scholarly journals Expression of NFAT-family proteins in normal human T cells.

1997 ◽  
Vol 17 (5) ◽  
pp. 2475-2484 ◽  
Author(s):  
L Lyakh ◽  
P Ghosh ◽  
N R Rice
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Interleukin 2 ◽  
Binding Assays ◽  
Myristate Acetate ◽  
Human T Cells ◽  
Specific Antisera ◽  
Predominant Form ◽  
Normal Human ◽  
Size Heterogeneity

NFAT proteins constitute a family of transcription factors involved in mediating signal transduction. Using a panel of specific antisera in immunoprecipitation assays, we found that NFATp (135 kDa) is constitutively expressed in normal human T cells, while synthesis of NFATc (predominant form of 86 kDa) is induced by ionomycin treatment. NFAT4/x was very weakly expressed in unstimulated cells, and its level did not increase upon treatment with activating agents. NFAT3 protein was not observed under any conditions. Higher-molecular-weight species of NFATc (of 110 and 140 kDa) were also detected. In addition, translation of NFATc mRNA apparently initiates at two different AUG codons, giving rise to proteins that differ in size by 36 amino acids. Additional size heterogeneity of both NFATc and NFATp results from phosphorylation. In contrast to ionomycin treatment, exposure of cells to phorbol myristate acetate (PMA) plus anti-CD28 did not induce NFATc, indicating that under these conditions, interleukin-2 synthesis by these cells is apparently independent of NFATc. In DNA binding assays, both PMA plus anti-CD28 and PMA plus ionomycin resulted in nuclear NFAT. Surprisingly, the PMA-ionomycin-induced synthesis of NFATc that was detected by immunoprecipitation was not mirrored in the DNA binding assays: nearly all of the activity was due to NFATp. This is the first study of expression of all family members at the protein level in normal human T cells.

INHIBITION OF INTERLEUKIN 2 RECEPTOR EXPRESSION IN NORMAL HUMAN T CELLS BY CYCLOSPORINE

1992 ◽  
Vol 53 (1) ◽  
pp. 146-150 ◽  
Author(s):  
BAOGUI LI ◽  
PRABODH K. SEHAJPAL ◽  
AJIT SUBRAMANIAM ◽  
ANTONIO JOSEPH ◽  
KURT H. STENZEL ◽  
...  
Keyword(s):  
T Cells ◽  
Interleukin 2 ◽  
Receptor Expression ◽  
Interleukin 2 Receptor ◽  
Human T Cells ◽  
Normal Human

scholarly journals Proliferation and apoptosis of human T cells during replicative senescence--a critical approach.

2000 ◽  
Vol 47 (2) ◽  
pp. 293-300 ◽  
Author(s):  
E Jaruga ◽  
J Skierski ◽  
E Radziszewska ◽  
E Sikora
Keyword(s):  
T Cells ◽  
T Cell ◽  
Replicative Senescence ◽  
Interleukin 2 ◽  
Critical Approach ◽  
Cd8 Cells ◽  
Human T Cells ◽  
Proliferation And Apoptosis ◽  
Normal Human

Normal human T lymphocytes growing in culture undergo replicative senescence. Previously, we have shown that in our conditions polyclonal T cells cease proliferation after about three weeks (Radziszewska et al., 1999, Cell Biol. Int. 23, 97-103). Now we present results of a more detailed analysis of in vitro growth as well as phenotypic changes of T cells. Cell cycle analysis showed that about 20% of cells were in the S phase until the 17th day of culture (young cells). The highest number of mitotic cells (phase G2/M; 10%) was observed during the first week of culture. All not dividing senescent cells were stopped in the G1 phase (after the 30th day of culture). The sub-G1 fraction which represents apoptotic cells did not exceed 8% during the whole period until the 30th day of culture. During in vitro T-cell growth, a rather rapid selection to CD3+ CD8+ cells occurs. In the presenescent (between the 17th and 30th day) and senescent populations the majority of cells (above 90%) were CD8 positive. We also have checked the expression of alpha-chain interleukin-2 (IL-2) receptor (CD25). In young and presenescent cells about one third of cells was CD25 positive, but only 15% in the pool of senescent cells. Immunoblotting analysis of p16 protein recognized previously as a marker of senescent T cells, showed its highest and transient expression in presenescent cells. A critical review of the polyclonal T cell replicative senescence model is presented.

scholarly journals Differential regulation of transforming growth factor beta and interleukin 2 genes in human T cells: demonstration by usage of novel competitor DNA constructs in the quantitative polymerase chain reaction.

1991 ◽  
Vol 174 (5) ◽  
pp. 1259-1262 ◽  
Author(s):  
B Li ◽  
P K Sehajpal ◽  
A Khanna ◽  
H Vlassara ◽  
A Cerami ◽  
...  
Keyword(s):  
T Cells ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Quantitative Polymerase Chain Reaction ◽  
Interleukin 2 ◽  
Tgf Beta ◽  
Human T Cells ◽  
Normal Human ◽  
Growth Factor Beta ◽  
Polymerase Chain

The regulation of mRNA encoding transforming growth factor beta (TGF-beta) and interleukin 2 (IL-2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-beta mRNA and IL-2 mRNA are regulated differentially in normal human T cells, quiescent or signaled with the synergistic combinations of: sn-1,2-dioctanoylglycerol and ionomycin or anti-CD3 monoclonal antibody (mAb) and anti-CD2 mAb; (b) the steady-state level of TGF-beta mRNA in the stimulated T cells, in contrast to that of IL-2 mRNA, is increased by the immunosuppressant cyclosporine (CsA); and (c) the paradoxical effect of CsA on TGF-beta mRNA levels is also appreciable at the level of production of functionally active TGF-beta protein. Our findings, in addition to demonstrating the utility of the competitor DNA constructs for the precise quantification of immunoregulatory cytokines, suggest a novel and unifying mechanistic basis for the immunosuppression and some of the complications (e.g., renal fibrosis) associated with CsA usage.

Microplate method for retrovirus-induced transformation of normal human T-cells

1984 ◽  
Vol 73 (2) ◽  
pp. 379-385 ◽  
Author(s):  
Kazuo Sugamura ◽  
Mitsuru Sakitani ◽  
Yorio Hinuma
Keyword(s):  
T Cells ◽  
Human T Cells ◽  
Normal Human

Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells

1991 ◽  
Vol 11 (5) ◽  
pp. 2794-2803
Author(s):  
A Zmuidzinas ◽  
H J Mamon ◽  
T M Roberts ◽  
K A Smith
Keyword(s):  
T Cells ◽  
Kinase Activity ◽  
Interleukin 2 ◽  
Constitutive Expression ◽  
Fold Increase ◽  
Activity Increase ◽  
Phosphorylation State ◽  
Fourfold Increase ◽  
Protein Levels ◽  
Human T Cells

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.

Interferon- Activates Multiple STAT Proteins and Upregulates Proliferation-Associated IL-2R, c-myc, and pim-1 Genes in Human T Cells

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 1980-1991 ◽  
Author(s):  
Sampsa Matikainen ◽  
Timo Sareneva ◽  
Tapani Ronni ◽  
Anne Lehtonen ◽  
Päivi J. Koskinen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Biology ◽  
Interleukin 2 ◽  
T Cell Proliferation ◽  
Stat Proteins ◽  
Human T Cells ◽  
T Cell Biology ◽  
Cytokine Stimulation

Interferon- (IFN-) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN- has an important role in T-cell biology. We have analyzed the expression ofIL-2R, c-myc, and pim-1 genes in anti-CD3–activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)–induced T-cell proliferation. Treatment of T lymphocytes with IFN-, IL-2, IL-12, and IL-15 upregulated IL-2R, c-myc, andpim-1 gene expression. IFN- also sensitized T cells to IL-2–induced proliferation, further suggesting that IFN- may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2R,pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-–induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN- was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN- enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN- as a T-cell regulatory cytokine.

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