IntroductionChemotaxis assays have been traditionally set up to analyse gross migration of cells across a membrane in response to a chemotractant either by manual counting or by the use of a cellular dye measuring gross cellular movement. For simple assay systems where a recombinant cell line is being used for basic efficacy measurement this system works well. However, Chemotaxis assays can also be used to monitor the migration of specific cell types from within a mixed population of cells. At Huntingdon Life Sciences we have worked with AstraZeneca on the development of a phenotyping Chemotaxis assay by the use of flow cytometry to monitor the migration of the specific cell types within human Peripheral Blood Mononuclear Cells (PBMCs) in response to MDC. Due to the nature of this novel assay it then allows the use of disease state PBMC samples (or different conditioned media from separate primary assay systems) to be used for mechanism of action (MOA) studies with compounds, or to allow the biological investigation of disease state samples and the cell types involved. Described within this poster is the development of a T Cell Chemotaxis assay utilising flow cytometry to phenotype cellular migration in response to MDC (macrophage derived chemokine; CCL22), an agonist of the human CCR4 chemokine receptor. In particular the assay has been used to specifically quantify the numbers of CD4+/CLA+ T cells migrating as well as the effect of inhibition of that response with an AstraZeneca CCR4 inhibitor

Assay systemThe HTS Transwell-96 System is composed of four components: ● A 96 well permeable support plate - with choice of membranes● Reservoir plate (single well feeder plate) - with removable

media stabilizer● A 96 well receiver plate - for use with cell growth or assay ● Lid - minimizes evaporation and protects against

contamination

MethodHuman PBMCs isolated from whole blood using Histopaque

PBMCs counted and prepared ready for T Cell isolation

CD4 T Cells isolated using a Milltenyi Biotech CD4 negative selection kit

CD4 Isolated T Cells counted

175 µl Chemotractant at desired concentrations and compound (where required) added to the bottom wells of a 96 well Transwell plate

1.25 x 105 CD4 Cells added to the filter layer of the Transwell insert

Transwell plate incubated for 2 hours at 37°C/5% CO2

The Transwell filter is discarded and the cells in the lower wells are stained for CD3, CD4, CD8, CLA and CD195. The appropriate

compensation controls and FMO’s are also prepared

Cell samples analysed and counted using a BD FACS Canto II flow cytometer

Phenotypic Chemotaxis by the use of flow cytometryA Freeman1, J Hincks1, Y Whitehead1, C Murray2, G Wilkinson2, M Fagura2

1Huntingdon Life Sciences, Huntingdon, Cambridgeshire, England. 2AstraZeneca, Macclesfield, Cheshire, England.www.huntingdon.com

Poster 661

Results

MDC CLA+ CCR4+

0.001 0.01 0.1 1 10 100 10000

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Figure 1b: 1nM MDC

DiscussionThe assay described within this poster has shown the development of a novel Chemotaxis based method to monitor the migration of specific cell populations within PBMC samples.Specifically we have shown the migration of CCR4+ CLA+ skin homing T cells in response to the CCR4 agonist MDC and the inhibition of that response with an AstraZeneca CCR4 antagonist. The migration of these cells in response to MDC yielded an EC50 of 1nM, which is in accordance with the literature for the effect of this chemokine at the CCR4 receptor.The efficacy of the AstraZeneca compound measured was also consistent with the potency observed in a human CCR4 receptor-binding assay (pIC50 = 8.3). Using this assay we have demonstrated that it is possible to accurately determine the numbers of cells migrating in response to receptor stimuli and suggest that this may be a useful method to quantify the effect of such ligands, and specific inhibitors and antagonists, on the migration of cells derived from disease samples such as atopic dermatitis.A major benefit of using flow cytometry to monitor the specific cell types that have migrated compared to a measure of gross cell migration, means that the assay system can be used as a format for investigating cellular mechanism within disease samples.

ConclusionThis poster describes the successful development of a Chemotaxis method at HLS for the phenotypic analysis of the migrated cells. Using flow cytometry with the Transwell system, has enabled the Chemotaxis assay to not only be used as a primary efficacy assay, but also be used as an assay system to investigate disease mechanism.Using flow cytometry with the Transwell system also enables:● Rare cell types to be identified● A cell count of cells migrated ● Phenotype of cells migrated● Use of low sample volumes

Figure 1d: Cell migration positive control

Figure 1a: 0.3nM MDC

CLA+CCR4+ chemotaxis

0.1 1 10 100

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800MDC only3uM AZ1uM AZ0.3uM AZ0.1uM AZ0.03uM AZ

[MDC] nMCLA

+CC

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Figure 2b: Concentration-effect curve for the migration of isolated CD4+ T cells in response to MDC in the presence or absence of the indicated concentrations of a CCR4 antagonist. Data are the mean +/- SD of 2 determinations and representative of 3 individual experiments.

Figure 1c: 10nM MDC

Upper compartment

Microporous membrane

Transwell insert

Lower compartment

CD8+

CD8+

CD8+

CD8+

Figure 2a: Concentration-effect curve for the migration of isolated CD4+ T cells in response to MDC. Data are the mean +/- SD of 2 determinations and are representative of 4 individual experiments.

EC50 = 1nM