Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Summary

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.

Abstract

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.

Introduction

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 disorders^6,7. 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 cessation^6,7. 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.

Protocol

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

1. 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.
2. 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.
3. Add 20 µL of 2% FBS in PBS.
4. Add 10 µL of human-specific FcR blocking reagent. Mix well and incubate for 15 min at room temperature.
5. Add 500 µL of red blood cell lysis buffer. Mix well and incubate for 10 min at room temperature.
6. 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.
7. Repeat the wash and aspiration process described in step 1.6.
8. Re-suspend cells in 100 µL of 2% FBS in PBS and divide into two tubes of 50 µL each.
9. 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.
10. Wash samples 2x as described in step 1.6.
11. Resuspend cells in 200 µL of 2% FBS in PBS.

2. Flow Cytometric Analysis

1. Insert tubes into the flow cytometer and acquire cells, basically following the recommended protocol⁹.
2. Record 10,000 events as the lymphocyte gate (Figure 1A) and export flow data as .fcs files for analysis.
3. 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).
4. 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).
5. 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).

Results

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, pembrol...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
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
FLOWJO	BD
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