We developed a simple flow cytometry assay for evaluating the binding of PD-1–blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.
Immune checkpoint inhibitors, including PD-1–blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1–blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1–blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1–blocking antibodies in cancer patients.
PD-1–blocking antibodies have become the standard choice for treatment of various types of cancer, including non-small cell lung cancer (NSCLC)1,2,3,4. They show a remarkable therapeutic effect in a subset of cancer patients who have not responded to conventional cytotoxic chemotherapies. However, immune checkpoint inhibitors (ICIs), which include PD-1–blocking antibodies, can cause a unique and distinct spectrum of adverse events, termed immune-related adverse events (irAEs)5. Although irAEs can affect almost all tissues, they are most commonly observed in the gastrointestinal tract, endocrine glands, skin, and liver, and they can cause pruritus, rash, nausea, diarrhea, and thyroid disorders6,7. In general, most irAEs appear within 1 to 2 months after the initiation of ICIs. However, in some cases, they can occur later than 1 year after the beginning of treatment or even after treatment cessation6,7. They also cause various symptoms that may be difficult to discriminate from other pathologies. Thus, it can be challenging to promptly diagnose irAEs and treat them appropriately. irAEs can affect all tissues, and their onset is strongly influenced by circulating immune cells, especially T cells bound to PD-1–blocking antibodies. Therefore, a straightforward and minimally invasive method to monitor antibody-targeted T cells is important in clinical settings.
Here, we developed a simple method to assess the binding of PD-1–blocking antibodies to T cells using a drop of peripheral whole blood from cancer patients who received nivolumab or pembrolizumab. Using this approach, we were able to monitor each of the following: 1) the duration of antibody binding to T cells, 2) the occupancy of T cell PD-1 molecules by therapeutic antibodies, and 3) the activation status and immunological features of T cells. This method is a modification of a previously reported technique8. The amount of blood required is almost the same as that needed for a glucose test, and the approach does not require mononuclear cell enrichment or co-culturing with PD-1–blocking antibodies. We confirmed that this method can also be performed using frozen samples, including peripheral blood mononuclear cells (PBMCs) and cells from pleural effusion, pericardial effusion, bronchoalveolar lavage fluid, and cerebrospinal fluid, suggesting that this strategy may be useful in the context of a multicenter study. This method may facilitate the early diagnosis of irAEs, and also help to determine the appropriate immunosuppressive treatments to control their symptoms and to identify the optimal times to initiate subsequent therapies after PD-1 inhibitors.
Sampling was performed during routine clinical procedures. All human samples were obtained after informed consent was provided by the subjects, in accordance with the Declaration of Helsinki and with the approval of the ethical review board of the Graduate School of Medicine, Osaka University, Japan (15383 and 752).
1. Whole Blood Sample Preparation and Staining
- Collect whole blood samples into blood collection tubes containing ethylene diamine tetra-acetic acid (EDTA).
NOTE: Blood collection can be performed using either a regular needle or a blood lancet.
- Transfer 20 µL of whole blood samples to 5 mL round-bottom polystyrene flow cytometry tubes.
NOTE: To reduce non-specific binding of cells to tubes, 1 mL of 2% fetal bovine serum (FBS) in phosphate buffered saline (PBS) is added into tubes and vortexed for 10 s before application to samples.
- Add 20 µL of 2% FBS in PBS.
- Add 10 µL of human-specific FcR blocking reagent. Mix well and incubate for 15 min at room temperature.
- Add 500 µL of red blood cell lysis buffer. Mix well and incubate for 10 min at room temperature.
- Add 4 mL of 2% FBS in PBS and spin down cells at 400 x g (1,500 rpm) for 5 min at 4 °C. Remove supernatant by aspiration.
- Repeat the wash and aspiration process described in step 1.6.
- Re-suspend cells in 100 µL of 2% FBS in PBS and divide into two tubes of 50 µL each.
- Add surface marker antibodies (Table 1). Mix well and incubate for 20 min at room temperature in the dark.
NOTE: When profiling the T cell immune status, the number of markers can be increased based on the flow cytometry machine quality.
- Wash samples 2x as described in step 1.6.
- Resuspend cells in 200 µL of 2% FBS in PBS.
2. Flow Cytometric Analysis
- Insert tubes into the flow cytometer and acquire cells, basically following the recommended protocol9.
- Record 10,000 events as the lymphocyte gate (Figure 1A) and export flow data as .fcs files for analysis.
- Open files in the analysis software. Visualize the cells on a forward scatter (FSC) (A) vs. side scatter (SSC) (A) plot and gate lymphocytes (Figure 1A).
- After selecting single cells using FSC (H) vs. FSC (W) and SSC (H) vs. SSC (W) (Figure 1B) and displaying them on a CD3 vs. CD8 or CD3 vs. CD4 plot, gate the CD8 T cells and CD4 T cells, respectively (Figure 1C).
- After selecting the gated cells and displaying them on a PD-1 vs. human IgG4 plot, identify PD-1–blocking antibody–bound CD8 and CD4 T cells based on isotype control (Figure 1D).
The gating strategy and flow cytometry analysis (Figure 1) can detect PD-1–blocking antibody binding to T cells obtained from a drop of NSCLC patient peripheral blood. Before PD-1–blocking antibody is administered, no human IgG4-positive CD8 or CD4 T cells are present, and PD-1 expression can be confirmed by a PD-1–detecting antibody (EH12.1) (Figure 2A). After nivolumab or pembrolizumab administration, IgG4 (nivolumab, pembrolizumab) can be detected on T cells by anti-IgG4 antibody (HP6025) whereas the PD-1–detecting antibody EH12.1 does not recognize any PD-1 on T cells because therapeutic PD-1 antibodies disturb EH12.1 binding. This means that we are indirectly measuring the therapeutic binding of PD-1–blocking antibody based on the lack of binding of PD-1–detecting antibody. Representative data show the different binding statuses of PD-1–blocking antibody (Figure 2A). Nivolumab and pembrolizumab binding and occupancy of PD-1 on T cells decrease over time10, and there is partial binding (PB), which is shown in the double-positive area, and finally complete loss of binding (LB) (Figure 2B).
Figure 1: Representative gating strategy to evaluate PD-1–blocking antibody binding to T cells from a drop of peripheral blood. (A) FSC (A) vs. SSC (A) plot and gating of lymphocytes. (B) Doublets are excluded by drawing gates around the main cell population on plots of FSC (H) vs. FSC (W) and SSC (H) vs. SSC (W). (C) CD3 vs. CD8 plot (upper) and CD3 vs. CD4 plot (lower) and gating of CD8 T cells and CD4 T cells, respectively. (D) PD-1 vs. human IgG4 plot and detection of the binding of nivolumab (a PD-1–blocking antibody) to CD8 and CD4 T cells. Orange dots and black dots indicate anti-IgG4 antibody and isotype control staining, respectively. Please click here to view a larger version of this figure.
Figure 2: Representative flow cytometry analysis indicating the change in binding status of PD-1–blocking antibodies. (A) Staining of PD-1 and human IgG4 in CD8 T cells from ICI-pretreated patient blood was evaluated by flow cytometry (left). Ten microliters of pretreated blood was treated with serial dilution of nivolumab for 15 min, and steps 1.4 to 1.10 of the protocol were completed. Complete binding (CB) (red), partial binding (PB) (blue), and loss of binding (LB) (green) are defined by the indicated gates. (B) The status of PD-1–blocking antibody binding to CD8 T cells was analyzed at the follow-up time points, as indicated, in NSCLC patients who discontinued nivolumab and pembrolizumab. Please click here to view a larger version of this figure.
|Isotype control||Isotype control||CD3||Isotype control||CD4||CD8|
Table 1: Antibodies used in flow cytometric analysis.
In this article, we report a method using a flow cytometer to detect PD-1–blocking antibodies bound to T cells derived from a drop of peripheral blood, which we originally developed for nivolumab detection10. Although this technique is very simple and easy to perform, two important points should be noted in order to obtain accurate results. One is that to detect PD-1 molecules, an appropriate antibody that competes with nivolumab and pembrolizumab should be used. This issue was evaluated in a previous study11. The other is that RBC lysis should be performed thoroughly before surface staining. As long as an isotype control is established for each assay to determine the gate of the IgG4-positive cluster, the frequency is unaffected. However, with this protocol, when RBC lysis is insufficient there may be reductions of both the T cell count in the lymphocyte gate and of the intensity of each surface marker. In addition, the RBC lysis step must be performed before surface staining, otherwise the anti-IgG4 antibody (HP6025) does not function properly.
The limitation of this method is that the population of T cells bound to PD-1–blocking antibodies include specific T cell clones responsible for therapeutic effects and irAEs; however, the frequencies of these specific clones among the antibody-bound population are quite low. Therefore, we still need to enrich the specific target using certain markers, for instance CD3912. Another limitation is that the fluorescence intensity of IgG4 is not high in some cases, which makes it difficult to determine binding status (i.e., PB and LB).
Other studies have monitored therapeutic PD-1 antibodies in the blood to evaluate pharmacokinetics. Measuring the plasma concentration of nivolumab or pembrolizumab is essential to determine how the residual amount of these antibodies in blood correlates with time. However, we previously reported that the concentration of nivolumab does not completely correlate with residual binding to T cells10. Compared to the measurement of plasma antibody concentration13, our method may be more appropriate for evaluating the residual binding of anti–PD-1 antibodies to T cells. The original method of monitoring nivolumab binding to T cells in blood required incubation with fluorescence-labeled nivolumab8. We simplified this approach and succeeded in substantially reducing the assay time and the amount of blood needed for analysis.
Frozen cells can also be analyzed using this assay, suggesting that this method is a good fit for multicenter studies. The usefulness of this approach will be further enhanced by combining monitoring binding status with other immunological markers, such as Ki-67, of T cells bound to PD-1–blocking antibodies10. Future studies should use our method in conjunction with single-cell RNA sequencing and T cell receptor sequencing to try to identify and characterize the specific subsets of T cells that are bound to PD-1–blocking antibodies and are responsible for irAEs.
The authors have nothing to disclose.
This work was supported by grants to S.K. from the Japan Society for the Promotion of Science KAKENHI (JP17K16045) and the Japan Agency for Medical Research and Development (JP18cm0106335 and 19cm0106310).
|10X RBC Lysis Buffer (Multi-species)||Thermo Fisher Scientific||00-4300-54||50 mL|
|APC/Cyanine7 anti-human CD4 Antibody||BioLegend||300518||Clone RPA-T4|
|BD FACS Canto II Flow Cytometer||BD|
|Brilliant Violet 510 anti-human CD8a Antibody||BioLegend||301048||Clone RPA-T8|
|Dulbecco's Phosphate Buffered Saline||nacalai tesque||14249-95||500 mL|
|Falcon Round-Bottom Polystyrene Tubes||STEMCELL Technologies||352058||5 mL|
|FcR Blocking Reagent, human||Miltenyi Biotec||130-059-901||2 mL|
|Gibco Fetal Bovine Serum||Thermo Fisher Scientific||12676029||500 mL|
|Mouse IgG1 monoclonal - Isotype control||abcam||ab81200|
|Mouse monoclonal Anti-Human IgG4 Fc||abcam||ab99825||Clone HP6025|
|Pacific Blue Mouse Anti-Human CD3||BD||558117||Clone UCHT1|
|PE-Cy7 Mouse anti-Human CD279 (PD-1)||BD||561272||Clone EH12.1|
|PE-Cy7 Mouse IgG1 κ Isotype Control||BD||557646|
- Gong, J., Chehrazi-Raffle, A., Reddi, S., Salgia, R. Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations. Journal for ImmunoTherapy of Cancer. 6, (1), 8 (2018).
- Borghaei, H., et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. The New England Journal of Medicine. 373, (17), 1627-1639 (2015).
- Brahmer, J., et al. Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer. The New England Journal of Medicine. 373, (2), 123-135 (2015).
- Herbst, R. S., et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. The Lancet. 387, (10027), 1540-1550 (2016).
- Postow, M. A., Sidlow, R., Hellmann, M. D. Immune-Related Adverse Events Associated with Immune Checkpoint Blockade. The New England Journal of Medicine. 378, (2), 158-168 (2018).
- Weber, J. S., et al. Safety Profile of Nivolumab Monotherapy: A Pooled Analysis of Patients With Advanced Melanoma. Journal of Clinical Oncology. 35, (7), 785-792 (2017).
- Champiat, S., et al. Management of immune checkpoint blockade dysimmune toxicities: a collaborative position paper. Annals of Oncology. 27, (4), 559-574 (2016).
- Brahmer, J. R., et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. Journal of Clinical Oncology. 28, (19), 3167-3175 (2010).
- Bommareddy, P. K., Lowe, D. B., Kaufman, H. L., Rabkin, S. D., Saha, D. Multi-parametric flow cytometry staining procedure for analyzing tumor-infiltrating immune cells following oncolytic herpes simplex virus immunotherapy in intracranial glioblastoma. Journal of Biological Methods. 6, (2), 112 (2019).
- Osa, A., et al. Clinical implications of monitoring nivolumab immunokinetics in non-small cell lung cancer patients. JCI Insight. 3, (19), 59125 (2018).
- Zelba, H., et al. Accurate quantification of T-cells expressing PD-1 in patients on anti-PD-1 immunotherapy. Cancer Immunology, Immunotherapy. 67, (12), 1845-1851 (2018).
- Simoni, Y., et al. Bystander CD8(+) T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature. 557, (7706), 575-579 (2018).
- Chiu, H. H., et al. Development of a general method for quantifying IgG-based therapeutic monoclonal antibodies in human plasma using protein G purification coupled with a two internal standard calibration strategy using LC-MS/MS. Analytica Chimica Acta. 1019, 93-102 (2018).