Identification and Functional Analysis of Human CD56neg NK Cells by Flow Cytometry

Summary Although scarce in the peripheral blood of healthy people, CD56neg NK cells are known to be expanded in some pathological conditions. However, studies on CD56neg NK cells had revealed contradictions, probably due to the lack of a specific NK cell surface marker that helps to identify this subset. This protocol details the step-by-step procedure for the identification and functional analysis of CD56neg NK cells, providing an improved gating strategy for the selection of this intriguing population. For complete details on the use and execution of this protocol, please refer to Orrantia et al. (2020).


MATERIALS AND EQUIPMENT
Alternatives: This protocol uses a MACSQuant Analyzer 10 flow cytometer for sample acquisition. Any other flow cytometer can be used as well. However, we recommend performing antibody titration to determine the optimal concentration of each antibody before starting the experiment. Total n/a 500 mL Add 400 mL of PBS to a beaker and heat it until 55 C-60 C. Add 40 g of PFA and let it dissolve (it will take time). Allow the solution to cool down. Measure and adjust pH to 7 (add 1 M NaOH or HCl as needed). Filter the solution if needed. Add PBS to adjust the final volume to 500 mL. Measure and adjust pH to 7 again (add 1 M NaOH or HCl as needed). Store at 4 C.

Reagent
Final Concentration Amount RPMI 1640 medium containing GlutaMAX n/a 89 mL Total n/a 100 mL We filter sterilize (22 mm filter) the R10 medium after combining all the reagents. Store at 4 C. Do not store more than a month. Note: Before use, we aliquot the plasmocin treatment in quantities of 50 mL. Store at À20 C.

Reagent
Note: The plasmocin treatment is added for preventing and eliminating mycoplasma contamination in cell cultures.
Note: When adding the DMSO to the FBS, an exothermic reaction occurs. We recommend preparing the medium and wait some minutes (5-10 min) before add it to the cells. This medium contains 20% of DMSO, but it will be diluted with FBS to a final concentration of 10% DMSO when freezing the cells.

STEP-BY-STEP METHOD DETAILS Peripheral Blood Mononuclear Cells (PBMCs) Isolation
Timing: 1.5-2 h This step details how to isolate PBMCs from buffy coats ( Figure 1A).
1. Add 10 mL of Ficoll Paque Plus to two conical 50 mL tubes ( Figure 1B). 2. Dilute the buffy coat 1:4 by adding sterile PBS and mix gently. In our protocol, we add 30 mL of sterile PBS to 10 mL buffy coat ( Figures 1C and 1D).
Note: If starting from whole blood, prepare a 1:2 dilution in sterile PBS.
CRITICAL: During all the steps of PBMC isolation, use PBS without calcium and magnesium to avoid cell aggregation.
3. Carefully add 20 mL of the diluted buffy coat on top of the Ficoll in each conical tube ( Figures 1E-1G).
CRITICAL: Avoid mixing the Ficoll and the diluted buffy coat. For that, tilt the conical tube with Ficoll and slowly add the diluted buffy coat or whole blood in the tube by sliding it through the tube wall.
CRITICAL: Use low brake and acceleration during the Ficoll density gradient centrifugation to avoid mixing of the layers.
5. Collect PBMC layer with a Pasteur pipette in a 50 mL conical tube ( Figures 1H and 1I). 6. Add 30 mL of sterile PBS and centrifuge cells at 200 3 g for 10 min at 20 C-22 C (RT).
Note: This washing step is done at lower speed to remove platelets that could remain after collecting the PBMC layer.

Reagent Final Concentration Amount
Total n/a 5 mL We recommend preparing this medium the day that it will be used and not to store it. ll OPEN ACCESS 7. After washing, discard the supernatant and resuspend the pellet in 30 mL of sterile PBS. Centrifuge samples at 300 3 g for 10 min at 20 C-22 C (RT). 8. Repeat step 7. 9. Resuspend pellet in 20 mL sterile PBS and filter cells with 70 mm cell strainers.

Note:
The volume in which you should resuspend the pellet may vary depending on the concentration of your starting sample.
10. Count viable cells by Trypan Blue exclusion. 11. PBMCs cryopreservation: a. Prepare cell cryopreservation medium. b. We usually freeze 10 7 cells per cryovial. Add the number of cells of interest in a conical 15 mL tube and centrifuge at 300 3 g for 10 min at 20 C-22 C (RT). c. Discard the supernatant and resuspend the pellet in sterile FBS at a concentration of 500 mL/ 10 7 cells. d. Place 500 mL of cells in each cryovial and add 500 mL of cell cryopreservation medium to obtain a final volume of 1 mL.

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e. Place all the vials in a freezing container and store it at À80 C for 48 h. Then, transfer the vials to an appropriate container and store them in liquid nitrogen.
Note: The cryopreservation step must be done as quickly as possible. Try to reduce the time that cells are exposed to DMSO before they are transferred to the freezer as this influences cell viability.
Note: The cryopreservation step can be omitted and fresh PBMCs can be used for phenotypic and functional assays. However, this protocol was optimized using cryopreserved samples. The cryopreservation of biological samples has some advantages such as, easier collection of the samples in locations far from where the study is performed, and possibility of analyzing samples in large batches, minimizing overall analytical variability.

Thawing Cryopreserved PBMCs
Timing: 2-3 h This step details how to thaw cryopreserved PBMCs before starting the phenotypical characterization and the functional assay.
12. Thaw the cryovial in a water bath at 37 C and place the cells into a conical 15 mL tube. 13. Add 3 mL of RPMI medium (previously warmed at 37 C) and centrifuge at 300-400 3 g for 5 min at 20 C-22 C (RT). 14. Discard the supernatant and resuspended the pellet in 3 mL of R10 medium. 15. Add DNase I from the stock solution to obtain a working concentration of 10U DNase I for up to 5 mL cell suspension. 16. Place the cells in the incubator at 37 C and 5% CO 2 for 1 h. 17. After the incubation, centrifuge the tubes at 300-400 3 g for 5 min at 20 C-22 C (RT). 18. Discard the supernatant and resuspended the pellet in 2 mL of NK cell medium. 19. Filter cell suspension with a 70 mm cell strainer and place it into a new 15 mL tube. 20. Count viable cells by Trypan Blue exclusion. 21. If needed, add the appropriate volume of NK cell medium to obtain the desired concentration (e.g., 10 6 cells/mL).
Note: If phenotypic analysis are performed with the same sample, at this point divide the cells for the phenotypic and the functional analysis before continuing with the protocol.

Functional Assay -Day 1
Timing: 1-2 h This step details how to prepare the cells for the functional assay after the thawing process.
Note: The functional assay consists of two different stimulation conditions: K562 cell line and IL-12+IL-15+IL-18 stimulation. In addition, a non-stimulated condition is needed as a control for each sample. Moreover, an unstained condition is needed for the whole experiment. The unstained condition serves as a control condition to check for cellular autofluorescence. Thus, this condition will be processed the same way as all the other conditions, except for the staining with the viability reactive dye and fluorochrome conjugated antibodies. (See Figure 2 for a plate set up example).
22. For each condition, plate 0.5 3 10 6 of the thawed PBMCs in NK cell medium in a 48 wells plate. If needed, add the appropriate volume of NK cell medium to obtain a final volume of 1 mL/well.
Note: We individually add each cytokine. However, preparing a master mix would also be fine.
25. Shake the plate very carefully by hand in a cross motion for 3 s to mix the wells. Then, incubate the cells in the incubator at 37 C and 5% CO 2 until the next day.
Note: In our protocol this incubation lasts for 20-21 h. However, shorter incubation time (17-18 h) may also work.  Note: We prepare a final volume of 20 mL/well of anti-CD107a mAb by diluting it 1:10 with NK cell media. However, the manufacturer's recommended antibody dilution is 1:50 for up to 10 6 cells or 100 mL. Therefore, we recommend doing a titration to determine the optimal antibody concentration.
31. Shake the plate very carefully by hand in a cross motion for 3 s to mix the wells. Then, incubate the plate in the incubator at 37 C and 5% CO 2 for 1 h. 32. Individually add 0.66 mL/mL of monensin (GolgiStop) and 1 mL/mL of brefeldin A (GolgiPlug) to all the conditions. On the other hand, a master mix could be prepared before adding the two compounds to the wells. 33. Shake the plate very carefully by hand, in a cross motion, for 3 s to mix the wells. Then, incubate the plate in the incubator at 37 C and 5% CO 2 for 5 more hours.
Note: This protocol was optimized to continue with the staining steps (see Staining Day-1 section). However, it is also possible to stop the experiment at this point and continue with the staining the next day. For that, after the incubation period is over, take out the plate from the incubator and store it in the fridge (4 C), covered to protect from light, until the next day. Then, continue with step 34 of the protocol.

Staining -Day 1
Timing: 3-4 h This step details how to perform the extracellular staining and the fixation.
34. After the incubation, label cytometry tubes for each condition. Pipette up and down the cell suspension and transfer it from each well into tubes.
35. Viability staining: a. Add 2 mL of PBS to each tube and centrifuge at 300-400 3 g for 5 min at 4 C. b. Discard the supernatant and resuspended pellet in 1 mL of PBS. c. If needed, reconstitute the LIVE/DEADä Fixable Aqua Dead Cell Stain Kit reactivity dye by adding 50 mL of DMSO to the vial. Mix well and visually confirm that all of the dye has dissolved.
Note: We highly recommend performing live/dead staining when working with cryopreserved samples. d. Add 1 mL of the reactivity dye to all the tubes, except to the unstained condition, and vortex them. Leftover reconstituted viability dye can be frozen and used in future experiments following manufacturer's recommendations. e. Incubate for 30 min on ice. Protect from light. f. Add 2 mL of cold PBS + BSA (2.5%) to all the tubes and centrifuge at 300-400 3 g for 5 min at 4 C. Discard the supernatant and resuspend the pellet in residual volume.
36. Prepare the antibody mix for the extracellular staining as follow (Table 1): Note: The volume of each antibody has been optimized to acquire cells in the MACSQuant Analyzer 10 flow cytometer. We recommend performing antibody titration before starting the experiments.

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Note: We recommend preparing a bit more antibody mix volume than the indicated to ensure that the mix will be enough for all the tubes. For that, when doing the calculations, calculate for one additional tube or add a bit more of PBS + BSA (2.5%) before adding the mix to the tubes.
Note: We recommend preparing the mix during viability dye incubation (step 35e) to save time.

CRITICAL: The BD Brillant Stain
Buffer is a solution that is added when using two or more antibodies conjugated with BD Horizon Brillant fluorescence polymer dyes (e.g., BV421, BV510, etc.) to avoid fluorescent dye interaction, as these interactions may cause staining artifacts and affect data interpretation.
37. Extracellular staining: a. Add the corresponding volume of antibody mix (77 mL) to each tube, except to the unstained condition, and vortex them. b. Incubate for 30 min on ice. Protect from light. c. Add 2 mL of cold PBS + BSA (2.5%) to all the tubes and centrifuge at 300-400 3 g for 5 min at 4 C. Discard the supernatant and resuspend pellet in residual volume. 38. Prepare 150 mL/tube of 4% PFA from the 8% PFA stock buffer by diluting it 1:2 with PBS.
Note: We recommend preparing a bit more volume of 4% PFA buffer than what is needed.

Cell fixation:
a. Add 140 mL/tube of 4% PFA and vortex. b. Incubate for 15 min on ice. Protect from light. c. Add 2 mL of cold PBS + BSA (2.5%) to all the tubes and centrifuge at 300-400 3 g for 5 min at 4 C. Discard the supernatant and resuspend pellet in residual volume. d. Repeat step 39c. 40. Resuspend pellet in 1 mL of cold PBS + BSA (2.5%) and vortex. 41. Place the tubes in the fridge (4 C) protected from light until next day.
Note: This protocol was optimized to stop the experiment at this point and continue the next day. However, it is also possible to continue performing the permeabilization and the intracellular staining steps. For that, omit steps 40 and 41 and continue as explained in step 44.

Staining -Day 2
Timing: 1.5-2.5 h a. Add 1 mL of 13 BD Perm/Washä Buffer to all the tubes and vortex. b. Incubate for 15 min at 20 C-22 C (RT). Protect from light. c. Centrifuge the tubes at 300-400 3 g for 5 min at 4 C. Discard the supernatant and resuspend pellet in residual volume. 46. Prepare the antibody mix for the intracellular staining as follow (Table 2): Note: The volume of each antibody has been optimized to acquire cells in the MACSQuant Analyzer 10 flow cytometer. We recommend performing antibody titration before starting the experiments.
Note: We recommend preparing a bit more antibody mix volume than the indicated to ensure that the mix will be enough for all the tubes. For that, when doing the calculations, calculate for one additional tube or add a bit more of 13 BD Perm/Washä Buffer before adding the mix to the tubes.
Note: We recommend preparing the mix during cell permeabilization (step 45b) to save time.

Intracellular staining:
a. Add the corresponding volume (58 mL) of antibody mix to each tube, except to the unstained condition, and vortex them. b. Incubate for 30 min on ice. Protect from light. c. Add 2 mL of 13 BD Perm/Washä Buffer to all the tubes and centrifuge at 300-400 3 g for 5 min at 4 C. Discard the supernatant. 48. Resuspend the pellet with appropriate volume of PBS (200-400 mL) before acquiring the samples in the flow cytometer.
Note: Before acquiring samples in the flow cytometer, we recommend performing fluorescence compensation to correct the emission spectra overlap of different fluorochromes following flow cytometer manufacturer's recommendations. However, compensation can be also done after sample acquisition with different flow cytometry data analysis software (e.g., FlowLogic, FlowJo, etc.).

EXPECTED OUTCOMES
In this protocol, we have developed a gating strategy for the correct identification of human CD56 neg NK cells in peripheral blood and the subsequent functional analysis of this intriguing subpopulation. First, we electronically gated lymphocytes based on their forward (FSC) and side (SSC) scatter parameters. Then single cells are selected based on FSC-area and FSC-height. An exclusion channel (viability dye and anti-CD3, anti-CD19 and anti-CD14 mAbs) was included in our gating strategy to specifically study non-T, non-B, non-monocytes viable cells. Thus, for NK cell identification, the negative population for the exclusion channel was selected. Lastly, CD56 neg NK cells were identified based on the expression of NKp80 and CD56 surface markers, as CD56 neg NKp80+ cells (Figure 3). This population is known to be expanded in some pathological conditions: during human immunodeficiency virus (HIV)-1 infection in both untreated ( Figure 3A) and in patients under combined antiretroviral therapy (cART) ( Figure 3B) (Alter et al., 2005;Hu et al., 1995;Vitallé et al., 2019), and in multiple myeloma patients (Orrantia et al., 2020) ( Figure 3C). CD56 neg NK cells can also be identified, although in lower frequencies, in healthy people ( Figure 3D) (Campos et al., 2014;Mü ller-Durovic et al., 2019).
Once CD56 neg NK cells are correctly identified, the effector functions of this subset can be assessed. For that, we include anti-IFNg and anti-TNF mAbs to determine cytokine production, and anti-CD107a mAb to measure degranulation capability of CD56 neg NK cells (Figure 4). This approach reveals that the effector functions of CD56 neg NK cells are not as diminished as previously described (Orrantia et al., 2020).

QUANTIFICATION AND STATISTICAL ANALYSIS
To assess the effector functions of human CD56 neg NK cells we measure degranulation and production of TNF and IFNg. We use FlowJo for the analysis of flow cytometry data.
1. Open the non-stimulated condition file and select CD56 neg NK cells as explained above (Figure 3). 2. Select the marker of interest (IFNg, TNF, CD107a) and the FSC-A parameter and make a gate selecting the region where the population ends (Figure 4, left column). This will be the positive population for the marker of interest.
Note: Alternatively, to determine the gate for the positive population, the fluorescence minus one (FMO) control can also be used. These controls contain all the fluorochrome conjugated mAbs, except for the one that is being measured in each case (IFNg, TNF, or CD107a mAbs). FMO control is used to identify and gate cells in the context of data spread due to the multiple fluorochromes in a given panel. If you decide to use FMO controls you will need to prepare one control per marker that you want to determine and per condition (non-stimulated, K562 cell line and IL-12+IL-15+IL-18 conditions). Once you have the FMO controls, select CD56 neg NK cells as explained above ( Figure 3) and determine the positive population for each marker in the corresponding FMO control.
3. Paste the positive gates for each marker in the K562 cell line and IL-12+IL-15+IL-18 conditions. 4. Calculate the percentage of positive cells for CD107a, IFNg and TNF on each stimulation condition by subtracting the non-stimulated condition. We usually use Excel for this step.

LIMITATIONS
This protocol uses NKp80 for the identification of CD56 neg NK cells. However, NKp80 is not a specific marker of NK cells because some CD8+ T cells also express it (Kuttruff et al., 2009), although these cells are gated out by using the exclusion channel. Within CD56 neg NKp80+ subset, a small percentage of cells (around 20%) do not express Eomesodermin (Orrantia et al., 2020), a transcription factor needed for the development and function of NK cells (Vivier et al., 2018). On the other hand, although NKp80 is not downregulated on our stimulation protocol (Orrantia et al., 2020), Klimosch et al. have described that NK cells stimulated with PMA or IL-2+IL-12+IL-18 downregulated the expression of this receptor after 24 h stimulation (Klimosch et al., 2013). This suggests that NKp80 is not the perfect surface marker to identify the intriguing CD56 neg NK cell subset and further studies are needed to identify a specific NK cell surface marker. Nevertheless, the gating strategy we proposed in this protocol is the best currently available for the identification and subsequent analysis of the effector functions of CD56 neg NK cells, especially when it concern to frozen samples.

Problem 1
Flow cytometer laser and filters configuration do not allow for the detection of the proposed fluorochromes (steps 36 and 46).

Potential Solution
To overcome this problem, try to find the same antibody clones conjugated with fluorochromes that your flow cytometer can detect. Moreover, we recommend choosing a fluorochrome with a high stain index for CD56 marker since it is important to be able to differentiate between CD56 dim and CD56 neg NK cells.

Problem 2
Inadequate cell number for functional assay (step 22).

Potential Solution
This protocol was optimized for using 1.5 3 10 6 PBMCs per sample for functional assay (0.5 3 10 6 PBMCs per condition). However, if you have less cells, we propose three different potential solutions: Using 96 U-bottom wells plates instead of 48 wells plates is also possible. In this event, 0.2 3 10 6 PBMCs are plated per condition in a final volume of 200 mL. Be aware that you will need to recalculate the volume of the three ILs and GolgiStop and GolgiPlug that you must add to each well. In addition, we recommend doing the titration of the anti-CD107a mAb, although adding 2 mL/well may work. Maintain the E:T ratio of the K562 cell line stimulation condition in 1:1. You can also decide to not include one of the stimulation conditions, as for example the IL-12+IL-15+IL-18 condition. This condition is principally used for the measurement of IFNg production but ll OPEN ACCESS STAR Protocols 1, 100149, December 18, 2020 you should also be able to measure production of this cytokine (although at lower levels) with the K562 cell line condition. Considering that the unstained condition is only used as a control, you can always plate fewer cells. Using 0.25-0.3 3 10 6 PBMCs for the unstained condition may also work.

RESOURCE AVAILABILITY Lead Contact
Further information and request for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Francisco Borrego (francisco.borregorabasco@osakidetza.eus).

Materials Availability
This study did not generate new unique reagents.

Data and Code Availability
This study did not generate datasets and codes.