In vitro evaluation of the effects of candidate immunosuppressive drugs

Flow cytometry and quantitative real-time PCR as two independent and correlated read-outs

Mona G. Flores, Sally Zhang, Ann Ha, Bari Holm, Bruce A. Reitz, Randall E. Morris, Dominic C. Borie

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Background: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan® RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan® RT-PCR). Methods: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-γ, IL-2, IL-4, and TNF-α and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan® RT-PCR. Results: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC 50's) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan® RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3+ gate to a lymphocyte gate improved correlation (r=0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r=0.97) was observed when drug IC50's were considered. Conclusions: Flow cytometry and TaqMan® RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

Original languageEnglish (US)
Pages (from-to)123-135
Number of pages13
JournalJournal of Immunological Methods
Volume289
Issue number1-2
DOIs
StatePublished - Jun 2004
Externally publishedYes

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Immunosuppressive Agents
Real-Time Polymerase Chain Reaction
Flow Cytometry
Cytokines
Polymerase Chain Reaction
Pharmaceutical Preparations
Lectins
Inhibitory Concentration 50
Interleukin-2
RNA
Immunologic Monitoring
Messenger RNA
Macaca fascicularis
Tacrolimus
Interleukin-4
Cyclosporine
In Vitro Techniques
Molecular Biology
Lymphocytes
Population

Keywords

  • CSA
  • cyclosporine A
  • Cytokines
  • Flow cytometry
  • IFN-γ
  • IL
  • Immunosuppression
  • interferon gamma
  • interleukin
  • phorbol myristate acetate
  • PMA
  • Quantitative real-time PCR
  • RT-PCR
  • tacrolimus
  • TNF-α
  • TRL
  • tumor necrosis factor alpha

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

In vitro evaluation of the effects of candidate immunosuppressive drugs : Flow cytometry and quantitative real-time PCR as two independent and correlated read-outs. / Flores, Mona G.; Zhang, Sally; Ha, Ann; Holm, Bari; Reitz, Bruce A.; Morris, Randall E.; Borie, Dominic C.

In: Journal of Immunological Methods, Vol. 289, No. 1-2, 06.2004, p. 123-135.

Research output: Contribution to journalArticle

Flores, Mona G. ; Zhang, Sally ; Ha, Ann ; Holm, Bari ; Reitz, Bruce A. ; Morris, Randall E. ; Borie, Dominic C. / In vitro evaluation of the effects of candidate immunosuppressive drugs : Flow cytometry and quantitative real-time PCR as two independent and correlated read-outs. In: Journal of Immunological Methods. 2004 ; Vol. 289, No. 1-2. pp. 123-135.
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AU - Flores, Mona G.

AU - Zhang, Sally

AU - Ha, Ann

AU - Holm, Bari

AU - Reitz, Bruce A.

AU - Morris, Randall E.

AU - Borie, Dominic C.

PY - 2004/6

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N2 - Background: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan® RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan® RT-PCR). Methods: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-γ, IL-2, IL-4, and TNF-α and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan® RT-PCR. Results: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC 50's) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan® RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3+ gate to a lymphocyte gate improved correlation (r=0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r=0.97) was observed when drug IC50's were considered. Conclusions: Flow cytometry and TaqMan® RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

AB - Background: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan® RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan® RT-PCR). Methods: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-γ, IL-2, IL-4, and TNF-α and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan® RT-PCR. Results: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC 50's) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan® RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3+ gate to a lymphocyte gate improved correlation (r=0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r=0.97) was observed when drug IC50's were considered. Conclusions: Flow cytometry and TaqMan® RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

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KW - phorbol myristate acetate

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KW - Quantitative real-time PCR

KW - RT-PCR

KW - tacrolimus

KW - TNF-α

KW - TRL

KW - tumor necrosis factor alpha

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