Isolation and initial characterization of multiple species of T-lymphocyte subset cDNA clones.

Abstract

A modified differential screening procedure was applied to analyze cDNA libraries of cloned helper T lymphocytes (Th) and cytolytic T lymphocytes (CTL). Negative and positive differential screening and RNA blot analysis were used to identify cDNA clones that were expressed preferentially in Th or CTL. Seven clones corresponded to previously described T-cell genes, and 16 additional types of cDNA clones were isolated, 9 from Th and 7 from CTL. Of these, 3 were expressed in both Th and CTL, 7 were expressed in only Th, and 6 only in CTL. These clones were analyzed for induction after stimulation by interleukin 2 or Con A or after stimulation of the T-cell antigen receptor (TCR). Three different patterns of expression were seen: (i) induction only by Con A, (ii) induction by Con A and interleukin 2, and (iii) induction by Con A and TCR stimulation. The approach is potentially useful for analyzing paths of T-cell differentiation and detecting cDNA clones encoding unrecognized cytokines.T lymphocytes play a central role in the immune network both as effectors and as regulators. They may be divided into subsets endowed with distinct properties such as helper, suppressor, and cytolytic activities. These activities may be mediated by subset-specific immune effectors, which are elaborated and secreted after stimulation with either lectin or specific antigen (1)(2)(3). Not all activities are correlated with cloned genes, and the regulatory mechanisms by which the same stimulus can activate different sets of genes in these closely related cell types are not known.Analysis of subset-specific genes requires pure populations of T cells. Glasebrook and Fitch (4) have developed two T-cell clones from the same mouse spleen; L2 shows helper activity and L3 displays cytolytic properties. These clones are well characterized on a cellular level (5, 6).The isolation of a broad representation of L2-and/or L3-specific transcripts is the object of this study. We have developed a protocol for a differential screening without prior selection, which allowed us to isolate even very rare mRNA species specific to a cell type, and applied the approach to the analysis of cDNA libraries from Con A-stimulated L2 and L3 cells. T-cell-specific cDNAs were analyzed to determine whether they were specific for L2 or L3 cells and whether they were inducible by Con A, interleukin 2 (IL-2), or stimulation of the T-cell antigen receptor (TCR).MATERIALS AND METHODS Cells. Methods for isolating and maintaining the cloned helper T lymphocytes (Th), L2, and the cloned cytolytic T lymphocytes (CTL), L3, have been described previously (7).To stimulate the cloned T cells, we resuspended them at 106-107 cells per ml and cultured them with Con A (Pharmacia) at 10 tkg/ml for L2 cells or 2 gg/ml for L3 cells or human recombinant IL-2 (rIL-2; Cetus) at 102-103 units/ml. Immobilized clonotypic monoclonal antibody 384.5, which reacts with the TCR of L3 cells (8, 9), was used to stimulate L3 cells.Mouse thymoma cells, EL4, and mouse B-cell lines, A20.2j and K46, ...

Regulation of Mouse 4-1BB Expression: Multiple Promoter Usages and a Splice Variant

“…4-1BB (CD137) is a receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily (Goodwin et al, 1993;Kwon et al, 1987;Lotz et al, 1996;Setareh et al, 1995) which is expressed in an activation-dependent manner on T cells Pollok et al, 1993;Schwarz et al, 1996;Shuford et al, 1997) and constitutively on CD4 + CD25 + regulatory T cells (McHugh et al, 2002). Although 4-1BB is mainly involved in regulating activated T lymphocyte functions, expression of 4-1BB has been noted on non-T cells such as B cells, monocytes, macrophages, dendritic cells, eosinophils, and neutrophils (Futagawa et al, 2002;Heinisch et al, 2000;Schwarz et al, 1995;Watts et al, 2005;Wilcox et al, 2002;Yoon et al, 2005).…”

Complexity of the primary genetic response to mitogenic activation of human T cells.

“…In the past, genes induced in activated T cells, such as those encoding IL-2 and IL-2 receptor, often have been identified on the basis of their encoded proteins. However, the transcriptional response of T cells is likely more complex than has been reported to date (2,4,15,24,29,33,42); therefore, we sought to characterize thoroughly the initial response of T cells to activation by cloning a maximal number of induced genes. Thus, we have used an unbiased approach to clone novel, mitogen-induced genes on the basis of the property of differential expression.…”

“…Immunol., in press). Beyond the limited number of genes isolated previously on the basis of being induced in various T-cell preparations, cell lines, or clones (2,4,15,23,29,33,42), more extensive research has been conducted on the serum-induced activation process in mouse 3T3 fibroblasts. Previous studies in which immediately activatable genes were cloned yielded a number much smaller than discovered here (10,31).…”

Interferon-regulatory factor 1 is an immediate-early gene under transcriptional regulation by prolactin in Nb2 T cells.

“…A number of early-response genes or competence genes have been isolated from diverse cell types, including growth factor-induced (5) or serum-induced (1,3,5,22,26,27,29,43) fibroblasts, growth factor-induced pheochromocytoma PC12 cells (33,34,43,44), and mitogen-induced T lymphocytes (25,30,39,49; reviewed in reference 6). In most cases, the protein synthesis inhibitor cycloheximide (CHX) was used in the cloning strategy to isolate mRNA sequences whose expression does not require de novo protein synthesis, as well as to stabilize those rare gene transcripts that may be rapidly but transiently induced.…”