Development of autoreactive transitional B cells induced by apoptotic Ag stimulation

Jennie Hamilton February 26, 2015

Research In Progress

1

Hypothesis: AC stimulation (and other factors eg. IL-17R signaling) of transitional B cells promotes development of autoreactive T1 B cells

2 Dörner et al. Arthritis Research & Therapy 2009 11:247 doi:10.1186/ar2780

X

X

C57BL/6 DBA/2

(B6XD2)F1 (B6XD2)F1

20 generations

BXD1 BXD2 BXD6 BXD41 BXD42

Fixed genome

(50% B6; 50% DBA/2)

MHC locus and Ig locus from B6

BXD2 (H&E) B6 (H&E) BXD2 IgG

BXD2 (PNA) B6 (PNA)

GCs

Spleen GC staining using PNA

…100 strains

BXD2 autoAb development follows similar pattern

Mountz et al, Scan J Immunol 2004

Hsu et. al. Arthritis Rheum. 2006

Hsu et al, J Immunol 2007 3

La and Ro cluster in apoptotic blebs

J Exp Med. 1994 Apr 1;179(4):1317-30.

What drives BXD2 autoAb formation to the ubiquitous AC? 4

2 mo 5 mo 9 mo

B6

BXD2

SIGN R1

Age-related decrease in MZM and an increase in uncleared apoptotic cells (ACs) in BXD2 mice

Li et al

Cutting Edge JI 2013 5

Decreased clearance of apoptotic cells (ACs) in BXD2 mice

Incubate for 6

hr with

glucocorticoid

Wash

Sacrifice

5, 10, 15 min

B6

BXD2

CFSE

CFSE

B6

BXD2

6 MARCO (MZM) CFSE (ACs) PNA (GC B cells)

Control

5 min

10 min

15 min

B6 BXD2 B6 BXD2

Li et al

Cutting Edge

JI 2013

Summary

• BXD2 mice exhibit defective clearance of AC

• Inhibiting AC generation leads to decreased anti-DNA titers

• The uncleared AC is in the vicinity of T1 B cells

7

Uncleared AC

Modified from Pillai et al Annu. Rev. Immunol. 2005. 23:161–96

Yurasov et al. JEM. 2005.

Defect identified between the T1/T2 to mature naïve stage in SLE

8

Suryani et al, Blood, 2010

How can we specifically study mechanisms leading to maturation of autoreactive T1 B cells

in autoimmune individuals?

9

Suryani et al, Blood, 2010

Use of a PE-Avidin – biotin-peptide tetramer strategy to identify

autoreactive B cells in BXD2

Auto-Ag Tetramer (La13-27 or snRNP357-373)

PE

Non-specific peptide

Tetramer

AF647

5.72

X Axis

Y A

xis

10

Chip Nuclear Centromere

DNA Damage Repair

Nucleosome

Polymerase

Topoisomerase

RNP

rRNA processing

Transc. Regulation

Other

Nuclear in top 100

54% 67%

Nuclear

ER/Mito Enzyme

Matrix

Membrane

Cytoplasm

Secreted

Lysosome Enzyme

Ig

52%* 31%

9% 14%* 17%

13%

Chip Ag Content Top 100 Ag

Pepperprint identification of peptides for tetramer generation

PEPperPRINT Peptide Autoimmunity Microarray probed with BXD2 serum

11 *Predominance of nuclear and RNP autoAg in BXD2 mice

Selecting peptides for tetramer generation: Anti-La, histone and RNP peptide autoAbs in BXD2 mice

IgG

La13-27

H1b

205-219

snRNP

357-373

Blank

Unstimulated PMA+Ionomycin

B6 BXD2

La13-27

H1b

205-219

snRNP

357-373

Blank

0

8

16

24

32

40

Unstimulated B6Unstimulated BXD2PMA + Iono B6PMA + Iono BXD2

Sp

ots

/ 1

06 c

ells

**

**

** ***

*** ***

** **

1

IgM

B6 BXD2 B6 BXD2

Unstimulated

0

5

10

15

20

25

La

H1b

sn

RN

P1

Sp

ots

/ 1

06 c

ells

B6

BXD2

*

*

12

La

BXD2 DN NS La Total

Serum Supernatant

B6

40

kDa

Antibody maturation of La tetramer+ B cells

Probed with serum or supernatant from tetramer sorted cells and detected with anti-mouse IgG

PE

AF

647

-bio

Non-specific

(NS)

La+

La13-27-PE

DN

13

Summary of autoreactive B cell analyses using Ag tetramer

• Overall % of La and snRNP tetramer reactive B cells not significantly different B6 vs BXD2

• Most autoreactive cells are FO, with a higher % and number in BXD2 mice.

• There is a skew in the IgMhiCD21hi population to the MZ-P (IgMhiCD21hiCD23+) in BXD2 mice.

• These autoreactive MZ-Ps also express activation markers CD69 and CD86

• Is MZ-P dysregulation a result of earlier B cell defect?

14

CD

19

CD23

68.9 20.4 24.7 72.1

CD

21

IgM

7.8 11.7

81.1 79.8

La13-27

PE

AF

64

7

La

2.7 2.4

B6 BXD2

Column enriched

14 20 18 22

CD

93

CD19

sIg

M

CD23

La13-27

B6 BXD2 B6 BXD2

1snRNP357-373

40 18

48 21

23

25 22

40

30 37 24 26 T1 T2

T3

Dysregulation of autoreactive cells at the T1 stage in BXD2 mice

*CD93 = C1q receptor

T1 T2 T3 FO B

sIgM ++ ++ + +/++

IgD - ++ ++ ++

CD93 ++ + + -

CD23 - + + +

T1 T2

T3

sIg

M

CD23

15

Summary

• BXD2 mice exhibit IgG response to peptides derived from common apoptotic cell antigens

• Established a general method to identify autoreactive B cells

• Identified skewing in the development of early transitional autoreactive B cells including T1 and MZ-P subsets

• Abnormal maturation in autoreactive T1 cells is found in SLE patients and BXD2 mice. What is the underlying mechanism?

Uncleared AC

Modified from Pillai et al Annu. Rev. Immunol. 2005. 23:161–96

Research in Progress

• Do transitional B cells from BXD2 mice exhibit immunoreactivities to apoptotic cell self antigens?

• And if so, is this related to defects in MZMs which then promoted the maturation of autoreactive B cells in BXD2 mice?

17

0

0.2

0.4

0.6

0.8

1

1.2

1.4

B6 BXD2 B6 BXD2

La La 7 days post boost

IgG

OD

450

ACCLAC/CLPBS

Anti-La IgG and chronic AC stimulation

B6

BXD2

± CL

4 weekly injections Analyze

sera for anti-La

B6

BXD2

La boost

18

B6 Vehicle zVAD

BXD2

GL-7

Fas 0.2±0.1 2.9 ±0.6 0.4±0.2

0.1

0.18

0.26

Anti-D

NA

(O

D45

0)

Age (wk-old)

BXD2 zVAD

BXD2 Vehicle

B6

4 8 12 16 20

Inhibition of apoptosis suppressed anti-DNA antibody

zVAD (10 uM)

19

• This will be done in mice in which ACs have been blocked with Z-VAD caspase inhibitor.

• EXPECTED RESULTS: Z-VAD treatment will shift the T1-T3 skewing to a more normalized ratio in BXD2 mice at young age. However, blocking of apoptotic responses in older BXD2 mice after disease onset may be an issue since this also prevent clonal deletion of autoreactive B cells.

20

Question-Does prevention of the generation of AC

before the onset of autoimmunity blocks the skew toward the more mature T3 phenotype

4 weekly injections

Sacrifice and analyze transitional populations by FACS

zVAD (10 uM)

4-, 12-, and 20-wk-old

Question – Does excessive ACs or elimination of

MZM induce follicular localization of transitional B

cells in autoimmune BXD2 mice

– EXPERIMENTAL DESIGN: The anatomical location of CD93+

transitional B cells will be determined by confocal imaging in

normal B6 and lupus BXD2 mice at various ages.

– EXPECTED RESULTS: There will be increased CD93+ B cells

within the follicle of BXD2 mice.

21

Hsu et al. Nature Immunology. 2008

More follicular entry of IgMhi CD93+ B cells in BXD2?

BXD2 B6

Future Experimental Plans

• Increased follicular entry of transitional B cells in BXD2 mice compared to B6.

• Features of anergy reversal in transitional cells from BXD2 mice, including lower basal levels of pTyr, increased pTyr response after anti-BCR stimulation, increased baseline intracellular Ca2+ levels, and increased Ca2+ levels after BCR stimulation. This will be associated with B-cell proliferative responses following anti-IgM stimulation.

• Elevated pSyk in the expanded T3 B cells of MZM depleted BXD2 mice.

• Abnormal phenotypes of transitional B cells in BXD2 mice will be further augmented by disruption of MZMs or exogenous ACs. This will suggest integrity of MZMs or normal clearance of ACs can prevent maturation of autoreactive T1 B-cells to maintain B-cell tolerance.

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Thank you

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