B-cell isotype switch and the longevity of the IgE-...

B-cell isotype switch and

the longevity of the IgE-

antibody response

Lone Hummelshøj

Allergy Clinic

Gentofte Hospital

Denmark

Antibodies (Immunoglobulins)

Variable region, rearranged VDJ

antibody specificity.

Constant region

antibody biological function.

Allergen (e.g. pollen)

The biological function can be replaced and still keeping the

specificity: Isotype switch/class switch recombination

B cell differentiation in type I allergy

IL-4 & IL-13

TGF-b

IL-10

IFN-g

IL-10

2

IgM

IgD

Immunoglobulin genes

Signalling through IL-4R and CD40

Somatic hypermutation

Hypothetic function of germline transcripts

GLT (germline transcripts)

AID (Activation-Induced Deaminase)

UNG (Urasil DNA glycosylase)

EN (Endonuclease)

Double stranded break

Class switch recombination

dsDNA break dsDNA break

2

Class switch recombination

Class switch recombination

Class switch recombination

Transcription factors in B cell differentiation

CXCR3 (CXCL9, CXCL10, CXCL11): Inflamed tissue.

CXCR4 (CXCL12): Bone marrow.

CCR9/CCR10 (CCL25/CCL28): Mucosal sites (IgA)

1-2 weeks after their generation, plasmablasts lose their ability to

migrate towards ligands.

Migration of plasmablasts

Survival niches

Number of plasma cells in the bone marrow

Steiner and Pearson, J. Pediatrics, 1966

Plasma cell replacement

Radbruch A, Nature Rev Immunology, 2006

Radbruch 2006, Nature reviews immunology:

Bone marrow: 0,1 – 1 % plasma cells Total of 109 plasma cells

Immunization studies: 1 x 106 specific plasma cells in the bone marrow.

That gives space for 1000 different main specificities.

Plasma cells for an old antigen would wane at a frequency of 0.1% for

each generation of cells with a new specificity.

50% reduction in plasma cell number: ~700 challenges

30 estimated challenges per year 23 years to reach 50% of the

original steady-state concentration.

Plasma cell repertoire – the rough calculations!

Factors that may influence the composition of the plasma cell

repertoire in the bone marrow:

Different antigens might induce different number of plasma cells –

some specificities might wash out sooner than others

Plasma cells of different antibody specificity might have different

migratory capacity (IgG >> IgE)

Plasma cells of different antibody specificity might have a different

capability in staying settled in the survival niches

Competition for survival niche space

Hiepe A, Nature Rev Rheumatol 2011

Strategies for targeting long-lived plasma cells

Method: Isolation and culture of IgD+ B cells

B cell culture, phenotype

Hummelshoj L, 2006

B cell culture, phenotype

Surface Ig Secreted Ig

Hummelshoj L, 2006

Plasma cell markers

Cord blood B cells

Hummelshoj L, 2007

Cord blood B cells

Hummelshoj L, 2007

Cord blood B cells vs. adult B cells

Hummelshoj L, 2007

Strategies for targeting long-lived plasma cells

IL-21

FcgRIIb

siRNA

+

The function of IL-21

1Mouse: IgG1 IgE

2Man: IgE

1Ozaki et al., Science, 2002. 2Wood et al., Cell Immunol, 2004.

IL-21 and germline transcripts

anti-CD40

IL-21 + anti-CD40

IL-4 + anti-CD40

IL-21 + IL-4 + anti-CD40

Germeline transcripts (GLTs)

Hummelshoj L, 2006

IL-21 and transcription factors

Hummelshoj L, 2006

IL-21 and immunoglobulin production

10000

1000

100

10

1

0.1

0 10 100

Imm

un

glo

bu

lin

(n

g/m

l)

IL-21 (ng/ml)

Anti-CD40

IgA

IgGtotal

IgG4

IgE

Hummelshoj L, 2006

10000

1000

100

10

1

0.1

0 10 100

IL-21 (ng/ml)

Anti-CD40 + IL-4

IgA

IgGtotal

IgG4

IgE

imm

un

glo

bu

lin

(n

g/m

l)

IL-21 conclusion

IL-21 alone increase the production of all immunoglobulins

• No GLTs

• No significant change in AID

• No significant change in XBP-1

Does IL-21 effect pre-shifted B cells by cell division?

IL-21 increase the IL-4 induced production of IgE and IgG4.

• Increased XBP-1

IL-21 increases the general plasma cell differentiation.

Plasma cells and FcgRIIb

ng/ml FcgRIIb blocking antibody

% p

osit

ive c

ells

00,

5 5 50

0

5

10

15

20

25

% CD19 positive cells

% CD138 positive cells

Hansen T, Hummelshoj L, 2011

Nimmerjahn, Nat Rev Immonol, 2008

Plasma cells and IgE siRNA

SiRNA concentration

To

tal Ig

E (

pg

/ml)

0 nM

20 n

M

50 n

M

Contr

ol

0

5000

10000

15000

*

*

Hansen T, Hummelshoj L, 2011

Once settled in their survival niches, long-lived plasma cells are

beyond the reach of available therapies.

Curative therapy might require elimination of long-lived IgE producing

plasma cells.

Take home message…