Non-invasive biomarker signatures as a new tool for stratifying …cybernephrology.ualberta.ca/banff/2011/Powerpoint... · 2011. 9. 28. · IGKC * IGLL3 * KRT18 MGC29506 * MS4A1 *

Non-invasive biomarker signatures as a new tool for stratifying

biopsy indications?

Blood B cell profile as Biomarker in kidney transplantation?

Sophie BrouardSophie Brouard

ITUN, INSERM U643-ITERT, Nantes, France

“The exposure of mice to foreign antigens at a foetal stage induced tolerance to a skin graft expressing the same antigens at the adult stage”

Well fonctioning graftLacking histological lesion of rejectionIn the absence of immunosuppressionIn an immunocompetent hostAccepting a second graft of the same donorWhile able to reject a third party graft

LimitsLimits in in humanhuman tolerancetolerance??

=> Several key elements of transplant tolerance in rodents cannot be demonstrated…• « clinical state » observed « by chance » and not in the context of planned tolerance

inducing protocols• Patients usually incompliant• Patients usually incompliant• Graft biopsies are often unavailable• Refuse investigative procedures

⇒Functional definition

“Well-functioning graft in the absence of exogenous immunosuppression in an

Immunocompetent host”

OperationallyOperationally toleranttolerant patientspatientsN=2710+17 new casescreatinemia < 150 µmol/Lproteinuria < 1g/dayabsence of immunosuppressive drugs for at least one year

400

DF-Tol Sta CR0

100

200

300

crea

tinem

ia (

µmol

/l)

Identification of blood biomarkers of operational Identification of blood biomarkers of operational tolerance: help for biopsy and clinical decision tolerance: help for biopsy and clinical decision

BenchClinic

High risk Low risk

ToleranceChronic rejection

A challenge for the future:To identify allograft recipients in whom IS withdrawal could be safely

conducted

Transcriptional profiling of blood samples from operationally

tolerant transplant recipients

published experiences

� Martínez-Llordella et al. Am J Transplant 2007 (16 tolerant patients)

Liver

� Brouard et al. PNAS 2007 (17 tolerant patients)

� Sagoo et al. J Clin Invest 2010 (11 tolerant patients)

� Newell et al. J Clin Invest 2010 (25 tolerant patients)

Kidney

� Martínez-Llordella et al. Am J Transplant 2007 (16 tolerant patients)

� Martínez-Llordella et al. J Clin Invest 2008 (28 tolerant patients)

Transcriptional profiling of blood samples from operationally

tolerant kidney transplant recipients17 tolerant recipients

8 healthy individuals

22 chronic rejection

10 minimally immunosuppressed

12 immunosuppressed stable kidney recipients

14 acute rejection Healthy individuals

Chronic

Training set

5 8Tolerantrecipients

Testing set12 Tolerant11 Chronic rejection

49-gene predictivesignature

Correct classification in 22/23

Chronicrejection

11

BrouardBrouard et alet al. . ProcProc NatlNatl AcadAcad SciSci 20072007

Gene Definition Fold TOL vs. CR Fold TOL vs. N

TK1* Thymidine kinase 1, soluble 6.36 6.95

DHRS2* Dehydrogenase/reductase (SDR family) member 2 5.63 7.48

CCNB2 Cyclin B2 5.10 3.91

CYR61* Cysteine-rich, angiogenic inducer, 61 5.08 4.55

AFP* Alpha-fetoprotein 4.86 6.52

COL3A1 Collagen, type III, alpha 1 3.76 4.28

STK6* Serine/threonine kinase 6 3.59 5.27

SERPINA3* Alpha-1 antiproteinase, antitrypsin member 3 3.43 5.20

NGFRAP1 TNFRSF16 associated protein 1 3.20 2.06

IFITM3 Interferon induced transmembrane protein 3 (1-8U) 3.16 2.10

CTGF Connective tissue growth factor 3.07 4.66

CCNB1 Cyclin B1 2.63 3.14

LTBP4 Latent TGF beta binding protein 4 2.62 2.04

SOX3* SRY (sex determining region Y)-box 3 2.50 4.18

8

SOX3* SRY (sex determining region Y)-box 3 2.50 4.18

MTB More than blood homolog 2.44 2.03

KIFC3 Kinesin family member C3 2.29 3.20

NKG7 Natural killer cell group 7 sequence 1.93 0.84

TCL1A T-cell leukemia/lymphoma 1A 1.80 0.52

CD79A CD79A antigen (Ig-associated alpha) 1.66 0.70

CD79B CD79B antigen (immunoglobulin-associated beta) 1.41 1.03

RPS5 Ribosomal protein S5 1.38 2.18

CD22 CD22 antigen 1.15 0.41

CD19 CD19 antigen 0.79 0.37

CD20 Membrane-spanning 4A1 (MS4A1) 0.73 0.13

BCNP1 B-cell novel protein 1 0.69 0.34

DPYD Dihydropyrimidine dehydrogenase 0.25 0.46

EGR1 Early growth response 1 0.15 0.42

EVI2A Ecotropic viral integration site 2A 0.11 0.46

Pangenomic microarray (Brouard et al. PNAS 2007)

Enrichement of genes related to B cell pathways (GSEA)Biological function analysis (Gominer)

7 major gene sets related to B cell pathways (Pallier et al. Kidney Int. 2010)

Z= 15

Gene set nameNumber of genes

NESNOM p-val

FDR q-val

Gene set description References

BASSO_REGULATORY_HUBS

61 1.80 0.006 0.21Genes highly interconnected in the reconstructed regulatory networks from expression profiles in human B cells.

Basso K et al., Nat Genet. 2005

HOFFMANN_BIVSBII_BI_TABLE2

100 1.73 0.005 0.19Genes with at least five fold change in expression between Pre-BI and Large Pre-BII cells.

Hoffmann R et al., Genome Res. 2002

TARTE_PLASMA_BLASTIC

160 1.71 0.014 0.20

Genes overexpressed in mature plasma cells isolated from tonsils and mature plasma cells isolated from bone marrow as compared to polyclonal plasmablastic cells.

Tarte K et al., Blood 2003

ZHAN_MM_CD138_PR_VS_REST

22 1.70 0.014 0.21

50 top ranked over-expressed genes in each of the seven subgroups of multiple myeloma in CD138-enriched plasma cells from 414 newly diagnosed

Zhan F et al., Blood 2006S_REST enriched plasma cells from 414 newly diagnosed

patients.Blood 2006

GREENBAUM_E2A_UP 15 1.54 0.014 0.24Genes up-regulated at least 3-fold in the E2A-deficient cell lines.

Greenbaum S et al., J Biol Chem. 2004

HOFFMANN_BIVSBII_LGBII

60 1.53 0.013 0.24Genes with at least five fold change in expression between large and small Pre-BII cells.

Hoffmann R et al., Genome Res. 2002

TARTE_PC 38 1.59 0.015 0.25

Genes overexpressed in polyclonal plasmablastic cells, mature plasma cells isolated from tonsils and mature plasma cells isolated from bone marrow as compared to B cells purified from peripheral blood and tonsils.

Tarte K et al., Blood 2003

⇒ Several B cell-associated subsets with up-regulation of genes related to cell cycle, proliferation, development and maturation. (Pallier et al. Kidney int 2010)

Tolerant patients are characterized by a higher number of peripheral B cells

B c

ells

Louis et al. Transplantation 2006

B c

ells

B cells modulate T cell immunity

•The presence of infiltration of B cell compartments is actua lly correlated withworse prognosis

•Antigen presentation by B cells•B cell cytokines•B cell costimulation of T cells•B cells form ectopic lymphoid organs•Antibody mediated immune enhancement

•B cells and regulation of alloimmune response?12

CR DF STA HV0.0

2.5

5.0

7.5

10.0

12.5*

*

CD

19+ B

cel

ls(F

requ

ency

)

CR DF STA HV0

50

100

150

200

250

300

350 * **

*

**

CD

19+ B

cel

ls/µ

l

Operationally tolerant patients display a higher number of memory B cellsPallier et al. Kidney Int. 2010

***

CR DF STA HV0

10

20

30

40

50

60

70

80

****

****

IgD

CD

38+

/- B

cel

ls/µ

l

CR DF STA HV0

10

20

30

40

50

60

70

80**

**

***

***

CD

19+ C

D27

+ B c

ells

/µl

0

1

2

3** ***

*

IgD

- CD

38+

/- C

D80

+ B c

ells

/µl

0.0

0.5

1.0

1.5

2.0 ** ***

IgD

- CD

38+

/- C

D86

+ B c

ells

/µl

DF patients display a high number of circulating B cells that express a memory phenotype and over-express costimulatory molecules.

CR DF STA HV0Ig

D

CR DF STA HV0.0Ig

D

Pallier et al. Kidney Int. 2010

CR Tol STA HV0

50

100*

IgD

- CD

38+

/- CD

40+ B

cel

ls/µ

l

CR DF STA0

1000

2000

3000

4000

5000

6000* **

CD

40/I

gD- C

D38

+/- E

BM

5 B

M5

mea

n flu

ores

cenc

e in

tens

ity

Sagoo et al. J. Clinical. Invest. 2010Newell et al. J. Clinical. Invest. 2010

CD79b

TCL1A

Rank120

5

10

15

***

units

/HP

RT

1.5

2.0 *

units

/HP

RT

1.0

1.5

2.0 *

units

/HP

RT

*****

Tolerant patients exhibited a distinct pattern of gene expression

•Significant enrichment of several B-lymphocyterelated pathways

•6/10 of the genes with most altered levels ofexpression areexpressed in B cells

BLKHS3ST1

SH2D1BTCL1A

Rank 329Rank 1

Rank 3 Rank 111

0.0

0.5

1.0

1.5

units

/HP

RT

0.0

0.5

1.0

units

/HP

RT

0.00

0.02

0.04

0.06

0.08

0.10 **

units

/HP

RT

0

1

2

3

**

units

/HP

RT

******

*****

Sagoo et al. J. Clinical. Invest. 2010

Newell et al. J. Clinical. Invest. 2010

•Blood B cell signature: Immunoglobulin variable region genes

•Urine B cell signature

524 6 433

Lozano et al.

[808]

Sagoo et al.

[946]

35

3971

ADAM28 *AFF3 *ASB2ASPMBACE2BTLA *CD19 *CD200 *

ID3 *IGHM *IGKC *IGLL3 *KRT18MGC29506 *MS4A1 *PEX3

Newell et al.

[4721]

CD200 *CD22 *CD79A *CNOT2DTX1 *EBF1 *FCER2 *FCRL2 *FNBP4 *GNG7 *HLA-DOB *

PEX3PLEKHG1 *PNOC *QRSL1 *RFC1RFC4RPS21SWAP70 *ZNF264ZNF606

* B cell genesLozano et al. Am. J. Transplant. In press

DiLillo et al. Ann. N. Y. Acad.Sci. 2010

CR DF STA HV0

5

10

15

20

25 *

CD

19+ C

D5+

B c

ells

(Fre

quen

cy)


CD

24

CD38

Memory CD27 +IgD -

Naïve/Mature

Transitional

Newell et al. J. Clinical. Invest. 2010

10

20 *

(CD

32a/

CD

32b)

mR

NA

/HP

RT

0.4

0.6

0.8

**

CD

32b

mR

NA

/HP

RT

*

B cells from operationally tolerant recipients display an inhibitory profile

DF-patients had a significantly decreased CD32a/CD32b ratio compared to patients with chronic rejection (p<0.05) in total blood.

CR DF STA HV0(C

D32

a/C

D32

b)m

RN

A/H

PR

T

CR DF STA HV0.0

0.2

CD

32b

mR

NA

/HP

RT

FcγγγγRIIA (CD32a) transduces an activator signalFcγγγγRIIB (CD32b) contains an immunoreceptor tyrosine-based inhibition motif, transducing an inhibitory signal upon co-ligation with the B cell receptor.The CD32a/CD32b ratio has been shown to be a relevant index of B cell activation/inhibition(Ravetch et al. Science, 2000; Ono et al. Nature, 1996)


Key leader gene

2

3

4* **

BA

NK

1 m

RN

A/H

PR

T

B cells from operationally tolerant recipients display an inhibitory profile

CR DF STA HV0

1

BA

NK

1 m

RN

A/H

PR

T

Modulator of hyperactive B cell responses by inhibi ting AKT activation upon CD40 signalling (Aiba et al. Immunity, 2006)


Sivozhelezov V. et al.JCB, 2008

FcγγγγR2b PBMC

Le Texier et al. Am. J. Transplant. 2010

What about B cell Transcriptional profiling of blood samples

from operationally tolerant liver recipients?

� Brouard et al. PNAS 2007

Kidney

Liver

6.7%

33.3%

60%

20

30

40

50

60

70

%

3-6 years

6-10 years

over 10

years

� Martínez-Llordella et al. Am J Transplant 2007

� Martínez-Llordella et al. J Clin Invest 2008

6.7%

0

10

A. Sanchez, Barcelona, personal communication

DF STA0

5

10

15*

CD

19+ B

-cel

ls (

Fre

quen

cy)

Patients and MethodsSTUDY SUBJECTS:

Liver-TOL (n=12): HCV-negative liver recipients off IS for >1 year maintaining stable

graft function liver histology.

Liver-NonTOL (n=12): HCV-negative liver recipients maintaining stable graft function

under maintenance IS but who underwent rejection during a previous weaning

attempt.

Kidney-TOL (n=12): kidney recipients without IS with stable graft function for at

least 3 years (serum creat <150µmol/L and proteinuria<1g/24h).

Kidney-STA (n=12): kidney recipients under maintenance IS with stable graft

function for at least 3 years (serum creat <150µmol/L and proteinuria<1g/24h).

Kidney-CR (n=12): kidney recipients with deteriorating graft function (serum creat

>150µmol/L and/or proteinuria>1g/24h) with either transplant glomerulopathy or C4d

deposits plus DSA.

HV (n=12): healthy individuals

METHODS:

� PBMC samples

� Gene expression analyses: Affymetrix U133 Plus 2.0 microarrays

Different PBMC subsets contribute to the kidney and liver tolerance-related expression patterns

LIVER

Z=3.685

all genesCD56-specific genes


Z=-22

Den

sity

CD56 CD19

Haematology Expression Atlas Gene Sets(Watkins NA et al. Blood 2009): CD14, CD56, CD4, CD8, CD19, CD16

KIDNEY


Z= 15


Z= -9


Z= -21

CD56CD19 CD14

Absence of overlap between the kidney and liver tolerance -related datasets

UPDOWN

2610

20

621372

2610

10

35472

Liver-TOLvs

Liver-NonTOL

Kidney-TOLvs

Kidney-Sta

DOWN0 0 372

FDR<10%

Liver-TOLvs

Liver-NonTOL

Kidney-TOLvs

Kidney-CR

0 0 472

Liver Kidney Liver Kidney

28

Liver Kidney

Liver Kidney

CD

19+C

D5+

B c

ells

Lordano et al, AJT In press

Conclusions

•B cells and B cell-related transcripts are enriched in blood from tolerant

kidney recipients.

•This is a highly reproducible observation common to all cohorts of kidney

tolerant recipients.

•There are no obvious similarities for B cells between blood from kidney and•There are no obvious similarities for B cells between blood from kidney and

liver tolerant recipients (at least similarities that are reproducible across all

studies).

•This analysis is restricted to peripheral blood and has a number of limitations

��B cells: markers or key players?

•Large studies

•Access to the biopsy…

Operationally tolerant Kidney recipients: An heter ogenous group…

Complex and multifaceted clinical concept with poss ibly truly tolerant recipients

Some patients keep a functional graft for decades ( >30years)No opportunistic infection (Roussey et al. Am. J. Tr ansplant 2005)Normal immune response (Ballet et al. Transplantati on 2007)17 patients without/ 8 patients with HLA Abs (4 wit h DSA)

Metastable in some patients

9/27 decline their functionTo what extend the rupture of the operational toler ance is driven by To what extend the rupture of the operational toler ance is driven by

immunological phenomena is not clear yet….

Histology C4d Banff scores DSA

Case 4 Transplant Glomerulopathy -g2, i0, t0, v0, ptc1,

cg2, mm2, ti2, ci3, ct3, cv1, ah3 ND

Case 5 Borderline -g0, i1, t1, v0, ptc0,

cg0, mm0, ti1, ci1, ct1, cv2, ah3 -

Case 7 Transplant Glomerulopathy -g2, i0, t0, v0, ptc0,

cg2, mm2, ti1, ci2, ct2, cv1, ah1 +

Case 9 IF/TA grade 1 -g0, i0, t0, v0, ptc0,


Case 10Transplant Glomerulopathy

and Chronic active TCMR

-g3, i0, t0, v0, ptc1,


Case 13 IF/TA grade 3 NDgx, i1, t0, v0, ptc3,

cgx, mmx, ti2, ci3, ct3, cvx, ah0 -

“Acceptation” rather than “tolerance”?

« Lack of a destructive immune response toward the graf t »

“Old” kidneys may actually be tolerated despite a slo w and physiological loss of function over years…

AknowledgementsInserm U643

Annaick PallierSophie Hillion

Richard DangerKarine Renaudin

Cécile Guillot-GuegenSandrine LerouxYohann FoucherNicolas Degauque

ITUN-INSERM U643 (France)

Nicolas DegauqueAnne Devys

Anne Cesbron-Gauthier

Jean-Paul Soulillou/Magali Giral

Coll. A. SanchezColl. A. Sanchez--FueyoFueyo, , BarceloneBarcelone, Spain, Spain

G. Roussey, J-F. Subra, F. Villemain, C. Legendre, F.J. Bemelman, G. Orlando, A. Garnier, H. Jambon, H. Le Monies De Sagazan, L. Br aun, C. Noël, E.

Pillebout, M-C. Moal, C. Cantarell, A. Hoitsma, M. Ranbant, A. Testa

Documents

Non-invasive biomarker signatures as a new tool for stratifying …cybernephrology.ualberta.ca/banff/2011/Powerpoint... · 2011. 9. 28. · IGKC * IGLL3 * KRT18 MGC29506 * MS4A1 *