Bookmark and Share
BioAssay: AID 492962

Mouse Th1 T cell differentiation assay for inhibitors of ROR gamma transcriptional activity

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma ..more
_
   
 Tested Compounds
 Tested Compounds
All(3)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Inactive(3)
 
 
AID: 492962
Data Source: NCGC (RORG529)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-12-06
Hold-until Date: 2011-12-01
Modify Date: 2011-12-02

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
2551qHTS for inhibitors of ROR gamma transcriptional activityconfirmatory
2604Quantitative high throughput screen for inhibitors of ROR gamma transcriptional activity: Summarysummary
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: R03 DA026211-01
Assay Provider: Dan Littman, New York University

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma

Assay Overview:

The retinoic acid-related orphan receptor (ROR) gamma is a transcription factor that has a central role in the differentiation of Th17 cells, a subset of T helper cells that secrete the inflammatory cytokines IL-17a, IL-17f, and IL-22. Th17 cells have been implicated in graft versus host disease, autoimmune disease and asthma. ROR gamma is induced in naive T helper cells in the presence of TGF-beta combined with IL-6, IL-21, or IL-23, and thereafter directs the expression of the Th17 lineage cytokines. To confirm the selectivity of ROR gamma inhibitors, compounds with strong inhibitory activities on Th17 cells were tested in a mouse Th1 T cell differentiation assay. T cells differentiate into Th1 cells in the presence of IL-12 and IL-2. Th1 differentiation does not require ROR gamma and thus selective ROR gamma inhibitors should not block this process. Th1 differentiation was tracked by the induction of interferon gamma (IFN-g) expression as measured by an anti-IFN-g antibody using flow cytometry. Selective ROR gamma inhibitors do not block IFN-g expression in this assay.
Protocol
Assay Protocol Summary:

Cells from lymph nodes and spleens derived from six to eight week old IL17a-GFP mice (Biocytogen LLC) were used for T cell purification. B220- cells were isolated on an autoMACS Pro with bead depletion of B220+ cells (Miltenyi). Naive CD4+ T cell were further purified as TCRb+CD8- DAPI- CD19- CD4+CD25- CD62L+CD44low/Int by cell sorting on a FACSAria (BD). Cells were cultivated in an incubator at 37 C and 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated FBS (Hyclone), penicillin-streptomycin, 2 mM glutamine and 0.1 mM nonessential amino acids. Cells were seeded on day 0 at a density of 0.4E5 cells per ml in 96-well plates coated with anti-CD3e (5 ug/ml) and anti-CD28 (10 ug/ml). Cells were cultured for 4~5 days in Th1 [IL-12 (10 ng/ml), IL-2 (100 U/ml), anti-IL-4] condition. At day 1, compounds dissolved in DMSO were added. For intracellular cytokine staining, cells were incubated for 5 h with phorbol ester (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma) and GolgiStop (BD). When needed, surfaces were stained with PECy7-conjugated CD4 (BD Biosciences) by incubating on ice for 15 min. The Cytofix/Cytoperm buffer set (BD) was used for intracellular staining. Cells were fixed, permeabilized for 30 min on ice and stained for 30 min on ice in permeabilization buffer with Alexa647-conjugated anti-IL17a (eBioscience) and PE-conjugated anti-IFN-g (eBioscience). An LSR II (BD Biosciences) and FlowJo software (Tree Star) were used for flow cytometry.

Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, nuclear hormone receptor, retinoic acid-related orphan receptor, ROR gamma
Comment
Compound Ranking:

All compounds are inactive and assigned a PUBCHEM_ACTIVITY_SCORE of 0.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1% Activity% Activity at test concentration.Float%
2Test concentration (uM)Concentration tested.FloatμM
3Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String
Additional Information
Grant Number: R03 DA026211-01

Data Table (Concise)
PageFrom: