analyzing immune response with flow cytometry
DESCRIPTION
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
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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
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Islet Transplant
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Islet Transplant
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Islet Transplant
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Cytokines
• Proteins related to immune response– TNF
• Fuad (2007)
– IFN• Augustine (2005), Bellisola (2006)
– IL-2
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First Goal
• To determine whether ICS is superior to ELISPOT
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ELISPOT
ELISPOT plate
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ELISPOT
Capture antibody ELISPOT plate
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ELISPOT
ELISPOT plate
Cells added
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ELISPOT
ELISPOT plate
Cytokines released
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ELISPOT
ELISPOT plate
Detection antibody
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ELISPOT
ELISPOT plate
Streptavidin-HRP
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Cell
Cytokine
Cytokine
Cytokine
ICS
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ICS
Cell
Cytokine
Cytokine
Cytokine
Permed
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ICS
Antibody-conjugated dye
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ICS
Detected using a flow cytometer
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Results: ELISPOT vs. ICS
• ICS– All readable– Two days
• ELISPOTs– Two readable results out of five– Five days
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Second Goal
• To modify ICS for use on allo-stimulated human cells– Determine sensitivity– Cytokine peaks
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Modified ICS Assay 1
• Extended BFA incubation time for shorter workday, more user-friendly
• Cells incubated for 24, 72, 96 hours post-stimulation
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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
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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)
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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)
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Modified ICS Assay 1
0
10
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
0
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
0
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
Day 1
Day 3 Day 4
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Modified ICS Assay 1
0
10
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
0
10
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70
Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
0
10
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Perm w/ stim PMA/Iono
Type of stimulation
Percent cytokine-positive cells
Day 1
Day 3 Day 4
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Modified ICS Assay 2
0
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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
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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
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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
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Acknowledgements
• Dr. Pratima Pakala
• Kelly Hire and Diem Ngo
• Ms. Lois Fruen
• Team Research
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Analyzing Immune Response With Flow Cytometry
Madelaine Taft-Ferguson (2007-2008)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.