Demonstration of in vivo and in vitro Changes of T Helper Lymphocytes by Using Cytokine Flow Cytometry

Kim, Yeom; Hahn, Youn Soo

Abstract

PURPOSE: The purpose of this study is to determine IFN-gamma and IL-4 production patterns of individual Th lymphocytes in various in vivo and in vitro conditions by cytokine flow cytometry.
METHODS
Using a multiparameter flow cytometric assay that allows simultaneous determination of surface CD4 and intracellular IFN-gamma or IL-4, we studied the emergence of Th1 or Th2 lymphocytes in peripheral blood mononuclear cells (PBMC) which were incubated under the influence of various stimuli for Th1/Th2 polarization or steroid. The same analysis was performed for PBMC from six healthy donors (three children and three adults) and three asthmatic children before and after rush immunotherapy.
RESULTS
Dominant Th2 lymphocytes arised from IL-2-stimulated PBMC in spite of dominant production of IFN-gamma in the supernatant, but the addition of IL-12 and/or IFN-gammainduced increased emergence of Th0 and Th1 lymphocytes. Steroid could not modify ongoing Th2 response arising from D. farinae or tetanus toxoid-stimulated PBMC. IFN-gamma-producing Th1 lymphocytes predominated over IL-4-producing Th2 lymphocytes in peripheral blood isolated from three healthy adults and three healthy children, which was also demonstrated in three children with allergic asthma. The early changes in peripheral Th lymphocytes after immunotherapy on asthmatic children were reduced frequency of IFN-gamma-producing Th1 lymphocytes and increased frequency of IL-4-producing Th2 lymphocytes.
CONCLUSION
We conclude that detection of intracellular cytokines by flow cytometry is an easy and specific method which may be used to study single cells in heterogeneous populations as well as to observe cytokine patterns of homogeneous cells. And the direct visualization of T cell effector responses shown by this flow cytometric approach allows the opportunity to evaluate the participation of Th cells in various human diseases.