In vitro evaluation of the effects of candidate immunosuppressive drugs: flow cytometry and quantitative real-time PCR as two independent and correlated read-outs.
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-gamma, IL-2, IL-4, and TNF-alpha 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 IC(50)'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.
Flores
Mona G
MG
Transplantation Immunology Laboratory, Department of Cardiothoracic Surgery, Falk Cardiovascular Research Center, Stanford University School of Medicine, CA 94305-5407, USA.
Zhang
Sally
S
Ha
Ann
A
Holm
Bari
B
Reitz
Bruce A
BA
Morris
Randall E
RE
Borie
Dominic C
DC
D000818:Animals; D001681:Biological Assay; D001773:Blood Cells / Q000187:drug effects* / Q000276:immunology; D016572:Cyclosporine / Q000494:pharmacology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D037102:Lectins / Q000494:pharmacology; D008252:Macaca fascicularis; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology
D016207:Cytokines; D007166:Immunosuppressive Agents; D037102:Lectins; D012333:RNA, Messenger; D016572:Cyclosporine; D016559:Tacrolimus