Because this method determines PD-1-blocking antibody-bound cells using peripheral blood from patients, the specific subset responsible for antitumor effects and immune-related adverse events can be identified. This method needs only a tiny amount of blood sample and the analyzing process is fairly simple and quick. It just needs a flow cytometry machine.
To begin, collect whole blood samples into blood collection tubes containing EDTA. Treat each five milliliter round bottom polystyrene flow cytometry tube with one milliliter of 2%fetal bovine serum in PBS. Vortex for 10 seconds.
Then transfer 20 microliters of whole blood samples to the treated five milliliter round bottom tubes. Add 20 microliters of 2%FBS in PBS and 10 microliters of human-specific FC receptor blocking reagent. Mix well and incubate for 15 minutes at room temperature.
Next, add 500 microliters of red blood cell lysis buffer. Mix well and incubate for 10 minutes at room temperature. Add four milliliters of 2%FBS in PBS and spin down cells at 400 times g for five minutes at four degrees Celsius.
Remove the supernatant by aspiration. Repeat the wash and aspiration process. Resuspend the cells in 100 microliters of 2%FBS in PBS and divide into two tubes of 50 microliters each.
Add surface marker antibodies. Mix well and incubate for 20 minutes at room temperature in the dark. Wash samples twice in 2%FBS in PBS as previously.
Resuspend the cells in 200 microliters of 2%FBS in PBS. Insert the tubes into the flow cytometer and acquire cells following the recommended protocol. Record 10, 000 events as the lymphocyte gate and export flow data as FCS files for analysis.
Open files in the analysis software and click on the file of isotype control to visualize the cells on a forward scatter versus side scatter plot and gate lymphocytes. Select single cells using FSCH versus FSCW and SSCH versus SSCW and display them on a CD3 versus CD8 or CD3 versus CD4 plot. Gate the CD8 T-cells and CD4 T-cells respectively.
Select the gated cells and display them on a PD-1 versus human IgG4 plot. And then based on isotype control, select Q3 to identify PD-1-blocking antibody-bound CD8 and CD4 T-cells. In this study, the gating strategy and flow cytometry analysis detected PD-1-blocking antibody binding to T-cells obtained from a drop of patient peripheral blood.
The PD-1 versus human IgG4 plot shows the binding status of PD-1-blocking antibodies on T-cells. Orange dots and black dots indicate anti-IgG4 antibody and isotype control staining respectively. Before PD-1-blocking antibody was administered, no human IgG4 positive CD8 or CD4 T-cells were present and PD-1 expression was confirmed by a PD-1 detecting antibody.
After nivolumab or pembrolizumab administration, IgG4 can be detected on T-cells by anti-IgG4 antibody whereas the PD-1 detecting antibody EH12.1 does not recognize any PD-1 on T-cells. The red, blue, and green gates indicate complete binding, partial binding and loss of binding respectively. After patients discontinued nivolumab and pembrolizumab, the status of PD-1-blocking antibody binding to CD8 T-cells decreased.
There was partial binding and finally complete loss of binding. Please make sure to mix the blood sample with RBC lysis as well. Also, mixing the RBC lysis before surface marker staining is the key step in this method.
If the researcher has access to an advanced flow cytometry machine, PD-1-blocking antibody-bound T-cells can be evaluated with more and more markers related to This strategy can be applied for profiling T-cell phenotypes responsible for adverse events caused by PD-1-blocking antibodies. Sampling from patients with developed adverse events is important to evaluate the utility of this strategy.
