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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