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)
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 | |
| Electrophoresis | Load 20-50μg total protein per lane. |
| Transferring | Use Ponceau S staining to determine if the transfer is successful |
| Maximizing signals | Treat samples with PNGase F to confirm the specificity of bands if necessary. 5 |
| Actual band size in Western Blot is between 40-60kD. | |
| Controls | Positive: 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 incubation | It is recommended to optimize antibody dilution in preliminary experiments according to datasheets. |
| Maximizing signal | Treat 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/ | |
| Controls | Positive: Human tonsil, head, and neck squamous cell carcinoma Placenta tissues Negative: MCF-7 5 |
Find full information on working with PD-L1:
- 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)
- 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)
- Mezzadra, R., Sun, C., Jae, LT., et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature. 549, 106-110 (2017)
- 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)
- 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)