analyzing immune response with flow cytometry
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Analyzing Immune Response With Flow Cytometry. Madelaine Taft-Ferguson (2007-2008). 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 - PowerPoint PPT PresentationTRANSCRIPT
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
Type of stimulation
Percent cytokine-positive cells
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
Day 1
Day 3 Day 4
Modified ICS Assay 1
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
Day 1
Day 3 Day 4
Modified ICS Assay 2
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Perm 1:1 stims 3:1 stims 5:1 stims PMA/Iono
Type of stimulation
Percent cytokine-positive cells
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Perm 1:1 Stims 3:1 Stims 5:1 Stims PMA
Type of stimulation
Percent cytokine-positive cells
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Perm 1:1 Stims 3:1 Stims 5:1 Stims PMA
Type of stimulation
Percent cytokine-positive cells
Day 1
Day 2 Day 3
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.