Influence of activating stimulus on functional phenotype: interleukin 2 mRNA accumulation differentially induced by ionophore and receptor ligands in subsets of murine T cells.

Abstract

We have investigated the linkage between CD4/CD8 phenotype and programing for specific responses in primary T-cell populations. In situ hybridization has been used to determine the frequency of cells competent to express the interleukin 2 (IL-2) gene after short-term stimulation with various polyclonal activators. The effects of the T-cell receptor ligands Con A and anti-CD3 monoclonal antibody were compared with those of a calcium ionophore that bypasses membrane receptors altogether. Induction with a calcium ionophore and phorbol ester revealed that potential IL-2 producers not only constitute >85% of the cells with a CD4' "helper/ inducer" phenotype but also constitute over half of the cells with a CD8' "killer/suppressor" phenotype. There is no defect in the ability of these CD8' cells to accumulate IL-2 transcripts under these conditions. By contrast, in response to phorbol ester and either Con A or anti-CD3, the CD8' cells show an abortive IL-2 production response with rapid disappearance of IL-2 mRNA. This results in substantially lower yields of IL-2 per cell than is made by CD4+ cells in response to the same stimuli. The extent to which these populations appear to have diverged in function thus depends on the stimulus used to trigger the response. The results suggest that differences in signal transduction or posttranscriptional regulatory mechanisms, rather than effector gene inducibility per se, may initially underlie the commitment of CD4+ and CD8+ cells to distinct functional roles.Mature T cells are functionally specialized in their responses to recognition of antigen. In general, T-cell lines that express the CD4 cell-surface glycoprotein are "helper" or "amplifier" cells that respond to the recognition of antigen by secreting lymphokines, often including the major T-cell growth factor interleukin 2 (IL-2) (1, 2). Cells that express CD8 include most killer T cells and show little helper activity (3-6). This suggests that the constitutive CD4/CD8 phenotypes of T cells are correlated with the inducibility of different, limited sets of functional response genes. Although the correlation is frequently observed in memory T cells and T-cell lines, it is not known how it is established during T-cell differentiation. When fresh CD8+ cells are activated and cloned, '10% of the clones respond to stimulation by expression of IL-2 (5, 6). This would suggest an intrinsic difference between CD8 + cells and CD4 + cells in programing for gene expression. However, in a variety of other studies, CD8 + cells appear to make IL-2 as well as CD4+ cells (7-11). In the work reported here, we used in situ hybridization and quantitative probe protection analyses to measure the accumulation of IL-2 mRNA in individual splenic T cells responding for the first time to different stimuli. We demonstrate that the majority of CD4+ and :50% of CD8+ splenocytes are competent to accumulate similarly high levels of IL-2 mRNA when treated with calcium ionophore and phorbol ester. However, the two cell types differ markedly in t...

Th0 Cells in the Thymus: The Question of T‐Helper Lineages

“…activation. E. Rothenberg et al originally reported that IL-2 gene induction, thought to be a specific marker of the T-!ymphocyte lineage, could be obtained by stimulation with the combination of a calcium ionophore and a phorbol ester in the presence of exogenously added IL-1, as early as at the double-negative stage of thymocyte development, before expression of the CD3-TCR complex (Rothenberg et al 1988(Rothenberg et al , 1990. We confirmed this result using FACS-purified cells from adult mice, and found, in addition, that IL-4 Is not yet detectable, but that small amounts of y-IFN can be produced by these CD3" cells (Table I).…”

“…This might explain why no functional activity has been convincingly demonstrated FACS-purified subsets of adult thymocytes (I x 10^) were stimulated in microculture wells in the presence of irradiated dendritic cells (3 x lO'') as accessory cells and the combination of ionomycin (I //M) and PMA (1 ng/ml), plus or minus recombinant IL-1 (50 units/ml). Double-positive thymocytes were separated into high and low density by centrifugation on a percoil gradient (60%/70%), in mouse double-positive cells; in particular, no iymphokine secretion can be obtained after ionomycin and PMA stimulation (Rothenberg 1988). Recently, however, the minor subset of large cells among the double-positive thymocytes was reported to generate single-positive thymocytes upon intrathymic transfer, suggesting that this subset includes the minority of cells that will be selected for further maturation (Guidos et al 1989).…”

Interleukin-1 synergy with phosphoinositide pathway agonists for induction of interleukin-2 gene expression: molecular basis of costimulation.

“…Mature TcR+ thymocytes resemble peripheral T cells in their insensitivity to IL-1 when they are stimulated to produce IL-2 in response to TPA plus A23187. These chemical proxies for mediators of the phosphatidylinositol bisphosphate hydrolysis pathway may be able to induce IL-2 expression in all mature T cells, for they can also elicit high-level responses in most fresh peripheral CD8+ cells (24), cells which generally do not express IL-2 mRNA in response to either antigen or mitogenic lectins. In contrast, however, immature TcRdouble-negative thymocytes do not make IL-2 in response to TPA and A23187 at all unless IL-1 is present (35,36).…”

cAMP inhibits induction of interleukin 2 but not of interleukin 4 in T cells.

“…The induction of either type of gene expression response depends on two steps: (i) the coupling of membrane receptors to intracellular signaling pathways and (ii) the effect of the resulting signaling mediators on transcription factors and/or on chromatin structure and RNA stability. It is known that the frequency of IL-2 producers in various populations and the levels of IL-2 mRNA that they accumulate are a function of the particular activating stimulus used (2)(3)(4). This suggests that the ultimate response phenotype of a T cell may not be heritably fixed but may be influenced by the type of external stimulus it receives.…”