Physiologic signaling in normal human T-cells: mRNA phenotyping by northern blot analysis and reverse transcription-polymerase chain reaction

Cell Immunol. 1990 Jun;128(1):41-51. doi: 10.1016/0008-8749(90)90005-c.


Synergy between ionomycin and sn-1,2-dioctanoylglycerol (diC8) was shown at the level of lymphokine gene transcription. Transcriptional activation of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and the protooncogene H-ras was accomplished by signaling highly purified normal human resting T-lymphocytes (T-cells) with diC8, a physiologic regulator of protein kinase C, and the calcium ionophore, ionomycin. Northern blot analysis of mRNA for early T-cell activation genes demonstrated the synergism between diC8 and ionomycin at the gene induction level. To amplify very low levels of IL-2 mRNA, sequential reverse transcription and polymerase chain reaction (RT-PCR) of T cell mRNA were used to demonstrate the capacity of the calcium signal (ionomycin) to promote low-level IL-2 transcription in normal human T-cells without additional signals. Cyclosporine (CsA) prevented diC8 and ionomycin-induced expression of IL-2, IFN-gamma, and H-ras genes. The completeness of its inhibitory effect was evident by the absence of IL-2 transcripts in CsA-treated cultures screened by the RT-PCR technique. CsA also prevented IL-2 and IFN-gamma gene expression in accessory cell-depleted T-cells stimulated by cross-linking the CD2 and CD3 antigens on the cell surface. Our observations demonstrate that a physiologic regulator of PKC, diC8, and cell surface crosslinking of the CD2 and CD3 antigen, promote gene expression in normal human quiescent T-cells independently of accessory cells, and that CsA prevents gene expression via a direct effect on T-cells.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Differentiation, T-Lymphocyte / physiology
  • Base Sequence
  • Blotting, Northern
  • CD2 Antigens
  • Cyclosporins / pharmacology
  • Diglycerides / pharmacology
  • Gene Expression / drug effects
  • Humans
  • In Vitro Techniques
  • Interferon-gamma / genetics
  • Interleukin-2 / genetics
  • Ionomycin / pharmacology
  • Molecular Sequence Data
  • Oligonucleotides
  • Polymerase Chain Reaction
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins p21(ras)
  • RNA, Messenger / genetics
  • Receptors, Immunologic / physiology
  • Signal Transduction / drug effects
  • T-Lymphocytes / physiology*


  • Antigens, Differentiation, T-Lymphocyte
  • CD2 Antigens
  • Cyclosporins
  • Diglycerides
  • Interleukin-2
  • Oligonucleotides
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • Receptors, Immunologic
  • 1,2-dioctanoylglycerol
  • Ionomycin
  • Interferon-gamma
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)