scholarly journals Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

1991 ◽  
Vol 11 (3) ◽  
pp. 1718-1723 ◽  
Author(s):  
Y Koltin ◽  
L Faucette ◽  
D J Bergsma ◽  
M A Levy ◽  
R Cafferkey ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Binding Protein ◽  
Interleukin 2 ◽  
Yeast Cells ◽  
Ppiase Activity ◽  
Resistant Mutants

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

scholarly journals Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein

1991 ◽  
Vol 11 (3) ◽  
pp. 1718-1723 ◽  
Author(s):  
Y Koltin ◽  
L Faucette ◽  
D J Bergsma ◽  
M A Levy ◽  
R Cafferkey ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Binding Protein ◽  
Interleukin 2 ◽  
Yeast Cells ◽  
Ppiase Activity ◽  
Resistant Mutants

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

scholarly journals Antigen-independent activation of naive and memory resting T cells by a cytokine combination.

1994 ◽  
Vol 180 (3) ◽  
pp. 1159-1164 ◽  
Author(s):  
D Unutmaz ◽  
P Pileri ◽  
S Abrignani
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Effector Function ◽  
Necrosis Factor Alpha ◽  
Naive T Cells ◽  
Naïve T Cells

We investigated whether human resting T cells could be activated to proliferate and display effector function in the absence of T cell receptor occupancy. We report that combination of interleukin 2 (IL-2), tumor necrosis factor alpha, and IL-6 activated highly purified naive (CD45RA+) and memory (CD45RO+) resting CD4+ T cells to proliferate. Under this condition, memory resting T cells could also display effector function as measured by lymphokine synthesis and help for immunoglobulin production by B cells. This novel Ag-independent pathway of T cell activation may play an important role in vivo in recruiting effector T cells at the site of immune response and in maintaining the clonal size of memory T cells in the absence of antigenic stimulation. Moreover, cytokines can induce proliferation of naive T cells without switch to memory phenotype and this may help the maintenance of the peripheral pool of naive T cells.

Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2181-2190 ◽  
Author(s):  
Maria Paola Martelli ◽  
Huamao Lin ◽  
Weiguo Zhang ◽  
Lawrence E. Samelson ◽  
Barbara E. Bierer
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Transcriptional Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Adapter Protein ◽  
Activated T Cells

Abstract Activation of T cells can be initiated through cell surface molecules in addition to the T-cell receptor-CD3 (TCR-CD3) complex. In human T cells, ligation of the CD2 molecule by mitogenic pairs of anti-CD2 monoclonal antibodies activates T cells via biochemical signaling pathways similar but not identical to those elicited on TCR engagement. This study describes a key role for the p36/38 membrane adapter protein linker for T cell activation (LAT) in CD2-mediated T-cell activation. Following ligation of CD2 on the surface of the Jurkat T-cell line and human purified T cells, LAT was tyrosine phosphorylated and shown to associate in vivo with a number of other tyrosine phosphorylated proteins including PLCγ-1, Grb-2, and SLP-76. Using Jurkat cell lines deficient in ZAP70/Syk (P116) or LAT (ANJ3) expression, CD2-dependent PLCγ-1 and SLP-76 tyrosine phosphorylation required expression both of ZAP70 or Syk and of LAT. As predicted, the absence of either LAT or ZAP70/Syk kinases correlated with a defect in the induction of nuclear factor of activated T cells (NFAT) transcriptional activity, activation of the interleukin-2 promoter, and ERK phosphorylation following CD2 stimulation. These data suggest that LAT is an adapter protein important for the regulation of CD2-mediated T-cell activation.

Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2181-2190 ◽  
Author(s):  
Maria Paola Martelli ◽  
Huamao Lin ◽  
Weiguo Zhang ◽  
Lawrence E. Samelson ◽  
Barbara E. Bierer
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Transcriptional Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Adapter Protein ◽  
Activated T Cells

Activation of T cells can be initiated through cell surface molecules in addition to the T-cell receptor-CD3 (TCR-CD3) complex. In human T cells, ligation of the CD2 molecule by mitogenic pairs of anti-CD2 monoclonal antibodies activates T cells via biochemical signaling pathways similar but not identical to those elicited on TCR engagement. This study describes a key role for the p36/38 membrane adapter protein linker for T cell activation (LAT) in CD2-mediated T-cell activation. Following ligation of CD2 on the surface of the Jurkat T-cell line and human purified T cells, LAT was tyrosine phosphorylated and shown to associate in vivo with a number of other tyrosine phosphorylated proteins including PLCγ-1, Grb-2, and SLP-76. Using Jurkat cell lines deficient in ZAP70/Syk (P116) or LAT (ANJ3) expression, CD2-dependent PLCγ-1 and SLP-76 tyrosine phosphorylation required expression both of ZAP70 or Syk and of LAT. As predicted, the absence of either LAT or ZAP70/Syk kinases correlated with a defect in the induction of nuclear factor of activated T cells (NFAT) transcriptional activity, activation of the interleukin-2 promoter, and ERK phosphorylation following CD2 stimulation. These data suggest that LAT is an adapter protein important for the regulation of CD2-mediated T-cell activation.

scholarly journals Targeted Disruption of LIGHT Causes Defects in Costimulatory T Cell Activation and Reveals Cooperation with Lymphotoxin β in Mesenteric Lymph Node Genesis

2002 ◽  
Vol 195 (12) ◽  
pp. 1613-1624 ◽  
Author(s):  
Stefanie Scheu ◽  
Judith Alferink ◽  
Tobias Pötzel ◽  
Winfried Barchet ◽  
Ulrich Kalinke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Lymphoid Organs ◽  
Lower Percentage ◽  
Skin Allograft ◽  
Mesenteric Lymph

The recently described tumor necrosis factor (TNF) family member LIGHT (herpes virus entry mediator [HVEM]-L/TNFSF14), a ligand for the lymphotoxin (LT)β receptor, HVEM, and DcR3, was inactivated in the mouse. In contrast to mice deficient in any other member of the LT core family, LIGHT−/− mice develop intact lymphoid organs. Interestingly, a lower percentage of LIGHT−/−LTβ−/− animals contain mesenteric lymph nodes as compared with LTβ−/− mice, whereas the splenic microarchitecture of LIGHT−/−LTβ−/− and LTβ−/− mice shows a comparable state of disruption. This suggests the existance of an additional undiscovered ligand for the LTβ receptor (LTβR) or a weak LTα3–LTβR interaction in vivo involved in the formation of secondary lymphoid organs. LIGHT acts synergistically with CD28 in skin allograft rejection in vivo. The underlying mechanism was identified in in vitro allogeneic MLR studies, showing a reduced cytotoxic T lymphocyte activity and cytokine production. Detailed analyses revealed that proliferative responses specifically of CD8+ T cells are impaired and interleukin 2 secretion of CD4+ T cells is defective in the absence of LIGHT. Furthermore, a reduced 3[H]-thymidine incorporation after T cell receptor stimulation was observed. This for the first time provides in vivo evidence for a cooperative role for LIGHT and LTβ in lymphoid organogenesis and indicates important costimulatory functions for LIGHT in T cell activation.

scholarly journals Evaluation of Production Protocols for the Generation of NY-ESO-1-Specific T Cells

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 152
Author(s):  
Wenjie Gong ◽  
Lei Wang ◽  
Sophia Stock ◽  
Ming Ni ◽  
Maria-Luisa Schubert ◽  
...  
Keyword(s):  
T Cells ◽  
Cost Effectiveness ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
T Cell Subsets ◽  
Transduction Efficiency ◽  
Cell Production

NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). However, standardized protocols for their generation are limited. Particularly, cost-effectiveness considerations of cell production protocols are of importance for conducting clinical studies. In this study, two different NY-ESO-1-specific T cell production protocols were compared. Major differences between protocols 1 and 2 include culture medium, interleukin-2 and retronectin concentrations, T cell activation strategy, and the transduction process. NY-ESO-1-specific T cells generated according to the two protocols were investigated for differences in cell viability, transduction efficiency, T cell expansion, immunophenotype as well as functionality. NY-ESO-1-specific T cells showed similar viability and transduction efficiency between both protocols. Protocol 1 generated higher absolute numbers of NY-ESO-1-specific T cells. However, there was no difference in absolute numbers of NY-ESO-1-specific T cell subsets with less-differentiated phenotypes accounting for efficient in vivo expansion and engraftment. Furthermore, cells generated according to protocol 1 displayed higher capacity of TNF-α generation, but lower cytotoxic capacities. Overall, both protocols provided functional NY-ESO-1-specific T cells. However, compared to protocol 1, protocol 2 is advantageous in terms of cost-effectiveness. Cell production protocols should be designed diligently to achieve a cost-effective cellular product for further clinical evaluation.

scholarly journals Autologous Tumor Infiltrating T Cells Cytotoxic for Follicular Lymphoma Cells Can Be Expanded In Vitro

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3806-3816 ◽  
Author(s):  
Joachim L. Schultze ◽  
Mark J. Seamon ◽  
Sabine Michalak ◽  
John G. Gribben ◽  
Lee M. Nadler
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytotoxic T Cells ◽  
Interleukin 2 ◽  
Interferon Γ ◽  
Autologous Tumor

Abstract Follicular lymphomas (FLs) rarely induce clinically significant T-cell–mediated responses. We showed that freshly isolated tumor infiltrating T cells (T-TILs) lack tumor-specific cytotoxicity. Stimulation of these T cells with FL cells in the presence of interleukin-2 (IL-2) and/or costimulation via CD28 does not lead to T-cell activation and expansion. In contrast, when stimulated with FL cells preactivated via CD40, autologous T-TILs can be expanded by the addition of exogenous IL-2. These T cells can be further expanded in vitro by the addition of exogenous IL-4, IL-7, or interferon-γ, but not IL-12. Once activated, these T cells showed FL-directed cytotoxicity in four of five patients tested. We concluded that autologous cytotoxic anti-FL–specific T cells exist, but can only be detected in vitro under optimized conditions for T-cell stimulation and expansion. This suggests that their frequency in vivo is either very low or that the microenvironment does not provide the necessary signals to activate these T cells. This model system allows dissection of the requisite conditions for activation and expansion of lymphoma-directed cytotoxicity and may permit expansion of previously activated cytotoxic T cells for adoptive transfer.

scholarly journals RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter.

1995 ◽  
Vol 15 (8) ◽  
pp. 4260-4271 ◽  
Author(s):  
J H Lai ◽  
G Horvath ◽  
J Subleski ◽  
J Bruder ◽  
P Ghosh ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Specific Antigen ◽  
Transcriptional Activator ◽  
Nf Kappa B ◽  
Enhancer Activity ◽  
Response Element

T-cell activation requires two different signals. The T-cell receptor's recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors.

scholarly journals CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production

1998 ◽  
Vol 188 (2) ◽  
pp. 287-296 ◽  
Author(s):  
Angela M. Thornton ◽  
Ethan M. Shevach
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Peripheral Tolerance ◽  
Cell Contact ◽  
Suppressor T Cells

Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4+CD25+ T cells represent a unique population of suppressor T cells that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4+ T cells. To analyze the mechanism of action of these cells, we established an in vitro model system that mimics the function of these cells in vivo. Purified CD4+CD25+ cells failed to proliferate after stimulation with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4+CD25− cells, the CD4+CD25+ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4+CD25+ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25− T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4+CD25+ T cells in normal mice may represent a distinct lineage of “professional” suppressor cells.

scholarly journals Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production.

1996 ◽  
Vol 183 (4) ◽  
pp. 1937-1943 ◽  
Author(s):  
D G Motto ◽  
S E Ross ◽  
J Wu ◽  
L R Hendricks-Taylor ◽  
G A Koretzky
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Sh2 Domain ◽  
Activated T Cells ◽  
Amino Terminal

Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.

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