a predictive assay for success rates of islet transplantation to treat type-1 diabetes tracy fuad...

A Predictive Assay for Success Rates

of Islet Transplantation to Treat Type-1 Diabetes

Tracy Fuad

2007

Diabetes Institute of Immunology and Transplantation

Objective• Engineer an assay to improve

success rates of islet transplantation in type-1 diabetics– As a tool to match patients to donors– As an in vitro monitoring tool

Type-1 Diabetes• An auto-immune

disease• Characterized by loss

of pancreatic islet cell function

Type-1 Diabetes• An auto-immune

disease• Characterized by loss

of pancreatic islet cell function

http://www.diabetesresearch.org

Islet Cells

Islet Transplantation

http://www.rsna.org/rsna/media/

Immune-Mediated Graft Rejection• Patient’s immune system recognizes

islets as foreign

• Immune system attacks and destroys transplanted tissues

HLA-Matching• Human leukocyte-antigen typing (HLA-

typing) matches tissues to minimize the immune response in a transplant

• Immune response can be also be measured by cytokine production

ELISPOT Assay

• Enzyme-linked immunospot assay

• Quantifies the cytokine production of lymphocytes (white blood cells)

• TNF is an inflammatory cytokine that is produced during transplantation and rejection

Goals• Optimize ELISPOT assay to test TNF

production in donor-stimulated patient cells

• Use the modified ELISPOT protocol to look for a correlation between failed transplants and elevated TNF production

Previous Studies

• Studies by Augustine et al. and Bellisola et al. have correlated heightened IFN production in an ELISPOT assay to increased rates of renal transplant rejection

Hypothesis

• Because IFN is an inflammatory cytokine often produced in conjunction with TNF, I hypothesized that heightened TNF production would correlate with failed islet transplants

Methods: ELISPOT Assay

• Uses a 96-well plate with nitrocellulose membranes

• Quantifies cytokines by capturing them locally and visualizing each cytokine

ELISPOT well

Capture antibody

Capture antibody

Blocked with protein serum

Leukocytes or splenocytes are added

Cytokines Released

Detection Antibody

Detection Antibody

Strepdavidan-HRP,

a colored substrate

Precipitation reaction

Reading the ELISPOT Plate

http://www.biosciencetechnology.com/images

Reading the ELISPOT Plate

www.elispot.com/index.html?elispot_reader/software.html

ELISPOT Modification

• Increased the protein concentration of blocking buffers

• Reduced the number of cells per well

• Decreased secondary detection antibody

incubation time

• Added more washes between steps

Cell-to-Cell ELISPOT• Used to find the optimal donor-to-patient cell-to-

cell ratioRow Description, Columns 1-3 Description, Columns 4-6

A Media control Responder cells alone

B Responder cells (R) + PHA Responder cells (R) + Con-A

C Matched stimulator (S1) Mismatched stimulator (S2)

D 9:1 (S1) to (R) cell to cell ratio 9:1 (S2) to (R) cell to cell ratio

E 3:1 (S1) to (R) cell to cell ratio 3:1 (S2) to (R) cell to cell ratio

F 1:1 (S1) to (R) cell to cell ratio 1:1 (S2) to (R) cell to cell ratio

G 1:3 (S1) to (R) cell to cell ratio 1:3 (S2) to (R) cell to cell ratio

H 1:9 (S1) to (R) cell to cell ratio 1:9 (S2) to (R) cell to cell ratio

TNF Allo-Titration ELISPOT

TNF Allo-Titration ELISPOT

TNF Allo-Titration ELISPOT

TNF Allo-Titration ELISPOT

Donor-to-Patient Induced Immune Response

• Islet transplant patient was selected

– First transplant failed

– Second transplant was successful

• Patient cells stimulated with cells from each donor

Donor-to-Patient Induced Immune Response

• Modified ELISPOT protocol used with the following plate map:

Row Description Cells/well

A Patient Cells Alone 90,000

B Patient cells with PHA 90,000

C Patient cells with Con-A 90,000

D Donor-Transplant 1 270,000

E Donor-Transplant 2 270,000

F Patient (P) + Donor 1 (D1) 90,000 (P) + 270,000 (D1)

G Patient (P) + Donor 2 (D2) 90,000 (P) + 270,000 (D2)

H Patient (P) + 3rd party (3P) 90,000 (P) + 270,000 (3P)

TNF Clinical Patient ELISPOT

TNF Clinical Patient ELISPOT

PositiveControls

TNF Clinical Patient ELISPOT

PositiveControls

TNF Clinical Patient ELISPOT

Negative Controls

TNF Clinical Patient ELISPOT

Failed Transplant

Successful transplant

0

10

20

30

40

50

60

70

80

Patient+Donor 1 Patient+Donor 2 Patient+3rd Party

spots/well

0

10

20

30

40

50

60

70

80

Patient+Donor 1 Patient+Donor 2 Patient+3rd Party

spots/well

Failed Transplant

0

10

20

30

40

50

60

70

80

Patient+Donor 1 Patient+Donor 2 Patient+3rd Party

spots/well

Successful Transplant

0

10

20

30

40

50

60

70

80

Patient+Donor 1 Patient+Donor 2 Patient+3rd Party

spots/well

p=0.0000892

Conclusion

• TNF ELISPOT assay is an effective means to measure immune response in transplantation patients

• Heightened TNF production has a statistically significant correlation to failed transplantation

Applications• A tool to match patients to donors

• An in vitro monitoring assay–Identify immunosuppression needs of

individual patients

–Reduce the use of immunosuppresant medications

Future Studies• Test samples from additional islet

transplant patients

• Test patient samples from different points in transplantation timeline

Acknowledgements• The University of Minnesota and Dr.

Bernard Hering

• Dr. Pratima Pakala, Kelly Hire, Adam Nettles, and Olivia Thai

• Ms. Fruen and the Science Research Class

A Predictive Assay for Success Rates

of Islet Transplantation to Treat Type-1 Diabetes

Tracy Fuad

2007