Cell culture

Human umbilical vein ECs (HUVEC, Lonza) and human aortic endothelial cells (HAEC, Lonza) were cultured in base EBMTM‑2 EC growth media supplemented with EGM Bullet kit containing human epidermal growth factor (hEGF), vascular endothelial growth factor (VEGF), R3‑insulin‑like growth factor‑1 (R3‑IGF‑1), ascorbic acid, hydrocortisone, human fibroblast growth factor‑beta (Hfgf-β), heparin, fetal bovine serum (FBS, Bovogen) and gentamicin/amphotericin B (GA) (Lonza). HUVECs /HAECs were passaged every 3–4 days and cultured in 75 cm² U‑shape cell culture flasks (Corning). All experiments were performed on ECs under ten passages. THP-1 human leukemia monocytic cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Life Technologies) supplemented with 5% (vol/vol) heat inactivated FBS and J774 mouse macrophages were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) heat inactivated FBS. Media for both cell lines was supplemented with streptomycin (50 µg/mL) and penicillin (50 U/mL) (Life Technologies). All cell lines were cultured in media containing MycoZap (Lonza) and incubated at 37 °C with 5% CO2 in a humidified CellXpert 170i incubator (Eppendorf). EC lines were passaged with 0.25% (weight/vol) trypsin with 0.5 mM EDTA (Thermo Fisher) and seeded 24 h before experimentation to allow them to adhere.

In vitro model of inflammatory cell death

HUVECs or HAECs were seeded onto 10–15 cm dishes 24 h prior to experimentation and allowed to adhere. To induce apoptosis, the BH3-mimetic cocktail of ABT-737 (2 µM)/S63845 (500 nM) was used, denoted ‘ABT-S’. Cells were treated with ABT-S in normal growth medium and incubated for 2 h. To induce apoptosis under inflammatory conditions, cells were pre-treated with recombinant TNF (50 ng/mL, Abcam) for 24 h prior to ABT-S treatment. Media was then removed, and cells were washed twice with 1x PBS before adding ABT-S treatment.

Animal studies

All animal experiments were approved by the La Trobe University Animal Ethics Committee (AEC21034). For plasma analysis experiments and efferocytosis experiments, wild type (WT) C57Bl/6 mice (aged between 6 and 20 weeks) were obtained from M. Hulett breeding lines (La Trobe University, Bundoora VIC Australia).

Analysis of EC-ApoBDs from whole mouse blood

WT C57Bl/6 mice aged 6 weeks or 20 weeks were humanely killed by CO2 asphyxiation and blood was collected immediately via cardiac puncture into heparinised collection tubes. Blood was diluted 1/5 with sterile 1 x PBS and centrifuged at 100 g for 20 min to separate erythrocytes. The top erythrocyte-free layer was carefully collected and centrifuged at 3,000 g for 10 min to sediment ApoBDs. Samples were stained with Vybrant FAM (‘FLICA’) Caspase‑3 marker (Thermo Fisher) as per manufacturer’s instructions, and resuspended in FACS buffer (2 mM EDTA, 10% FBS, 1x PBS). Samples were incubated with Fc Block for 15 min at 4 °C followed by antibody staining panel to determine endothelial cell-derived ApoBDs: CD45.2-APC (clone 104, Thermo Fisher), CD11b-e450 (clone M1/70, Thermo Fisher), CD41-PerCP Cy5.5 (clone MWReg30, Biolegend), CD146-PE Cy7 (clone ME-9F1, Biolegend), CD31-PE (clone 390, Biolegend) for 20 min at 4 °C and analysed by flow cytometry (BD 3-laser Canto II). Events identified as CD45.2−/CD11b−/CD41−/FSClow/CD146+/CD31+/FLICAhigh were defined as endothelial cell-derived apoptotic bodies. Spherotech AccuCount counting beads were used to determine absolute number of events per mL whole mouse blood.

Flow cytometry-based analysis of apoptotic ECs

Following the induction of apoptosis, samples were gently pipetted to lift any remaining cells and stained with A5‑FITC (1:200, BD Biosciences) and TO-PRO-3 (1 µM, Life Technologies) in 1x A5 binding buffer for 10 min at 4°C. Cell populations (i.e. viable, apoptotic, necrotic) were determined using flow cytometry gating as previously described [15]. For pan caspase‑inhibition assays, HUVECs or HAECs were seeded into a 24‑well plate at a density of 5 × 104 cells per well. Cells were pre-incubated with Q-VD-OPh (50 µM, SigmaAldrich) or DMSO for 1 h at 37 °C, 5% CO2 before induction of apoptosis with ABT-S. In adhesion molecule expression experiments, samples were stained with antibodies against ICAM-1 (clone HA58, Thermo Fisher), VCAM-1 (clone STA, Thermo Fisher), E-selectin (clone P2H3, Thermo Fisher) and P-selectin (clone PselK02.3, Thermo Fisher) following TO-PRO-3 and A5 staining. In HLA surface expression assays, samples were derived from single donor HUVECs (donor #809, Lonza) and were stained with HLA-I‑BV421 Ab (clone W6/32, BD Biosciences) following TO-PRO-3 and A5 staining. All antibodies were stained for 20 min at 4°C.

ApoBD isolation via differential centrifugation

After apoptosis induction as described above, any remaining adhered cells were lifted from dish via gentle pipetting, transferred to 14 mL polypropylene tubes and centrifuged at 300 g for 10 min. Cell pellets were discarded, supernatants were transferred to clean tubes and centrifuged at 3,000 g for 10 min. Supernatants were discarded, cell pellets were gently resuspended and washed with 10 ml 1x PBS, followed by a repeated centrifugation at 3,000 g for 10 min. Supernatants were discarded and cell pellets were collected. ApoBDs collected from cells treated with ABT-S are denoted ‘ApoBDs’ and ApoBDs collected from cells pre-treated with ABT-S and TNF are denoted ‘iApoBDs’.

Normalisation of ApoBD counts

For each preparation of ApoBDs, equal volumes of ApoBDs and iApoBDs in EGM-2 were prepared in combination with Spherotech AccuCount counting beads (20 µl/20,000 beads per sample) and analysed by flow cytometry to determine absolute number of ApoBDs per condition. Where specified, an equal number of ApoBDs/iApoBDs was then used per experiment.

Confocal laser scanning microscopyApoBD/iApoBD generation by HUVECs

2 × 104 HUVECs per well were seeded into a Nunc™ Lab‑Tak™ II 8‑well chamber slide in EGM-2 media (Nunc). Apoptosis was induced in cells as described above with the addition of A5-FITC (1:200, BD Biosciences) in 0.5x A5 binding buffer (BD Biosciences). Stains and drugs were added simultaneously, and endpoint images were taken using the Zeiss 900 confocal spinning disk microscope equipped with an electronically switchable illumination and detection (ESID) module (Zeiss). To observe blebbing formation, images were taken using a Zeiss Axio Observer Z1 microscope equipped with an Axiocam 506 monochrome digital camera and Zen software (Carl Zeiss Ltd). All confocal microscopy experiments were conducted at 37 °C in humidified atmosphere with 5% CO2. Images were processed using ZEN imaging software (Zeiss).

Quantification of ApoBDs/iApoBDs via live cell imaging

ApoBDs/iApoBDs were generated and isolated as described above. NuncTM Lab‑TakTM II 8‑well chamber slides were pre-coated in 0.01% (w/v) poly-L-lysine to allow vesicles to adhere. Vesicles were stained with A5‑FITC (1:50, BD Biosciences) and TO-PRO-3 (1 µM, Life Technologies) in 1x A5 binding buffer. 5 × 5 tile regions were imaged using the Zeiss 900 confocal spinning disk microscope. Vesicle diameters were quantified based of A5 staining, where regions of interest were exported as binary images to measure feret diameter in ImageJ (version 2.14.0).

Engulfment assay via live cell imaging

5 × 104 phagocytes per well (J774 murine macrophages) were seeded into Nunc™ Lab‑Tak™ II 8‑well chamber slide in complete DMEM media one day prior to allow adherence to slide. HUVECs were seeded two days prior at a ratio of 5:1 (EC: phagocyte). For preparation of iApoBDs, HUVECs were treated with TNF one day prior, as described above. On day of experiment, HUVECs were stained with CypHer 5E (4 mg/ml, Cytiva) in serum-free RPMI for 45 min at 37 °C, washed twice with warm PBS and treated with ABT-S followed by isolation of ApoBDs or iApoBDs as described above. Phagocytes were stained with CFSE for 20 min at 37 °C and washed twice with PBS. ApoBDs or iApoBDs were then resuspended in complete DMEM. ApoBD/iApoBD counts were normalised as described above and equal numbers were added to phagocytes. Samples were centrifuged for 5 min at 1000 g and co-incubated for 1 h, washed twice gently with PBS to remove non-engulfed particles and imaged using the Zeiss 900 confocal spinning disk microscope.

Immunoblotting

HUVECs were seeded into 15 cm plates and induced to undergo apoptosis as described above. Samples were lysed in cell lysis buffer containing 1% Nonidet P‑40, 10% glycerol, 1% TX‑100, 150mM NaCl, 20mM HEPES (pH 7.4) and protease inhibitor cocktail (Sigma Aldrich) on ice for 30 min and centrifuged at 13,000 g for 10 min at 4 °C to remove insoluble material. Cell lysates were collected and stored in ‑80 °C until needed. Protein concentration was determined using a Pierce Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher) as per the manufacturer’s instructions. Absorbance was read at 562 nm on a SpectraMax M5e plate reader (Bio‑Strategy). Prior to SDS-PAGE gel electrophoresis, cell lysates were incubated with dithiothreitol (DTT, Astral Scientific) and lithium dodecyl sulphate (LDS, Life Technologies) at 70 °C for 10 min, then separated on a 4–12% BisTris gel at 120 V for 5 min followed by 190 V for 30 min. Polyvinylidene fluoride (PVDF) membranes were activated with 100% methanol before protein transfer in NuPAGE transfer buffer (Life Technologies) at 20 V for 60 min. Following transfer, the membrane was blocked with 5% skim milk powder in 1 x PBS/0.1% Tween (0.1% PBS‑T) for 2 h at RT. Membranes were washed thrice with 0.1% PBST before being incubated with the primary antibody overnight at 4 °C diluted in 3% BSA/0.1% PBS‑T. Primary antibodies include rabbit Pannexin-1 (1:1000, clone D9M1C, Cell Signalling Technology), rabbit caspase-3 (1:1000, clone 8G10, Cell Signalling Technology), rabbit cleaved caspase-3 (1:1000, clone Asp175, Cell Signalling Technology) and mouse β-actin (1:5000, clone AC-74, Sigma Aldrich). Membranes were washed three times again with 0.1% PBS‑T and incubated 1 h at RT with a HR‑conjugated secondary antibody diluted in 3% BSA/0.1% PBST. Secondary antibodies include donkey anti-rabbit-HRP (1:5000, GE Healthcare) and sheep anti-mouse-HRP (1:5000, GE healthcare). Secondary antibodies were removed with a final three 10 min washes and detected using enhanced chemiluminescence (ECL) prime reagent (Bio-Strategy) and imaged using a G:BOX Chemi XL 1.4 system and GeneSys software (Syngene).

Cytokine antibody array

Human Cytokine Antibody Array (42 targets, Abcam) was used as per manufacturer instructions. Briefly, ECs were grown in 15 cm round tissue culture plates and ApoBDs and iApoBDs were generated and collected as described above. ApoBD/iApoBD lysates were prepared using cell lysis buffer provided by the kit. Protein concentration was determined via BCA assay and 200 µg protein per condition were loaded onto membranes, incubated and probed with HRP-antibodies. Membranes were imaged on G: Box (Syngene). Densitometry analysis was performed in ImageJ and analysed in Prism (GraphPad).

Cytometric bead array

Equal numbers of iApoBDs were incubated over 1–7 h at 37 °C, 5% CO2. Samples were centrifuged at 3,000 g for 20 min to pellet iApoBDs and supernatants were collected. Positive control supernatants were generated by lysing iApoBDs with three consecutive rounds of freeze‑thawing in liquid N2/37°C water bath. Following manufacturer’s instructions, cytokine concentrations were measured using the BD™ Cytometric Bead Array (CBA) Human Soluble Protein Master Buffer kit (cat#558264) and Human IL-6 (cat#558276), IL-8 (cat#558277) and MCP-1 (cat#558287) flex sets (BD Biosciences). Briefly, equal volumes of sample supernatant and mixed capture beads from the flex sets were incubated together in a 96‑well plate for 1 h at room temperature. Following the addition of the detection reagent, the plate was incubated for a further 2 h. The plate was pelleted, supernatant removed, and beads resuspended in wash buffer to be analysed by flow cytometry. Flow cytometry analysis was performed using FCAP Array software (version 3.0.1, BD Biosciences).

Caspase Glo 3/7 assay

1 × 104 HUVECs or HAECs were seeded per well into a black, clear bottom 96 well plate. 2 h following apoptosis induction, 50 µL of CaspaseGlo® 3/7 assay reagent (Promega, cat #G8091) was added to the wells, shaken for 5 min, and incubated at RT for 1 h. Luminescence was recorded on the SpectraMax M5e plate reader (Bio‑Strategy) and data was analysed by subtracting media only background control measurement from the samples.

Transwell migration assay

Equal numbers of ApoBDs or iApoBDs (based on a 5:1 ratio of EC starting cells to THP-1 monocytes) in 5% RPMI media were added to each lower well of a 24‑well Transwell plate (Corning), with 6.5 mm diameter and 5.0 μm pore size polycarbonate membrane inserts. 2 × 105 THP‑1 cells in normal 5% RPMI growth medium were transferred to the upper chambers of the plates and incubated across a time series at 37 °C, 5% CO2. Negative (untreated) and positive (1 µM ATP) control wells were loaded with fresh 5% RPMI media or 1 µM ATP in 5% RPMI respectively. Migrating cells were collected at 3, 6 and 9 h from the lower well and incubated with Fc Block (4 °C, 10 min), stained with CD45.2 mAb (4 °C, 20 min, clone 5B1, Miltenyi Biotec) and analysed by flow cytometry. In neutralising assays, ApoBDs were preincubated with MCP-1 Ab (25 µg/mL, clone 2H5, Thermo Fisher) or IgG Armenian hamster isotype control (25 µg/mL, clone eBio299Arm, Thermo Fisher) for 10 min at RT before being loaded into the bottom chamber.

LDH cytotoxicity assay

ApoBD lysis was measured using a lactate dehydrogenase kit (LDH, Abcam, cat #ab65393). Following manufacturer’s instructions, 10 µl of supernatant was added to the LDH reaction mix and incubated for 60 min at RT. Spectral absorbance was read using a SpectraMax M5e plate reader (Thermo Fisher) at OD 450 nm.

Static adhesion assay

Target cells (THP-1s or Jurkats) were prepared at 1 × 106/mL in low glucose DMEM supplemented with 0.5% BSA. Endothelial cells were seeded to a ratio of 10:1 (EC: target cells) and stained with CellTrace Violet. ApoBDs or iApoBDs were generated via differential centrifugation as described above. Target cells were then co-incubated with equal numbers of ApoBDs or iApoBDs at 37 °C (30 min). Prior to flow cytometry analysis, samples containing THP-1s were incubated with Fc block (4 °C, 15 min) prior to staining with CD45.2 mAb (4 °C, 20 min) and samples containing Jurkats were stained with CD3 Ab (4 °C, 20 min, clone SK7, BD Biosciences). Samples were then analysed by flow cytometry (BD Canto II) and adhesion events were based on CD45.2+/CTV+ (THP-1) or CD3+/CTV+ (Jurkat) gating.

Proteomics

For sample preparation, ApoBDs and iApoBDs were generated as described above, washed in 1 x PBS, lysed with cell lysis buffer and incubated on ice for 30 min before centrifugation at max speed to collect supernatants. Three biological replicates of each condition were collected. Protein concentrations were determined by BCA assay and 30 µg of protein per condition was used per sample. In-gel digestion and subsequent liquid chromatography with tandem massspectrometry (LCMS/MS) was then performed as previously described[16]. Data set was analysed by the La Trobe Proteomics Platform using Uniprot as the reference proteome database with one sequence per gene (human database March 2021 version). The common repository of adventitious peptides was used as a marker database to identify laboratory contaminants. Spectra were searched with MS Fragger 3.3 and Sequest search engines using Proteome Discoverer 2.4. Search parameters included (Trypsin (Full), fixed modification of carbamidomethyl of cysteine, variable modifications were oxidation of M, deamidation of N/Q, and N-terminal Acetylation. Precursor mass tolerance was 50 ppm and fragment tolerance was 0.05 Da. Label-free quantitative measurement of peptide precursor intensity was calculated from unique and razor peptides. intensities were normalised on total peptide amount. Peptides were validated at peptide spectral match (PS) and peptide levels according to a strict criteria (0.01 False Discovery Rate (FDR)) and a relaxed criteria (0.05 FDR). The Differential Enrichment analysis of Proteomics data (DEP) R package was used to process and complete statistical tests. Proteins were retained for analysis if detected in at least 2 out of 3 replicates for one treatment. Protein abundance values underwent variance stabilising normalisation using the vsn-package. Missing values were imputed using MinProb function with a q value of 0.01. DEP uses protein-wise linear models combined with empirical Bayes statistics for the differential enrichment analysis.

In vitro engulfment assay

Phagocytes (J774 murine macrophages) were seeded one day prior to allow adherence to tissue culture plates. Phagocytes were stained with CTV or CFSE for 20 min at 37 °C and washed twice with PBS. ApoBDs/iApoBDs pre-stained with CypHer 5E were prepared as described above and added to phagocytes. Either DMSO or Cytochalasin D (5 µM) was added to each phagocyte well and samples were co‑incubated for 1 h. Media was then removed, and wells were washed gently twice with PBS to ensure removal of non-engulfed particles. Phagocytes were suspended with Trypsin/EDTA solution and neutralised with equal volume of complete DMEM. Samples were analysed by flow cytometry (BD 3‑Laser Canto II). Engulfing populations were determined as double‑positive for CFSE/CTV and CypHer 5E staining.

In vivo engulfment assay

HUVECs were stained with CypHer 5E for 40 min at 37 °C and 5% CO2, in serum free RPMI and washed twice with 1x PBS before isolation of ApoBDs/iApoBDs. 1.5 × 105 ApoBDs/iApoBDs were injected into the intraperitoneal cavity of C57BL/6 mice. After 1 h, mice were culled via CO2 and a peritoneal lavage was performed using 5% RPMI media. Samples were passed through a 70 μm cell strainer and centrifuged at 300 g for 5 min. Pellets were resuspended in FACS buffer, incubated with Fc Block for 20 min at 4 °C, then stained with CD45.2 Ab (clone 5B1, Miltenyi Biotec) and F4/80 Ab (clone T45-2342, BD Biosciences) for 20 min at 4 °C, and analysed by flow cytometry. Engulfing macrophages were identified as CD45.2+/F4/80+/CypHer 5E+ cells. For neutralising experiments, iApoBDs isolated from 1.6 × 107 HUVECs per mouse were preincubated with ICAM-1 neutralising Ab (10 µg/mL, clone HA58, Thermo Fisher) or mouse IgG1κ isotype control (clone P3.6.2.8.1, Thermo Fisher) for 10 min at 4 °C before injections. Engulfing macrophages were defined as F4/80+/CypHer 5E+ events. Due to variance in absolute values between independent experiments, isotype control samples were normalised to 100% and ICAM-1 treated samples were expressed as ‘percentage decrease’ compared to isotype controls.

Antigen presentation studies

Whole peripheral blood was obtained from volunteer donors following COVID-19 vaccination. Peripheral blood mononuclear cells (PBMCs) were seperated using Ficoll density gradient centrifugation as previously described [17, 18]harvested and stored at −80 °C until use. Donor PBMCs were HLA typed by CareDx Pty Ltd (Fremantle, WA). Research was approved by the La Trobe University Human Ethics Committee (HEC21097) and was conducted in accordance with the declaration of Helsinki.

PBMCs from 3 x HLA-A*02:01+ donors who had received > 3 COVID-19 vaccinations, were used to generate peptide-specific CD8+ T cell lines as previously described [19]. Briefly, 1/3 of the PBMCs were pulsed with 10 µM of the known immunodominant “YLQ” peptide (YLQPRTFLL; Genscript) derived from the spike protein of SARS-CoV-2 in FBS free media for 90 min. These were washed twice and added to the remaining 2/3 of PBMCs in RPMI (Gibco) supplemented with 1x Penicillin Streptomycin Glutamine (Gibco), 1x non-essential amino acids (100 x NEAA, Gibco), 2 mM L-glutamine (Sigma-Aldrich), 5 mM HEPES (Sigma-Aldrich), 50 lM β-mercaptoethanol (Sigma-Aldrich) and foetal bovine serum (FBS, Bovogen), herein referred to as RF10 media. On day 4, 10 IU mL−1 of recombinant human IL‑2 (Peprotech) was added, then again twice weekly.

Single donor HUVECs (Lonza) were pulsed for 1 h at 37 °C and 5% CO2 with S269‑277 (YLQPRTFLL) SARS-CoV-2 spike‑derived peptide (10 µM, GenScript). Cells were washed thrice with 1x PBS to ensure the removal of free peptide before inducing apoptosis as described above. Isolated ApoBDs were resuspended in 5% RPMI media. Equal numbers of ApoBDs or iApoBDs were co‑incubated with 1 × 106 HLA-A*02:01 typed human donor T lymphocytes following a previously published protocol [20, 21]. ApoBDs/iApoBDs and T cells were co-incubated with brefeldin A (10 µg/mL, Sigma Aldrich) for 5 h (37 °C, 5% CO2). The non-specific T cell activator X500 containing 5 ng/mL phorbol myristate acetate and 500 ng/mL ionomycin was used as a positive control. Following incubation, cells were pelleted by centrifugation (300 g, 5 min) and washed with 1x PBS. Samples were stained with CD3‑APC Ab (1:100, clone SK7, BD Biosciences), CD4‑FITC Ab (1:100, clone RPAT4, BD Biosciences), CD8‑PerCPCy5.5 Ab (1:50, clone SK1, BD Biosciences), LIVE/DEADTM‑APC Cy7 (1 µL/mL, Thermo Fisher), CD14‑APC Cy7 (1:10, cloneMoP9, BD Biosciences) and CD19‑APC Cy7 (1:10, clone SJ25C1, BD Biosciences) for 30 min at 4 °C. Cells were washed with 1x PBS and fixed with 1% paraformaldehyde (PFA, SigmaAldrich) at RT for 20 min. Following fixation, samples were washed with 0.2% (weight/vol) saponin (Thermo Fisher) and stained with IFN-?-BV510 (1:300 in 0.2% (weight/vol) saponin, clone B27, BD Biosciences) for 30 min at 4 °C. A final 1x PBS wash step was performed before cells were analysed by flow cytometry. Activation of T cells was determined by gating CD3+/CD8+/IFN-? events.

As a control, the proportion of CD8+ T cell activation was assessed in an intracellular cytokine staining (ICS) assay as previously described [20]. Briefly, C1R-A2 cells were peptide pulsed with 10 µM of the YLQ peptide (or no peptide control) for 60 min in FCS free media, were washed twice and added in a 1:2 C1R-A2: CD8+ T cell line ratio. Alternatively, 10 µM of the YLQ peptide (or no peptide control) was added to CD8+ T cell lines directly. An additional well with the non-specific T cell activator X500 containing 5 ng/mL phorbol myristate acetate and 500 ng/mL ionomycin was used as a positive control. CD8+ T cell lines were left to stimulate for 5 h at 37 °C with 5% CO2 in the presence of Golgi-Plug (1:1000, BD Biosciences), Golgi‑Stop (1:1400, BD Biosciences) and CD107a-AF488 Ab (1:200, Clone eBioH4A3, BD Biosciences). Following this, cells were washed and surface stained for 30 min at 4 °C with CD14-APCH7 Ab(1:200; Clone MϕP9, BD Biosciences) CD19-APCH7 Ab (1:200, Clone HIB19, BD Biosciences), CD3-BV480 Ab (1:100, Clone UCHT1, BD Biosciences), CD4-BV650 Ab (1:100, Clone SK3, BD Biosciences), CD8‑PerCpCy5.5 Ab (1:50, Clone SK1, BD Biosciences) and LIVE/DEAD NIR (1:1000, Molecular Probes). Cells were washed and Fix/Perm buffer (BD Biosciences) was added for 20 min at 4 °C. Cells were then washed and intracellular stained for 30 min at 4 °C with anti‑human IFN‑γ‑BV421 (1:100, Clone B27, BD Bioscience), TNF-PeCy7 Ab (1:100, Clone Mab11, BD Biosciences), MIP-1β-APC Ab (1:100, Clone D21-1351, BD Bioscience) and IL-2-PE Ab (1:100, Clone MQ1-17H12, BD Bioscience). Samples were acquired on the BD FACSymphony A3 analyser (BD Biosciences) and were analysed using Flowjo software (version 10, BD Biosciences). Samples were gated as per Figure S4.

Statistical analyses

Statistical significance was measured using various relevant statistical tests including normality testing, where appropriate, as specified within the figure legends of each figure. All analysis was performed using GraphPad Prism (Version 10.2.3, GraphPad Software).