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BioAssay: AID 2052

HTS for developing T Cell Immune Modulators

When naive T cells encounter antigen presenting cells (APC) displaying cognate MHC-peptide complexes (pMHCs), the T cell and the APC form a highly organized structure at the contact site termed the 'immunological synapse', which is critical not only for prolonged and stable T/APC contact but also for the efficient and organized T cell signaling required for cell cycle progression and development more ..
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 2052
Data Source: NMMLSC (UNM_LFA-1_T-Cell_Immune_Modulators_MLPCN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-27
Modify Date: 2010-03-02

Data Table ( Complete ):           Active    All
BioActive Compounds: 221
Depositor Specified Assays
2087Summary report of screening for developing T Cell Immune Modulatorssummary
University of New Mexico Assay Overview
Assay Support: 1 X01 MH085707-01
Project Title: HTS for developing T Cell Immune Modulators
PI: Inkyu Hwang,PhD The Scripps Research Institute (La Jolla)
Assay Implementation: Mark K. Haynes PhD, Chelin Hu MS, Anna Waller PhD, Mark Carter MS
University of New Mexico Center for Molecular Discovery PI: Larry Sklar PhD

Assay Background and Significance:
When naive T cells encounter antigen presenting cells (APC) displaying cognate MHC-peptide complexes (pMHCs), the T cell and the APC form a highly organized structure at the contact site termed the 'immunological synapse', which is critical not only for prolonged and stable T/APC contact but also for the efficient and organized T cell signaling required for cell cycle progression and development of effector functions. Several membrane proteins and their ligand partners are involved in the formation and stability of the immunological synapse, including the pMHC/TCR complex, as well as LFA-1/ICAM-1 and the secondary signaling pair B7/CD28. This assay takes advantage of the processes leading to the formation of the immunological synapse to probe for small molecules that interfere with its formation. High affinity LFA-1/ICAM-1 interactions are central to sustained synapse formation [Reichardt, et al., 2007]. The LFA-1 molecule is a T cell integrin that plays unique and diverse roles in T cell immunity acting as both an adhesion and signaling molecule during cognate recognition of APC displayed pMHC by T cells [Smith, et al., 2007].

Integrins (including LFA-1) are highly regulated during immune responses, and ligands for integrins, such as ICAM-1 for LFA-1, are expressed on a wide variety of cell types and tissues. The adhesive activity of LFA-1 requires high-affinity binding and it is well established that the functional activity of LFA-1 is profoundly increased by molecular signals triggered by other receptors (eg TCR/CD3 complex, chemokine receptors, CD28) or chemical compounds such as phorbol esters [Wojcikiewicz, et al. 2003]. The signaling process controlling LFA-1 function has been termed 'inside-out' signaling (intracellular signals generated by other receptor/ligand interactions prompt LFA-1 to alter its molecular properties). Several signaling molecules are known to be involved in the process, including adaptor proteins [Kim, et al., 2003] (eg ADAP24, VASP, and SLP-76) and small GTP-binding proteins25 (eg Rap 1). The actin cytoskeleton also plays a critical role in 'inside-out' signaling.

The important role of LFA-1 in T cell immunity makes it an attractive target for developing drugs and probes for treatment and understanding of inflammatory diseases, autoimmune diseases, allergy, and organ transplantation. Moreover, over-expression of ICAM-1 and LFA-1 in tissues that experience chronic inflammation implicates LFA-1/ICAM-1 involvement in the initiation and/or progression of certain diseases [Rychly and Nebe, 2006]. Extensive efforts have been made to develop reagents that block the LFA-1/ICAM-1 interaction [Anderson and Siahaan, 2003]. The reagents developed include humanized monoclonal antibodies that selectively bind to LFA-1 or ICAM-1 and the cyclic peptides that bind to the LFA-128,30 epitope. Despite the reported effectiveness of these reagents in blocking the LFA-1/ICAM-1 interaction, their clinical use has been limited [Li, et al. 2009].

The purpose of this screening campaign is to find small chemical compounds that modulate the LFA-1 / ICAM-1 interaction. The assay detects the interaction of fluorescently labeled vesicles with murine CD8+ T lymphocytes resulting from associations between the TCR complex/CD28/LFA-1 (T cells) and the MHC Class I + QL9 peptide complex/B7-1/ICAM-1 (vesicles). Small molecules of interest could act as inhibitors of the extracellular molecular interactions. Alternatively, inhibitory compounds could cause changes in the T cell-vesicle interaction by acting via intracellular signaling pathways that are known to influence the T cell surface TCR complex assembly and the LFA-1 activation state.
This is a flow cytometry, cell-based assay using freshly isolated T cells from 2C TCR transgenic mice. Direct stimulation of naive, transgenic murine CD8+ T cells is achieved using membrane micro-vesicles produced from transgenically-engineered drosophila cells expressing murine membrane proteins involved in naive T cell activation. It has been determined that ICAM-1/LFA-1 recognition as well as TCR/QL9 recognition are essential for stable binding of micro-vesicles to naive CD8+ T cells [Kovar et al., 2006; Hwang et al. 2003].

The assay is conducted in a 384-well microplate format. Drosophila micro-vesicles expressing B7-1 (the ligand partner for T cell-expressed CD28), ICAM-1 (the ligand partner for T cell expressed LFA-1) and the Ld class I murine MHC molecule, are incubated with the antigenic peptide, QL9 (QLSPFPFDL, single letter amino acid sequence), that binds to Ld and is specifically recognized by 2C TCR transgenic CD8+ T cells. QL9-loaded micro-vesicles are incubated (1.5 hour/37degrees C) with MACS-purified CD8+ T cells, followed by anti-CD8 (Alexa647) and anti-B7-1 (PE) staining (20 minutes on ice). The stained, CD8+ T cell/micro-vesicle mixture is fixed by the addition of paraformaldehyde (1%/10minutes), diluted with PBS and analyzed by flow cytometry using the HyperCyt (Intellicyt, USA) high-throughput analytical platform.

Negative control wells on the plate contain micro-vesicles that are not loaded with QL9 antigenic peptide (naked micro-vesicles), resulting in low binding of micro-vesicles to CD8+ T cells. Positive control wells on the plate contain QL9 loaded vesicles and compound vehicle (0.7% DMSO).


The flow cytometric data are analyzed by HyperView(R) (IntelliCyt, USA) software and are initially gated on forward and side scatter parameters to distinguish the cell population from debris. Additional quadrant gating is made by placing calculation markers on FL2 (parameter used to measure PE conjugated anti-B7.1) versus FL8 (parameter used to measure APC conjugated anti-CD8-APC). This distinguishes CD8+ T cells from CD8- cells (FL8) and high vesicle binding from low vesicle binding (FL2). The distinction of CD8+ versus CD8- is made during the initial analysis. The distinction between high and low vesicle binding is calculated based on pooling the negative control wells on a given plate and allowing for 10 percent of the CD8+ T cells to be in the double positive quadrant (i.e. the negative control population is defined as a population where 10% of the CD8+ T cells have bound naked micro-vesicles). Based on these gates, HyperView parses the temporal event clusters of the entire data file and produces an annotated fluorescence summary for each well. These data sets are merged with compound worklist files generated by HyperSip(R) (IntelliCyt, USA) software. The parsed data are then processed through an Excel (R) (Microsoft, USA) template file constructed specifically for this assay to calculate the percent response.

Out of the various parameters collected and calculated from the flow cytometric data, we focused on three: FL2 mean channel fluorescence of the CD8+ T cell population, percent events present in the double positive quadrant (CD8+ and vesicle binding+), and the 90th percentile FL2 fluorescence intensity for the CD8+ population.
Due to variations over the plate, the percent response was calculated on a per-row-basis with the measurement of the positive control sample of a particular row being 100%, as per the following equation:
%Response = 100* Sample_CD8+_Mean/Row_PCntr_CD8+_Mean
where Sample_CD8+_Mean is the FL2 mean channel fluorescence of the CD8+ T cells in the test sample well and Row_PCntr_CD8+_Mean is the FL2 mean channel fluorescence of CD8+ T cells from the positive control well in the same row as the sample well. Note the use of mean channel fluorescence values rather than median. With this method of calculating a response, a compound that blocks vesicle binding to CD8+ T cells would result in a %Response less than 100%.

The other two parameters were used as calculated from HyperView.

Compounds were considered Active if they exhibited values outside 1 standard deviation for all three of the assessed parameters. Thus compounds were identified as active if the %Response < 81, %Events in the double positive quadrant (CD8+ and vesicle binding+ population) < 18, and CD8+ 90th PFI < 36.

The PUBCHEM_ACTIVITY_SCORE was calculated from a weighted function of these three parameters and given to any Active compound:
SCORE = 0.4*(100 - %Response) + 0.4 *(100 - %Events) + 20*(36 - 90th PFI)/36
where %Response is calculated based on FL2 mean channel fluorescence of the CD8+ T cell population, %Events are the percentage of events the double positive quadrant (CD8+ and vesicle binding+ population), and 90th PFI is the 90th percentile fluorescence intensity for the CD8+ population.
Inactive compounds were given a score of 0.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1RESPONSE (7μM**)Percent response based on mean channel fluorescence of CD8+ cellsFloat%
2CD8POSITIVE_90_PFIFluorescence intensity of 90th percentile of the CD8+ population Float
3DOUBLEPOSITIVE_EVENTSPercentage of Events in the double positive population (CD+ and vesicle binding+)Float%
4TOTAL_EVENTSTotal number of events collected from that well with sample compoundInteger

** Test Concentration.
Additional Information
Grant Number: 1 X01 MH085707-01

Data Table (Concise)