Anti-PDL-1 Antibody Treatment Reduces Expression of the Exhaustion Marker PD-1 in T Cells

November 25, 2015

Surgery
George Washington University, Washington DC
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

PE anti-mouse CD279 (PD-1) Antibody

Catalog Number:

135206

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Elevated expression of the exhaustion marker PD-1 is a major cause of programmed cell death of activated T cells. We hypothesized blocking the PD-1/PDL-1 interaction by treatment with anti-PDL-1 antibody in-vitro would down-regulated PD-1 expression in activated T cells and rescue them from apoptosis. Splenocytes isolated from mice were activated in an antigen-specific manner in presence or absence of mouse anti-PDL-1 polyclonal antibody. CD8 T cells were then stained for PD-1 expression using PE Anti-mouse PD-1 fluorochrome conjugated antibody and analyzed for PD-1 expression in untreated vs. PDL-1 treated group

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse splenocyes (activated)

Primary Incubation

Splenocytes were activated in an antigen specific manner. 1 million splenocytes were seeded in each well of a 24 well plate. Polyclonal anti-PDL-1 antibody (CD274) was added to the treatment group in the concentration of 1ug/ml for 48 hours.

Blocking Agent

NA

Secondary Incubation

After 48 hours, cells from both treated and un-treated groups were washed twice with PBS and stained with PE-Anti-PD-1 antibody in the concentration of 1:50 in staining buffer for FACS. Staining was done for 30 mins at 4 degree C. Cells were then washed twice and resuspended in 500ul of FACS buffer (PBS containing 0.1% NaN3 and 1% bovine serum albumin) and analyzed.

Tertiary Incubation

NA

Detection

Flow cytometry

Results Summary

Reduced expression of PD-1 (CD279) was observed in activated T cells which had been treated with anti-PDL-1 (CD274) antibody.

Additional Notes

PD-1 is expressed as an exhaustion marker which directs the cell towards apoptosis in different cells types including T cells, B cells and APC's. When performing experiments for different cell types, levels of stimulation a and concentration of antibodies used should be standardized prior to actual experiment. Experiment should be carefully designed.

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Summary

The Good

Excellent bright staining for a very useful surface marker.

The Bad

Nothing about the product

The Bottom Line

Studies related to exhaustion markers are becoming more important by the day as it is of prime importance in deciding the fate of a cell. Using PE-conjugated anti PD-1 antibody is a useful, reliable and fairly simple technique to estimate the exhaustion state of an activated cell by flow cytometry.

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