High-Throughput 13-Parameter Immunophenotyping Identifies Shifts in the Circulating T-Cell Compartment Following Reperfusion in Patients with Acute Myocardial Infarction

Rationale With the advent of primary PCI (PPCI), reperfusion is achieved in almost all patients presenting with acute myocardial infarction. However, despite multiple trials, reperfusion injury has not been successfully dealt with so far. In mouse models, CD4+ T lymphocytes (T cells) have been shown to be crucial instigators of reperfusion injury. Objective Our goal was to investigate the role of CD4+ T cells during myocardial reperfusion following PPCI by developing a protocol for high-throughput multiplexed flow cytometric analysis and multivariate flow clustering. Methods and Results 13-parameter immunophenotyping and hierarchical cluster analysis (HCA) identified a unique CD4+CD57+ T-cell population in PPCI patients that reflected acute proliferation in the CD4+ T-cell compartment. CD4+CCR7+ T cells were specifically depleted from peripheral blood during the first 30 min of myocardial reperfusion after PPCI, suggesting a potential role for the chemokine receptor CCR7 in T-cell redistribution to either peripheral tissues or migration to the infarcted heart during ischemia/reperfusion following PPCI. Conclusions High-throughput polychromatic flow cytometry and HCA are capable of objective, time and cost efficient assessment of the individual T-cell immune profile in different stages of coronary heart disease and have broad applications in clinical trials.

for 15 minutes with 1500 µl ammonium chloride-based lysing buffer (BD PharmLyse,Cat. No. 555899,BD Biosciences). Data acquisition was performed on BD FACS Canto II flow cytometer (BD Biosciences) using FACSDiva software (BD Biosciences). Threshold was set on CD45 PerCp channel and 10000 of CD45 positive lymphocytes were counted for the stop gate. Spectral overlap between different channels was calculated automatically by the FACSDiva software after measuring single-colour compensation controls. Optimal compensation was achieved using antibody capture beads (Anti-Mouse Ig, κ CompBeads, Cat. No. 552843 BD Biosciences) and the corresponding conjugated antibodies (see below). Data was analyzed using FACS-Diva Software and absolute cell counts pro µl PB were calculated according to manufacturer's protocol.

PBMCs isolation, cell cryopreservation and storage ("PBMC Cryobank")
Peripheral blood mononuclear cells (PBMCs) were obtained after density gradient centrifugation using Ficoll-Hypaque (Biochrom, Cat. No. #L6115). PB blood was transferred from EDTA collection tubes into 50 ml conical tubes (BD Falcon) and gently resuspended with PBS in a ratio 1:1. Subsequently, 15 ml of Ficoll-Hypaque were placed at the bottom of a second 50 ml Falcon tube, and 35 ml of the blood/PBS-mix were then slowly layered above Ficoll. After being centrifuged for 20 min at 800xg at RT (without brake), the following layers were visible in the Falcon tube, from top to bottom: plasma with platelets and PBS, a yellow-whitish layer of mononuclear cells (PBMCs interphase), a clear layer of Ficoll-Paque, whitish layer of granulocytes and cell-debris covering the dark-red pellet of erythrocytes. The PBMC interphase was than carefully aspirated using a slow-speed automatic 10 ml pipette and transferred to a fresh Falcon tube. Following two washes with PBS/2mM EDTA PBMCs were counted with trypan blue using Neubauer haemocytometer, carefully resuspended in ice-chilled freezing medium containing RPMI 1640 (Cat. No. 21875034, Invitrogen) with 10% FBS (Cat. No. A15-151, PAA), 1% P/S (Penicillin/Streptomycin 5000Units /ml, Cat. No. 15070063, Invitrogen) and 10% DMSO (Cat. No. D2650, Sigma) (100µl medium pro 1x10 6 cells) and aliquoted into 1 ml cryovials Thermo Fisher Scientific,Denmark). Cell aliquots were imediately frozen using 1ºC freezing containers with isopropyl alcohol Thermo Fisher Scientific), and stored at -80ºC New Brunswick Scientific,Edison,NJ,USA).

Cell staining for 11-colour flow cytometry
Frozen PBMC aliquots (2x10 6 PBMC/aliquot) were quickly thawed in water bath (37ºC for 1 min) and immediately transferred into polystyrene round bottom 12 x 75 mm Falcon tubes (BD Biosciences). After adding 1 ml wash buffer containing PBS (Cat. No. 18912-014, Gibco), 2mM EDTA and 0,1% azide, samples were washed once using automatic cell washer (BD Lyse Wash Asistant,Cat. No. 337146). This high-throughput washing technique provides much better cell recovery while maintaining higher viability of thawed PBMCs as compared with traditional centrifugebased washing. Following primary wash step, the cell viability and cell numbers were analysed using video imaging system with automated trypan blue exclusion protocol (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter,). 1x10 6 PBMCs were then transferred into new FACS tube and the total sample volume ('stain volume') was adjusted up to 100 µl with PBS/2mM EDTA. For staining, master mix containing primary antibodies was added into tubes and samples were incubated light protected for 20 minutes at RT. Quantum-dot-conjugated antibodies (CD3-QDot-605) were added separately into samples, prior to addition of the primary antibody mix. This ensured optimal staining results as the used nanocrystals turned to get unstable when premixed with other primary antibodies (supplementary figure 3). Following incubation with primary antibodies, 1 ml of wash buffer per sample was added and samples were washed three times using lyse wash assistant. Following wash steps, appropriate amounts of secondary antibodies (2 µl of Streptavidin-AF532 and 5 µl of rat anti-mouse-AF350) and 1 µl of Aqua Dye were added into the total sample volume of 300 µl. Samples were then incubated light protected for another 20 minutes at RT. Following incubation, 1 ml of wash buffer per sample was added and cells were washed again three times using lyse wash assistant. Following these final wash steps, unfixed samples (total end volume -300 µl) were immediately subjected to analysis by flow cytometry.

Flow cytometry data acquisition
Samples were measured in our flow core facility on a BD LSR II cytometer (see below for detailed description) using BD FACSDiva acquisition software. PMT voltages and compensation values were set as described below. At least 100000 viable cell events per sample were acquired.

Flow cytometry data analysis
The analysis of acquired data was performed using MATLAB (for Hierarchical Cluster Analysis, see below) or FlowJo software (Version 9.4.1 for Macintosh). Twodimensional plots (pseudo-color or dot plot) were created using biexponential transformation and sequential gating of viable T cells was performed according to the model of T-cell memory differentiation as described by Sallusto and Romero [2,3,4] (see supplementary figure 4).

Instruments setup
The Becton Dickinson LSR II flow cytometer was equipped with 5 spatially and time delayed lasers: blue (488nm), violet (407nm), UV (355nm), red (638nm) and green (532nm). The detailed configuration of our LSR II can be seen below (see supplementary table 1). Prior to any analysis the instrument was checked for correct laser delay settings and fluidics stability by consistency of results using cytometer tracking and set up beads using an acceptable tolerance of +/-10 volts (Becton Dickinson), and single peak Spherotech Ultra Rainbow beads.

PMT voltage settings
At the time of experimental set up the PMT voltages were adjusted for each fluorochrome with the objective of optimizing the signal to noise ratio, based on single stained samples.

Spectral compensation
Spectral overlap between different fluorochromes was calculated automatically by the Compensation matrix used in the study can be seen below (supplementary table 3).
Only minor experiment-to-experiment adjustments of this set up were needed in the course of the present study.

Variability of the multiparameter flow-cytometry panel
The variability of the multiparameter immunophenotyping method was calculated by analysing the repeated measurements of the frozen PBMC aliquots from the same healthy donor. We used 28 independent measurements, each performed on a steps were carried out. Compensation matrix (as present in .fcs files) was applied to the data followed by biexponential-like transformation and normalisation (zscore).
After this the data were ready for HCA, which was performed using our new algorithm described previously [5] .

8-colour flow sorting of CD4 T Cells (Aria II)
3x10 7 cryopreserved PBMCs were used for fluorescence-activated cell sorting. Cell aliquots were thawed and processed as described above. Prior to staining, cells were evaluated for size, viability and concentration using the Vi-Cell analyzer. PBMC staining was performed using carefully pre-titrated antibody mix containing antihuman-CD3-FITC, CD4-PE-Cy7, CD8-APC-H7, CCR7-PE CD45 (BD Bioscience), CD45-Pacific Blue and Aqua dead cell dye (Invitrogen). Cell sorting was performed in our flow core facility on a BD FACS AriaII cell sorter equiped with 4 lasers: 638nm Red, 355nm UV, 405nm Violet and 488nm saphire lasers. The sorting conditions were 70micron nozzle at 70 psi, using a high purity mask and acquisition speed below 10000 events per second. 3-5x10 5 CD4 + T cells were sorted directly into 1.5 ml eppendorf tubes. The sorted aliquots were spun down and the flow buffer supernatant was completely removed with a vacuum suction. Dry T-cell pellets were stored at -80ºC freezer until further processed with DNA isolation protocol (see below).

DNA isolation and telomere length RT-PCR assay
DNA was extracted from sorted CD4 + T cells with the QIAamp DNA Mini Kit (Cat. No. 51304, Qiagen Ltd, Crawley, UK). DNA concentration and quality were monitored by agarose gel electrophoresis. Samples were discarded if DNA degradation (smear <20 kb) was visible. Telomere length was measured as the ratio of the starting quantity for telomeres versus the starting quantity for the single copy gene of glyceraldehyde-3-phosphate dehydrogenase (as control) by quantitative real-time 8 polymerase chain reaction (PCR) [6] with modifications as described previously [7].
Measurements were performed in quadruplicates. Three DNA samples with known telomere lengths (3.0, 5.5 and 9.5 kb pairs) were run as internal standards together with each batch of 16 study samples to convert the ratios of starting quality into telomere lengths in base pairs. The intra-assay coefficient of variation for this PCR method in our lab is 2.65% and the inter-assay coefficient of variation is 5.12%.

Statistical analysis
In the text, data are reported as mean±SE. Comparison of 3 means was performed by ANOVA, followed by Tukeys' post-hoc test. Comparison of 2 groups was calculated using an unpaired t-test, if normal probability plots (P-P plots) demonstrated approximate normality. All statistical tests were performed using GraphPad Prism version 5 for Macintosh (www.graphpad.com).

Supplementary figure 10.
High-Throughput multiparameter immunophenotyping and hierarchical cluster analysis as a novel biomarker screening tool for multicenter trials in cardiology. The selected PBMC subsets of interest may be additionally purified from remaining frozen aliquots using meultiparameter flow sorting and subjected to further molecular profiling (e.g. telomere length analysis, gene-or micro-RNA arrays, etc.).