Immune Profiling in Renal Transplantation: Biopsy

Correlations with Urine and Plasma PCR Studies

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

Weill Cornell Medical CollegeNew York-Presbyterian Hospital

New York, USA

Renal Dysfunction in Transplants

● Acute tubular injury● Drug toxicity● Infections – viral, bacterial● Allergic interstitial nephritis● Perioperative or post-operative

complications

Acute

ChronicRejection

T cell Antibody mediated

Tubulo-interstitial

Vascular

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 J Transplant 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 Renal Transplantation

● Diagnosis● Therapeutic decision making● Acquire prognostic information● Monitor/surveillance of allograft

function● Elucidate pathogenetic mechanisms and

molecular pathways of cell activation and tissue 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 ag based

Acute Cellular Rejection

Acute T Cell Rejection : Molecular events Invitation: IP10

Contact: CD103

Induced suicide: Granzyme B/ Perforin

Collateral Protection: PI-9

Damage control: FoxP3

Pre-Amplification Enhanced Kinetic Quantitative (RT) PCR Assay: Designed & Validated at Suthanthiran Laboratory

50 cc urine sampleCentrifuge - Pellet Total RNA isolated - RNA quantifiedQuality checkedReverse transcribed to cDNAcDNA concentration: 1µg/100µl TE bufferApprox: 1-2 ug cDNA (cf. Biopsy 5-10 ug, Blood 10-15 ug)

Design of gene specific primers & fluorogenic probesStep 1: 10 cycle PCR with multiple primer pairs of interestStep 2: Kinetic quantitative PCR with single primer pair & probe A standard curve is generatedcDNA 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

Tatapudi RR et al, Kidney Int 2004

Immunohistochemical Localization of IP-10 and CXCR3 in Renal Allografts

Ding R et al, Transplantation 2003

Acute Allograft Rejection: mRNA Profiling of Urinary Cells for CD103 – Contact gene

The ‘Suicide Inducer’ Genes in Acute Rejection

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

Acute Cellular RejectionCD3

TIAGr-B

Tissue Microarray and Proteomic Analysis in Acute

Cellular Rejection

Cross-Platform Comparisons

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

Schaub S et al, J Am Soc Nephrol 2004

Proteomic Based Detection of Urine Proteins Associated with Acute Renal Failure

Anglicheau D et al PNAS 2009

Micro RNA transcripts in renal transplants can identify acute rejection

Anglicheau D et al PNAS 2009

Micro RNA in renal transplants

Anglicheau D et al PNAS 2009

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 ENDOTHELIAL GENE EXPRESSION IN KIDNEY TRANSPLANTS WITH ALLOANTIBODY TRANSPLANTS WITH ALLOANTIBODY

INDICATES ANTIBODY-MEDIATED DAMAGE INDICATES ANTIBODY-MEDIATED DAMAGE DESPITE LACK OF C4D STAININGDESPITE LACK OF C4D STAINING

Sis et al. AJT 2009 Sis et al. AJT 2009

C4d+ Ab+ BiopsiesC4d Ab+ Biopsies

Controls (normal nephrectomies)

C4d Ab Biopsies

Bx type

Biopsy type: Histopathologic Diagnosis:

C4d+ ABMRTCMR

Other

Normal nephrectomy

Borderline changesMixed TCMR and C4d+ ABMR

BK virus nephropathy

Diagnosis

Cluster with high ENDAT expressionCluster with high expression

Clustering endothelial transcripts detects Clustering endothelial transcripts detects C4d negative C4d negative samplessamples with increased endothelial transcript expression with increased endothelial transcript expression

Population= 165 biopsies with or without circulating AbPopulation= 165 biopsies with or without circulating Ab

This heatmap indicates:This heatmap indicates:1.1. All C4d+ ABMR biopsies (black) have high Endothelial All C4d+ ABMR biopsies (black) have high Endothelial

transcript expressiontranscript expression2.2. There are also C4d negative biopsies with high There are also C4d negative biopsies with high

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

Red: high expressionRed: high expressionBlue: low expressionBlue: low expression

Sis et al. AJT 2009 Sis et al. AJT 2009

Infections in Renal Transplant

Dadhania D et al, Transplantation 2003

Acute Rejection vs. Urinary Tract Infection: mRNA Profiling of Urinary Cells for Granzyme

CD3 CD20

Granzyme B

Pyelonephritis in Renal Allograft

Drachenberg et al. AJT 2

Graft Loss is a Significant Risk in Individuals with BKV Nephropathy

INCREASED FIBROSIS and INFLAMMATION

32

Current Non-Invasive Diagnostic Tools

Decoy cells – ground glass intranuclear 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 nephritis is often associated with histological findings consistent with acute rejection such as mononuclear 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

Urinary cell Granzyme B mRNA (a cytotoxic attack molecule) levels, but not BKV VPI mRNA levels are a correlate of allograft damage in patients with BKV nephritis (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.

BKV Nephritis: mRNA Profiling in Urinary cells for 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 response against 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

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

and quantitative assessment of allograft immune response as well as and quantitative assessment of allograft immune response as well as differentiate from other forms of allograft dysfunction.differentiate from other forms of allograft dysfunction.

Considering the limitations of the biopsy tissue, significant reduction in the Considering the limitations of the biopsy tissue, significant reduction in the sources of variability can be achieved.sources of variability can be achieved.

Such data may complement the routine histological examination in the grand Such data may complement the routine histological examination in the grand scheme of immune profiling in renal transplantationscheme 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