2.1 The Anti-Human P2X7 mAb Inhibits Ca²⁺ Responses in HEK-P2X7 Cells

To confirm the functionality of the anti-P2X7 mAb, the ability of this mAb to block ATP-induced Ca²⁺ responses in HEK-P2X7 cells was examined. First, to confirm the presence of human P2X7 on HEK-P2X7 cells, cells were incubated with fluorochrome-conjugated anti-P2X7 (clone L4) and isotype control (MPC-11) mAbs and analysed by flow cytometry with a consistent gating strategy as demonstrated in Figure S1. Non-transfected HEK-293 cells, which do not express P2X7 [18], were also examined for comparison. Compared with the isotype control mAb, the anti-P2X7 mAb showed a right shift in fluorescence for HEK-P2X7 but not HEK-293 cells (Figure 1A). The mean fluorescence intensity (MFI) for the anti-P2X7 mAb on HEK-P2X7 cells was 98,291. Conversely, the MFI for anti-P2X7 mAb on HEK-293 cells was like that of the isotype control mAb values, 3,063 and 1,625, respectively.

Prior to mAb blockade studies, the half maximal effective concentration (EC₅₀) for the P2X7-mediated Ca²⁺ response in HEK-P2X7 cells was determined. Cells were incubated with increasing concentrations of ATP and Ca²⁺ responses measured using a fluorescent plate reader (Figure 1B,C). ATP induced Ca²⁺ responses in Fura-2 AM loaded HEK-P2X7 cells in a concentration-dependent manner with a maximal response occurring at 2 to 5 mM and an EC₅₀ of 759 ± 15 µM (Figure 1C). Finally, to determine if the anti-P2X7 mAb could inhibit the P2X7-mediated Ca²⁺ response, Fura-2 AM loaded HEK-P2X7 cells were preincubated with increasing concentrations of the anti-P2X7 or isotype control mAb, and then incubated with 760 µM ATP, approximate to the EC₅₀ obtained above (Figure 1D,E). The anti-P2X7 mAb inhibited ATP-induced Ca²⁺ responses in HEK-P2X7 cells in a concentration-dependent manner with maximum blockade (∼85% inhibition) at 5 µg/mL and with a half maximum inhibitory concentration (IC₅₀) of 0.48 ± 0.24 µg/mL (Figure 1E). Conversely, the isotype control showed minimal to no blockade of ATP-induced Ca²⁺ responses in HEK-P2X7 cells (Figure 1E).

2.2 The Anti-Human P2X7 mAb Induces CDC in Human P2X7 Expressing Cell Lines

To determine if the anti-human P2X7 mAb (clone L4) could mediate CDC, HEK-P2X7 cells were incubated with either Dulbecco's phosphate-buffered saline (PBS) (mAb diluent), the anti-P2X7 or isotype control mAb in the presence of complement. Cell cytotoxicity was assessed using a spectrophotometric lactate dehydrogenase (LDH) release assay. Non-transfected HEK-293 cells were studied as a negative control. Percent cytotoxicity at 2 h was calculated as a percentage of maximum LDH release (obtained using the provided cell lysis solution).

HEK-P2X7 cells incubated in the presence of PBS or isotype control mAb showed similar mean cytotoxicities of 25.1 ± 2.7% and 18.8 ± 3.1%, respectively (Figure 2A). HEK-P2X7 cells incubated in the presence of the anti-P2X7 mAb revealed a mean cytotoxicity of 60.8 ± 4.4%, which was significantly greater than that of cells incubated in the presence of PBS (p < 0.001) and the isotype control mAb (p < 0.0001; Figure 2A). In contrast, HEK-293 cells incubated in the presence of PBS, isotype control or the anti-P2X7 mAb revealed similar cytotoxicities of 27.2 ± 3.8%, 21.0 ± 3.7%, and 19.8 ± 2.8%, respectively, with no significant differences observed between treatments (Figure 2B).

Next, the ability of the anti-P2X7 mAb to mediate CDC was assessed using a flow cytometric cell death assay with human RPMI 8226 cells, known to express endogenous cell surface P2X7 [19]. Mouse J774 cells, which also express cell surface P2X7 [20], were studied as a negative control. Cells were incubated as above, but with cell viability at 2 h assessed using a viability dye, 7-aminoactinomycin D (7AAD), and flow cytometry. The gating strategy used to identify live and dead cells is demonstrated in Figure S2. Human RPMI 8226 cells incubated in the presence of PBS or isotype control mAb showed mean cytotoxicities of 13.7 ± 1.7% and 8.1 ± 1.9%, respectively (Figure 2C). RPMI 8226 cells incubated in the presence of the anti-P2X7 mAb revealed a mean cytotoxicity of 39.4 ± 2.7%, which was significantly greater than that of cells incubated in the presence of PBS (p < 0.001) or the isotype control mAb (p < 0.001) (Figure 2C). In contrast, mouse J774 cells incubated in the presence of PBS, isotype control or the anti-P2X7 mAb revealed low amounts of cytotoxicity of 3.6 ± 3.1%, 16.7 ± 5.6%, and 11.8 ± 6.2%, respectively, with no significant differences observed between treatments (Figure 2D).

2.3 Cell Surface P2X7 is Present on Human Blood Mononuclear Leukocyte Subsets

Previous studies with the anti-human P2X7 mAb (clone L4) or the anti-human P2X7 nanobody (Dano1) have shown that P2X7 is present on various human mononuclear blood leukocyte subsets [6-10, 21]. Before determining whether the anti-P2X7 mAb can mediate CDC in these cells, the presence of cell surface P2X7 on blood mononuclear leukocyte subsets was first examined. Human PBMCs from four healthy donors were labelled with fluorochrome-conjugated mAbs (Table S1) and analysed by flow cytometry using consistent gating strategies as demonstrated in Figures S3–S6. Relative cell surface P2X7 expression was determined as the difference between the MFI of anti-P2X7 mAb and the MFI of the corresponding fluorescence minus one (FMO) control. P2X7 was present on all blood mononuclear leukocyte subsets analysed (Figure 3).

The relative cell surface P2X7 expression (reported as mean MFI ± SEM) was first compared on the main subtypes of PBMCs. This analysis revealed a rank order of expression (highest to lowest) of CD14⁺ monocytes (1,044.0 ± 141.3), myeloid DCs (mDCs) (736.3 ± 109.7), plasmacytoid DCs (pDCs) (606.0 ± 104.7), CD16⁺ NK cells (288.5 ± 25.6), CD3⁺ T cells (223.8 ± 21.3), and CD19⁺ B cells (154.3 ± 7.7; Figure 3A). CD14⁺ monocytes had significantly greater cell surface P2X7 expression compared with CD3⁺ T cells (p < 0.0001), CD19⁺ B cells (p < 0.0001), CD16⁺ NK cells (p < 0.0001), and pDCs (p = 0.02). mDCs had significantly greater cell surface P2X7 expression compared with CD3⁺ T cells (p = 0.006), CD19⁺ B cells (p = 0.002), and CD16⁺ NK cells (p = 0.02). Lastly, pDCs had significantly greater cell surface P2X7 expression compared with CD19⁺ B cells (p = 0.02).

Next, relative cell surface P2X7 expression was examined on subsets of lymphocytes and monocytes. Amongst CD3⁺ T cells, P2X7 expression was similar on CD4⁺ (149.3 ± 20.8), CD8⁺ (204.8 ± 33.4), and CD4⁻CD8⁻ cell subsets (342.0 ± 156.3), with greater expression on CD4⁺CD8⁺ cells (521.8 ± 130.3), but this did not reach statistical significance (Figure 3B). Analysis of total CD4⁺ T cell subsets revealed similar P2X7 expression between conventional T cells (Tcons) (152.0 ± 20.9), T regulatory cells (Tregs) (110.0 ± 18.2), and CD39⁺ Treg cells (204.5 ± 27.2), with slightly but not significantly greater P2X7 expression on Th17 cells (281.8 ± 61.3; Figure 3C). Analysis of CD8⁺ T cell subsets showed greater expression on T cytotoxic 17 (Tc17) cells (402.0 ± 73.9) compared with total CD8⁺ T cells (204.8 ± 33.4; p = 0.05; Figure 3D).

A comparison of naive and memory CD4⁺ and CD8⁺ T cells was also undertaken. CD4⁺ T cell memory subsets showed similar P2X7 expression between naive (202.8 ± 12.5), effector memory (264.0 ± 35.9), central memory (265.3 ± 34.8), and terminally differentiated effector memory (TEMRA) cells (170.8 ± 33.3; Figure 3E). Likewise, CD8⁺ T cell memory subsets showed similar P2X7 expression between naive (313.8 ± 64.2), effector memory (239.8 ± 33.9), central memory (311.3 ± 18.8), and TEMRA cells (254.0 ± 47.7; Figure 3F).

P2X7 expression on memory B cells (205.3 ± 6.0) was significantly greater compared with total B cells (154.3 ± 7.7) (p = 0.002; Figure 3G). P2X7 expression was significantly greater on CD56⁻CD16⁺ NK cells (462.3 ± 45.6) compared with both CD56⁺CD16⁺ (272.0 ± 26.2; p = 0.008) and CD56^hiCD16⁻ NK cells (273.8 ± 28.2; p = 0.009), which had similar expression (Figure 3H). Notably, P2X7 expression on invariant (i) NK T cells (687.5 ± 75.9), identified as Vα24-Jα18 T cell receptor⁺, was significantly greater than total NK T cells (331.5 ± 36.9) (p = 0.006; Figure 3I). Monocyte cell subsets revealed a rank order of P2X7 expression on intermediate (1275.0 ± 216.6; p = 0.003 to non-classical), classical (1040.0 ± 144.0; p = 0.02 to non-classical) and non-classical monocytes (284.0 ± 26.4; Figure 3J).

A limited number of studies indicate the presence of P2X7 on mouse MDSCs [22-24], but whether this receptor is present on human MDSCs has not been reported. Analysis of MDSC subsets revealed the presence of cell surface P2X7 on early (E-) (206.0 ± 36.1), granulocytic (G-) (265.0 ± 53.8), and monocytic (M-) MDSCs (163.0 ± 30.0) with similar expression between all three subsets (Figure 3K).

2.4 The Anti-Human P2X7 mAb Mediates CDC of Human Blood Leukocyte Subsets That Display Relatively High Cell Surface P2X7

To determine if the anti-P2X7 mAb can mediate CDC in leukocytes, human PBMCs were incubated with either the anti-P2X7 or isotype control mAb in the presence of complement for 2 h. Cell subsets were analysed by flow cytometry using the same gating strategies as above, with the number of cells quantified using count beads.

Analysis of T cell subsets showed that numbers of CD3⁺ (p = 0.94; Figure 4A), CD4⁺ (p = 0.81; Figure 4B), and CD8⁺ T cells (p = 0.99; Figure 4C), CD4⁺CD8⁺ (p = 0.78; Figure 4D), and CD4⁻CD8⁻ T cells (p = 0.85; Figure 4E)_, Tcons (p = 0.81) (Figure 4F), Tregs (p ≥ 0.99; Figure 4G) and CD39⁺ Tregs (p = 0.61; Figure 4H) were comparable between treatments with minimal percent change. In contrast, Th17 cell numbers were significantly decreased by 47.1 ± 11.2% (mean ± SEM) in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells (p = 0.02; Figure 4I). Tc17 cells were partially but not significantly decreased by 23.0 ± 31.7% in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells (p = 0.31; Figure 4J).

The numbers of B cells were similar between both treatment groups (p = 0.85; Figure 4K). CD56⁻CD16⁺ NK cell numbers were significantly decreased by 38.3 ± 11.8% in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells (p = 0.02; Figure 4M). Conversely, the numbers of both CD56⁺CD16⁺ NK cells (p = 0.10; Figure 4L) and CD56^hiCD16⁻ NK cells (p = 0.38; Figure 4N) did not differ between treatment groups. NK T cell numbers were decreased by 31.8 ± 7.4% in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells, a difference which approached statistical significance (p = 0.06); Figure 4O), while iNKT cells were significantly reduced by 57.4 ± 4.9% in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells (p = 0.01; Figure 4P).

Both DC subsets also showed a significant reduction in numbers in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells, with mDCs decreased by 78.8 ± 4.6% (p = 0.006; Figure 4Q) and pDCs decreased by 33.2 ± 7.8% (p = 0.02; Figure 4R). Numbers of classical monocytes were decreased by 66.9 ± 15.4% in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells, a difference which approached statistical significance (p = 0.06; Figure 4S). Intermediate monocyte cell numbers were significantly decreased by 69.4 ± 11.3% between treatment groups (p = 0.01; Figure 4T). Conversely, numbers of non-classical monocytes did not differ between both treatment groups (p = 0.14; Figure 4U).

Finally, numbers of all three MDSC subsets were significantly decreased in anti-P2X7 mAb-treated cells compared with isotype control mAb-treated cells, with E-MDSCs decreased by 33.7 ± 8.0% (p = 0.02; Figure 4V), G-MDSCs decreased by 37.9 ± 9.3% (p = 0.03; Figure 4W), and M-MDSCs decreased by 32.4 ± 6.5% (p = 0.04; Figure 4X).

3.1 Data Limitations and Perspectives

This study has several limitations. First, P2X7 mAb-mediated CDC in human leukocytes was assessed indirectly by measuring changes in the numbers of cell subsets, rather than by measuring cell death directly within these populations. Second, although the anti-P2X7 mAb was used in the CDC assays at a concentration (2 µg/mL) that induced maximal inhibition of ATP-induced Ca²⁺ responses in HEK-P2X7 cells it was not titrated for the CDC assays, which may have resulted in suboptimal mAb-mediated CDC of some cell types. Finally, studies of P2X7 expression and CDC were conducted using different cohorts of PBMC donors, preventing direct comparisons between these parameters.

This study provides the most comprehensive single analysis of P2X7 expression on human blood leukocyte subsets to date. Further, this study demonstrates for the first time that a biologic targeting P2X7 mediates CDC in cells expressing P2X7 from any species. Use of anti-P2X7 mAb-mediated CDC and subsequent depletion of P2X7⁺ cells may represent a new approach to preventing P2X7-mediated processes in experimental and clinical settings.

4.1 Cell Lines

All cell culture reagents were from Thermo Fisher Scientific (Waltham, USA) unless stated otherwise. HEK-293 cells were from the American Type Culture Collection (Manassas, USA) and were maintained in DMEM/F-12 medium containing 10% foetal calf serum (FCS), 2 mM GlutaMAX, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 95% air/5% CO₂. HEK-P2X7 cells were originally provided by Dr. Leanne Stokes (University of East Anglia, Norwich, UK) and were maintained in the same medium as HEK-293 cells but containing 400 µg/mL geneticin. Human multiple myeloma RPMI 8226 cells were from the European Collection of Cell Cultures (Wiltshire, UK). Mouse macrophage J774 cells were obtained from the American Type Culture Collection (Rockville, USA). Both cell lines were maintained in RPMI-1640 medium containing 10% FCS and 2 mM GlutaMAX. The anti-P2X7 mAb-producing mouse hybridoma cell line (clone L4) was originally obtained from the Glaxo Institute for Applied Pharmacology (Cambridge, UK) and maintained in IMDM (Sigma Aldrich, St Louis, MO, USA) containing 20% FCS and 2 mM GlutaMAX. A mouse multiple myeloma cell line producing an IgG2_b isotype control mAb (clone MPC-11) was obtained from CellBank Australia (Westmead, Australia) and maintained in DMEM (Sigma Aldrich) containing 20% horse serum (Sigma Aldrich) and 2 mM GlutaMAX. Cell lines were assessed for Mycoplasma spp. contamination using the Myco Alert Mycoplasma Detection Kit (Lonza, Switzerland) as per the manufacturer's instructions and were found to be routinely negative.

4.2 Purification of the Anti-P2X7 and Isotype Control mAbs

Anti-P2X7 and isotype control mAbs were purified as described [35]. Briefly, tissue culture supernatant (TCSN) was collected from clone L4 and MPC-11 cell lines. IgG from TCSNs was then purified using a Pierce Protein A Agarose IgG Purification Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. TCSN was incubated with 1 mL Protein A resin for 90 min with gentle rocking. The mixture was re-packed into a supplied chromatography column and the sample was allowed to flow through to form a bed. The column was washed with the supplied binding buffer, and 1 mL fractions were collected. The absorbances of the fractions were measured at 280 nm using a Suprasil quartz cuvette (Hellma, Müllheim, Germany) in a SpectraMax Microplate Reader spectrophotometer (Molecular Devices, San Jose, CA, USA) to confirm unbound protein was removed from the column. Bound IgG was then eluted with the supplied elution buffer, with 1 mL fractions collected into microfuge tubes containing 50 µL of 1 M Tris solution (pH 9.5) and the absorbances measured as above. Fractions with the highest absorbances, indicating the elution of IgG, were pooled and concentrated to 1 mg/mL in PBS, using a 50 kD molecular cut-off centrifugal device (Thermo Fisher Scientific) as per the manufacturer's instructions. The purity of IgG preparations was confirmed by protein electrophoresis using 4–10% Mini-PROTEAN TGX Stain-Free Protein gels (Bio-Rad Hercules, CA, USA), which were visualised using a Gel DocTM XR+ Imager (Bio-Rad).

4.3 Human PBMC Isolation

Collection and utilisation of human blood were done in accordance with approval by the University of Wollongong Human Ethics Committee (HE 12/290). PBMCs were isolated by density centrifugation as described [36]. Briefly, peripheral blood was collected from healthy donors (four males and three females; age range 24–52 years) and diluted in an equivalent volume of sterile PBS (Thermo Fisher Scientific). Samples were underlaid with Ficoll-Paque PLUS (GE Healthcare; Uppsala, Sweden) and centrifuged (560 × g for 30 min with deceleration set to 0). PBMCs were recovered from the gradient interface, washed twice with PBS, and resuspended at a final concentration of 1 × 10⁶ cells/mL for P2X7 expression assays or 3 × 10⁶ cells/mL for CDC assays.

4.4 Detection of P2X7 on Cell Lines by Immunolabelling and Flow Cytometry

The presence of P2X7 on HEK-P2X7 and HEK-293 cells was assessed as described [13]. Briefly, HEK-P2X7 and HEK-293 cells were washed in PBS containing 10% FCS (300 × g for 5 min). Cells (1 × 10⁶ cells/100 µL) were incubated with DyLight488-conjugated anti-P2X7 (clone L4) or isotype control (clone MPC-11) mAbs, prepared as described [13, 35], and the cell viability dye 7AAD (1 µg/mL; Cayman Chemical, Ann Arbor, USA) for 20 min in the dark on ice. Cells were then washed once and resuspended in PBS. Data were acquired with a BD Biosciences (San Diego, USA) LSR Fortessa X-20 flow cytometer using an excitation wavelength of 488 nm for DyLight488 and 7AAD and detection wavelengths of 525/50 and 675/20 nm for DyLight488 and 7AAD, respectively. A consistent gating strategy was used as demonstrated in Figure S1. The MFI of mAb labelling was determined using the geometric mean function of FlowJo software v10.7.1 (BD Biosciences). The relative P2X7 expression on cells was determined as the difference between the MFI of anti-P2X7 mAb and the isotype control mAb. Gating strategies for these and the flow cytometric studies below were based on journal guidelines [37].

4.5 Fura-2 AM Ca²⁺ Response Assay

P2X7 activity was assessed using a Ca²⁺ response assay as described [38]. HEK-P2X7 and HEK-293 cells were plated in a black clear-bottom 96-well plate (5 × 10⁴ cells per well; Greiner Bio-One, Krems Master, Austria) overnight at 37°C and 95% air/5% CO₂. Cells were washed twice using an extracellular Ca²⁺ solution (ECS; 145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM KCl, 13 mM glucose, and 10 mM HEPES, pH 7.4), then preincubated with Fura-2 AM loading buffer [2.5-µM Fura-2 AM (Thermo Fisher Scientific)/0.2% pluronic acid (Sigma Aldrich, St Louis, USA) in ECS] in the dark for 30 min at 37°C and 95% air/5% CO₂. Cells were then washed once and incubated with ECS for a further 20 min to allow complete de-esterification of the Fura-2 AM. Fura-2 AM fluorescence was measured at 510 nm every 5 s using a FlexStation 3 (Molecular Devices) following excitation at 340 and 380 nm. Baseline recordings were taken for 30 s, and then following the addition of ATP (Sigma Aldrich) or ECS (basal), recordings were measured for up to 3 min. Where indicated, anti-P2X7 (clone L4) or isotype control (clone MPC-11) mAbs were added to wells at regularly spaced 1 min intervals at 37°C for 10 min prior to the addition of ATP or ECS. Data was acquired using SoftMax Pro version 7.0 (Molecular Devices). Relative changes in intracellular Ca²⁺ were calculated using the ratio of Fura-2 AM fluorescence (F340 nm/380 nm). Ca²⁺ responses were normalised to the baseline recordings using the formula ∆Ca²⁺ = ∆F/Frest = (F − Frest)/Frest, where F is the F340 nm/380 nm ratio in a well at a particular time point and Frest is the mean fluorescence of a well from 0 to 30 s (prior to ATP or ECS addition). Ca²⁺ responses over time were plotted in Prism software v8.0.2 (GraphPad Software, La Jolla, USA). To determine relative P2X7-mediated Ca²⁺ responses, the area under the curve from 100 to 180 s was calculated with GraphPad Prism. ATP-induced responses were calculated by subtracting the corresponding basal response. These responses were normalised to the maximal ATP-induced response within their respective experiment.

4.6 CDC Assay (Determined Using LDH Release)

CDC of HEK-P2X7 and HEK-293 cells was assessed using a modification of an assay described previously [39]. Cells were plated (5 × 10⁴ cells per well) in a flat-bottom 96-well plate (Greiner Bio-One). Plates were incubated overnight at 37°C and 95% air/5% CO₂. Cells were washed twice with RPMI-1640 medium containing 2 mM GlutaMAX and 0.1% bovine serum albumin (BSA; Amresco, Solon, USA; RPMI-BSA). Then, 50 µL RPMI-BSA, 50 µL of PBS, anti-P2X7 or isotype control mAb (2 µg/mL), and 50 µL of rabbit complement (at a final dilution of 1:4) (Cedarlane Laboratories, Burlington, Canada) were added per well in triplicate. To determine maximal LDH release, 135 µL RPMI-BSA and 15 µL 10x Lysis Solution (Promega, Madison, USA) were added to wells in triplicate (maximum release wells). Plates were incubated for 2 h at 37°C and 95% air/5% CO₂. Following incubation, 50 µL from all wells were transferred to corresponding wells of a new flat-bottom 96-well plate. To three other wells, 50 µL of RPMI-BSA was added (medium-only wells). LDH release was assessed using a 96 Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer's instructions. Briefly, a total of 50 µL of CytoTox96 reagent was added per well and plates were incubated for 30 min at room temperature in the dark. Incubations were stopped by the addition of 50 µL of stop solution to each well. Absorbances of each well were read at 490 nm on a SpectraMax Microplate Reader (Molecular Devices) with the mean of medium-only wells used to define background absorbances, which were subtracted from all other absorbance values. Percent cytotoxicity was determined as the percent of LDH release in sample wells compared with the mean of maximum release wells.

4.7 CDC Assay (Using 7AAD Exclusion)

CDC of RPMI 8226 and J774 cells or freshly isolated PBMCs was performed using a variation of the method above. Cells were washed twice (300 × g for 5 min) in RPMI-BSA and resuspended in RPMI-BSA (3 × 10⁶ cells/mL). Then 150 µL of cell suspension, 150 µL of PBS, anti-P2X7 or isotype control mAb (2 µg/mL), and 150 µL of rabbit complement (at a final dilution of 1:8) were added per well to a flat-bottomed 24-well plate (Greiner Bio-One). Plates were incubated for 2 h at 37°C 95% air/5% CO₂. RPMI 8226 and J774 cells were incubated with 7AAD (1 µg/mL) for 5 min at room temperature in the dark. Data were directly acquired from wells with a BD Biosciences Accuri flow cytometer using an excitation wavelength of 488 nm and detection wavelength of 675/20 nm. Percent cytotoxicity was determined using FlowJo v10.7.1 software with the gating strategy demonstrated in Figure S2. PBMCs were scraped and collected from duplicate wells and transferred to 5 mL polystyrene tubes (Sarstedt, Nümbrecht, Germany) and centrifuged (300 × g for 5 min). PBMCs were also immunolabelled with fluorochrome-conjugated mAbs (Table S1), with Precision Count Beads (BioLegend, San Diego, USA) added to each tube. Data were acquired using a BD LSR Fortessa X-20 flow cytometer at the corresponding excitation and emission wavelengths. Numbers of immune cell subsets were assessed using FlowJo software version 10.7.1. Percent changes in the number of cell subsets were compared between anti-P2X7 mAb-treated cells and isotype control mAb-treated cells.

4.8 P2X7 Expression on PBMCs by Immunolabelling and Flow Cytometry

Freshly isolated PBMCs were washed (300 × g for 5 min) and resuspended in PBS. Cells (1 × 10⁶ cells/mL) were incubated with Zombie Dye NIR (BioLegend; 1 µg/mL) for 15 min in the dark on ice. Cells were washed with PBS containing 2.5% FCS and centrifuged (300 × g for 5 min). Cells were incubated with 10 µL of human FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min on ice and then with fluorochrome-conjugated mAbs (Table S1) for 15 min in the dark on ice. Cells were washed twice with PBS. Data were acquired using an LSR Fortessa X-20 flow cytometer at the corresponding excitation and emission wavelengths (Table S1). The MFI of anti-P2X7 mAb labelling was determined using the geometric mean function of FlowJo software v10.7.1. The relative P2X7 expression on cells was determined as the difference between the MFI of anti-P2X7 mAb stained cells and the corresponding FMO control cells.

4.9 Data Presentation and Statistical Analysis

Data are represented as the mean ± standard error of the mean (SEM). All statistical analyses were conducted, and graphs were assembled, using GraphPad Prism software v8.0.2. Data were tested for normality using a Shapiro–Wilk test. Statistical differences were calculated using an unpaired (or paired as indicated) Student's t-test (two-tailed; parametric) or Mann–Whitney test (non-parametric) for single comparisons or one-way analysis of variance (ANOVA; parametric) or Kruskal–Wallis (non-parametric) with a Tukey's post hoc test for multiple comparisons. For all analyses, differences were considered significant if p < 0.05.