scholarly journals Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors.

1994 ◽  
Vol 14 (3) ◽  
pp. 2159-2169 ◽  
Author(s):  
P A Garrity ◽  
D Chen ◽  
E V Rothenberg ◽  
B J Wold
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cyclosporin A ◽  
Interleukin 2 ◽  
Cell Types ◽  
Regulatory Elements ◽  
In Vitro Assays ◽  
Nuclear Extracts

Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.

scholarly journals Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors

1994 ◽  
Vol 14 (3) ◽  
pp. 2159-2169
Author(s):  
P A Garrity ◽  
D Chen ◽  
E V Rothenberg ◽  
B J Wold
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cyclosporin A ◽  
Interleukin 2 ◽  
Cell Types ◽  
Regulatory Elements ◽  
In Vitro Assays ◽  
Nuclear Extracts

Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.

scholarly journals A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katherine E. Harris ◽  
Kyle J. Lorentsen ◽  
Harbani K. Malik-Chaudhry ◽  
Kaitlyn Loughlin ◽  
Harish Medlari Basappa ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Bispecific Antibody ◽  
T Regulatory Cells ◽  
Tumor Regression ◽  
Interleukin 2 ◽  
In Vivo Studies ◽  
Preferential Binding

AbstractThe use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαβγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rβγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule’s in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

scholarly journals In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

2006 ◽  
Vol 74 (7) ◽  
pp. 3817-3824 ◽  
Author(s):  
Karen L. Wozniak ◽  
Jatin M. Vyas ◽  
Stuart M. Levitz
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Mouse Lung

ABSTRACT Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

scholarly journals The critical immunosuppressive effect of MDSC-derived exosomes in the tumor microenvironment

2020 ◽  
Author(s):  
Mohammad H. Rashid ◽  
Thaiz F. Borin ◽  
Roxan Ara ◽  
Raziye Piranlioglu ◽  
Bhagelu R. Achyut ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Suppressor Cells ◽  
Cell Types ◽  
Biological Macromolecules

AbstractMyeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME), and our perception regarding the role of MDSCs in tumor promotion is attaining extra layer of intricacy in every study. In conjunction with MDSC’s immunosuppressive and anti-tumor immunity, they candidly facilitate tumor growth, differentiation, and metastasis in several ways that yet to be explored. Alike any other cell types, MDSCs also release a tremendous amount of exosomes or nanovesicles of endosomal origin and partake in intercellular communications by dispatching biological macromolecules. There has not been any experimental study done to characterize the role of MDSCs derived exosomes (MDSC exo) in the modulation of TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant amount of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those are in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper activating or exhausting CD8 T-cells and induce reactive oxygen species production that elicits activation-induced cell death. We confirmed the depletion of CD8 T-cells in vivo by treating the mice with MDSC exo. We also observed a reduction in pro-inflammatory M1-macrophages in the spleen of those animals. Our results indicate that immunosuppressive and tumor-promoting functions of MDSC are also implemented by MDSC-derived exosomes which would open up a new avenue of MDSC research and MDSC-targeted therapy.

scholarly journals TRAF6 inhibits Th17 differentiation and TGF-β–mediated suppression of IL-2

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4750-4757 ◽  
Author(s):  
Pedro J. Cejas ◽  
Matthew C. Walsh ◽  
Erika L. Pearce ◽  
Daehee Han ◽  
Gretchen M. Harms ◽  
...  
Keyword(s):  
T Cells ◽  
Transforming Growth Factor ◽  
Interleukin 2 ◽  
Transforming Growth Factor Β ◽  
Main Function ◽  
Normal Numbers ◽  
T Helper 17 ◽  
Th17 Differentiation

Abstract Transforming growth factor-β (TGF-β) has an essential role in the generation of inducible regulatory T (iTreg) and T helper 17 (Th17) cells. However, little is known about the TGF-β–triggered pathways that drive the early differentiation of these cell populations. Here, we report that CD4+ T cells lacking the molecular adaptor tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) exhibit a specific increase in Th17 differentiation in vivo and in vitro. We show that TRAF6 deficiency renders T cells more sensitive to TGF-β–induced Smad2/3 activation and proliferation arrest. Consistent with this, in TRAF6-deficient T cells, TGF-β more effectively down-regulates interleukin-2 (IL-2), a known inhibitor of Th17 differentiation. Remarkably, TRAF6-deficient cells generate normal numbers of Foxp3-expressing cells in iTreg differentiation conditions where exogenous IL-2 is supplied. These findings show an unexpected role for the adaptor molecule TRAF6 in Smad-mediated TGF-β signaling and Th17 differentiation. Importantly, the data also suggest that a main function of TGF-β in early Th17 differentiation may be the inhibition of autocrine and paracrine IL-2–mediated suppression of Th17 cell generation.

“In vivo” activated splenic T cells are refractory to interleukin 2 growth “in vitro”

1987 ◽  
Vol 17 (7) ◽  
pp. 901-908 ◽  
Author(s):  
António Bandeira ◽  
Eva-Lotta Larsson ◽  
Luciana Forni ◽  
Pablo Pereira ◽  
António Coutinho
Keyword(s):  
T Cells ◽  
Interleukin 2

scholarly journals Fused protein domains inhibit DNA binding by LexA.

1992 ◽  
Vol 12 (7) ◽  
pp. 3006-3014 ◽  
Author(s):  
E A Golemis ◽  
R Brent
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
In Vitro Assays ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2084-2093 ◽  
Author(s):  
Alexander D. McLellan ◽  
Michaela Kapp ◽  
Andreas Eggert ◽  
Christian Linden ◽  
Ursula Bommhardt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Antigen Expression ◽  
Cell Receptor ◽  
In Vitro Assays ◽  
Peptide Complexes ◽  
Marginal Zones

Abstract Mouse spleen contains CD4+, CD8α+, and CD4−/CD8α− dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule–associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8α+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8α+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)–transgenic T cells. CD8α− or CD8α+DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I–peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8α DCs was responsible for the low allostimulatory capacity of CD8α+ DCs in vitro. Surprisingly, both CD8α+ DCs and CD4−/CD8− DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8α+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8α+ DCs and CD8+ T cells.

scholarly journals Signaling function of PRC2 is essential for TCR-driven T cell responses

2018 ◽  
Vol 215 (4) ◽  
pp. 1101-1113 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Joon Seok Park ◽  
Jonas Marcello ◽  
Michael T. McCabe ◽  
Richard Gregory ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Therapeutic Potential ◽  
Cell Types ◽  
Polycomb Repressive Complex 2 ◽  
Histone Lysine Methyltransferases

Differentiation and activation of T cells require the activity of numerous histone lysine methyltransferases (HMT) that control the transcriptional T cell output. One of the most potent regulators of T cell differentiation is the HMT Ezh2. Ezh2 is a key enzymatic component of polycomb repressive complex 2 (PRC2), which silences gene expression by histone H3 di/tri-methylation at lysine 27. Surprisingly, in many cell types, including T cells, Ezh2 is localized in both the nucleus and the cytosol. Here we show the presence of a nuclear-like PRC2 complex in T cell cytosol and demonstrate a role of cytosolic PRC2 in T cell antigen receptor (TCR)–mediated signaling. We show that short-term suppression of PRC2 precludes TCR-driven T cell activation in vitro. We also demonstrate that pharmacological inhibition of PRC2 in vivo greatly attenuates the severe T cell–driven autoimmunity caused by regulatory T cell depletion. Our data reveal cytoplasmic PRC2 is one of the most potent regulators of T cell activation and point toward the therapeutic potential of PRC2 inhibitors for the treatment of T cell–driven autoimmune diseases.

scholarly journals Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

2020 ◽  
Vol 21 (13) ◽  
pp. 4804
Author(s):  
Vincent van Duinen ◽  
Wendy Stam ◽  
Eva Mulder ◽  
Farbod Famili ◽  
Arie Reijerkerk ◽  
...  
Keyword(s):  
Drug Screening ◽  
Cell Formation ◽  
Cell Types ◽  
Culture Conditions ◽  
In Vitro Assays ◽  
Vegf Receptor ◽  
Screening Assay ◽  
Angiogenesis Assay

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

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