Identification of mRNAs Differentially Expressed in Lymphocytes Following Interleukin-2 Activation Giuseppe Damiani, Enrica Capelli, 1 Sergio Comincini, Elena Mori, Simona Panelli, and Mariaclara Cuccia Dipartimento di Genetica e Microbiologia, Universita ` di Pavia, 27100 Pavia, Italy; and IDVGA-CNR, Milano, Italy We investigated genes involved in the interleukin-2 activation of cultured lymphocytes using a differential display reverse transcription PCR technique. Three cDNA fragments corresponding to mRNAs differentially amplified in the activated lymphocytes were sequenced and identified. These fragments were identical to the 3 region of the mRNAs encoding for the tumor rejection antigen TRA 1 that is the human homologue of the mu- rine heat shock protein gp96, the DAP12 protein that possesses an immunoreceptor tyrosine-based activation motif, and the human motor protein p87/89 expressed in the heart. These proteins are involved, respectively, in cellular communication, in signal transduction, and in cellular movements. Our findings suggest that the acti- vation of cellular immune response by interleukin-2 is a process analogous to other known phenomena of activa- tion of catabolic reactions of energy transduction for activities which allow adaptation of cells to stress con- ditions. © 1998 Academic Press Key Words: interleukin-2; lymphocytes; cell activa- tion; mRNA; differential display. INTRODUCTION Interleukin-2 (IL-2) is a cytokine activating the im- mune response against pathogens and tumor cells. Several studies demonstrated that IL-2 is an autocrine growth factor for the population of T-helper1 (Th1) lymphocytes which polarize the immune response in the direction of cell-mediated type immunity [1]. IL-2 can promote tumoricidal activity through generation of lymphokine-activated killer (LAK) cells both in vivo and in vitro [2– 4]. In particular IL-2 induces the pro- liferation of lymphocytes with a non-MHC-restricted activity against transformed tumoral cells. It is not yet established if these lymphocytes belong to the NK sub- set or are a different cellular population [5–7]. The difficulties in the understanding of the developmental pathway of LAK cells are due mainly to the heterogen- ity of this cellular type and of the experimental condi- tions used for the production of LAK cells. For example IL-2 dose seems to be critical in determining lympho- cytes proliferation or death [8, 9]. Differing data were reported by various authors regarding the immune CD phenotype of activated lymphocytes and it was hypoth- esized that LAK activity represents a function rather then a precise cell type. Using a treatment with phy- tohemagglutinin (PHA) and IL-2, we have developed an in vitro system for the production of lymphocytes with reproducible and well-defined LAK activities and morphologies [10, 11]. The characterization of the cel- lular and molecular mechanisms of this differentiative process should lead to rational strategies of immune response manipulation for prophylaxis and therapy. The identification of mRNAs differentially transcribed or modified before and after the IL-2 treatment is of fundamental importance for the understanding of the molecular bases of lymphocyte transformation into LAK cells. Several methods allowing the identification of dif- ferentially transcribed or modified mRNA are based on arbitrarily primed PCR fingerprinting [12, 13]. Liang and Pardee named these methods differential display reverse transcription polymerase chain reaction (DDRT-PCR) [14]. Using these methods either a few cultivated cells or tissue samples may be used as starting material for the isolation of mRNA. After the generation of cDNA, a PCR is performed using different arbitrary primers and there- fore, only a defined subset of the cDNAs is amplified with each primer. Electrophoretic pattern of the amplified cDNA fragments produced starting from different mate- rial, e.g, quiescent and growth-induced cells can be com- pared. Bands which are present under one condition and absent in the other can be cloned, sequenced, and used for further characterizations. In the present report, we describe an improved method which has been applied to the study of the mRNAs differentially expressed in lymphocytes after in vitro induction with IL-2 and three identified se- quences. MATERIALS AND METHODS Cell culture. Peripheral blood mononucleated cells (PBMC) from six healthy subjects were obtained by centrifugation of heparinized 1 To whom reprint requests should be addressed. Fax: 0382 528496. E-mail: capelli@ipvgen.unipv.it. 0014-4827/98 $25.00 27 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. EXPERIMENTAL CELL RESEARCH 245, 27–33 (1998) ARTICLE NO. EX984230