Ecp2009 Precongress Transpl Path Svseshan

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Immune Profiling in RenalTransplantation: Biopsy Correlationswith Urine and Plasma PCR Studies

Surya V. Seshan, T. Muthukumar, D, Dadhania,

M. Suthanthiran

Weill Cornell Medical College

New York-Presbyterian Hospital

New York, USA





Banff 07 Update: Diagnostic Categories for Renal

Allograft Biopsies ● 1 – Normal

● 2 – Antibody Mediated Changes

● C4d deposition with no morphological e/o active rejection

● Acute AMR

● Chronic AMR

● 3 – Borderline Changes – suspicious for acute T cell mediated rejection

● 4 – T cell Mediated Rejection

● Acute

● Chronic active (Chronic allograft arteriopathy)

● 5 - Interstitial Fibrosis & Tubular Atrophy NOS

● 6 – Others

● Chronic hypertension

● Calcineurin inhibitor toxicity

● Chronic obstruction

● Bacterial pyelonephritis

● Viral infection K Solez Am JTransplant 2008



Limitations of Renal Biopsy

● Specimen adequacy

Banff 1997 – 9-11 glomeruli, 1-2 arteries

● Need for the presence of cortex

● Patchy distribution of disease

● Borderline lesions

● Prior treatment

● Chronic parenchymal scarring



Immune Profiling in RenalTransplantation

● Diagnosis

● Therapeutic decision making

● Acquire prognostic information

● Monitor/surveillance of allograft function

● Elucidate pathogenetic mechanisms andmolecular pathways of cell activation andtissue injury

Samples used: Blood, urine and tissue



Levels of Monitoring

Initiating Event End Point

Molecular

Histological

Biochemical

Clinical



Methods of Immune Profiling

● Immunohistochemistry

Immunofluorescence

Immunoperoxidase

● Polymerase chain reaction (PCR) studies

Viral proteins

Inflammatory cells

Immune mediators Tissue Microarray studies- cDNA,

Oligonucleotide

● Serum and urine proteomics

● Allo-antibodies – Cell & soluble a based



Acute Cellular Rejection



Acute T Cell Rejection : Molecular events Invitation: IP10

Contact: CD103

Induced suicide:

Granzyme B/

Perfor in

Collateral

Protection: PI-9

Damage control:

FoxP3

P A lifi ti E h d Ki ti Q tit ti (RT) PCR



Pre-Amplification Enhanced Kinetic Quantitative (RT) PCR

Assay: Designed & Validated at Suthanthiran Laboratory

50 cc urine sample

Centrifuge - Pellet

Total RNA isolated - RNA quantifiedQuality checked

Reverse transcribed to cDNA

cDNA concentration: 1µg/100µl TE bufferApprox: 1-2 ug cDNA (cf. Biopsy 5-10 ug, Blood 10-15 ug)

Design of gene specific primers & fluorogenic probes

Step 1: 10 cycle PCR with multiple primer pairs of interest

Step 2: Kinetic quantitative PCR with single primer pair & probeA standard curve is generated

cDNA quantity expressed as copies/ ug total RNA

Urine Cells

DNA mRNA

cDNA

Exon 1 Intron Exon 2

Exon 1 Exon 2

Exon 2Exon 1



Tatapudi RR et al, Kidney Int 2004

Non-Invasive Detection of Renal Allograft Inflammation:

mRNA Profiling for IP-10 & CDCR3 in Urine Cells –

Invitation genes

I hi t h i l L li ti f IP 10



Tatapudi RR et al,

Kidney Int 2004

Immunohistochemical Localization of IP-10

and CXCR3 in Renal Allografts

ll f j i fili f i



Ding R et al, Transplantation 2003

Acute Allograft Rejection: mRNA Profiling of Urinary

Cells for CD103 – Contact gene

Acute Allograft Rejection: mRNA Profiling of Urinary Cells for Perforin and



The ‘Suicide Inducer’

Genes in AcuteRejection

Li B NEJM 2001,

Muthukumar T

Transplantation 2003

Acute Allograft Rejection: mRNA Profiling of Urinary Cells for Perforin and

Granzyme B. They have a high degree of accuracy in distinguishing AR from

other causes of allograft dysfunction

A t C ll l R j ti



Acute Cellular RejectionCD3

TIAGr-B



Tissue Microarray and Proteomic

Analysis in Acute CellularRejection

C Pl tf C i



Cross-Platform Comparisons

DNA Microarray Profiling Showing Molecular



Sarwal M et al, N Eng J Med 2003

DNA Microarray Profiling Showing Molecular

Heterogeneity in Acute Renal Allograft Rejection



ProteomicsBlood, urine &

Biopsy tissue

Surface enhanced

or Matrix assisted

Laser desorption

Mass Spectroscopy

Proteomic Based Detection of Urine Proteins Associated



Schaub S et al, J Am Soc Nephrol 2004

Proteomic Based Detection of Urine Proteins Associated

with Acute Renal Failure

Mi RNA t i t i l t l t id tif t j ti



Anglicheau D et al PNAS 2009

Micro RNA transcripts in renal transplants can identify acute rejection

Micro RNA in renal transplants




Micro RNA in renal transplants






A tib d M di t d R j ti



C4d Positive PTC

Antibody Mediated Rejection

Abs to HLA Class I & II

Non MHC ags in

endothelium

ABO

Other

Fluctuations in

DSA levels

Variability in C4d

staining



ENDOTHELIAL GENE EXPRESSION IN KIDNEY

TRANSPLANTS WITH ALLOANTIBODY

INDICATES ANTIBODY-MEDIATED DAMAGE

DESPITE LACK OF C4D STAINING

Sis et al. AJT 2009

Clustering endothelial transcripts detects C4d negative



C4d+ Ab+ Biopsies

C4d Ab+ Biopsies

Controls (normal nephrectomies)

C4d Ab Biopsies

Bx type

Biopsy type: Histopathologic Diagnosis:

C4d+ ABMR

TCMR

Other

Normal nephrectomy

Borderline changes

Mixed TCMR and C4d+ ABMR

BK virus nephropathy

Diagnosis

Cluster with high ENDAT expressionCluster with high expression

Clustering endothelial transcripts detects C4d negative

samples with increased endothelial transcript expression

Population= 165 biopsies with or without circulating Ab

This heatmap indicates:

1. All C4d+ ABMR biopsies (black) have high Endothelial

transcript expression

2. There are also C4d negative biopsies with high

expression which Ab+ (blue) or Ab negative (gray)

Red: high expression

Blue: low expression

Sis et al. AJT 2009



Ac te Rejection s Urinar Tract Infection: mRNA



Dadhania D et al, Transplantation 2003

Acute Rejection vs. Urinary Tract Infection: mRNA

Profiling of Urinary Cells for Granzyme

Pyelonephritis in Renal Allograft



CD3 CD20

Granzyme B

y p g

G ft L i Si ifi t Ri k i I di id l



Drachenberg et al. AJT 2

Graft Loss is a Significant Risk in Individuals

with BKV Nephropathy

INCREASED FIBROSIS and INFLAMMATION

Current Non Invasive Diagnostic Tools



32

Current Non-Invasive Diagnostic Tools

Decoy cells – ground glassintranuclear viral inclusion

bodies

EM of negatively stained urine

sample Icosahedral spherical shape

of 40nm

Urinary Haufen – cast like viral aggregates

PCR

Amplification of

DNA or cDNA

•Blood

•Urine

Pathology Evaluations – Decoy cells, EM contrast Molecular Evaluations

Urine Haufen

BKV (Polyoma virus) Associated Nephritis



BKV (Polyoma virus) Associated Nephritis

• BKV nephritis is often associated with histological

findings consistent with acute rejection such asmononuclear infiltration and tubulitis

• In the setting of BKVN, management decisions are

contingent upon accurate differentiation from acute

allograft rejection (AR).

• Differentiation of BKV nephritis from concurrent

rejection process

– Considerable tubulitis - C4d staining

– Vascular rejection - HLA – DR

expression

BKV Nephritis: mRNA Profiling in Urinary cells for



Urinary cell Granzyme B mRNA (a cytotoxic attackmolecule) levels, but not BKV VPI mRNA levels are acorrelate of allograft damage in patients with BKVnephritis (as defined by the presence or absence of

tubulitis).

(Manuscript under preparation)

•Allograft failure in patients with BKV nephritis can be predicted by urinary gene expression profiles.

Granzyme B



Gr-B HLA-Dr

Active tubulo-interstitial inflammation in BKV nephritis case

BKV Nephritis and tubulitis and HLA-DR staining





Conclusions

• Real Time Quantification of Urinary Cell mRNA levels allows for – accurate diagnosis of BKVN

– allows for measurement of prognostic markers simultaneously

• Individuals with increased levels of cytotoxic T cell molecule, granzyme B,have initial higher levels of creatinine and increased risk of subsequent

decline in renal allograft function.

• Whether elevated levels of granzyme B reflect adaptive immune responseagainst BK virus or against the allograft or both remains to be determined,thus modifying the management of BKVN.

(Manuscript in preparation)



K Solez, AJ T 2007

Genomics/Proteomics/PCR vs. Histopathology



Genomics/Proteomics/PCR vs. Histopathology

Molecular screening may be an attractive alternative for immune

surveillance and early diagnosis of acute rejection

These advanced molecular studies could have a potential for a more objective

and quantitative assessment of allograft immune response as well as

differentiate from other forms of allograft dysfunction.

Considering the limitations of the biopsy tissue, significant reduction in the

sources of variability can be achieved.

Such data may complement the routine histological examination in the grand

scheme of immune profiling in renal transplantation



Thank You

Special thanks to the

urine gene expression group in

Suthanthiran Laboratory

C Chang

R Ding

M Lagman

V Sharma

B Li

C Snopkowski

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Ecp2009 Precongress Transpl Path Svseshan

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