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Measurement of intracellular cytokines by flow cytometry in normal and immunodeprived subjects

University of British Columbia

Cytokines are polypeptide protein hormones which are transiently produced by cells of the immune system and act as both autocrine and paracrine mediators to modulate the growth, activation, differentiation and proliferation of target cells. They play an important immuno-regulatory role in normal health, and their production is substantially disturbed in disorders such as transplantation and acquired immunodeficiency. This study has examined the use of flow cytometry as a rapid, sensitive and clinically relevant method to measure the intracellular production of IL-2 and IFNƴ, key cytokines in the human immune response. PBMC were activated with PMA and ionomycin in the presence of monensin. Fluorochrome conjugated monoclonal antibodies were used to define the intracellular cytokine and surface phenotype. In normal controls IL-2 and IFNƴ were expressed in 34% and 26% of T cells respectively after 5 hours of stimulation. IL-2 and IFNƴ expression did not differ between patients on hemo- and peritoneal dialysis (IL-2: 40% and 26%, IFNƴ: 22% and 29%>) nor with normal controls. Expression of IL-2 was significantly reduced in stable renal transplant patients receiving immunosuppressive pharmacological agents ( p<0.001). The expression of IFNƴ was similar to normal controls after 5 hours of stimulation, the fluorescence of IFNy was reduced (p<0.05). The proportion of IL-2 and IFNy in CD3+ T cells was found to vary with each individual immuno compromised renal transplant patient in an elapsed period of 6 months . In HIV positive patients, IFNƴ expression was similar to normal controls but the fluorescence of IFNƴ was significantly reduced (p<0.001). The expression of IL-2 was also significantly reduced (p<0.001); although differential expression in CD4+ and CD8+ T cells parallelled that in normal controls. The measurement of intracellular IL-2 and IFNƴ by flow cytometry is a novel, adaptable in vitro method for quantification of the immune competence of cells in a heterogeneous population.

Thesis/Dissertation

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2009-07-29

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http://hdl.handle.net/2429/11478

Experimental Medicine

University of British Columbia

UBCV

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