AID	Name	Type	Comment
652024	qHTS of IL-2 Activators: Summary	summary

The adaptive immune response against any infectious agent begins with the activation of an antigen specific T cell. This relatively rare and inert ("naive") T cell undergoes a massive proliferative expansion and differentiation process to generate sufficient number of potent pathogen-specific effector T cells. The effector T cells fight the infection by making cytokines and helping other cells to become fully armed as well. If any naive T cell has antigen receptors (TCRs) that can be activated by self peptides, then a variety of regulatory mechanisms kick in to prevent this cell from performing to its full potential. These processes collectively constitute the phenomenon of immunological tolerance and their failure leads to autoimmunity. One arm of tolerance involves generation of biochemical negative feedback downstream of the TCR (tuning). This is especially relevant to self antigens present in the body over long periods of time (chronic), which form the bulk of our proteome. The negative feedback serves to tune the TCR signaling, such that the ambient antigenic stimulation is nullified intracellularly. Unfortunately, this critical machinery serves as a target for chronic viral, bacterial and parasitic infections (as well as tumors), which can evade a robust immune response, simply by allowing T cells to tune down their signaling to pathogen or malignancy derived antigens. As such, disrupting the tuning in such cases would allow the tolerant T cell to function again and deliver a robust effector response.
If a therapeutic can restore responsiveness to the tuned down T cell, even for a short time, they could combat the pathogen without any additional immunization. To that end, we have developed a sensitive HTS T cell based assay based on IL-2. This assay will be screened against the Molecular Libraries Small Molecule Repository (MLSMR) to find small molecules that could elicit this response.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH096572
Assay Submitter (PI): Ron Schwartz, NIAID

4 microL (750 cells/well) of unstimulated transgenic mouse T cells (columns 1-3,5-48) are dispensed into Aurora LoBase, black clear-bottom 1536-well assay plates; column 4 receives an identical concentration of transgenic memory T cells as a non-tolerant control. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). Following addition of compound and a 60 minute incubation, 1 microL of bead stimulation solution ((0.04 micron Fluospheres (Life technologies, CA) coated with 10ug/ml each anti-CD3 and anti-CD4 (clones 2C11 and GK1.5, BD Biosciences, CA) and mixed with 1ug/ml anti-CD28 (clone PV-1, BD biosciences, CA)). columns 2-48) is added to stimulate the IL-2 response pathway; media is instead added to column 1 as an unstimulated control, while PMA/ionomycin (Sigma Aldrich, MO) is added to column 3 as a control for IL-2 tolerance bypass. The plates are then incubated at 37 degrees C for 18 hours. EGFP and DsRed signal are then detected using an Acumen eX3 Explorer fluorescent plate cytometer. All cells are dispensed in cell media containing EHAA (Quality Biologicals, MD), RPMI (Quality Biologicals, MD), Glutamine (Life Technologies, CA), Penicillin/Streptomycin (Quality Biologicals, MD), 2-ME (Sigma Aldrich, MO) and 10% FBS (Gemini Bioproducts, CA) .

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Grant Number: MH096572