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77 Comparison of Multiple Analytical Methods to Measure Cytokine Production from Single T Cells
R. P. Bucy*1, G. Marshall1, P. Goeptfert1, J. Jacobson2, and J. M. Kilby1
1Univ. of Alabama at Birmingham, USA and 2Mount Sinai Med. Ctr., New York, NY, USA
Background: The accurate quantitative assessment of T-cell cytokine production from antigen-stimulated PBMC is critical for assessment of vaccine responses.
Methods: Several independent assays of IL-2 and IFN-gamma production were compared using PBMC from HIV-infected subjects in different clinical states. These assays included a novel amplified immunohistochemical (IHC) intracellular cytokine staining method, the EliSpot assay, intracellular staining with analysis by flow cytometry, the amount of cytokine mRNA detected by real-time RT-PCR, the amount of cytokine protein in the supernatant measured by ELISA, and the frequency of cells containing mRNA by both in situ hybridization and limiting dilution analysis with real-time RT-PCR.
Results: The frequencies of intracellular cytokine-expressing cells was equivalent with either IHC or flow cytometry methods at frequencies above 0.1% cells. Below this limit the IHC method could still accurately detect frequencies down to 0.001% of cells, based on dilution of cytokine expressing cells into unstimulated PBMC and comparison to frequencies with mRNA expression. A striking difference in frequencies of cytokine-expressing cells was detected between flat bottom and round bottom culture wells and with different densities of cells per well. Since the EliSpot assay must be done in flat bottom wells, often at cell densities too low to accurately count spots/well, this difference in the efficiency of response with cell density resulted in often significantly lower EliSpot responses compared with IHC and flow responses performed after high density stimulation cultures. Independent of the absolute frequency, there was a significant correlation between frequencies of cytokine protein-expressing cells and frequencies of mRNA and total amount of mRNA/105 cells.
Conclusions: The frequency of cytokine producing T cells is dependent on cell density during stimulation and correlates well with the frequency of cytokine mRNA+ cells. Assessment of vaccine responses depends on the conditions of T-cell stimulation and the frequency of antigen-specific precursors.
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