Inhibitory effects of interleukin 12 on retroviral gene transduction into CD34+++ cord blood myeloid progenitors mediated by induction of tumor necrosis factor-α

Mang Xiao, Zhi Hua Li, Jon McMahel, Hal Broxmeyer, Li Lu

Research output: Contribution to journalArticle

Abstract

Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34+++ cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34+++ granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which could potentially have been induced by IL-12 from CD34+++ cells. TNF-α protein release was significantly increased in CD34+++ cells incubated with IL-12. No detectable levels of IFN-γ were noted. Anti-TNF-α, but not anti-IFN-γ, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-α, but not IFN-γ, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-α.

Original languageEnglish
Pages (from-to)171-177
Number of pages7
JournalJournal of Hematotherapy and Stem Cell Research
Volume5
Issue number2
StatePublished - Apr 1996

Fingerprint

Interleukin-12
Fetal Blood
Tumor Necrosis Factor-alpha
Genes
Interferons
Stem Cell Factor
Interleukin-3
Granulocyte-Macrophage Colony-Stimulating Factor
Erythropoietin
Myeloid Progenitor Cells
Granulocyte-Macrophage Progenitor Cells
Erythroid Precursor Cells
Product Packaging
Granulocytes
Northern Blotting
Blood Cells
Suspensions
Enzyme-Linked Immunosorbent Assay
Macrophages
Cytokines

ASJC Scopus subject areas

  • Immunology
  • Hematology

Cite this

@article{080621b13d4046ee91b05b398229002f,
title = "Inhibitory effects of interleukin 12 on retroviral gene transduction into CD34+++ cord blood myeloid progenitors mediated by induction of tumor necrosis factor-α",
abstract = "Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34+++ cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34+++ granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which could potentially have been induced by IL-12 from CD34+++ cells. TNF-α protein release was significantly increased in CD34+++ cells incubated with IL-12. No detectable levels of IFN-γ were noted. Anti-TNF-α, but not anti-IFN-γ, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-α, but not IFN-γ, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-α.",
author = "Mang Xiao and Li, {Zhi Hua} and Jon McMahel and Hal Broxmeyer and Li Lu",
year = "1996",
month = "4",
language = "English",
volume = "5",
pages = "171--177",
journal = "Stem Cells and Development",
issn = "1547-3287",
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T1 - Inhibitory effects of interleukin 12 on retroviral gene transduction into CD34+++ cord blood myeloid progenitors mediated by induction of tumor necrosis factor-α

AU - Xiao, Mang

AU - Li, Zhi Hua

AU - McMahel, Jon

AU - Broxmeyer, Hal

AU - Lu, Li

PY - 1996/4

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N2 - Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34+++ cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34+++ granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which could potentially have been induced by IL-12 from CD34+++ cells. TNF-α protein release was significantly increased in CD34+++ cells incubated with IL-12. No detectable levels of IFN-γ were noted. Anti-TNF-α, but not anti-IFN-γ, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-α, but not IFN-γ, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-α.

AB - Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34+++ cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34+++ granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which could potentially have been induced by IL-12 from CD34+++ cells. TNF-α protein release was significantly increased in CD34+++ cells incubated with IL-12. No detectable levels of IFN-γ were noted. Anti-TNF-α, but not anti-IFN-γ, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-α, but not IFN-γ, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-α.

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