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Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T CellsA primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.
Document ID
20020066645
Acquisition Source
Marshall Space Flight Center
Document Type
Preprint (Draft being sent to journal)
Authors
Karr, Laurel J.
(NASA Marshall Space Flight Center Huntsville, AL United States)
Marshall, Gwen
(Alabama Univ. Birmingham, AL United States)
Hockett, Richard D.
(Lilly (Eli) and Co. Indianapolis, IN United States)
Bucy, R. Pat
(Alabama Univ. Birmingham, AL United States)
Curreri, Peter A.
Date Acquired
August 20, 2013
Publication Date
January 1, 2002
Subject Category
Life Sciences (General)
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.

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