Interleukin-12 induces gene expression in interleukin-2 stimulated human T lymphocytes

IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses by inducing the differentiation of Th1 cells. To better clarify the molecular basis of IL-12 action, we compared the gene expression in human T lymphocytes activated by IL-2 and IL-12. mRNAs from T lymphocytes activated by either IL-2 alone or IL-2 plus IL-12 were transcribed into cDNAs. A differential mRNA display was conducted. As a result, differential display of five cDNA fragments was obtained. Sequence analysis suggests that they had high homology with recorded genes as found by a computer search against GenBank. Two full genes of the five fragments were cloned, which activation-induced C-type lectin and glucose transporter-like protein. Interestingly, these proteins were expressed in the T cells stimulated by IL-2 and IL-12, but not in the T cells stimulated by IL-2 alone. These results suggest that C-type lectin and glucose transporter-like protein may play an important role in the T lymphocyte activation induced by IL-12.     

Accessory cell requirement for activation antigen expression and cell cycle progression by human T lymphocytes

Stringent accessory cell (AC) depletion by a three-step procedure--plastic adherence, nylon wool adherence, followed by simultaneous treatment with two anti-AC monoclonal antibodies + complement--has allowed the demonstration of several AC-dependent stages in the T cell activation pathway. Simultaneous analysis of DNA content and cell surface immunofluorescence (correlation of activation antigen expression with cell cycle position) or DNA and RNA content (cell cycle position) of cultured cells was accomplished by dual parameter flow cytometry. AC-depleted, PHA-stimulated human peripheral blood T lymphocytes (PBTL) failed to exhibit "early" indicators of activation, including increased RNA content, expression of three activation-associated cell surface proteins (IL 2 receptor, transferrin receptor, and 4F2 protein), and the production of IL 2. The AC-depleted PBTL that failed to express these "early" markers of activation also failed to progress into the "late" phase of activation, DNA synthesis. All indicators of PHA responsiveness were fully replenished upon addition of AC but were only reconstituted to intermediate levels by addition of excess quantities of either highly purified IL 1 or crude AC-conditioned medium with lymphocyte-activating factor activity. These data suggest that the AC membrane plays a key and as yet undefined role in the stimulation of T cells by PHA.     

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.     

Regulation of varicella-zoster virus gene expression in human T lymphocytes.

Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, is the etiologic agent of chicken pox and shingles (zoster) in humans. Using an in vitro transient expression assay, we have evaluated the ability of the putative immediate early VZV genes, ORF4, ORF61, and ORF62 (the analogs of the herpes simplex virus alpha 27, alpha 0, and alpha 4 genes, respectively), to modulate the expression of VZV genes of different putative kinetic classes in a human T lymphocyte cell line. These cells are of the type in which VZV can be readily detected in the viremic phase of human infection. We present evidence to indicate that, in this system, the gene product of ORF62 (IE62) is a major regulatory protein in VZV and is capable of activating VZV genes of all putative kinetic classes. In addition, we demonstrate that the gene product of ORF4 and, unlike the apparent situation in Vero cells, the gene product of ORF61 may play an accessory regulatory role in synergizing the activation of VZV genes induced by the gene product of ORF62 in human T lymphocytes.     

Undermethylation of interferon-gamma gene in human T cell lines and normal T lymphocytes.

The relative levels of DNA methylation at CCGG sequences within and around the interferon-gamma (IFN-gamma) gene in normal human tissues and cell lines were examined by Southern blot analysis using isoschizomeric restriction enzymes, HpaII and MspI. On the test of normal tissues, the IFN-gamma gene was undermethylated only in a small population of T lymphocyte, whereas the gene was fully methylated in T cell-depleted lymphocytes and uterus cells. In TCL-Fuj cell line which is a T cell line producing a high level of IFN-gamma spontaneously, the IFN-gamma gene was undermethylated. Moreover, the extent of DNA methylation was inversely correlated to the level of expression of the IFN-gamma gene in several T cell lines including sublines derived from TCL-Fuj cells. However, partial or complete unmethylation at the CCGG sites of IFN-gamma gene was observed in a promyelocytic leukemia cell line and two epithelial cell lines that fail to produce IFN-gamma irrespective of induction. These results suggest that undermethylation of IFN-gamma gene is necessary but not sufficient for its efficient expression.     

A novel glycoprotein expressed on sheep T and B lymphocytes is involved in a T cell activation pathway

We have produced a new mouse mAb that identifies a sheep T cell activation Ag. The mAb B5-5 is specific for low m.w. components on nearly all sheep thymocytes and peripheral T and B lymphocytes but does not label immature B cells in Peyer's patches or germinal centers. After cross-linking of target structures either directly by plastic-bound mAb or indirectly using anti-Ig reagents, peripheral T cells, but not thymocytes or peripheral B cells, were activated. IL-2 was secreted by T cells after cross-linking and activation was strongly augmented in the presence of PMA. The addition of soluble B5-5 mAb to mitogen-stimulated cultures of sheep lymphocytes resulted in a suppression of PHA responses and augmentation of PWM responses and had a variable effect on Con A responses but had no effect on LPS- or protein A-induced proliferation. When added to alloantigen-stimulated cultures, B5-5 augmented the proliferative response. The B5-5 membrane component consists of 14- to 19-kDa glycoproteins but the banding patterns obtained during SDS-PAGE analysis of 125I-labeled Ag differed between thymocytes, peripheral T cells, and peripheral B cells. On the basis of its range of expression on lymphoid cells and known biochemical and functional properties, we conclude that the B5-5 component on sheep lymphocytes is different from T cell activation Ag in other species.     

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.     

Effect of interleukin-2 on the expression of cell cycle genes in human T lymphocytes

We have studied the expression of seven cell cycle-dependent genes in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells, in macrophage-depleted cultures and in macrophage-depleted cultures plus Interleukin-2 (IL-2). The expression of all seven genes is increased in PHA stimulated peripheral cells. Only two (2F1 and the IL-2 receptor) are increased in PHA-stimulated macrophage depleted cultures. Addition of IL-2 to these cultures increased the RNA levels of four genes (KC-1, c-myc, beta-actin and IL-2R), but has no effect on three others (4F1, 2F1, and JE-3). The results indicate that the expression of these cell cycle genes is regulated by different components of the mitogenic stimulus.     

Inhibition of NFkappaB induces caspase-independent cell death in human T lymphocytes

Nuclear factor kappaB (NFkappaB) regulates the expression of various genes essential for cell survival. Here we demonstrate that suppression of NFkappaB nuclear import with SN50 peptide carrying the nuclear localization sequence (NLS) of the NFkappaB p50 subunit induces apoptosis in human peripheral blood T lymphocytes (T-PBL), which can be blocked with the pan-caspase inhibitor Z-VAD.fmk. However, even when caspase function is blocked, the addition of SN50 induces irreversible cell loss due to the reduction in the mitochondrial transmembrane potential (DeltaPsim) followed by disruption of the cell membrane, hallmarks of necrosis. These observations demonstrate that although inhibition of NFkappaB nuclear translocation by SN50 peptide can induce caspase-dependent apoptosis in T-PBL, cell death may still proceed in the absence of functional caspase activity. The availability of downstream caspases appears to determine the mode of cell death in NFkappaB defective cells.     

PPARdelta activation induces COX-2 gene expression and cell proliferation in human hepatocellular carcinoma cells

Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. Recently, many studies have shown increased expression of COX-2 in a variety of human malignancies, including hepatocellular carcinoma (HCC). Therefore, it becomes important to know more about what determines COX-2 expression. In this work, we have studied the effect of PPARdelta activation on COX-2 expression using a selective agonist (GW501516) in human hepatocellular carcinoma (HepG2) cells. Activation of PPARdelta resulted in increased COX-2 mRNA and protein expression. The mechanism behind the induction seems to be increased activity of the proximal promoter of the COX-2 gene, spanning nucleotides -327 to +59. The increased COX-2 protein expression and promoter activity induced by the GW501516 was also confirmed in the monocytic cell line THP-1. Induced levels of COX-2 have previously been associated with resistance to apoptosis and increased cell proliferation in many cell types. In HepG2 cells, we observed a dose-dependent increase in cell number by GW501516 treatment for 72h. The levels of PCNA, used as an indicator of cell division were induced, and the cell survival promoting complex p65 (NF-kappaB) was phosphorylated under GW501516 treatment. We conclude that PPARdelta activation in HepG2 cells results in induced COX-2 expression and increased cellular proliferation. These results may suggest that PPARdelta plays an important role in the development of HCC by modulating expression of COX-2.     

A novel homodimeric molecule involved in human T cell activation

A mAb, 10D1, was obtained by fusing spleen cells from BALB/c mice immunized with a CD3/TCR- human T cell line, P12/ichikawa, to mouse myeloma cells, P3X63-Ag8-653. 10D1 mAb is specific for T cells in that it reacted with all the T cell lines tested, but not with B or myeloid cell lines. A small fraction of normal peripheral blood T cells, preferentially CD4+, was also reactive with 10D1 mAb. Biochemical studies revealed that 10D1 mAb recognizes a disulfide-linked homodimeric molecule composed of 90-kDa polypeptide. 10D1 mAb induced a substantial proliferation of peripheral blood T cells when cross-linked with goat anti-mouse Ig antibody. The elimination of CD4+ cells totally abrogated the proliferative response induced by 10D1 mAb, whereas the elimination of CD8+ cells rather enhanced it. The proliferative response of peripheral blood T cells induced by 10D1 mAb was almost completely inhibited after modulation of the CD3/TCR complex with anti-CD3 mAb. In addition, a prompt increase in intracellular [Ca2+] was observed in a CD3+ T cell line, Jurkat but not in its surface CD3- mutant when 10D1 mAb was added. These results indicate that the 10D1 molecule is involved in a novel pathway of human CD4+ T cell activation, which is associated with the CD3/TCR-mediated pathway.     

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.     

Gene expression in the cell cycle of human T lymphocytes: I. Predicted gene and protein networks

The key genes involved in the cell cycle of human T lymphocytes were identified by iterative searches of gene-related databases, as derived also from DNA microarray experimentation, revealing and predicting interactions between those genes, assigning scores to each of the genes according to numbers of interaction for each gene weighted by significance of each interaction, and finally applying several types of clustering algorithms to genes basing on the assigned scores. All clustering algorithms applied, both hierarchical and K-means, invariably selected the same six "leader" genes involved in controlling the cell cycle of human T lymphocytes. Relations of the six genes to experimental data describing switching between stages of cell cycle of human T lymphocytes are discussed.