PD-L1

​

PD-L1 (programmed cell death 1 ligand 1) is a protein encoded by the CD274 gene. PD-L1 binds to its receptor, PD-1, to modulate immune activation or inhibition and is a target in cancer immunotherapy.

​Overview

​​Protein function, expression, and isoforms

Protein function:

  • PD-L1 plays a critical role in induction and maintenance of immune tolerance to self and, as a ligand for the inhibitory receptor PDCD1/PD-1, modulates the activation threshold & limits the effector response of T-cells. 1 2 3 
  • Through a yet unknown activating receptor, PD-L1 may co-stimulate T-cell subsets that predominantly produce interleukin-10 (IL10). 4
  • The PDCD1-mediated inhibitory pathway is exploited by tumors to attenuate anti-tumor immunity and escape destruction by the immune system, thereby facilitating tumor survival. 2 3
Expression

PD-L1 is highly expressed in the heart, skeletal muscle, placenta and lung, and is weakly expressed in the thymus, spleen, kidney and liver
PD-L1 is also expressed on activated T- and B-cells, dendritic cells, keratinocytes and monocytes 4 1
PD-L1 is up-regulated on T- and B-cells, dendritic cells, keratinocytes and monocytes after LPS and IFNG activation
It is also up-regulated in B-cells activated by surface Ig cross-linking 4 1 3

Expression

  • PD-L1 is highly expressed in the heart, skeletal muscle, placenta, and lung, and is weakly expressed in the thymus, spleen, kidney, and liver
  • PD-L1 is also expressed on activated T- and B-cells, dendritic cells, keratinocytes, and monocytes 4 1
  • PD-L1 is up-regulated on T- and B-cells, dendritic cells, keratinocytes, and monocytes after LPS and IFNG activation
  • It is also up-regulated in B-cells activated by surface Ig cross-linking 4 1 3

Isoforms

  • Human: Isoform I-3: 20-33kD (predicted)
  • Mouse: 32.8kD (predicted)

​​​​Applications

PD-L1 can be a challenging target to work with in some applications. 

Western blot

PD-L1 is heavily glycosylated, therefore the actual band size in western blot is between 40-60kD, different from the predicted 20-33kD. 



Sample preparation

Add adequate protease inhibitors (or phosphatase inhibitors for proteins modified by phosphorylation) to avoid target protein degradation.


Ultrasonicate samples to enrich more target proteins

Keep samples on ice during the whole WB process

Perform a Bradford assay, a Lowry assay, or a bicinchoninic acid (BCA) assay to determine the protein concentration
ElectrophoresisLoad 20-50μg total protein per lane.
TransferringUse Ponceau S staining to determine if the transfer is successful
Maximizing signalsTreat samples with PNGase F to confirm the specificity of bands if necessary. 5

Actual band size in Western Blot is between 40-60kD.
ControlsPositive: 
B-CPAP, MD-MB-231, U87-MG
Negative: 
A549 SW480









Immunohistochemistry

PD-L1 is highly expressed in lung, ovary, and colon carcinoma and in melanomas. However, not all samples show a positive signal, and the glycosylation of PD-L1 may interfere with antibody binding.



Sample preparation The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18–24h is suitable for most samples.

Under-fixation can lead to edge staining, with a strong signal on the edges of the section and no signal in the middle.

Over-fixation can mask the epitope. Antigen retrieval can help reverse this masking, but if the tissue has been fixed for a long period of time (ie over a weekend), there may be no signal even after antigen retrieval.
Antibody incubationIt is recommended to optimize antibody dilution in preliminary experiments according to datasheets.
Maximizing signalTreat samples with PNGase F may increase signal intensity.5

To find PD-L1 expression in lung cancer tissues, please refer to the table here (PD-L1 IHC 28-8 pharmDx, Dako). For PD-L1 expression in other tumors, please refer to: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991103/
ControlsPositive: 
Human tonsil, head, and neck squamous cell carcinoma
Placenta tissues
Negative:
MCF-7 5

Find full information on working with PD-L1: 

​​References

  1. Freeman, GJ., Long, AJ., Iwai, Y., et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 192 (7), 1027-34 (2000)
  2. Burr, ML., Sparbier, CE., Chan, YC., et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature. 549, 101-105 (2017)
  3. Mezzadra, R., Sun, C., Jae, LT., et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature. 549, 106-110 (2017)
  4. Dong, H., Zhu, G., Tamada, K., Chen, L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 5, 1365-9 (1999)
  5. Lee, HH., Wang, YN., Xia ,W., et al. Removal of N-Linked Glycosylation Enhances PD-L1 Detection and Predicts Anti-PD-1/PD-L1 Therapeutic Efficacy. Cancer Cell. 36, 168-178 (2019)