Analyzing Immune Response With Flow Cytometry

Madelaine Taft-Ferguson (2007-2008)

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Islet Transplant• Type-1 diabetes: autoimmune disease

– Targets islet cells– Can be cured with islet transplant

• 88% of islet transplants fail after five years– Immune system attacks foreign cells– Immune response can be measured by

cytokine production

Islet Transplant

Islet Transplant

Islet Transplant

Cytokines

• Proteins related to immune response– TNF

• Fuad (2007)

– IFN• Augustine (2005), Bellisola (2006)

– IL-2

First Goal

• To determine whether ICS is superior to ELISPOT

ELISPOT

ELISPOT plate

ELISPOT

Capture antibody ELISPOT plate

ELISPOT

ELISPOT plate

Cells added

ELISPOT

ELISPOT plate

Cytokines released

ELISPOT

ELISPOT plate

Detection antibody

ELISPOT

ELISPOT plate

Streptavidin-HRP

Cell

Cytokine

Cytokine

Cytokine

ICS

ICS

Cell

Cytokine

Cytokine

Cytokine

Permed

ICS

Antibody-conjugated dye

ICS

Detected using a flow cytometer

Results: ELISPOT vs. ICS

• ICS– All readable– Two days

• ELISPOTs– Two readable results out of five– Five days

Second Goal

• To modify ICS for use on allo-stimulated human cells– Determine sensitivity– Cytokine peaks

Modified ICS Assay 1

• Extended BFA incubation time for shorter workday, more user-friendly

• Cells incubated for 24, 72, 96 hours post-stimulation

Modified ICS Assay 2

• Extended BFA time

• Cells incubated for 24, 48, 72 hours post-stimulation

• Stimulator:responder ratios 1:1, 3:1, 5:1

BD Bioscience Assay

(T-test for TNF w/ stims vs. PMA/Ionomycin: p=0.11; on IFN p=0.15; on Il-2 p=0.39)

BD Bioscience Assay

(T-test for TNF w/ stims vs. PMA/Ionomycin: p=0.11; on IFN p=0.15; on Il-2 p=0.39)

Modified ICS Assay 1

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Perm w/ stim PMA/Iono

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Perm w/ stim PMA/Iono

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Modified ICS Assay 1

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Perm w/ stim PMA/Iono

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Modified ICS Assay 2

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Perm 1:1 stims 3:1 stims 5:1 stims PMA/Iono

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Perm 1:1 Stims 3:1 Stims 5:1 Stims PMA

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Perm 1:1 Stims 3:1 Stims 5:1 Stims PMA

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Conclusion• ICS is capable of detecting effect of

allo-stimulation on cytokine production

• Cells can survive extended incubation times in ICS

• TNF and IFN peak within 24 hours, IL-2 not expressed until after 72 hours

• 3:1 stimulator:responder ratio is best

Future Work

• Further modify ICS

• Test correlation suggested by the Fuad study (2007) of TNF with transplant rejection

• Investigate possible correlation with IFN, IL-2

Acknowledgements

• Dr. Pratima Pakala

• Kelly Hire and Diem Ngo

• Ms. Lois Fruen

• Team Research

Analyzing Immune Response With Flow Cytometry

Madelaine Taft-Ferguson (2007-2008)

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.