Individualisation of Imunosupression after Kidney transplantation Clinical Utility of the ELISPOT Assay for IFN? and the Cylex

1
Individualisation of Imunosupression after Kidney
transplantationClinical Utility of the ELISPOT
Assay for IFN? and the Cylex/ImmuKnow Assay

• Donald E. Hricik, M.D.
• Chief, Division of Nephrology and Hypertension
• Case Western Reserve University
• Cleveland, Ohio
• Postgraduate Education Director,
• American Society of Transplantation

2
Utility of Immune Monitoring Assays

• Surrogate for acute rejection (replacement for
  renal biopsy)
• Prediction of immune injury (acute rejection and
  chronic allograft dysfunction)
• Guide to immunosuppression

3
ELISPOT ASSAY
Primary coating antibody
Synthetic white membrane
Responding lymphocytes
Donor splenocytes or lymphocytes
Secreted cytokine (e.g. interferon-gamma)
Detection,enzyme-linked second antibody
h
Precipitated enzyme substrate which forms a spot
h
4
Hricik DE, et al. AJT 2003 3878
5
Relationship Between Early IFN-ELISPOTS and
Long-term Serum Creatinine (follow-up 12-68
months, mean 38 months, n65)
n 29 15
12 9
Correlation between Mean
IFN-ELISPOTS and Creatinine R0.36, p0.003

6
Multiple Regression Serum Creatinine at Last
Follow-up
Variable Beta Coefficient p
Mean IFN-ELISPOTS 0.329 0.006
African American 0.245 0.04
Acute Rejection 0.212 0.073
Age, gender, body weight, delayed graft function, PRA, donor source NS
7
ROC Curve Analysis

• Serum Creatinine gt2.0 mg/dl at last follow-up
• Threshold IFN-ELISPOTS gt 30/300 K cells
• Sensitivity 62
• Specificity 76

8
Hricik DE, et al. AJT 2003 3878


p0.16 p0.001
9
No Induction therapy


300
200
Acute Rejection in 11/24 (46)
100
Total IFN-? Spots
90
80
70


60
50
40
30
20
10
0


Pretransplant
1 mos
3 mos
2 mos
4 mos
5 mos
6 mos

Posttransplant
Augustine JJ, et al. Transplantation 86 529-534
10
Induction therapy


300
200
Acute Rejection in 0/8
100
Total IFN-? Spots
90
80
70


60
50
40
30
20
10
0


Pretransplant
1 mos
3 mos
2 mos
4 mos
5 mos
6 mos

Posttransplant
11
Pretransplant IFN-ELISPOTS Correlate with
Posttransplant Rejection
N 14
N 36
P 0.024
Adult AA kidney recipients treated with
sirolimus, tacrolimus, prednisone
Augustine JJ, et al. Am J Transplant 2005 51971
12
Logistic Regression Analysis for Acute Rejection
Variable Odds ratio 95 CI p Value
HLA match 1.3 0.7 to 2.4 ns
Percent PRA 1.0 0.9 to 1.0 ns
Induction Tx 0.2 0.1 to 8.2 ns
DGF 20.8 1.9 to 224 0.012
Elispot () 10.9 1.7 to 71.1 0.012
13
100
p0.001
80
60
MDRD GFR, ml/min
40
20
0
Positive n14
Negative n36
ELISPOT assay
14
Multivariate Analysis for GFR at 12 months
Variable Slope 95 CI p Value
Deceased donor 1.7 -9.1 to 12.4 ns
Older donor age (yr) -0.4 -0.7 to 0.1 0.021
DGF -17.2 -28.7 to 5.7 0.004
Acute rejection -4.6 -16.4 to 7.4 ns
Elispot () -19.8 -30.6 to 9.0 0.001
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Other Experiences

• Nather BJ, et al. Transplant Immunol 2006 16
  232-237
• Early measurements of posttransplant ELISPOTs for
  IFN predict graft function at 6 and 12 months
• Kim SH, et al. Transplant Proc 2007 39
  3057-3060
• Measures of pretransplant donor-specific (but not
  3rd party) ELISPOTS for IFN predict acute
  rejection and graft function in the first 6
  months posttransplant
• Bestard O et al. J Am Soc Nephrol 2008
  191419-1429

16
Early Posttransplant Donor Specific
Responsiveness Correlates with Late Rejection and
Poor Graft Function
MDRD-GFR (ml/min)
Donor-specific ELISPOT/300K cells
NO (n20) Yes (n14) POSITIVE
ELISPOT IFN? ASSAY
NO (n22) YES (n12) LATE
ACUTE REJECTION
Bestard O, et al. J Am Soc Nephrol 2008 191419
17

12mos 24mos
36mos 48mos 60mos 120mos
n 23
42 53 65
74 97
Time on Hemodialysis
18
Recipient 36 yo AA female
42 yo WM 56 yo AAM
35 yo AAF HLA match 4/6
2/6 3/6
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IFN-g ELISPOT PRT Assay Plate
Responder 1 2 3 4 5
Media Stimulator 1 Stimulator 2 Stimulator
3 Stimulator 4 Stimulator 5 Stimulator
6 Stimulator 7 Stimulator 8 Positive Control
(PHA)
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Distribution of PRT / PRA Status in Transplant
Recipients
n30
PRT / PRA
7
PRT / PRA
PRT / PRA
30
56
7
PRT / PRA
Poggio ED et al. Transplantation 2007 83847-852
21
PRT is Associated with BPAR
PRT
100
plt0.01
75
PRT percent
50
25
0
Rejection-free
Biopsy-proven AR
22
The Cylex Assay
Incubate
Wash
Lymphocyte Stimulation
Magnetic Separation
ATP
Luminometer
ATP
ATP Detection Reagents
ATP
Cell Lysis to Release ATP
Measure Light Intensity
23
Immune Response (ATP ng/ml)
High gt525
Normal 225-525
Low lt225

Median in healthy subjects 408
Median in transplant patients 259
Kowalski R, et al. Clin Transplant 2003
1777-88
24
Rejections and Infections by Transplanted Organ
Type of allograft Patients Acute rejections Median ATP (ng/ml) Infections Median ATP (ng/ml)
Kidney 243 22 462 31 164
Liver 150 7 471 27 60
Heart 86 3 620 2 160
Small Bowel 25 7 769 6 127
Kowalski RJ, et al. Transplantation 2006 82
663-668
25
Aggregate Immune Response Distributions of Solid
Organ Transplant Recipients
Plt0.001
ATP (ng/ml)
600
Plt0.001
488
400
249
200
111
0
Rejection (n39) Stable (n504)
Infection (n111)
Kowalski RJ, et al. Transplantation 2006 82
663-668
26
Use of Cylex assay to titrate immunosuppression
during viral infections

• Gautam A, et al. Transpl Infect Dis 2007
• Lymphocytic choriomeningitis
• Gautam A, at al. Pediatr Transplantation 2006
• EBV/PTLD
• Gautam A, et al. International
  Immunopharmacology 2006
• BK (3) EBV (3) CMV (2) HCV (2) LCMV (2)

27
Impact of Cylex Immune Cell Function Assay in
Predicting Acute Cellular Rejection and
Recurrence of HCV in Liver Transplantation

• Recurrence of HCV difficult to distinguish from
  acute cellular rejection
• Retrospective review of 85 patients with liver
  biopsies and Immunknow assays

Hashimoto K et al. Cleveland Clinic Foundation
28
ImmuKnow Categorized by Biopsy Findings after
Liver Transplant for Hepatitis C
Low (lt225) Medium (226-524) High (gt 524)
ACR 4 (20) 14 (70) 2 (10)
HCV Recurrence 35 (90) 4 (10) 0 (0)
Intermediate 4 (67) 2 (33) 0 (0)
Hashimoto K, et al. 2008 ATC
29
Comparison of ATP Levels
P lt 0.0001
ACR HCV Rec. Indeterminate
30
Pretransplant Immune Risk Assessment Associated
with Posttransplant Outcomes
No BPAR (n8) BPAR (n18) p-value Unstable Cr (n20) Stable Cr (n69) p-value
ATP (ng/ml) 334156 414139 0.04 348129 304151 0.22
ELISPOTS 2931 4728 0.30 3020 3632 0.85
donor-specific IFN? ELISPOTs/300K cells
Reinsmoen N, et al. Transplantation 2008 85
462-470
31
Summary I

• ELISPOT Assay for IFN gamma
• Most helpful for assessing immune risk
• Both pre- and post-transplant values predictive
  of posttransplant acute rejection
• Both pre- and post-transplant values predictive
  of long-term renal function
• Probably not a surrogate for acute rejection
• Can be donor specific
• Only preliminary evidence that the assay can
  guide immunosuppression
• Prospective interventional trials lacking

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Summary II

• Cylex (ImmunoKnow Assay)
• Not a surrogate for acute rejection
• Not donor specific
• May help guide immunosuppression
• May be helpful in guiding over-immunosuppression,
  especially in patients with existing infection
• Little evidence that the assay is helpful in
  guiding under-immunosuppression
• Prospective interventional trials lacking