Maria Gerbase de Lima

UNIFESP- Escola Paulista de MedicinaSão Paulo, Brazil

Immunogenetics Division

Immune response gene expression in

cardiac transplantation

Acute Rejection in Cardiac

Transplantation

•occurs in more than 50% of the recipients

• 90% occur in the first 6 months after Tx

•is an important cause of death

The gold standard is

the histologic examination

of biopsies.

EndomyocardialEndomyocardial BiopsiesBiopsies

TX1 32 5 64

Months

Diagnosis of Rejection

.....and biopsies are an invasive procedure, not devoided of risks for the patient.

the sensitivity of the biopsy for the diagnosis of rejection is not 100%.

However................

Correlation of endomyocardial biopsy findings with autopsy

findings in human cardiac allografts.Nakhleh et al, J Heart Lung Transplant 11:479, 1992

98-99 %98-99 %Specificity

79 %79 %Sensitivity

All Biopsies(grades 0 - 4)

97 %97 %

50%50%

Biopsies with grades 1, 2 and 3

Comparison of the diagnosis based on 5 biopsy specimens of cardiac allografts, obtained at autopsy, with the findings in

the whole heart.

Therefore, other methods are needed to improve the sensitivity of the histological examination of biopsies.

Tests that could predict rejection onset would be most helpful.

The ideal test would be sensitive, accurate and noninvasive.

And....

• Within the graft• In the Blood

Where?

How?

protein expression

ELISAimmunohistochemistry

antibodiesanti-HLAanti-myosin

differential gene

expression

Post-transplant Immunological MonitoringPost-transplant Immunological Monitoring

Strategies for searching the genes to be studied

YYYY YYYYYYYY

YYYY

CD8CD8APC

CD4CD4

B

YYYY

M

Knowledge of the Immune response

Candidate genes

Individual RT-PCR for several candidate genes

Microarray: differentially expressed genes

Validation by RT-PCR of some of the genes

Selection of the most informative combination

of markers

Baan et al, 1994

Increase of protein or mRNA levels during rejection

IL-2, IL-4

Granzyme B

Alpert et al,1995

Perforin

IL-6Zhao et al, 1993

Kimball et al., 1996

IL-8

TNF

Abdallah et al., 1997

Kimball et al., 1996

CD8APCCD4CD4

M

Heart TxHeart Tx:: Rejection Markers in Biopsies Rejection Markers in BiopsiesSome of the first observationsSome of the first observations

Increased mRNA levels during rejection (semiquantitative RT-PCR)

PerforinTNF-

IL-8

CD40L

FasL

IFN-

Granzyme B

Heart Tx: Rejection Markers in BiopsiesData from our GroupData from our Group

Transplantation 67: S266, 1999

0

100

200

300

400

Rejection No rejection

p<0.004

Fas mRNA

ag F

as m

RN

A /

ag

PO

LR

2K m

RN

A)

X 1

05

FasEx6del mRNA

0

50

100

150NS

Rejection No rejection

ag F

asE

x6d

el m

RN

A /

ag

PO

LR

2K m

RN

A)

X 1

05

FasL mRNA

0

10

20

30

40

50

Rejection No rejection

p<0.04

ag F

asL

mR

NA

/ ag

P

OL

R2K

mR

NA

) X

105 FasL

Fas

APOPTOSIS

soluble Fas (FasEx6del)

NO APOPTOSI

S

Heart Tx: Rejection Markers in BiopsiesHeart Tx: Rejection Markers in Biopsies

Are there markers capable of predicting rejection?

Classification of the samplesClassification of the samples

no R no R

ISHLT classification

1B, 2, 3A, 3B, 4RR

R - rejectionR - rejection

grade 0, 1A

pre-R post-R

7-15 days 7-15 days

grade 0, 1A

No Rej

Pre-Rej

Rej

Biopsies with increased mRNA expression

Braz J Med Biol Res 34:779-784, 2001

CD40L IFN-

10

20

30

40

50

60

70

80

90

100

%

FasL

IFN- kineticsIF

N-

mR

NA

/-a

ctin

mR

NA

0.25

0.50

0.75

No Rej. Pre-Rej. Rej.

B

mRNA expression (QC-RT-PCR) of cytotoxic effector molecules

Transplantation 72:1705-1708, 2001

% of biopsies with

increased mRNA of any

two out of the three markers

0

20

40

60

80

100* *

Perforin

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35 *FasL

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7 * *

Granzyme B

0

0,1

0,2

0,3

0,4

0,5

0,6

**

* p<0.05 in comparison

with no rejection

Without R, n=11Pre-R, n=6R, n=12

Are there markers in the blood?

mRNA levels in blood mononuclear cells

Transplant Proc 33:1610, 2001

TNFp<0.03

IL-8p<0.08

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Med

ian

m

RN

A l

evel

s

Granz. Bp<0.04

IFN-p<0.08

Perforinp<0.08

Rej.

No Rej.

Detection of cardiac allograft rejection by multi-parameter gene expression analysis on circulating mononuclear cells using real-time RT-PCR.Schoels et al, 2003(ISHLT Meeting, April 2003)

Gene expression patterns in peripheral bloood mononuclear cells

45 cardiac Tx patients - 66 blood samples

RT-PCR for MCP 3 (CCL-7), aminoacid transporter (xCT), MCP 1 (CCL-2), and IL-1 beta gene expression

Multi-Gene-Expression Score (MGES)sensitivity: 100% for grade 3 R,

93%for grade 2 or lower R

TCR/CD3

APC T

MHC

B7 CD28

CD40 CD40L

? TIRC7

4-1BBL 4-1BB

activated T

CD27 CD70 T

T CELL COSTIMULATION

Human Immunol 62:342, 2001

Mirror image of intragraft and blood TIRC7 expression during acute cardiac allograft rejection

biopsies

0

1

2

3

No R Pre-R R

TIR

C7/

GA

PG

H r

atio

s

*

*

0

1

2

3

4

5

6

7

blood

No R Pre-R R

*

•TIRC7: T cell immune response cDNA 7•T cell costimulatory molecule (Utku et al., Immunity 9:509, 1998)

Intragraft CD27 mRNA levels

Transplant Proc 34:747-5, 2002

0

20

40

60

80

p<0.02

p<0.02

CD

27/G

AP

GH

x1

000

Rej. No Rej. No Rej.Rejectors

At least one biopsy graded 1B duringthe first six months after transplantation

Non-rejectors

CD70 (CD27L) and 4-1BB mRNA expression

CD70 and 4-1BB transcripts were not detected in any biopsy

These transcripts were readily detected in the cells activated in mixed lymphocyte culture

CD704-1BB

0 h 24 h 48 h 72 h C- 0 h 24 h 48 h 72 h C-MLC

Transplant Proc 34: 474, 2002

STUDY OF ENDOMYOCARDIAL BIOPSIES BY MICROARRAY

• RNA isolation, amplification, labeling with Cy3 or Cy5

• Hybridization to 14K oligo-arrays (NIAID, NIH Microarray Facility)

• Two independent subsets of samples analyzed

Class prediction: rejection vs. no rejection

100%

88%

Sensitivity

77%

87%

Specificity

“winter”

“summer”

Subset

based on 70 discriminating genes

p<0.014

Composition of classifier: 70 genes significant at 0.001

Dendrogram for clustering genes, using eucledian distance and complete linkage (BRB-Array Tools Software, NCI, NIH)

rejection samples no rejection samples

rejection samples no rejection samples

Most genes: unknown function in rejectionSome examples:

•alpha-actinin-2-associated LIM protein •microsomal glutathione S-transferase 3

•chaperonin containing TCP1, subunit 3 (gamma) •Shwachman-Bodian-Diamond syndrome protein

•BCL2-associated athanogene •crystallin, mu

•small nuclear ribonucleoprotein polypeptide N •RYK receptor-like tyrosine kinase

•kinesin family member 22 •fumarate hydratase

•cytochrome P450, family 2, subfamily J •protein kinase H11

Cluster #1: genes upregulated in no rejection samples (n=23):

•syndecan 4 (amphiglycan, ryudocan) •ATP synthase, H+ transporting, mitochondrial

rejection samples no rejection samples

Clusters # 2-# 6: genes upregulated in rejection samples (n=47)

Examples (some known genes):•TCR beta locus

•MHC class I, E

•minor histocompatibility antigen HA-1

•integrin beta 2

•STAT1

•chemokine C-C ligand 19 (ELC)

•chemokine C-C ligand 5 (RANTES)

•lymphotoxin beta

•CD74 (invariant chain, MHC class II-associated)

•class I cytokine receptor

Non-invasive molecular screening for acute rejection: a multicenter prospective clinical study.Eisen et al, 2003 (ISHLT Meeting, April 2003)

•study of peripheral blood mononuclear cells (PBMC), n=158

•8000 gene custom PBMC array

> 50 differentially expressed

genes

Test (20%) 100% 56%

Training (80%) ? ?

Test, after exclusion of CMV+ and early samples

100% 100%

Sensitivity SpecificitySubset

PCR validation of expression of 40 markers

10-gene PCR algorithm with sensitivity of 94% and specificity of 89% for rejection

In Summary..................

Diagnosis of rejection:

Gene expression analysis of immune activation molecules by RT-PCR in cardiac transplantation

Markers in peripheral blood:Fewer studies but promising results

Markers in biopsies:High sensitivity is achieved with the use of several markers

Possible solution

Serial evaluation and combined analysis of

several markers?

Individual variability in expression levels

Problem

Short-termPrediction of acute rejection onset within 7-15 days

expression of IFN-, TIRC7, Granzyme B, Perforin, FasL in the graft

Gene expression analysis of immune activation molecules by RT-PCR in cardiac transplantationGene expression analysis of immune activation molecules by RT-PCR in cardiac transplantation

Prognosis of transplant evolution

expression of IFN-, TIRC7 in the blood

expression of IL-8, TNF in the blood

Gene expression analysis of immune activation molecules by RT-PCR in cardiac transplantationGene expression analysis of immune activation molecules by RT-PCR in cardiac transplantation

Prognosis of transplant evolution

Long-term

AIF-1 (allograft inflammatory factor 1): development of cardiac allograft vasculopathy (Autieri et al, 2002)

bFGF (basic fibroblast growth factor) in the first week post-Tx : acute rejection during the first year after TX (de Groot-Kruseman, 2001)

UNIVERSIDADE FEDERAL DE SÃO PAULO Escola Paulista de Medicina

Rosiane VZ DinizDirceu R Almeida

João NR Branco Antonio C CarvalhoCardiology Division

Medicine Department

Márcia M SouzaMarcello Franco

Pathology Department

Natalia Shulzhenko

Andrey Morgun

Angela P Chinellato

Gisele F Rampim

Maria Gerbase-DeLima

Immunogenetics DivisionPediatrics Department

Xin X. ZhengNaili Ma

Terry B. StromDivision of ImmunologyHarvard Medical School

Ainhoa Perez-DiezPolly MatzingerGhost Lab, LCMI

NIH, NIAID

Elizabeth C P Hurtado