An Imaging-Based Assay to Measure the Location of PD-1 at the Immune Synapse for Testing the Binding Efficacy of Anti-PD-1 and Anti-PD-L1 Antibodies

Materials and reagents

Biological materials

1. Jurkat T cells (ATCC, catalog number: TIB-152)

2. Raji B cells (ATCC, catalog number: CCL-86)

3. Staphylococcus enterotoxin E (SEE) ET404 1 mg (Toxin Technology, catalog number: NC1467973)

Reagents

1. RPMI 1640 (Thermo Fisher Scientific, Gibco^TM, catalog number: 31800)

2. Opti-MEM (Thermo Fisher Scientific, catalog number: 11058021)

3. Heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10082147)

4. Penicillin/streptomycin (P/S) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140)

5. Human CD3/CD28/CD2 T-cell activator (StemCell Technologies, ImmunoCult^TM, catalog number: 10970) (optional; see protocol)

6. 10× Poly-L-Lysine (PLL) solution (0.1% w/v in H₂O) (Sigma-Aldrich, Millipore, catalog number: P8920)

7. Trypan Blue stain (0.4%) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: T10282)

8. Molecular biology grade water (Corning, catalog number: 46-000-CI)

9. pHR-PD-1-GFP vector and pHR-PD-L1-mCherry vector (AddGene, catalog number: 180819) [19]

1. 1× Poly-L-Lysine solution (see Recipes)

2. RPMI (w/ FBS) media (see Recipes)

3. Staphylococcus enterotoxin E (SEE) working stock (see Recipes)

1. 1× Poly-L-Lysine solution

   Reagent	Final concentration	Amount
   10× PLL [0.1 % (w/v) in H2O]	1×	100 µL
   ddH2O	n/a	900 µL
   Total	n/a	1,000 µL




2. RPMI (w/ FBS) media

   Reagent	Final concentration	Amount
   RPMI 1640	n/a	500 mL
   FBS	10%	55 mL
   P/S	1%	5.5 mL
   Total	n/a	560.5 mL




3. Staphylococcus enterotoxin E (SEE) working stock (stored at -20 °C)

   Reagent	Final concentration	Amount
   Staphylococcus enterotoxin E	1 mg/mL	20 µg
   ddH2O	n/a	20 µL
   Total	n/a	20 µL

Laboratory supplies

1. 15 mL and 50 mL conical tube (Falcon, catalog numbers: 38009, 38010)

2. 12-well cell culture plate (Thermo Fisher Scientific, catalog number: 150200)

3. Untreated cell culture flask with vent cap (Thermo Fisher Scientific, Corning^TM, catalog number: 431463)

4. MatTek Plates (50 mm dish, No. 0 coverslip, 14 mm glass diameter, uncoated) (MatTek, catalog number: P50G-0-14-F)

5. Single-channel pipette, Pipetman 0.2–2 µL, 1–10 µL, 2–20 µL, 20–200 µL, 100–1,000 µL (Gilson, catalog numbers: F144054M, F144055M, F144058M, F144059M)

6. Filtered pipette tips (USA Scientific)

7. 1.5 mL microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-129)

8. Immersol autofluorescence-free immersion oil 518 F (Zeiss, catalog number: 444960-0000-000)

9. Aluminum foil (Amazon brand)

10. Cell counting chamber slides (Countess^TM, catalog number: C10228)

11. KOVA^TM Glasstic^TM slide 10 with grids (Fisher Scientific, catalog number: 22-270141)

12. Cell culture multiwell plate 24 wells (Grenier Bio-One, catalog number: 662160)

Equipment

1. Confocal microscope (Zeiss, model: LSM 900)

2. Heracell 150i CO₂ incubator (37 °C, 5% CO₂) (Thermo Fisher Scientific, catalog number: 50116048)

3. Digital miniblock heater (VWR, catalog number: 460-0334)

4. Automated cell counter (Invitrogen^TM, Countess^TM, catalog number: AMQAX2000)

5. Fisherbrand^TM manual differential counter (Fisher Scientific, catalog number: 13-684-141)

6. 5425 centrifuge (Eppendorf, catalog number: 5405000646)

7. 5810R centrifuge (Eppendorf, catalog number: 022625501)

Software and datasets

1. Image J (Version 1.54)

2. Zen (Version 3.3 Blue Edition)

Procedure

1. Preparation of T and B cells

   Note: For optimal imaging, the assay should be done with a fluorescent PD-1-expressing T-cell line (such as Jurkat T cells expressing pHR-PD-1-GFP) and a fluorescent PD-L1-expressing antigen-presenting cell line (such as Raji B cells expressing pHR-PD-L1-mCherry). However, primary T cells can also be used following transfection/viral transduction with a fluorescent PD-1 expression vector in combination with Raji cells [20]. Fluorescent T and B cell lines can be generated as described in Lerrer et al. [21].

   
   

   Day 1

   Note: If activation via CD3/CD28/CD2 ImmunoCult T-cell activator for T cells is needed, the steps delineated for day 1 and below for day 2 must be done separately. Otherwise, the steps for day 1 and day 2 can be combined into a single day. See Figure 1.

   
   

   
   Figure 1. Timeline of the cell preparation protocol. Timelines for the protocol A) with T-cell activation or B) without T-cell activation are given. Steps shown on the same tick mark are to be completed in parallel. Steps should be completed in the order shown and according to the protocol written.

   
   

   1. Maintain T and B cells in T75 flasks with 12–15 mL of RPMI (w/ FBS) media (see Recipes) at the appropriate optimal growing concentrations.

      1. ~2 × 10⁵ to 2 × 10⁶ cells/mL are optimal growing concentrations for Jurkat.

      2. ~3 × 10⁵ to 2 × 10⁶ cells/mL are optimal growing concentrations for Raji.

   2. Collect around 5 mL from flasks and ensure that any clumps are broken up with pipetting.

   3. Count viable T cells manually or using an automated cell counter with Trypan Blue.

      1. Take 10 µL of cells and 10 µL of Trypan Blue.

      2. Mix with a pipette.

      3. If counting using an automated cell counter:

         i. Add 10 µL of the mixture to a cell counting chamber slide.

         ii. Insert the slide into an automated cell counter, with the side containing the cell/Trypan mixture inserted first.

         iii. Focus the machine, press scan, and record cell viability as reported.

      4. If counting manually:

         i. Take 10 µL of the mixture and add it to a single chamber of the KOVA^TM Glasstic^TM slide.

         ii. Place the slide under a microscope and count the number of viable cells per grid using an appropriate magnification to visualize cells in each box.

         1) Viable cells will appear shiny or bright, whereas nonviable or dead cells will be permeable to the Trypan Blue dye and appear darker.

         iii. Calculate the number of viable cells by converting 10 viable cells/box = 2 million viable cells/mL.

         1) Count multiple boxes, convert, and then average the resulting viable cell numbers together to get the most accurate estimate of total cell viability.

   4. Calculate the appropriate volume needed for 10⁶ viable cells (or more, if desired) and centrifuge the cells at 500× g for 5 min in a microcentrifuge tube.

      If needed, centrifuge in a 15 mL or 50 mL tube at 500× g, and then resuspend and transfer to a microcentrifuge tube.

   5. Remove the supernatant and leave around 50 µL of media.

   6. Add fresh RPMI (w/ FBS) media to a final concentration of 10⁶ cells per milliliter (950 µL of media if 10⁶ cells were used in step A5).

   7. Move isolated 10⁶ cells (total volume 1 mL) to one well of a 24-well cell culture plate for overnight culture.

   8. Add 12.5 µL of CD3/CD28/CD2 ImmunoCult T-cell activator for T cells per milliliter and incubate overnight (not necessary for PD-1-expressing Jurkat cells) at 37 °C in a tissue culture incubator.

   
   

   Day 2

   Note: Both day 1 and day 2 steps can be done on the same day if overnight T-cell activation is not necessary, e.g., when using PD-1-expressing Jurkat cells. See Figure 1.

   1. Repeat steps 2–6 from Day 1 for B cells or perform in parallel for B and T cells if overnight T-cell activation was not performed.

      1. Collect around 5 mL from flasks and ensure that any clumps are broken up with pipetting.

      2. Count viable cells manually or using an automated cell counter with Trypan Blue, as described in the steps for day 1.

      3. Calculate the appropriate volume needed for 10⁶ viable cells (or more, if desired) and centrifuge them in a microcentrifuge tube at 500× g for 5 min.

         If needed, centrifuge in a 15 mL or 50 mL tube at 500× g, and then resuspend and transfer to a microcentrifuge tube.

      4. Remove the supernatant and leave around 50 µL of media.

      5. Add Opti-MEM media to a final concentration of 10⁶ cells per milliliter (950 µL of media if 10⁶ cells were used in step 1d).

   2. If T-cell activation is required, change the media with the T-cell activator solution to fresh media.

      1. Collect cells from the well into a microcentrifuge tube.

      2. Spin cells down at 500× g for 5 min.

         If desired, this spin can be done at the same time as the spin for step 1.

      3. Remove supernatant, leaving up to 50 µL above the cells, and resuspend in 1 mL Opti-MEM media.

   3. Add 5 µL of Staphylococcus enterotoxin E (SEE) working stock (see Recipes) for each 1 mL of B cells and incubate at 37 °C in a tissue culture incubator for 30–60 min in the microcentrifuge tube.

   4. Add the desired amount of anti-PD-1/PD-L1 antibodies to the appropriate tube and incubate at 37 °C in a tissue culture incubator for 30–60 min in the microcentrifuge tubes:

      1. If an anti-PD-1 antibody is to be tested, add the antibody to the T-cell tube.

         i. Add the anti-PD-1 antibody in parallel with the Staphylococcus enterotoxin E (SEE) so that both T and B cells will incubate for 30–60 min.

      2. If an anti-PD-L1 antibody is to be tested, add the antibody to the B-cell tube.

         i. Add the anti-PD-L1 antibody in parallel with the Staphylococcus enterotoxin E (SEE) so that both will incubate with the B cells for 30–60 min.

         ii. Keep the T-cell tube incubating at 37 °C during this process.

   5. During T-cell and B-cell incubation, coat MatTek plates with 200 µL of 1× Poly-L-Lysine (PLL) (see Recipes) solution for 30–45 min at room temperature or 37 °C in a tissue culture incubator.

   6. Remove PLL from the plate via vacuum before plating cells.

      Note: No wash is needed after removing the PLL.

   7. Add 100 µL (10⁵ cells) of treated B cells to a MatTek dish and let them settle for 5 min at room temperature.

      Note: This step does not have to be done in a sterile hood.

   8. Add 100 µL (10⁵ cells) of T cells on top of the previously plated B cells.

      Note: Ensure that T cells are added dropwise from the air, gently and evenly over the plate, to help maximize cell dispersion and minimize air bubble formation.

   9. Let the combined solution settle for at least 10 min at room temperature before imaging.

   10. Store microcentrifuge tubes with remaining treated B and T cells in a mini block heater at 37 °C, covered with tin foil to prevent photobleaching.




2. Imaging of T and B cells synapse formation

   Note: Before proceeding with this protocol portion, ensure that the microscope is turned on and calibrated and that Zen Blue software is functional.

   1. Load MatTek plate with cell solution (200 µL total) onto the microscope stage and center above 10× objective for initial observations of cell settling in the plate.

      Note: Be gentle with the transfer and ensure that the cell solution is not disturbed from the center of the plate.

   2. Locate the area of interest on the MatTek plate using the 10× objective and eyepiece to ensure that cells have fully settled in.

   3. Remove the MatTek plate from the microscope stage and change to 63× objective with a small amount of oil on the lens.

      Note: To prevent damage to the lens, ensure it is at the lowest Z-level before reloading the MatTek plate.

   4. Adjust the objective height to ensure cells are in focus.

      Note: The oil must touch the plate and the objective lens for clear images (Figure 2).

      
      

      
      Figure 2. Oil is applied to the 63× objective lens, and contact is formed with the MatTek plate. Areas of interest are highlighted using white circles. A) The microscope objective is at the lowest Z-position, with nothing placed on the lens. B) The objective is at the lowest Z-position with a small amount of Immersol autofluorescence immersion oil applied to the lens. C) The MatTek plate is centered above the lens without contact between the plate, oil, and lens. D) MatTek plate centered above the lens with contact between the plate, oil, and lens formed by adjusting using the focus knobs.

   
   

   5. Select wavelengths in the fluorescence setting to be imaged.

   6. Adjust gain, intensity, and wavelength absorbance until the background is sufficiently low, but PD-1 and PD-L1 fluorescent signals can be imaged.

   7. Press Capture to take an image for further analysis when settings are acceptable.

   8. The colocalization of both fluorescent signals will evidence synapse formation in a “straight line” between B and T cells (Figure 3).

      1. The number of synapses formed for each antibody tested can be used to visually analyze synapse abundance and quality.

      2. Count the number of synapses formed per X of pairs of T and B cells in contact with one another and compare the number of synapses counted between antibody treatment groups and the control group.

         Note: Synapses should be counted only when GFP-PD-1 is enriched in the contact area.

      
      

      
      Figure 3. Conjugates formation between Jurkat T cells and Raji B cells. Jurkat T cells expressing GFP-PD-1 were cocultured with Raji B cells expressing mCherry-PD-L1 and pulsed with SEE. The cells on the left panel (A) were imaged after 20 min of coculturing, while the cells on the right (B) were imaged after treatment with anti-PD-1 antibodies. Without anti-PD-1 antibodies, GFP-PD-1 is enriched in the synapse between the Raji and the Jurkat cells (yellow arrows). Scale bar in 20 μm.




3. Quantification of synapse intensity using microscopic colocalization analysis

   Note: A proper version of Zen Blue is recommended for this step. Other software, such as AIM or Image J, can also be utilized, but these steps are outlined explicitly for Zen Blue. For best results, use a .czi file. See Figure 4.

   
   

   
   Figure 4. Colocalization analysis using ZEN Blue software. Areas of note are highlighted in white (colocalization tab, threshold adjustment bars, quadrant numbers, region of interest indicator, colocalization coefficients, region of interest tool, Costes auto threshold button, table option). For the example image, the colocalization coefficient to be recorded is 0.725, and the weighted colocalization coefficient to be recorded is 0.767, indicating that 72.5% of GFP-PD-1 signal colocalizes with mCherry-PD-L2 without taking into account pixel intensity, and 76% of GFP-PD-1 signal colocalizes with mCherry-PD-L2 when taking into account pixel intensity. A) Click on the colocalization option on the sidebar. B) Select the region of interest (ROI) using the shape tools. C) Appropriately position the ROI over the synapse and ensure it remains selected. D1) Either manually adjust the bars on the cytofluorogram, or D2) Click the Costes button to auto-set the threshold bars with the selected region of interest using the mouse. E) Click the table option to view the values for the localization coefficients. F) Unweighted and weighted colocalization coefficient values will be listed from left to right (Colocalization Coefficient 1, Colocalization Coefficient 2, W Colocalization Coefficient 1, W Colocalization Coefficient 2). G) Colocalization coefficient values for the region of interest (synapse) will be displayed in the table.

   
   

   1. After capturing the images, restart Zen Blue, select image processing, and open the images to be quantified in the image processing suite.

   2. Select colocalization on the sidebar.

   3. Markdown channels associated with PD-1 and PD-L1 fluorescence in the colocalization tab (e.g., GFP and mCherry).

      Note: Channels will usually be automatically labeled according to how they were captured during the image acquisition step.

   4. Utilize the region of interest (ROI) tool to mark areas where contact between T and B cells is observed.

      Notes:

      1. ROIs should not be limited to GFP-PD-1-enriched contact areas, especially in antibody treatment groups.

      2. This step can also be excluded if a whole image analysis is desired, in which case the rest of the protocol can be conducted similarly without an ROI.

   5. To ensure reproducibility, utilize the Costes auto threshold option by clicking the Costes button. Alternatively, if desired, select the crosshair option to set proper quadrants for use in colocalization analysis. Quadrants are labeled on the cytofluorogram.

      1. Quadrant 4 will contain pixels with low-intensity levels for both channels.

      2. Quadrants 1 and 2 will have high-intensity pixels in one of the channels.

      3. Quadrant 3 will contain pixels with high intensity in both channels.

   6. Cytofluorogram threshold bar placements can be determined using single fluorescence control samples to determine the optimal boundaries to eliminate background signals.

   7. Check the table option to view values for colocalization coefficients and compare between groups with higher colocalization coefficients indicative of more pixels with signals from both channels.

      1. The unweighted colocalization coefficient values (labeled Colocalization Coefficient 1 and Colocalization Coefficient 2) for the region of interest can be utilized to analyze colocalization. Values for weighted colocalization coefficients will range from 0 to 1, with 0 indicating no colocalization and 1 indicating complete colocalization.

         i. Unweighted colocalization coefficients are derived from Mander’s coefficients as described in Manders et al. [22].

         ii. Weighted colocalization coefficient values (labeled as W Colocalization Coefficient 1 and W Colocalization Coefficient 2 on Zen Blue) consider pixel intensity in their calculation but otherwise use the same methodology as unweighted colocalization coefficient values.

         iii. Either coefficient can be utilized at one’s discretion.

         1) Colocalization Coefficient 1 (or W Colocalization Coefficient 1) displays the percentage of the cytofluorogram horizontal axis channel that overlaps with the vertical axis channel.

         2) Colocalization Coefficient 2 (or W Colocalization Coefficient 2) displays the percentage of the cytofluorogram vertical axis channel that overlaps with the horizontal axis channel.

         3) Record the colocalization coefficient (or W Colocalization Coefficient) that correlates with the channel for PD-1.

         a) If PD-1 is the horizontal axis channel, use Colocalization Coefficient 1 (or W Colocalization Coefficient 1).

         b) If PD-1 is the vertical axis channel, use Colocalization Coefficient 2 (or W Colocalization Coefficient 2)

      2. Higher colocalization coefficient values mean increased colocalization of PD-1 and PD-L1 in the synapse, which indicates poorer antibody efficiency due to the lessened exclusion of PD-1 from the synapse.

         Note: Colocalization coefficients can be plotted in GraphPad Prism to analyze quantified synapse intensity values between antibody treatment groups.

      3. Multiple images for each antibody are used to ensure the colocalization coefficient derived is accurate.




4. Alternative ImageJ quantification of synapse intensity using microscopic colocalization analysis

   Note: The JACoP plugin can be utilized as an alternative method for quantifying colocalization. Image processing can be optimally done using a Czi or TIFF file. If not already integrated, the JACoP plugin must be downloaded to the plugin folder in ImageJ. A region of interest cannot easily be specified using the JACoP plugin, so specific synapses must be preemptively isolated for analysis before using ImageJ. Please see the ImageJ website for more details regarding the usage of JACoP.

   1. Following capturing images, open the image to be quantified using ImageJ.

   2. Select Hyperstack for Stack viewing.

   3. Select Default for Color options and check the Autoscale box.

   4. Select Split channels to split into separate windows.

      Note: This will separate the PD-L1 and PD-1 fluorescent channels, allowing the JACoP plugin to work correctly.

   5. Select the Plugins tab and click JACoP.

   6. On the JACoP menu, select M1 and M2 coefficients, which will give Mander’s coefficients as calculated in Manders et al. [22].

      1. M1 will give the percentage of channel 1 that overlaps with channel 2.

      2. M2 will give the percentage of channel 2 that overlaps with channel 1.

      3. Record the Mander’s coefficient that correlates with the channel for PD-1.

         For example, if PD-1 is labeled as channel 1, look at M1.

   7. Click the threshold tab and adjust thresholds for each channel.

      The area utilized for the colocalization analysis will appear in red when the threshold is adjusted.

   8. Once thresholding is satisfactory, click analyze.

      1. Results for M1 and M2 will appear in a separate window.

      2. M1 and M2 can be interpreted in the same manner as described for Zen colocalization analysis.

Validation of protocol

This protocol or parts of it has been used and validated in the following research article(s):

1. Hao et al. [1]. Exclusion of PD-1 from the immune synapse: A novel strategy to modulate T cell function. Molecular Therapy: Oncology (Figures 1–3, S1).

General notes and troubleshooting

1. This protocol outlines the process for Zen Blue software analysis of confocal microscopy images, which is commonly utilized in conjunction with the Zeiss confocal microscope. Image analysis can also be conducted in other programs, such as ImageJ (the most recent version is preferred), but the steps taken to quantify colocalization will differ. The rest of the steps involving plating and imaging remain the same. Confocal microscopes, such as the Leica SP8, can also be utilized. In this case, the analysis would need to be done using the JACoP plugin for ImageJ.

2. The amount of SEE added to B cells for incubation can be increased as needed to help form immune synapses. SEE concentration can vary, with the minimum recommended volume added being 0.5 µL. It was found that 5 µL of SEE was necessary to optimize this protocol, but the amount can be altered as needed.

3. Utilizing the CD3/CD28/CD2 ImmunoCult T-cell activator does not harm imaging quality when using a cell line such as PD-1-expressing Jurkat T cells to enhance results further. Still, it is unnecessary, as adding the activator only sometimes leads to significant improvements in synapse formation and image.

4. Optimal synapse formation begins after cells settle for at least 10 min. Synapses are mostly readily noted by a bright line of PD-1 GFP signal in the contact area between Raji and Jurkat cells.

5. Following the 10-min settling time, there is an approximately 40–50 min window for capturing images. Synapses will begin to form after this settling time, but the optimal imaging window for synapses will be 20–60 min after coculture. Cells have been observed to dissociate 1 h after being cocultured together (Figure 5).

   
   

   
   Figure 5. Image capture timeline and synapse formation. Synapses can be observed to begin forming at 10 min after coculture. The optimal image window for more developed synapses is 20–60 min after coculture. Cells are observed to dissociate at times greater than 60 min after coculture or greater than 50 min after initial synapse formation.

Acknowledgments

Grants from the NIH AI125640, AI150597, and AI175498 supported this work.

Competing interests

The authors report no competing interests.

Ethical considerations

No animal or human subjects were used in this protocol.

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