dfg-forschergruppe 832 regulatoren der humoralen immunantwort b cell club july 6, 2007 function of...

DFG-Forschergruppe 832Regulatoren der humoralen Immunantwort

B Cell Club July 6, 2007

Function of IRF-4 in humoral immunity

Hans-Martin JäckAbteilung für Molekulare Immunologie

an der Medizinische Klinik III

Division of Molecular Immunology, Nikolaus-Fiebiger-Center, FAU Erlangen-Nürnberg 2

Interferon regulatory factor 4 (IRF-4)

• Member of the IRF transcriptin factor family

• Contains DNA binding domain with five conserved tryptophan repeats that interacts to regulatory elements in IFN-inducible genes

• Expression is limited to lymphoid (B and T) and myeloid lineages (DZ and MФ)

• Most closely related to IRF-8 in structure and expression pattern

• Functions in proliferation, apoptosis and differentiation

• Binding activity is modified by Ets (PU.1 and Spi-B) or helix-loop-helix (E2A) factors as well as Stat-6 and IRF-8

• Biphasic expression in B lymphoid cells


LatePro-B

LatePre-B

ImmatureB

Early Pre-B

MatureB

(+/- TH)

PrimaryFocus

Secondaryfollicle

Germinal center

Mantel zonePrimaryfollicle

(+TH)

Centrocytes(CSR)

Centroblasts(SHM)

MemoryB

Secondaryplasma blast

Expression of IRF-4 in B Cell Lineage

IRF-4 ↑ ↑IRF-4 ↑ ↑

IRF-4 ↑ ↑

IRF-4 ↑ ↑


LatePro-B

LatePre-B

ImmatureB cellEarly Pre-B

Stemcell

EarlyPro-B

μHCfct?LC ?

Igα KO Igβ KO µHCmem KO

μHCdys

Pre-BCR: A Passport for Pre-B Cells to Multiply

IRF4/8 KO Blink KO




• Contains DNA binding domain with five conserved tryptophan repeats that bind to regulatory elements in IFN-inducible genes

• Expression is limited to lymphoid and myeloid lineages (DZ and MФ)





• Acts redundantly with IRF-8 in B cell maturation


Function of IRF-4 in humoral immunity

• Requirement of the Transcription Factor LSIRF/IRF4 in Mature B and T Lymphocytes (Mittrücker …….Mak, Science 27, 540, 1997)

• Graded Expression of Interferon Regulatory Factor-4 Coordinates Isotype Switching with Plasma Cell Differentiation(Sciammas …. Singh, Immunity 25, 225–236, 2006)

• Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination(Klein …. Dalla-Favera, NATURE IMMUNOLOGY 7, p773ff 2006)


IRF-4 deficient mouse (IRF-4-KO)

• Normal B and T cell maturation with normal numbers of peripheral B and T cell numbers

• Impairment at the transition of immature to mature B cell subsets


• Low serum Ig levels (99% reduced)

• No germinal centers

• Fail to elicit T cell-dependent antigen-specific antibodies

• Reduced LPS-induced B cell proliferation

Recall against DNP-KLH



• No CD8 response against virus

• No tumor rejection





• Contains DNA binding domain with five conserved tryptophan repeats that bind to regulatory elements in IFN-inducible genes

• Expression is limited to lymphoid and myeloid lineages (DZ and MФ)





• Acts redundantly with IRF-8 in B cell maturation

• Acts nonredundantly in B and T cell activation and generation of effector cells


Role of IRF-4 in post-GC B cell development

C1-Cremouse

eGFP

IRF4

eGFPXTargeted

IRF-4 mouse

IRF-4 -/- and GFP+

ImmunisierungIn vitro deletion with tat-cre

(IRF-4 -/+ and GFP-)

Klein et al., 2006


Role of IRF-4 in post-GC B cell development

• Normal GC formation !!!

• Plasma cell generation requires IRF-4

• IRF-4 deficient B cells fail to switch IgH isotype (low AID induction)

Elispot 5d after immuniation for detection of IgG secreting plasma blast

eGF

P p

lus

eGF

P m

inus

IgG

1-se

cret

ing

ce

lls

IRF4-minus

IRF4-plus

IgG1

GFP


• IRF-4 may not be required for B memory formation since IgG1-positive GFP-positive B cells with somatic hypermutation could be detected

• IRF-4 not required for reactivation of memory B cells since recall increase IgG1- and GFP-positive B cells

• However, required for differentiation of memory B cells in plasma blasts, since IgG1-/GFP and CD138-postive cells were not detected after recall with NP-KLH

Problem: You never know whether IRF4 had an effect before switch or whether small amounts of IRF4 are still present.

Solution: Inducible IRF4 mouse

IRF-4 and Memory B Cells


Mechanism by which IRF4 controls switchung and plasma cell differentiation

IRF-4-KO mouse from Tak mak

p-IRF4

IRF4

LPS

Anti-CD40/IL4

GFPB cells

IRF4-

Retroviral transduction

• Proliferation• Differentiation• Expression of AID • and Blimp1

Singh labImmunity 2006


• IRF-4 restores Ig secretion by upregulating Blimp1

• IRF-4 transduction restores IgH switch by upregualtion AID

IRF-4 controls swirch and Ig secretion

IgG1

IRF4-GFP Control-GFP

GFP


Problem: IRF-4 induces AID and Blimp1 program

AID and Blimp1 are key components of antagonistic developmental stages


Solution: Graded expression

• Plasma blast (CD138) produce more IRF-4 than activated B cells

• Elevated abundance of IRF-4 in normal B cells upregulates Blimp-1 and down-regulates AID

Blimp1AID

Transduced WT B cells after anti-CD40/IL4 stimulation


LatePro-B

LatePre-B

ImmatureB cell

Early Pre-B

Stemcell

EarlyPro-B

μHCfct?LC ?

MatureB cell

Vettermann et al.Sem. Immunol, 2006

D→ JHVH →DJH VL → JL

B Cell Maturation and Ig Repertoire


Plasmazelle

Naive, reifeB-Zelle

Ag

(+/- TH)IgM

Primärer FokusIgD

IgM

(in peripherären lymphatischen Organen)

(+/- TH)

z.B. Lymphknoden

Ext

rafo

lliku

lär

Fol

likul

är

Sekundärer Follikel

Keimzentrum

Mantelzone

PrimärerFollikel(+TH)

dfg-forschergruppe 832 regulatoren der humoralen immunantwort b cell club july 6, 2007 function of...

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