Eur. J. Immunol. 1989.19: 803-808 Enhanced receptor-mediated activation of memory cells zy 803 Martin E. Sanders', Malegapuru W. Makgoba', Carl H. Junev, Howard A. Youngo and Stephen Shaw' The ExperimentalImmunology Branch, National Cancer Institute', National Institutes of Health, The Naval Medical Research Institute', Bethesda and the Biological Response Modifiers National Cancer InstituteIFrederick Cancer Research Facility', Frederick hogram, Enhanced responsiveness of human memory T cells to z CD2 and CD3 receptor-mediated activation Previous investigations have defined phenotypic differences between unprimed (naive) and antigen-primed (memory) T cells from human peripheral blood. We now report that memory T cells proliferate much more than naive cells when stimulated with anti-CD3 monoclonal antibody or pairs of antLCD2 monoclonal antibodies. Enhanced responsiveness to receptor-mediated triggering is a novel mechanism for T cells which could facilitate memory cell response to specific antigen. Furthermore, when triggered via either CD2 or CD3, memory T cells produce substantial amounts of interferon gamma while naive cells produce virtually none; this suggests that differ- entiation from naive to memory state is accompanied by a stable change in regulation of the gene for interferon-y. We conclude that naive and memory T cells are dramati- cally different in function as well as phenotype. 1 Introduction During cell differentiation, critical changes occur in cellular responsiveness to external stimuli. Memory T cells differenti- ate from naive cells zyxwvutsr in vivo following stimulation with antigen, persist in the host and mediate an enhanced immune response upon subsequent stimulation with the same antigen. The basis for the vigorous responses of memory T cells to antigen restimulation is incompletely understood and is the subject of this report. Recent studies have identified putative phenotypic markers for memory and naive T cells in both human and murine sys- tems. Several strong lines of evidence indicate that CD45R (2H4) is a marker of human naive T cells while high expression of at least six cell surface molecules (CD2, LFA-3, LFA-1, CDw29 [4B4], UCHLl and Pgp-1) identifies human memory T cells [l-91. These lines of evidence include: (a) the finding that essentially all T cells in cord blood from a neonate are of the pJtative naive phenotype [2]; (b) the uniform unidirec- tional and stable conversion of phenotype of cord blood T cells into the putative memory phenotype following activation [2-41; (c) the finding of increase in percentage of peripheral blood T cells of the putative memory phenotype with increas- ing age [3]; (d) the finding of proliferative responses to anti- gens to which the donor has previously been immunized only in the putative memory subset [2, 5-91; (e) the finding that proliferative response to alloantigens presented in a primary mixed lymphocyte culture is recovered in a secondary mixed lymphocyte culture with the same alloantigens only in the putative memory subset and not in the putative naive subset [4] and (f) substantial in vivo immunization data showing high Pgp-1 expression as a marker of murine memory T cells and the parallel finding of high Pgp-1 expression on the putative human memory subset [2, 91. Based on these lines of evi- dence, we will refer to these subsets as memory and naive in the remainder of this report. It is of particular note that several of the molecules with enhanced expression on the memory [I 72111 Correspondence: Martin E. Sanders, The Upjohn Company, 301 Hen- rietta Street, 7214-24-2 Kalamazoo, MI 49007, USA Abbreviations: IFN: Interferon IL: Interleukin mAb: Mono- clonal antibody(ies) PHA: Phytohemagglutinin subset, zyxwvu i.e. CD2, LFA-3, LFA-1 and CDw29, have been dem- onstrated or implicated to have roles in cellular adhesion and/ or signal transduction, two processes which may be of critical functional importance for the production of a strong memory response [lo-161. Clonal expansion, resulting in increased numbers of respon- sive cells, is an established mechanism for increased intensity of memory responses [17]. It has also been suggested that memory cells recognize antigen with increased affinity. The present results establish an additional important mechanism, namely that memory cells have enhanced responsiveness to receptor-mediated stimulation; memory cells respond much better than naive cells to anti-CD3 monoclonal antibodies (mAb) or anti-CD2 mAb combinations. The process of differentiation also specifies the range of se- creted products which particular T cells can make. Murine memory cells produce more interferon-y (IFN-y) than do naive cells in response to a variety of stimuli [MI; we have previously reported similar results for human cells, but only in response to phytohemagglutinin (PHA) [2]. The present studies confirm and extend the distinction between naive and memory cells by demonstration that production of IFN-y is almost exclusively by memory cells in response to CD2- and CD3-mediated activation, and that this response is associated with increased IFN-y message in memory cells. 2 Materials and methods 2.1 Preparation of subsets Preparation of human peripheral blood T cells was performed as previously described, as was electronic sorting for subsets of T cells differing in expression of LFA-3 [2]. Magnetic immunoselection was performed using previously described procedures [19] but different mAb: T cells were prepared from peripheral blood mononuclear cells by incubation with a cock- tail of anti-CD20, anti-CDllb and anti-CD16 mAb, subse- quent incubation with anti-IgG-coated beads (Dynal Inc., Fort Lee, NJ) and removal of the beadmAb-coated B cells, mono- cytes and natural killer (NK) cells with a magnet. Memory and naive T cell subsets were then prepared by a second round of selection using anti-LFA-3 mAb TS2/9 (kindly provided by Dr. T. Springer, Boston, MA) [15] to negatively select naive cells z 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989