Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry

Louis J. Picker, Manoj K. Singh, Zoran Zdraveski, John R. Treer, Sharr L. Waldrop, Paul R. Bergstresser, Vernon C. Maino

Research output: Contribution to journalArticlepeer-review

486 Scopus citations


The array of cytokines produced by T cells in effector sites is a primary means by which these cells mediate host defense. It is well recognized that cloned T cells are heterogeneous with regard to cytokine synthesis and, thus, in their ability to mediate specific immune responses, but the extent to which the patterns of cytokine secretion observed in cloned cells reflect actual populations of memory/effector T cells existing in vivo is largely unknown. Here, we report our findings using a multiparameter flow cytometric assay that allows simultaneous determination of an individual T-cell's ability to produce multiple cytokines and its phenotype after only short (4 to 8 hours) in vitro incubation with an activating stimulus and the secretion inhibitor Brefeldin A. This assay shows a rapid accumulation of interleukin- 2 (IL-2), IL-4, and γ-interferon (γ-IFN) in the cytoplasm of CD4+ cells after stimulation with either accessory cell-independent (phorbol 12- myristate 13-acetate [PMA] + ionomycin [I]) or accessory cell-dependent (staphylococcal enterotoxins [SE] A and B) T-cell-activating stimuli. Further analysis showed that production of γ-IFN and IL-4 is predominantly, if not exclusively, restricted to the CD45RO(high) memory/effector T-cell subset, whereas IL-2 may be produced by both the CD45RO(high) and CD45RO(low) subsets. Simultaneous determination of IL-2 and γ-IFN production among CD45RO(high)/CD4+ T cells showed distinct subsets that produce each of these cytokines alone (an average of 30% for IL-2 alone, 8% for γ-IFN alone), both (16%), or neither (46%). Similar analyses with the small IL-4-producing memory/effector T-cell subset (only 4.3% of total CD4+/CD45RO(high) T cells) showed that an average of 51% of these IL-4-producing cells also synthesize IL-2, 23% synthesize only IL-4, 16% synthesize all three cytokines, and 9.6% synthesize IL-4 and γ-IFN. These patterns of cytokine synthesis were found to be similar with both PMA + I and SEA/SEB stimulation and were observed in both peripheral blood memory/effector CD4+ T cells and in T cells of similar phenotype obtained from cutaneous delayed-type hypersensitivity sites. Taken together, these date strongly support the in vivo existence of human memory/effector T-cell subsets with 'preprogrammed' cytokine synthesis potential, although they suggest that these subsets may be more complex than originally proposed in the TH1/TH2 hypothesis.

Original languageEnglish (US)
Pages (from-to)1408-1419
Number of pages12
Issue number4
StatePublished - Aug 15 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology


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