CELLULAR IMMUNOLOGY 128,4 l-5 1 (1990) Physiologic Signaling in Normal Human T-cells: mRNA Phenotyping by Northern Blot Analysis and Reverse Transcription- Polymerase Chain Reaction’ KENNETHJ. WIEDER,*GERDWALZ,*BERND ZANKER,* PRABODHSEHAJPAL, VIJAY K. SHARMA,EDWARDSKOLNIK, TERRY B. STROM,* AND MANIKKAM~UTHANTHIRAN *Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215 and The Rogosin Institute, Department ofMedicine and Biochemistry, Cornell University Medical College, New York, New York 10021 Received November IO. 1989; accepted January 16, 1990 Synergy between ionomycin and sn- l,2-dioctanoylglycerol (diCs) was shown at the level of lymphokine gene transcription. Transcriptional activation of interleukin-2 (IL-2), interferon-r (EN-r), and the protooncogene H-ras was accomplished by signaling highly purified normal human resting T-lymphocytes (T-cells) with diCs, a physiologic regulator of protein kinase C, and the calcium ionophore, ionomycin. Northern blot analysis of mRNA for early T-cell activa- tion genes demonstrated the synergism between diCs 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 ofthe calcium signal (ionomycin) to promote low-level IL-2 transcription in normal human T-cells without additional signals. Cyclosporine (CsA) prevented diCs and ionomycin-induced expression of IL- 2, IFN-7, 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 pre- vented IL-2 and IF%-, 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, diCs, and cell surface crosslinking of the CD2 and CD3 antigen, promote gene expression in normal human quiescent T-cells independently of accessorycells, and that CsA prevents gene expression via a direct effect on T-cells. o 1990 Academic PXSS, IX. INTRODUCTION An increase in the concentration of intracellular free calcium and the activation of protein kinase C (PKC) are thought to function as synergistic intracellular signals for the induced expression of growth-promoting genesin T-cells (1,2). However, much of the data that support this unifying theme for transcriptional activation have come largely from studies where preactivated T-cells (e.g., T-cell clones, T-leukemic lines etc.) rather than normal T-cells and nonphysiologic rather than physiologic activators of PKC have been utilized (3-5). Because the signaling requirements for normal, ’ This work was supported by Grants ROl-CA43977 and ROl-DK32929 (K.J.W., G.W., B.Z., and T.B.S.) from the National Institute of Health. 41 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.