Spectral biophysical cytometry with nanosensors reveals remodelling of immune cells in atherosclerosis

Preparation of BSLBs

1,2-Diarachidonoyl-sn-glycero-3-phosphocholine (20:4-20:4 (DAPC)), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (18:1-18:1 Δ9cis (DOPC)), 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (18:1-18:1 Δ6cis), 1,2-dielaidoyl-sn-glycero-3-phosphocholine (18:1-18:1 Δ9trans), 1,2-dipalmitoleoysl-sn-glycero-3-phosphocholine (16:1-16:1 Δ9cis), 1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine (16:1-16:1 Δ9trans), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0-18:1), 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0-18:1 (POPC)) and ldl cholesterol have been from Avanti Polar Lipids. As a primary step, 10 µl of every lipid at 25 mg ml⁻¹ (in chloroform) was dried beneath a mild stream of nitrogen to type a skinny lipid movie. The dried movie was rehydrated in 1 ml of SLB buffer (150 mM NaCl, 10 mM HEPES, pH 7.4), vortexed briefly and sonicated utilizing a tip sonicator (energy, 3; responsibility cycle, 40%) for 10 min to generate small unilamellar vesicles (SUVs). To type BSLBs, 45 µl of the SUV answer was blended with 5 µl of silica beads (Bangs Labs) in a last quantity of 500 µl SLB buffer. The combination was bath-sonicated at room temperature for 30 min to facilitate bilayer formation on the bead surfaces. The ensuing lipid-coated beads have been washed 3 times with SLB buffer to take away unbound lipids. NR12S and Pro12A have been synthesized as described beforehand^9,10. For move cytometry evaluation, beads have been incubated with the membrane order probes Pro12A or NR12S (last focus, 250 nM) for 1 min in move cytometry tubes instantly previous to acquisition on a Cytek Aurora spectral move cytometer (5 lasers, 64 channels). For imaging, beads have been plated in an 18-well glass-bottom chamber (ibidi) and excited with a 405-nm laser utilizing a Zeiss LSM 780 confocal microscope geared up with 32-channel spectral detectors.

Drug remedies on CEM cells

CEM cells (ATCC, CRL-2265) have been cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) beneath customary circumstances. The next compounds have been used for drug therapy experiments: CK-666 (5 µg ml⁻¹), latrunculin B (1 µg ml⁻¹), methyl-β-cyclodextrin (12 µg ml⁻¹), sphingomyelinase (1/100 dilution of 25 models), oseltamivir phosphate (40 µg ml⁻¹), swainsonine (1 µg ml⁻¹), digitonin (5 µg ml⁻¹) and valinomycin (25 µM) (all from Sigma-Aldrich), and CCCP (20 µM) (Thermo Fisher). For every situation, 3 × 10⁵ cells have been used. If not said in any other case, all remedies have been carried out in full medium at 37 °C for 30 min–1 h. Following incubation, cells have been washed 3 times with PBS. Cell viability was assessed utilizing BD Horizon Fixable Viability Stain 660 (FVS660, Reside/Lifeless), utilized for 15 min at room temperature, adopted by two PBS washes. For membrane order evaluation, cells have been stained with Pro12A dye (0.6 µl of 100 µM dye in 300 µl PBS for 300,000 cells) for 1 min instantly within the move cytometry tubes instantly earlier than the acquisition. For mitochondrial depolarization measurements, JC-1 dye (Thermo Fisher) was incubated with the cells for 30 min both previous to or throughout drug therapy. For CCCP remedies, JC-1 (0.5 µl of 250 µM dye in 500 µl medium for 300,000 cells) was added over the past 5, 15 or 30 min of drug publicity, whereas for Digitonin, JC-1 was added throughout the last 5 min of therapy. Cells have been washed twice previous to Reside/Lifeless staining. For membrane potential measurements with CV1 dye (CytoCybernetics), cells have been incubated for 15 min in various ratios of high-potassium buffer (138 mM KCl, 2 mM NaCl, 2 mM CaCl2, 20 mM HEPES, pH 7.5) and low-potassium buffer (2 mM KCl, 138 mM NaCl, 2 mM CaCl2, 20 mM HEPES, pH 7.5), adopted by therapy with valinomycin for five min. The Nernst potential for potassium was calculated by the equation:

the place R is the gasoline fixed, T is the temperature, F is Faraday’s fixed and [K_out] is the focus of potassium within the exterior answer, assuming an intracellular potassium focus of 140 mM. Cells have been then stained with CV1 (0.5 µl of fifty µM dye in 300 µl PBS for 300,000 cells) for 1 min instantly previous to acquisition. For spectral imaging, cells have been plated in an 18-well glass-bottom chamber and imaged utilizing a Zeiss LSM 780, as described beforehand for Pro12A¹¹. Cells with JC-1 have been excited with a 488-nm laser, and 19 detectors have been set, ranging between 494 and 665 nm with 9-nm intervals. Normalized ratios ((monomer − aggregates)/(monomer + aggregates)) have been calculated utilizing wavelengths of 539 nm (monomers) and 592 nm (aggregates). For imaging of CV1, cells have been excited with wavelengths of 633 nm and picked up between 632 and 694 nm.

Drug remedies on PBMCs

PBMCs (coated by moral allow Dnr 2025-01255-01, 2025-02-20) have been remoted from 45 ml of buffy coat obtained from wholesome donors. The buffy coat was diluted 1:1 with PBS and evenly distributed into two 50-ml Falcon tubes. After thorough mixing with a serological pipette, 30 ml of the diluted pattern was fastidiously layered over 15 ml of Ficoll-Paque (Sigma-Aldrich) in three separate 50-ml tubes. Samples have been centrifuged at 400g for 25 min with out making use of the brake. The PBMC layer was collected and transferred into contemporary tubes, adopted by two PBS washes (300g, 5 min, brake on). After the ultimate wash, cells have been resuspended in 20 ml PBS, filtered by means of a 70-µm cell strainer (Thermo Fisher), and centrifuged at 200g for 10 min. The supernatant was discarded, and the cell pellet was resuspended in cryopreservation medium (90% FBS, 10% DMSO). Cells have been aliquoted (1 ml per vial) and saved at –150 °C till use.

For experiments, frozen PBMCs have been thawed by gradual addition of a 10-fold quantity of prewarmed RPMI 1640 medium supplemented with 10% FBS, adopted by centrifugation at 400g for six min. Cells have been processed in Eppendorf tubes at a last focus of 6 × 10⁵ cells per situation. For mitochondrial depolarization assays, cells have been first incubated with JC-1 dye for 30 min at 37 °C, adopted by two PBS washes. Cells have been then handled with CCCP for five min to induce mitochondrial depolarization and washed twice with PBS. For membrane order evaluation, cells have been handled with sphingomyelinase (Sigma-Aldrich) for 1 h at 37 °C and subsequently washed twice with PBS. Whereas antibody staining was carried out after remedies and JC-1 labelling for mitochondrial depolarization, it was carried out earlier than Pro12A or/and CV1 labelling.

Following remedies, cells and their untreated controls have been used to organize each single-stained controls and multicolour antibody panels for spectral move cytometry. Antibody titrations have been optimized primarily based on staining index calculations. Every pattern was both unstained, stained with a single antibody or probe (for spectral unmixing) or with a full antibody cocktail along with probe(s) in staining buffer (Thermo Fisher). Staining was carried out at 4 °C at midnight for 30 min, adopted by two PBS washes. Cells have been then resuspended in 500 µl PBS, and viability was assessed by including 1 µl of a Reside/Lifeless dye (1:100 dilution), incubating for 15 min at room temperature at midnight, adopted by two last PBS washes. Cells have been then resuspended in 300 µl PBS in move tubes for acquisition.

For Pro12A-only experiments, PBMCs have been stained with the next antibodies: anti-human CD3-PerCP-Cy5.5 (clone SK7, BioLegend), CD56-PerCP-eFluor 710 (clone CMSSB, Thermo Fisher), CD4-NovaFluor Yellow 660 (clone SK3, Thermo Fisher), CD197 (CCR7)-PE-Vio770 (clone REA108|TG8, Miltenyi Biotec), CD14-APC (clone M5E2, BioLegend), CD19-Alexa Fluor 647 (clone HIB19, BioLegend), CD11c-APC-R700 (clone 3.9, BD Biosciences), CD45RA-APC-H7 (clone HI100, BD Biosciences), CD8a-APC-Hearth 810 (clone SK1, BioLegend) and Fixable Viability Stain 570 (BD Biosciences). For JC-1-only and Pro12A–JC-1 mixture experiments, the next antibodies have been used: CD3-PerCP-Cy5.5, CD56-PerCP-eFluor 710, CD4-NovaFluor Yellow 660, CD197 (CCR7)-PE-Vio770, CD14-APC, CD11c-APC-R700, CD45RA-APC-H7, CD8a-APC-Hearth 810 and Fixable Viability Stain 660 (BD Biosciences). For CV1-only and Pro12A–JC-1–CV1 mixture experiments, the antibody panel included: CD56-PerCP-eFluor 710, CD19-Alexa Fluor 647, CD45RA-APC-H7 and CD8a-APC-Hearth 810.

PBMCs from people with atherosclerosis

This examine was accepted by the Lokman Hekim College Scientific Analysis Ethics Committee (approval quantity 2024/46; approval date 30 January 2024) and by Ethics Overview Authority Stockholm (Dnr 2025-02560-01, 2025-04-11). Written knowledgeable consent was obtained from donors previous to the examine. All procedures involving human members adhered to the moral ideas outlined within the 1964 Helsinki Declaration and its subsequent amendments or equal moral requirements. PBMC samples have been obtained from sufferers with angiographically confirmed coronary artery illness, however with out prior myocardial infarction, as a part of the inclusion standards. Intercourse- and age-matched wholesome topics had no recognized heart problems or drug therapy for it, had by no means smoked, had no historical past of diabetes and had no household historical past of most cancers. Complete blood from 38 sufferers and 26 wholesome controls was drawn into EDTA tubes (Becton Dickinson) and PBMCs have been remoted instantly. No statistical strategies have been used to predetermine pattern sizes. The samples weren’t randomized, and knowledge assortment and evaluation weren’t carried out blind to the circumstances of the experiments to have the ability to measure the identical variety of wholesome and affected person materials on every day. No donors have been excluded. Peripheral blood was diluted 1:1 with 1× DPBS and thoroughly layered over an equal quantity of Ficoll-Paque answer in a 50-ml conical tube, tilted at ∼45°, to take care of part separation. The tubes have been centrifuged at 400g for 30 min at room temperature with minimal acceleration and no brake to protect interface integrity. The mononuclear cell layer (buffy coat) was collected, washed sequentially with 1× DPBS and IMDM medium (Thermo Fisher), and centrifuged at 300g for 10 min between washes. After the ultimate wash, cells have been cryopreserved at 1 × 10⁶ cells per cryovial in freezing medium (90% FBS + 10% DMSO). The cryovials have been cooled at a charge of 1 °C min⁻¹ in a Mr Frosty and saved in a single day at −86 °C. All vials have been subsequently transferred to liquid nitrogen till evaluation. The dealing with of the PBMCs and the evaluation pipeline for membrane order and mitochondrial depolarization with Pro12A and JC-1 is described above.

SBC measurements and evaluation

Spectral move cytometry knowledge obtained with a Cytek Aurora (Supplementary Information 1) have been analysed utilizing FCS Categorical 7 (DeNovo Software program), and downstream computational analyses have been carried out utilizing Python-based pipelines. Spectral unmixing was carried out inside FCS Categorical utilizing unstained, autofluorescence for lymphocytes and monocytes, single-stained controls for every fluorochrome, and probes to precisely resolve sign overlap throughout channels. Spectral signatures have been fastidiously curated and reviewed to attenuate spillovers and preserve optimum separation between fluorochromes.

To establish the optimum fluorescence channel pair for separating experimental circumstances, we computed ratiometric values for all dual-channel combos. Every pair was evaluated utilizing the Kolmogorov–Smirnov statistic and absolutely the distinction in median ratios. An general rating combining each metrics was used to rank channel pairs, with changes for top intragroup variance. The highest-ranking pair was chosen as probably the most discriminative along with excitation/emission properties of the probes. For BSLBs stained with Pro12A or NR12S, V1–V9 (428–598 nm) and YG2–YG6 (598–697 nm) channels have been used, respectively. For mobile knowledge, Pro12A emission was captured in V1–V7 (428–542 nm), JC-1 in B3–B5 (542–598 nm) and CV1 in B7–R5 (661–738 nm). When Pro12A and JC-1 have been coincubated, V1–V5 (428–508 nm) have been chosen for membrane order quantification. When all probes have been mixed (Pro12A, JC-1, CV1), V1–V5 have been used for Pro12A and B9–R5 (697–738 nm) for CV1. ROUT 1% outlier was utilized to get rid of instrument-related faulty or disproportionately massive values. Gating methods for all circumstances, together with singlet discrimination, avoiding internalized probes, viability gating, and main immune lineage identification, are detailed (Supplementary Figs. 10–15).

To discover high-dimensional spectral options and visualize population-level variations, UMAP was used for dimensionality discount. Uncooked fluorescence depth values have been first normalized on a per-cell foundation by dividing every channel’s worth by the utmost depth throughout all channels for that cell. This per-cell normalization preserved the relative spectral composition whereas mitigating absolute depth variability. The intensities have been then normalized utilizing StandardScaler (scikit-learn), remodeling every function to have zero imply and unit variance. UMAP was applied utilizing the umap-learn package deal with parameters tuned to steadiness international and native construction preservation (for instance, n_neighbors, min_dist). These parameters have been chosen to take care of separation between biologically significant subpopulations whereas preserving finer construction within the knowledge. For classification and predictive modelling duties, we utilized Excessive Gradient Boosting (XGBoost) by way of the xgboost Python library. Previous to modelling, enter knowledge have been scaled utilizing StandardScaler and labels have been numerically encoded utilizing LabelEncoder (scikit-learn). Bayesian hyperparameter optimization was carried out utilizing the skopt package deal (Bayesian optimization with Gaussian Processes), with search house definitions spanning key XGBoost hyperparameters (for instance, learning_rate, n_estimators, max_depth, subsample, colsample_bytree, reg_alpha, reg_lambda, gamma, min_child_weight). Mannequin efficiency was evaluated utilizing stratified ok-fold cross-validation (sometimes 10-fold; 5-fold for small pattern units), with classification accuracy and confusion matrices.

scRNA-seq of biophysically chosen PBMCs

PBMC samples (4 atherosclerosis and 4 sex- and age-matched wholesome controls (A. Wholesome)) have been processed for scRNA-seq utilizing the 10x Genomics Chromium Single Cell 3′ v.4 platform (Supplementary Information 2), following the producer’s protocol. Briefly, after thawing and washing, cells have been resuspended in PBS containing 0.04% BSA, counted and assessed for viability. Cell suspensions have been loaded into the Chromium Controller to generate gel bead-in-emulsions, adopted by reverse transcription, cDNA amplification and library building utilizing the Single Cell 3′ v.4 Reagent Equipment. Remaining libraries have been quality-checked utilizing an Agilent Bioanalyzer and quantified by quantitative polymerase chain response. Sequencing was carried out on an Illumina NovaSeq X Plus system (NovaSeq X Collection Management Software program v.1.2.2.48004) utilizing a 151 nucleotide (nt) (learn 1) – 10 nt (index 1) – 10 nt (index 2) – 151 nt (learn 2) configuration on a ‘25B’ mode move cell.

Sequencing knowledge have been processed with Cell Ranger v.9.0.1 utilizing the GRCh38-2024-A reference genome to generate filtered gene-barcode matrices. Downstream evaluation was carried out in R v.4.4.3 utilizing the Seurat v.5.2.1 pipeline²⁶. Briefly, customary high quality management excluded cells with >20% mitochondrial content material or MALAT1, mitochondrial (MT-), ribosomal (RPS, RPL), and haemoglobin (HB) genes have been additionally excluded. Pattern-specific anticipated doublet charges (starting from 1.2% to 7.2%) have been utilized utilizing DoubletFinder to establish and exclude potential doublets. The ultimate dataset was normalized, scaled and subjected to PCA and UMAP for dimensionality discount, adopted by graph-based clustering utilizing the Louvain algorithm and GSEA-informed cell-type prediction. To deal with immune dynamics, we particularly chosen T cells and redefined identities primarily based on illness situation (Atherosclerosis versus A. Wholesome). Differential gene expression evaluation was carried out utilizing the Wilcoxon rank-sum check with conservative filtering (logfc.threshold = 0.25, min.pct = 0.15), figuring out genes considerably up- or downregulated within the illness context. These gene units have been analysed for pathway enrichment utilizing the GO Organic Course of 2023 database by way of enrichr. Enrichment outcomes have been filtered for pathways containing key phrases related to lipids and mitochondria and visualized with −log₁₀(P worth) bar plots to focus on condition-specific adjustments in T-cell biology.

Mass spectrometry of biophysically chosen PBMCs

We carried out direct infusion shotgun lipidomics evaluation on chosen atherosclerosis (n = 4) and sex- and age-matched wholesome management (n = 4) samples, utilizing the identical donor materials as for the scRNA-seq evaluation. Previous to lipidomics, main human T cells have been remoted from PBMCs by way of adverse magnetic choice utilizing the MojoSort Human CD3 T Cell Isolation Equipment (BioLegend), following the producer’s protocol. The ensuing cell populations exhibited >90% purity. Cells have been counted utilizing a BioRad TC20 cell counter and assessed for viability with trypan blue staining. Subsequently, cells have been washed twice with PBS, adopted by a last wash with 140 mM ammonium formate. Cell pellets have been then snap-frozen and saved −80 °C till the evaluation. Previous to sampling, the cells have been quickly thawed and lysed utilizing methanol:H₂O 9:1 v/v (0.1% formic acid), focusing on a last cell focus of 1000 cells µl⁻¹ in every pattern. The cell lysates have been centrifuged for 1 min at 8,000 r.p.m., and the supernatant was instantly analysed by immersing the direct infusion probe²⁷ within the solvent. The electrospray voltage was instantly utilized on the metal capillary and was set to 2,150 V. Mass spectrometry was carried out in positive-ion mode on an Orbitrap IQ-X (Thermo Fisher Scientific) instrument the place the AGC goal was 40% and scans have been acquired at 240,000 decision (at m/z 200) within the mass vary of m/z 250–1,200. For every measurement, a 1-min-long direct infusion was carried out, comparable to 120 particular person mass spectra on common. The acquired RAW knowledge information have been transformed to .mzML utilizing MSConvertGUI (ProteoWizard, v.3.0.22285). Analytes have been recognized primarily based on correct mass match (3 ppm tolerance) to our in-house library utilizing custom-made MATLAB scripts and the total-ion-current-normalized depth of molecular options have been extracted from every spectrum and averaged by means of all scan occasions. Subsequent, the identities of detected molecules have been confirmed utilizing tandem mass spectrometry measurements on pooled samples. Particularly, we pooled equal volumes of all 4 management and all 4 atherosclerotic samples after which carried out higher-energy collisional dissociation fragmentation utilizing normalized collisional energies of 30, 40 and 50. All tandem mass spectrometry spectra have been manually curated for structural annotation, and within the case of a number of adduct types of the identical molecular function, solely the best depth one was used for additional knowledge evaluation. Sparse partial least squares (PLS) discriminant evaluation was carried out to establish options distinguishing between atherosclerosis and wholesome T-cell lipidomes. Lipid depth values have been standardized first and the highest 30 discriminative options have been chosen utilizing univariate evaluation of variance (ANOVA) (SelectKBest, f_classif). PLS discriminant evaluation was carried out utilizing two elements, and the defined variance per element was calculated. Analyses have been carried out utilizing Python’s scikit-learn and matplotlib libraries.

Reporting abstract

Additional info on analysis design is on the market within the Nature Portfolio Reporting Abstract linked to this text.