm

Nucleus

Canonical NF-B1 pathway

Non- canonical

NF-B1 pathway

REL P100 Anti-apoptotic proteins

Such as BCL-X_Land MCL1 LYN

Plasma membrane

TRAF3 TRAF2 BAFFR CD19

NF-B2

Co-stimularory pathways of BcR

signaling

(13)

LYN LYN SYK

SYK BCR

P P

Iga Igβ

P P

PLC2 BTK

BLNK VAV GRB2 SOS RAS ERK

DAG PKC

IP3 Ca²⁺ Transcription factors Proliferation, activation and antibody secretion SHIP1

DOK

Migration

Survival

CXCR4 BAFFR

? Pl (3,4)P₂

Pl(3,4,5)P3

Pl(4,5)P2

PI3K

Long term BcR stimulation

(14)

C3d

P P P

antigén

Lyn a b a

Syk Syk

b Lyn

P P P P

P CD19 CD81

CD21(CR2)

Lyn

Vav P

P

PI3-K PI3-K

PIP₃

BCR

Gab1 PIP₃ Ras/MAPK

The positive (CR2) and negative

(FcgRIIb) B cell regulation model

(15)

Role for lipid rafts in B-cell activation

BCR BCR

LYN LYN

CD45

Iga/β CD22

ITIM ITAM

BCR

P P

BCR

P P

LYN LYN

CD45

Iga/β CD22

SYK SYK

Signal transduction Internalization

Antigen targeting Receptor

downregulation Lipid raft

Antigen binding

(16)

Lipid rafts

• The plasma membrane is composed primarily of

sphingolipids, (glycerol)phospholipids and cholesterol.

• Sphingolipids differ from most phospholipids in that they have long, largely saturated acyl chains that allow them to pack tightly in a bilayer, forming a gel phase in which there is very little lateral movement or diffusion.

• The gel phase of the sphingolipids is altered by the

association of cholesterol, which condenses the packing of the sphingolipids by occupying the spaces between the acyl chains.

• So, cholesterol-containing sphingolipid microdomains exist

in a liquid-ordered phase that is significantly more fluid than

the gel phase.

(17)

BCR

Iga Igb

Antigen

RapL Riam

Rap

Dok-1 ezrin

Bam32 clathrin

Cbl

Bam32

BLNK GRB2 LAB

Dok-3 Shc GRB2

TSC2

lB Lyn

c-RAF

MEK1/2

Erk1/2

PKC

TAK1

IKK

CaMK

Akt

GSK-3

mTOR

P70 S6K

Akt Btk

P13K p110

SOS

Ras GRP

Ras GAP

CaM Nck BLNK

PRK2 VAV

Rac/

cdc42 Rho

Rac

Ras

RhoA SHIP1

SHIP2 SHIP2

PTEN Gab BCAP

p85

Rheb IP3

DAG

Pl(4,5)P₂

DAG

PLC2

CARMA1 Bcl 10 MALT1

lB

NF-B NFAT

NF-B

FoXO MEKKs

PKC

PIR-B SHIP2

Cytoskeletal rearrangments and integrin activation

Proteasomal degradation

Protein synthesis

Glucose uptake Glycolysis

ATP generation Lipid raft

aggregation

BCR Internalization

CD22

CD19

CD40

FcgRIIB1

Ca²⁺

ER IP3R Intracellular

Ca²⁺ store STIM1

Transcription Growth arrest,

apoptosis Ca²⁺

Calcineurin Bam32

MKK3/4/6

p38

MKK4/7

JNK1/2

JNK

NFAT Ets-1

Oct-2 Bfl-1

ATF-2 Bcl-6 Egr-1 Elk-1 Bcl-xL

CREB Jun

CD19 CD19

Transcription Cytoplasm

Nucleus

CD19 CRAC channel

PIP₃

Syk Lyn

CD45

PIP₃

FcRIIB1

SHIP1

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

B-CELL RECEPTOR SIGNALING

B-cell development

• Haematopoietic development is a highly regulated multistep process in which pluripotent HSC

differentiate through intermediate progenitors to mature cells in the blood

• These processes are regulated by transcription factors and signaling pathways

• The generationof lymphoid progenitors depends on:

c-Kit, Flt3, and IL-7R

Early lymphopoiesis depends on:

• PU.1: crucial for myeloid and lymphoid progenitors

• IKAROS: controls development of lymphoid progenitors

• Bcl11a: zinc finger transcrition factor, in its absence development is blocked in CLP

• E2A: helix loop helix protein, B-cell fate determinant, turns on B-cell specific genes

• EBF: early B-cell factor, B-cell fate determinant, turns on B- cell specific genes

• Pax5: in its absence cells are blocked at pro-B stage, self

renew, broad developmental potential. Pax5 represses

non-B genes

The stage specific crucial events

involved in B lymphoid commitment

Genetic control of lineage

commitment in early lymphopoiesis

Early B-cell development in the bone marrow

The Ig gene rearrangement

Expression pattern of

transcription factors involved in B cell commitment and differentiation

Immunreceptor Tyrosin-based Activation Motif

Acute antigen signaling

Co-stimularory pathways of BcR

signaling

Long term BcR stimulation

The positive (CR2) and negative

(FcgRIIb) B cell regulation model

Role for lipid rafts in B-cell activation

Lipid rafts

• The plasma membrane is composed primarily of

sphingolipids, (glycerol)phospholipids and cholesterol.

• Sphingolipids differ from most phospholipids in that they have long, largely saturated acyl chains that allow them to pack tightly in a bilayer, forming a gel phase in which there is very little lateral movement or diffusion.

• The gel phase of the sphingolipids is altered by the

association of cholesterol, which condenses the packing of the sphingolipids by occupying the spaces between the acyl chains.

• So, cholesterol-containing sphingolipid microdomains exist

in a liquid-ordered phase that is significantly more fluid than

the gel phase.

Overview of BcR signaling