Polystyrene beads are broadly applied in flow cytometry. Implementing bead-based assays in mass cytometry is desired but hampered by the lack of an elemental label required for their detection. In this study, we introduce stable osmium tetroxide labeling as a universal approach for generating functionalized beads readily detectable by mass cytometry. We demonstrate the utility of osmium-labeled beads for signal spillover compensation in mass cytometry, and, strikingly, their application in quantitative Ab-binding capacity assays combined with high-dimensional profiling of human PBMC enabled the systematic assessment of receptor expression profiles across large numbers of cellular phenotypes. This analysis confirmed increased monocytic Siglec-1 expression in active systemic lupus erythematosus patients and, additionally, revealed interrelated reductions of CD4 expression by regulatory and memory CD4 T cells and HLA-DR expression by myeloid dendritic cells, pointing toward defective cross-talk at the immunological synapse that may limit immune responses in systemic lupus erythematosus. By converting conventional flow cytometry beads into beads suitable for mass cytometry, our approach paves the way toward the broad implementation of bead-based assays in high-dimensional cell profiling studies by mass cytometry in biomedical research.

This article is featured in In This Issue, p.2821

Flow and mass cytometry are employed to characterize complex cellular systems with single-cell resolution. In conventional, fluorescence-based flow cytometry, small nanoscale-sized microspheres (beads) are routinely used as a reference to assist in instrument setup and performance tracking. Beads equipped with Abs on their surface, (i.e., functionalized beads) are used in a variety of tools and assays for data curation and gaining additional information from the acquired cell samples. For example, compensation beads with Ab capture functionality are used to assess fluorescence spillover for subsequent correction of raw data (commonly termed compensation) (1). Likewise, quantification beads, such as Quantum Simply Cellular (QSC) beads, equipped with known, gradually increasing amounts of Ab-binding sites capturing a particular, receptor-specific, fluorescence-tagged Ab of interest, serve as reference for approximating the absolute number of Ab targets on the cell surface (Ab-binding capacity; ABC) (2). In other assays, beads serve as a spike-in reference for normalizing cell counts (e.g., in the BD Trucount assay) (3), as the solid phase for multiplexed ELISAs implemented in the Luminex platform or the cytometric bead assay (4) or in phagocytosis assays (5).

In conventional flow cytometry, functionalized beads are detected based on their distinctive light scatter properties and/or inherent fluorescence, whereas the fluorescence of the captured Ab conjugates is usually determined in an independent cytometric channel. Although the application of such bead-based assays in mass cytometry is highly desired, the adoption of respective applications from flow cytometry is challenged by the fundamentally different means of signal detection. Mass cytometry has no analog to light scatter, and all particles to be detected by mass cytometry have to be associated to a minimum amount of at least one element or isotope within the mass cytometer’s detection range for their recognition. Particles not or insufficiently labeled with such elements are not reliably detected. Consequently, beads designed for flow cytometry assays are per se not detectable by mass cytometry, and functionalized beads equipped with an inherent metal signal are commercially not available. In contrast, metal-containing europium or normalization beads, regularly used in mass cytometry, are not functionalized and not suitable for immunoassays (68). Nevertheless, functionalized beads intended for fluorescence-based flow cytometry have been employed in mass cytometry (i.e., for quality control of Ab conjugates and compensation) (811), in which, however, the detection of the beads relied on the signal elicited by the captured metal-conjugated Ab. In this study, we exemplify that this approach can lead to selective detection or ignorance of beads by the mass cytometer, which may importantly confound the readout signal. To overcome this limitation, we introduce labeling of functionalized beads with the tetroxide of osmium, a polyisotopic element with an average mass of 190 Da, enabling their detection in mass cytometry. We exemplify the application of osmium-labeled beads for compensation of signal spillover and for combining the power of high-dimensional immune profiling by mass cytometry with the quantification of the cell surface receptors, providing new insight into immune aberrations in patients with the autoimmune disease systemic lupus erythematosus (SLE).

Millipore-filtered, deionized water was used as CyTOF sample carrier and to prepare 1× PBS from 10× PBS (Rockland Immunochemicals, Gilbertsville, PA; pH was adjusted to 7.4). CyTOF staining medium (CSM) was prepared from 1× PBS supplemented with 0.5% (w/v) BSA (PAN-Biotech, Aidenbach, Germany) and 0.02% sodium azide (Sigma-Aldrich, St. Louis, MO). Buffers were filtered through 0.22-μm membranes (EMD Millipore, Billerica, MA) and stored in Stericup single-use bottles (EMD Millipore) at 4°C. Four percent (weight per volume) osmium tetroxide (OsO4) solution was prepared from crystalline OsO4 (no. 19100; Science Services, Munich, Germany) and distilled water. The stock solution was diluted in 1× PBS to generate OsO4 solutions ranging from 0.01 to 0.0001%. Stocks were stored in brown glass vials at −80°C, working solutions were stored in brown glass vials at −20°C. Native Abs and Ab conjugated to lanthanides or fluorochromes (Supplemental Table I) were purchased from Fluidigm (South San Francisco, CA), BioLegend (San Diego, CA), eBioscience (San Diego, CA), Miltenyi Biotec (Bergisch-Gladbach, Germany), and Novus Scientific AB (Uppsala, Sweden) or were produced in house (German Rheumatism Research Centre Berlin, Berlin, Germany). Rituximab was kindly provided by Dr. B. Hoyer, Charité Berlin. In-house Ab conjugations with lanthanide and indium isotopes were carried out using Maxpar X8 labeling kits (Fluidigm) according to the manufacturer’s instructions. Platinum or palladium labeling was performed as described previously (10, 12). Highly isotopically enriched metal salts not available through Fluidigm were purchased from Trace Sciences (Richmond Hill, ON, Canada). Cisplatins carrying isotopically enriched Pt were purchased from or kindly provided by Fluidigm. Cell events were detected using an iridium-containing DNA intercalator (Fluidigm). mDOTA-103Rh, used for discrimination of dead cells, was prepared from DOTA-maleimide (Macrocyclics, Dallas, TX) and rhodium chloride (Sigma-Aldrich) as described previously (13) and stored at 4°C. Polyisotopic cisplatin (Enzo Life Sciences, Lörrach, Germany), alternatively used for discrimination of dead cells, was dissolved at 25 mM in DMSO and stored at −20°C.

Polystyrene beads were aliquoted dropwise into new 1.5-ml reaction vials (Safe Seal; Sarstedt, Nümbrecht, Germany) from homogenously dispersed bead stocks. In experiments comparing numbers of acquired beads, equal numbers of drops were used per aliquot. Prior to OsO4 staining, polystyrene beads were pelleted by centrifugation (according to the individual manufacturers’ recommendations, 1500 – 3000 × g) and storage buffer was removed by aspiration. For osmium staining, beads were resuspended in 20 μl of 0.01–0.0001% OsO4 solution per 1 × 106 beads and incubated for 30 min at room temperature (RT) in 1.5-ml reaction tubes (Safe Seal). After incubation, 1 ml of CSM was added, beads were pelleted, and supernatant was discarded (“washing”). Beads were washed at least three times with 1 ml of CSM to remove unbound OsO4. The beads were then available for further protocols or resuspended in Millipore water for direct acquisition at the CyTOF instrument.

OsO4 is a hazardous chemical that requires specific safety measures to avoid skin and eye contact and aspiration, such as working in a fume hood. We suggest discussing the osmium-labeling procedure and waste disposal with the laboratory safety officer to implement safety precautions meeting the individual laboratories’ safety requirements. All work involving fresh/unbound OsO4 was conducted in a chemical hood. The resulting OsO4-labeled beads were further processed in a normal laboratory environment.

Peripheral blood samples were drawn by venipuncture from healthy individuals and patients diagnosed with SLE or inclusion body myositis (Table I) under approval by the local ethic committees in accordance with the Helsinki II Declaration (approval identifier EA1/132/116). All donors gave written informed consent prior to blood donation. For some experiments, PBMC were isolated from buffy coats obtained from the Institute of Transfusion Medicine, Charité University Medicine, Berlin, Germany.

PBMCs were prepared from anticoagulated whole blood samples by density gradient centrifugation using Ficoll (800 × g, 20 min; GE Healthcare; Chicago, IL). PBMC were subsequently washed twice in 45 ml of CSM, counted by volumetric flow cytometry (MACSQuant; Miltenyi Biotec), cryopreserved in freezing medium (10% DMSO [Sigma-Aldrich]/90% FCS [Biowest, Nuaillé, France]) and stored in liquid nitrogen gas phase until use. For each experiment, PBMC were gently thawed at RT, transferred to 50 ml of prewarmed (37°C) RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FCS and 5 U/ml benzonase (Sigma-Aldrich). To counteract metabolic activity, PBMC were immediately pelleted for 10 min at 500 × g and 4°C and washed once in 15 ml of cool PBS (500 × g, 4°C), followed by volumetric cell counting by flow cytometry. Finally, PBMC were resuspended in CSM and kept on ice for further use.

Ab–metal conjugates were used at concentrations optimized to separate cell population expressing or lacking a particular Ag. Ab conjugates applied in ABC assays were used at saturating conditions, which were determined by escalating the Ab concentration in the staining reaction. The Ab concentration at which the maximum separation between stained and unstained cell populations was observed, while not increasing background staining, was considered as optimal [QSC Manual, Bangs Laboratories (14)]. Ab mixtures were prepared fresh and kept on ice until use or were cryopreserved at −80°C. For experiments involving multiple samples of different donors, live-cell barcoding was performed before staining of cell surface Ags using a set of palladium and platinum isotope-labeled β2-microglobulin (B2M) Abs (10, 12). For live-cell barcoding, PBMC were incubated for 30 min at 4°C with combinations of B2M conjugates in a 5-choose-2 barcode scheme. Next, the barcoded cells were washed four times with 1.2 ml CSM, pooled and further processed together. For staining cell surface Ags by isotope-tagged Abs, up to 2 × 106 PBMC were resuspended in 50 μl Ab mixture diluted in CSM and incubated for 30 min at 4°C in 1.5 ml reaction vials. The cell pellet (500 × g, 4°C, 5 min), was resuspended and incubated in 1 ml 10 μM mDOTA-103Rh solution in PBS for 5 min at RT, to label dead cells for their later exclusion. The sample volume was filled up to 1500 μl with CSM followed by cell pelleting and supernatant aspiration. Next, the cells were washed once with 1 ml CSM, once with 1 ml PBS, and finally resuspended in 2% paraformaldehyde (PFA) solution (diluted from 16% stock with PBS; Electron Microscopy Sciences, Hatfield, PA) and incubated at 4°C overnight. On the next day, 500 μl CSM was added, cells were pelleted (700 × g, 5 min, 4°C) and subsequently washed once with 1 ml CSM. Next, the samples were incubated for 25 min at RT in 500 μl 1 × permeabilization buffer (diluted with Millipore water from 10 × permeabilization buffer; Thermo Fisher Scientific) supplemented 1:500 v/v with 0.125 μM iridium-based DNA intercalator (Fluidigm). Cells were then washed with 1 ml CSM. Thereafter, samples were resuspended in 1 ml PBS and counted by flow cytometry. Prior to acquisition on the CyTOF instrument, samples were washed twice with 1 ml Millipore water and pelleted by centrifugation at 800 × g, 5 min, 4°C. Cells were then resuspended in an appropriate volume of water to a maximum of 5 – 7.5 × 105 cells/ml supplemented with EQ Four Element beads (1:10, v/v) (Fluidigm) and were optionally filtered through 35 μm cell strainer cap tubes (BD, San Jose, CA) prior to acquisition.

For flow cytometry, up to 2 × 106 PBMC were resuspended in 50 μl of fluorescent Ab mixture diluted in CSM. Assay concentrations of Ab conjugates intended for ABC assays were optimized to saturate their respective Ag without increasing the nonspecific background staining. Cell staining was performed in 1.5-ml reaction vials (30 min, 4°C), washed once with 1 ml of CSM, and pelleted (500 × g, 5 min). Directly before acquisition on the flow cytometer, DAPI (Sigma-Aldrich) was added to label dead cells for their latter exclusion by gating. The data were acquired on a MACSQuant flow cytometer (Miltenyi Biotec).

Mass cytometry was performed on CyTOF version 1 [operated as described before (9, 15)] and Helios instruments (Fluidigm). Instruments were daily prepared for acquisition by tuning and cleaning according to the manufacturer’s advice, using tuning and cleaning solutions (Fluidigm). Data of cells or bead suspensions were acquired in cell acquisition mode at event densities of <5 × 105 events/ml for CyTOF v1 and <7.5 × 105 events/ml for the Helios instrument.

The sample supply was set to 45 μl/min for CyTOF version 1, or to 30 μl/min for the Helios instrument. Both instruments were run in dual calibration mode, with noise reduction turned on and event length thresholds set to 10 and 75. The lower event length limit was set to three in some experiments where indicated. The lower convolution threshold was kept at the default value of 300.

OsO4-labeled beads were detectable at atomic mass channels 186, 187, 188, 189, 190, and 192, and were typically evaluated or gated according to signal in the channel with the highest isotopic abundance (41%, 192Os). After acquisition of more than 50 osmium-labeled bead samples, osmium signals typically returned to baseline levels after routine washing with Fluidigm washing solution, HNO3, and water. At a single instance, additional injections of 3% HNO3 were required to wash out remaining osmium.

For Ab capturing assays, aliquots of up to 2 × 106 Ab capture beads from different vendors labeled or not with OsO4 were stained in 50 μl of CSM containing a single Ab conjugate (1:50, v/v) for 30 min at 4°C. Next, beads were washed three times with 1 ml CSM and finally resuspended and incubated in 500 μl 2% PFA solution in PBS overnight at 4°C. Fixation was stopped by adding 1 ml of CSM followed by centrifugation and aspiration of supernatant. Finally, beads were washed twice in 1 ml of CSM, once in 1 ml of PBS, and twice in 1 ml of Millipore water before acquisition. Unless otherwise indicated, BD CompBeads (anti-mouse Ig, anti-rat Ig) were applied for compensation (Supplemental Table I). For the experiment shown in Fig. 3, a large batch of beads was labeled with OsO4, aliquoted into cavities of a 96-well Deep Well Plate (Corning, Corning, NY), and incubated with individual Ab conjugates (Supplemental Table I). After loading of beads with Ab conjugates and washing, bead aliquots were pooled and either stored at −80°C (Supplemental Fig. 1B) or directly combined with cell suspensions previously stained with Intercalator-Ir and the same set of isotope-tagged Abs (1:4, v/v) for simultaneous acquisition.

For absolute quantification experiments (ABC assays), QSC quantification beads (Bangs Laboratories, Fisher, IN) were implemented according to the manufacturer’s instructions after labeling with OsO4. To distinguish different bead population in mass cytometry data, OsO4 labeling was performed with the following different concentrations of OsO4 to achieve distinct osmium signal intensities: bead 0, 0.01% OsO4; bead 1, 0.0075% OsO4; bead 2, 0.005% OsO4; bead 3, 0.0025% OsO4; and bead 4, 0.001% OsO4. After OsO4 labeling, beads (except bead 0) were incubated with the Ab–metal conjugate targeting the cellular receptor of interest for 30 min at 4°C. The amount of applied Ab conjugate was determined by escalating the Ab conjugate concentration to achieve target saturation. For this, the concentration at which the maximum cytometric separation of stained bead 4 from unstained beads in the channel of interest, while not increasing background, was considered optimal and applied to beads 1–4 in the ABC assay. After incubation, the supernatant was removed by centrifugation (2500 × g, 5 min). Different from Ab capture beads used for compensation, QSC beads were not exposed to PFA to mirror conditions applied to the beads in the flow cytometry–based ABC assays. QSC beads were always prepared freshly at the day of the experiment. For acquisition, the different bead populations were pooled and in some cases combined with PBMC and prepared for mass cytometric ABC assay (1:4, v/v).

Beads for quantification experiments performed by flow cytometry were not labeled with OsO4. The amount of Ab-fluorochrome conjugates necessary for saturation was determined in a similar manner as for mass cytometric quantification beads. The beads were processed according to the manufacturer’s instructions and acquired on a MACSQuant flow cytometer (Miltenyi Biotec).

Raw mass cytometry data were converted to Flow Cytometry Standard 3.0 files during acquisition. Data were normalized based on EQ Calibration Bead signals using CyTOF 6.7 software (Fluidigm). Flow Cytometry Standard files were analyzed using FlowJo (version 10.4; TreeStar, Ashland, OR), Cytobank Premium (http://www.cytobank.org) and CytoSplore (16). Data from beads and cells were distinguished according to their osmium (192Os at CyTOF 1, 188Os at Helios) and iridium (191/193Ir) signal. The retrieval of single Ab conjugate–positive Os+ beads was achieved by manual gating or by gating in t-distributed stochastic neighbor embedding (t-SNE) plots (17, 18). The Catalyst algorithm was used for calculating spillover matrices; channels occupied by osmium isotopes were not considered in this analysis, and bead data were downsampled to <15,000 events (11). Manual gating of PBMC subsets is represented in Supplemental Fig. 2. Statistical analyses were performed with GraphPad Prism (version 5.4; GraphPad, San Diego, CA). Receptor quantifications were based on median signal intensities of manually gated cell and bead populations or FlowSOM clusters.

For FlowSOM clustering, each Flow Cytometry Standard file containing data of live, single CD45+ cells was loaded into R (R version 3.5.1) in RStudio (version 1.1.463) using the flowCore Bioconductor package, and, in particular, the flowSet data structure, as it was done before in the CyTOF workflow Bioconductor package (19). Raw expression data were first arcsinh-transformed with a scale argument of five, as is typical for mass cytometry data preprocessing (20). Combined preprocessed data were clustered using FlowSOM (21), based on all markers listed in Panel C1 (Supplemental Table I) except CD45, B2M, CCR6, CD4, Siglec-1, and HLA-DR and MetaClustered as described before (22). We used the default 10 × 10 self-organizing map to generate 100 clusters, followed by consensus hierarchical MetaClustering down to 60 clusters. For both these functions, we used the FlowSOM Bioconductor package. The clustered data were converted into a cluster frequency table, in which the rows are clusters 1–60, the columns represent individual samples, and each element is the sample-specific percentage of CD45+ cells residing within that cluster. From this frequency table, Mann–Whitney U tests were performed between sample groups of interest. The resulting p values were adjusted for multiple comparisons using the Benjamini­–Hochberg procedure within the R Stats package. The frequency table and the median marker expressions per cluster were plotted as heatmaps using the pheatmap function in R.

First, the feasibility of labeling beads with natural abundance OsO4 solution was tested to create a general bead identification signal suitable for mass cytometry that is applicable to commercially available polystyrene beads. Osmium labeling was achieved by incubating Ab capture beads with OsO4 diluted in PBS for 30 min at RT, followed by the removal of unbound OsO4 by washing with PBS. Osmium-labeled beads were readily detectable by the mass cytometer, based on their uniform osmium labeling and independent of the captured Ab conjugate, exhibited no background in non-osmium channels and, at most, minimal background in osmium isotope channels in spectra recorded between the passing of beads (Fig. 1A). The distinct osmium signal enabled the clear discrimination of the OsO4-labeled beads from 4-element normalization beads or Ir–­DNA intercalator–labeled cells recorded simultaneously (Fig. 1B, Supplemental Fig. 2). Osmium staining solutions with concentrations of up to 0.01% OsO4 were evaluated for the labeling of beads. Only the incubation of beads with solutions containing at least 0.001% OsO4 was sufficient to generate a bead-associated osmium signal with event length values >10, securing their reliable detection with default acquisition settings on the mass cytometer (Figs. 1C, 2B). Hence, for all the following experiments, 0.005–0.01% OsO4 staining solutions were used unless indicated differently. Osmium-labeled beads and their osmium signal were stable for at least 4 wk when stored in CSM at −80°C or at 4°C (Fig. 1D).

FIGURE 1.

Osmium tetroxide labeling facilitates detection of functionalized polystyrene beads by mass cytometry. (A) Representative acquisition view (“rain plot”) of an OsO4-labeled Ab capture bead eliciting signals in all channels detecting osmium isotopes with a natural abundance >1% (186Os, 187Os, 184Os, 190Os, and 192Os) and the expected signal of the captured CD14–148Nd conjugate. More than 50 samples containing Os-labeled beads were acquired throughout the study. (B) OsO4-labeled Ab capture beads were clearly discriminable in a mixture with EQ Four Element Normalization Beads. No significant carry-over of osmium to normalization beads was observed. All combined cell/osmium-labeled bead samples acquired in this study were spiked with EQ Beads prior to data acquisition; a representative analysis is shown. (C) A concentration of at least 0.001% OsO4 in the staining solution is required to label beads with sufficient amounts of Os for detection by mass cytometry. Median signal intensities (MSI) are indicated. All beads captured a CD3–170Er conjugate. The experiment was performed twice with an anti-CD4–144Nd, and an anti-CD3–170Er Ab conjugate. The dataset for the CD3 conjugate is shown. (D) The OsO4 label on Ab capture beads is stable for at least 4 wk when stored at −80 or 4°C. The experiment was performed once. A single batch of capture beads was labeled with 0.01% OsO4 solution and acquired directly or after storage, as indicated. CV, coefficient of variation.

FIGURE 1.

Osmium tetroxide labeling facilitates detection of functionalized polystyrene beads by mass cytometry. (A) Representative acquisition view (“rain plot”) of an OsO4-labeled Ab capture bead eliciting signals in all channels detecting osmium isotopes with a natural abundance >1% (186Os, 187Os, 184Os, 190Os, and 192Os) and the expected signal of the captured CD14–148Nd conjugate. More than 50 samples containing Os-labeled beads were acquired throughout the study. (B) OsO4-labeled Ab capture beads were clearly discriminable in a mixture with EQ Four Element Normalization Beads. No significant carry-over of osmium to normalization beads was observed. All combined cell/osmium-labeled bead samples acquired in this study were spiked with EQ Beads prior to data acquisition; a representative analysis is shown. (C) A concentration of at least 0.001% OsO4 in the staining solution is required to label beads with sufficient amounts of Os for detection by mass cytometry. Median signal intensities (MSI) are indicated. All beads captured a CD3–170Er conjugate. The experiment was performed twice with an anti-CD4–144Nd, and an anti-CD3–170Er Ab conjugate. The dataset for the CD3 conjugate is shown. (D) The OsO4 label on Ab capture beads is stable for at least 4 wk when stored at −80 or 4°C. The experiment was performed once. A single batch of capture beads was labeled with 0.01% OsO4 solution and acquired directly or after storage, as indicated. CV, coefficient of variation.

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FIGURE 2.

Osmium labeling of beads is critical for achieving representative readouts from captured Ab conjugates. (A) The impact of the sequence of OsO4 labeling and Ab capturing on signal intensity of the captured Ab conjugate was analyzed. In comparison with beads not labeled with OsO4, Ab capturing after OsO4 labeling had little if any impact on the CD3–170Er signal. When the Ab capturing was followed by the OsO4 labeling, median signal intensities (MSI) of the Ab conjugate was ∼2-fold increased. Similar data were obtained in a second experiment with an anti-CD4–144Nd Ab conjugate. (B) Palladium, lanthanide, and platinum Ab conjugates, representing three different conjugation strategies, were successfully captured by OsO4-labeled Ab capture beads. Bead event length values, signals of the captured Ab conjugates, and numbers of beads detected (only available for Pd and Nd conjugates) were determined for beads that were labeled with OsO4 (green), or not (gray). Omitting the OsO4 labeling led to critically low event length values of the beads, equal to or only slightly higher than the event length cut-off value (set to three in this experiment) and consistently incomplete detection of the bead samples. The lower event length cut-off value of 10, typically used in routine mass cytometry data acquisitions, is indicated as a dotted line. Consistent with a selective detection of beads in the absence of the osmium label, MSI of the identical captured Ab conjugates differed depending on whether the beads were labeled with OsO4. Moreover, signals of Ab conjugates showed Gaussian distributions when derived from OsO4-labeled beads but not when derived from beads lacking the osmium label. Data of three out of five Ab conjugates is shown. NA, not available.

FIGURE 2.

Osmium labeling of beads is critical for achieving representative readouts from captured Ab conjugates. (A) The impact of the sequence of OsO4 labeling and Ab capturing on signal intensity of the captured Ab conjugate was analyzed. In comparison with beads not labeled with OsO4, Ab capturing after OsO4 labeling had little if any impact on the CD3–170Er signal. When the Ab capturing was followed by the OsO4 labeling, median signal intensities (MSI) of the Ab conjugate was ∼2-fold increased. Similar data were obtained in a second experiment with an anti-CD4–144Nd Ab conjugate. (B) Palladium, lanthanide, and platinum Ab conjugates, representing three different conjugation strategies, were successfully captured by OsO4-labeled Ab capture beads. Bead event length values, signals of the captured Ab conjugates, and numbers of beads detected (only available for Pd and Nd conjugates) were determined for beads that were labeled with OsO4 (green), or not (gray). Omitting the OsO4 labeling led to critically low event length values of the beads, equal to or only slightly higher than the event length cut-off value (set to three in this experiment) and consistently incomplete detection of the bead samples. The lower event length cut-off value of 10, typically used in routine mass cytometry data acquisitions, is indicated as a dotted line. Consistent with a selective detection of beads in the absence of the osmium label, MSI of the identical captured Ab conjugates differed depending on whether the beads were labeled with OsO4. Moreover, signals of Ab conjugates showed Gaussian distributions when derived from OsO4-labeled beads but not when derived from beads lacking the osmium label. Data of three out of five Ab conjugates is shown. NA, not available.

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In summary, OsO4 labeling converts polystyrene beads, originally intended for use in conventional flow cytometry, into beads readily applicable in mass cytometry.

Next, we confirmed that labeling beads with OsO4 had only minor, if any, impact on the Ab-capturing functionality of the beads, as evidenced by the successful capture of CD3–170Er after osmium labeling (Fig. 2A). Moreover, osmium-labeled beads not only successfully captured lanthanide Ab conjugates but also palladium Ab conjugates and platinum Ab conjugates, confirming the compatibility of osmium labeling with different types of metal Ab conjugates (Fig. 2B). Labeling beads with OsO4 after Ab capturing also preserved the signal of the captured Ab conjugate. However, as OsO4 prelabeling of Ab capture beads did not impede the captured Ab signal, provided a higher degree of flexibility for application in various assays and minimized handling of toxic OsO4, we choose to prelabel beads with OsO4 throughout all the following experiments. That strategy proved successful for polystyrene beads from multiple vendors with different capturing specificities (Supplemental Fig. 1A).

We then compared bead event length values, signals of the captured Ab conjugates, and numbers of beads detected by the CyTOF software between beads labeled with OsO4 or not. Omitting the OsO4-labeling lead to critically low event length values of the beads, equal to or only slightly higher than the event length cut-off value, even when that cut-off value was lowered to three, indicating that ion clouds of these beads did not contain sufficient amounts of ions of the mass cytometers measurement range in a sufficient number of consecutive mass spectra to trigger their detection as “events” by the CyTOF software. Consistently, bead samples were only incompletely detected in the absence of the osmium label. Less than 28% of bead events were detected in the suspension as compared with beads that were labeled with osmium (n = 2). When labeled with osmium, more than 99% of the beads exceeded an event length value of 10, typically used as a lower cut-off in routine mass cytometry sample acquisitions. Consistent with a selective, and nonrepresentative detection of beads in the absence of the osmium, median signal intensities of the captured identical Ab conjugates differed depending on whether the beads were labeled with OsO4 (reductions of 18 and 66%, gain of 36%, Fig. 2B). Moreover, histograms of Ab conjugate signal intensities showed Gaussian distributions when captured by OsO4-labeled beads, but not when captured by beads lacking osmium (Fig. 2B).

Taken together, labeling of beads with an identifier tag, such as osmium, is required for obtaining accurate results from bead-based assays. Osmium labeling was proven successful for a variety of beads and did not interfere with capturing Ab conjugates.

Recently, the concept of compensation for signal spillover between channels resulting from metal oxide formation and isotopic impurities of metal labels has been introduced to mass cytometry by Chevrier et al. (11). To generate a spillover matrix, the authors stained aliquots of Ab capture beads (compensation beads) individually with a specific Ab conjugate from a mass cytometric Ab panel, pooled them, and acquired the bead convolute separately from cells. In this study, we extend this approach using osmium-labeled Ab capture beads, which can be combined with the cell sample for joint acquisition (Fig. 3A). The osmium label discriminated the bead fraction from DNA intercalator–labeled cells, and all bead populations carrying individual Ab conjugates were detectable within the bead fraction (Fig. 3B). Catalyst (11) was applied to bead data to calculate a spillover matrix, which was used to compensate spillover effects in bead and cell data. For example, a representative Ab panel used CD20–113In with an 113In purity of 93.1% beside CD3–115In for detection of B and T cells, respectively. As expected, the majority of impurity in the 113In was explained by the presence of 115In, which resulted in an artifactual signal by B cells in the CD3–115In detection channel (calculated spillover, 6.5%). Compensation using osmium-labeled beads and Catalyst properly corrected the spillover artifact, thereby improving data accuracy and facilitating proper interpretation (Fig. 3C, 3D). Importantly, once prepared, compensation bead pools were storable at −80°C for at least 2 wk without affecting the signal intensity of the captured Ab conjugates (Supplemental Fig. 1B), simplifying its use as a routine spike-in compensation control in mass cytometry.

FIGURE 3.

Osmium labeling facilitates joint acquisition of cells and Ab capture beads for spillover correction. (A) Ab capture beads were first labeled with OsO4, split into aliquots, which were individually stained with 47 different Ab conjugates of an Ab panel for analyzing PBMC. All beads were pooled and combined with PBMC stained with the same Ab panel for acquisition by the mass cytometer. Compensation with osmium-labeled beads was performed four times; for two (B0, B1), the applied panel is indicated. (B) Osmium-labeled beads were always discriminable from nucleated, iridium–DNA intercalator–labeled cells. Os+Ir+ events represent bead/cell doublets. t-SNE graphs of all bead pools (top) and CD45+ PBMC (bottom) are shown. Colors indicate 113In or 115In signals prior to spillover correction (from blue, no signal to red, high signal). Note that the 113In preparation used in the CD20 Ab conjugate contains 6.9% 115In (according to the manufacturer), explaining the spillover detectable in the 115In channel (used for detection of CD3) at the example of beads and PBMC. Compensation with osmium-labeled beads acquired simultaneously with cells was conducted two times. We show, in this study, the spillover correction for the panel referred to as B1 (Supplemental Table I). (C) Catalyst was used to calculate a spillover matrix based on bead data acquired together with cells. After signal correction (compensation) according to the calculated spillover matrix, beads that had captured the CD20 Ab conjugate and CD20+ B cells show the expected absence of a CD3 signal, as indicated by the blue color of the indicated populations. t-SNE maps were generated based on signal of all markers except barcoding channels and CD3–115In. The data shown refer to one compensation experiment with osmium-labeled beads and cells stained with panel B1 (Supplemental Table I). (D) Bivariate plots of the same data shown in (B and C) before and after compensation.

FIGURE 3.

Osmium labeling facilitates joint acquisition of cells and Ab capture beads for spillover correction. (A) Ab capture beads were first labeled with OsO4, split into aliquots, which were individually stained with 47 different Ab conjugates of an Ab panel for analyzing PBMC. All beads were pooled and combined with PBMC stained with the same Ab panel for acquisition by the mass cytometer. Compensation with osmium-labeled beads was performed four times; for two (B0, B1), the applied panel is indicated. (B) Osmium-labeled beads were always discriminable from nucleated, iridium–DNA intercalator–labeled cells. Os+Ir+ events represent bead/cell doublets. t-SNE graphs of all bead pools (top) and CD45+ PBMC (bottom) are shown. Colors indicate 113In or 115In signals prior to spillover correction (from blue, no signal to red, high signal). Note that the 113In preparation used in the CD20 Ab conjugate contains 6.9% 115In (according to the manufacturer), explaining the spillover detectable in the 115In channel (used for detection of CD3) at the example of beads and PBMC. Compensation with osmium-labeled beads acquired simultaneously with cells was conducted two times. We show, in this study, the spillover correction for the panel referred to as B1 (Supplemental Table I). (C) Catalyst was used to calculate a spillover matrix based on bead data acquired together with cells. After signal correction (compensation) according to the calculated spillover matrix, beads that had captured the CD20 Ab conjugate and CD20+ B cells show the expected absence of a CD3 signal, as indicated by the blue color of the indicated populations. t-SNE maps were generated based on signal of all markers except barcoding channels and CD3–115In. The data shown refer to one compensation experiment with osmium-labeled beads and cells stained with panel B1 (Supplemental Table I). (D) Bivariate plots of the same data shown in (B and C) before and after compensation.

Close modal

Finally, we exemplify the utility of osmium-labeled beads for combining cell surface receptor quantification assays with high-dimensional immune cell phenotyping by mass cytometry. We analyzed five SLE-active patients and four control donors using 42-dimensional mass cytometry (Table I). After analyzing the abundance of FlowSOM clusters in SLE versus control PBMC, revealing multiple aberrations, including reduced frequencies of CD8+ T cell, CD4+ T cell and B cell clusters, and increased frequencies of a monocyte cluster (Supplemental Fig. 3G, 3H) consistent with known lymphopenia in SLE (23, 24), we used the same data to analyze the expression of four different receptors (CD4, HLA-DR, Siglec-1, and CCR6) at the level of absolute ABC quantification across all clusters in high-dimensional phenotypic space. For each target, one set of QSC beads equipped with known, gradually increasing numbers of Ab-binding sites were labeled with OsO4, incubated with the Ab conjugate, pooled, and acquired directly before acquisition of the cell sample by the mass cytometer. Bead data served as reference for the determination of ABC (Fig. 4A, 4B). Notably, calibration curves exhibited very similar slopes for the CD4 and HLA-DR assays that were performed by both flow and mass cytometry (flow cytometry: 0.9168; mass cytometry: 0.9817, Fig. 4C, Supplemental Fig. 3C). This shows that despite employing biophysically fundamentally different detection methods, the assay calibration worked comparably well in flow and mass cytometry. In addition, mass cytometric ABC assays were reliably reproducible (Supplemental Fig. 3D), confirming the robustness of the approach.

Table I.
Patient and control donor characteristics
CodeDonationGenderAgeDiseaseNo. of American College of Rheumatology CriteriaModified SLEDAI-2k (SLEDAI-2k)Anti-dsDNA (<20 U/ml)TreatmentMatched ControlBarcode NumberBarcode Metals
Pat_001 Whole blood 55 SLE 5/11 6 (6) 18.4 HCQ 200 mg/d, Pred. 5 mg/d ND_051 104Pd/106Pd 
Pat_002 Whole blood 35 SLE 6/11 10 (14) 57.9 HCQ 200 mg/d, Pred. 5 mg/d ND_050 104Pd/108Pd 
Pat_003 Whole blood 35 SLE 8/11 12 (16) >200 HCQ 200 mg/d, Pred. 20 mg/d ND_049 104Pd/110Pd 
Pat_004 Whole blood 35 SLE 7/11 6 (10) 27 MTX 15 mg/wk, Pred. 5 mg/d, HCQ 200 mg ND_049 104Pd/196Pt 
Pat_005 Whole blood 39 SLE 7/11 20 (24) >200 Pred. 30 mg/d, HCQ 200 mg/d ND_051 106Pd/108Pd 
Pat_006 Whole blood 72 IBM — — — Baricitinib  106Pd/110Pd 
ND_048 Whole blood 48 —      106Pd/196Pt 
ND_049 Whole blood 39 —      108Pd/110Pd 
ND_050 Whole blood 31 —      108Pd/196Pt 
ND_051 Whole blood 42 —      10 110Pd/196Pt 
CodeDonationGenderAgeDiseaseNo. of American College of Rheumatology CriteriaModified SLEDAI-2k (SLEDAI-2k)Anti-dsDNA (<20 U/ml)TreatmentMatched ControlBarcode NumberBarcode Metals
Pat_001 Whole blood 55 SLE 5/11 6 (6) 18.4 HCQ 200 mg/d, Pred. 5 mg/d ND_051 104Pd/106Pd 
Pat_002 Whole blood 35 SLE 6/11 10 (14) 57.9 HCQ 200 mg/d, Pred. 5 mg/d ND_050 104Pd/108Pd 
Pat_003 Whole blood 35 SLE 8/11 12 (16) >200 HCQ 200 mg/d, Pred. 20 mg/d ND_049 104Pd/110Pd 
Pat_004 Whole blood 35 SLE 7/11 6 (10) 27 MTX 15 mg/wk, Pred. 5 mg/d, HCQ 200 mg ND_049 104Pd/196Pt 
Pat_005 Whole blood 39 SLE 7/11 20 (24) >200 Pred. 30 mg/d, HCQ 200 mg/d ND_051 106Pd/108Pd 
Pat_006 Whole blood 72 IBM — — — Baricitinib  106Pd/110Pd 
ND_048 Whole blood 48 —      106Pd/196Pt 
ND_049 Whole blood 39 —      108Pd/110Pd 
ND_050 Whole blood 31 —      108Pd/196Pt 
ND_051 Whole blood 42 —      10 110Pd/196Pt 

—, does not apply.

F, female; HCQ, hydroxychloroquine; IBM, inclusion body myositis; M, male; MTX, methotrexate; Pred., prednisone.

FIGURE 4.

Application of osmium-labeled beads for absolute quantification of four cell surface receptors in high-dimensional immune cell profiles of patients with SLE and healthy controls. (A) PBMC of five SLE patients, one inclusion body myositis (IBM) patient, and four age- and gender-matched normal controls were barcoded with a set of B2M Ab conjugates, pooled, and stained with a 42-parameter Ab panel (Supplemental Table I), including CD4-114Nd (clone RPA-T4) for the determination of CD4 ABC, and acquired by the mass cytometer. Five different bead preparations with known ABC serving as reference were labeled with OsO4 at gradually decreasing concentrations and incubated with the same CD4–144Nd Ab conjugate, pooled, and acquired by the mass cytometer. Reference data for Abs targeting HLA-DR, Siglec-1, and CCR6 were obtained in the same manner. The experiment was performed once. (B) Five reference bead populations were distinguishable according to their gradually increasing CD4–144Nd and decreasing osmium signals. (C) Comparison of ABC assay calibration data obtained by reference beads loaded with the same CD4 Ab clone (RPA-T4) in flow (black; Alexa Fluor 488) and mass cytometry (blue; 144Nd). Data in (B) and (C) are representative of at least four similar analyses. (D) Quantification of CD4, CCR6, HLA-DR, and Siglec-1 ABC in FlowSOM clusters of 42-dimensional mass cytometric immune profiles of five SLE patients and four controls prepared as described in (A). For each quantitated target, clusters exceeding the indicated ABC threshold are superimposed on a t-SNE projection of the entire dataset (upper row; see Supplemental Fig. 3F, 3G for marker expression). Scatter diagrams (lower row) show ABC values of the selected clusters of the individual SLE patients (red) and healthy controls (blue) and their group medians and interquartile ranges. Clusters with significantly different ABC values (p < 0.05) between SLE patients and controls are indicated by an asterisk (Mann–Whitney U test). Color boxes match with cluster overlays in the respective t-SNE projections.

FIGURE 4.

Application of osmium-labeled beads for absolute quantification of four cell surface receptors in high-dimensional immune cell profiles of patients with SLE and healthy controls. (A) PBMC of five SLE patients, one inclusion body myositis (IBM) patient, and four age- and gender-matched normal controls were barcoded with a set of B2M Ab conjugates, pooled, and stained with a 42-parameter Ab panel (Supplemental Table I), including CD4-114Nd (clone RPA-T4) for the determination of CD4 ABC, and acquired by the mass cytometer. Five different bead preparations with known ABC serving as reference were labeled with OsO4 at gradually decreasing concentrations and incubated with the same CD4–144Nd Ab conjugate, pooled, and acquired by the mass cytometer. Reference data for Abs targeting HLA-DR, Siglec-1, and CCR6 were obtained in the same manner. The experiment was performed once. (B) Five reference bead populations were distinguishable according to their gradually increasing CD4–144Nd and decreasing osmium signals. (C) Comparison of ABC assay calibration data obtained by reference beads loaded with the same CD4 Ab clone (RPA-T4) in flow (black; Alexa Fluor 488) and mass cytometry (blue; 144Nd). Data in (B) and (C) are representative of at least four similar analyses. (D) Quantification of CD4, CCR6, HLA-DR, and Siglec-1 ABC in FlowSOM clusters of 42-dimensional mass cytometric immune profiles of five SLE patients and four controls prepared as described in (A). For each quantitated target, clusters exceeding the indicated ABC threshold are superimposed on a t-SNE projection of the entire dataset (upper row; see Supplemental Fig. 3F, 3G for marker expression). Scatter diagrams (lower row) show ABC values of the selected clusters of the individual SLE patients (red) and healthy controls (blue) and their group medians and interquartile ranges. Clusters with significantly different ABC values (p < 0.05) between SLE patients and controls are indicated by an asterisk (Mann–Whitney U test). Color boxes match with cluster overlays in the respective t-SNE projections.

Close modal

Because CD4 ABC have been quantified in various studies before (3, 2528), we first analyzed CD4 ABC in select PBMC subsets of our data to enable a direct comparison with previous data. We determined a median of 140,677 CD4 Abs per cell on Th cells (minimum: 126,861; maximum: 160,378 Abs per cell; 10 donors, Supplemental Fig. 3A). As expected, the CD4 ABC of monocyte subsets and plasmacytoid dendritic cells was much lower than that of Th cells (median: 20,367–44,886 Abs per cell, Fig. 4A; gating in Supplemental Fig. 2). Because those data were largely consistent with other studies (Table II) (25, 26, 28, 29), providing confidence in the approach, we then performed a systematic comparison of CD4, HLA-DR, Siglec-1, and CCR6 ABC across all PBMC subsets derived from FlowSOM clustering between five SLE patients and four healthy controls. Despite the few individuals analyzed, this analysis revealed several distinct clusters with significantly reduced CD4 or HLA-DR ABC or enhanced Siglec-1 ABC in patients with SLE. CCR6 ABC showed high interindividual variation and were not found significantly dysregulated in SLE (Fig. 4D). HLA-DR ABC were found diminished in cluster 7, showing the phenotype of myeloid dendritic cells (mDC) (high expression of CD36, CD11c, and CD1c; low or no expression of CD3, CD19, CD56, CD16, and CD14, Supplemental Fig. 3G), and the increase of Siglec-1 ABC was documented in seven clusters with dominant monocyte or mDC features.

Table II.
Comparison of published CD4 ABC values with data generated in this study
TargetSpecificationsMedian ABCInstrumentAssayReference
CD4+ T lymphocytes Cryopreserved 47,000 Flow cytometer QuantiBrite (25
Cryopreserved, fixed 42,000 Flow cytometer QuantiBrite (25
Cryopreserved, fixed 46,000 Mass cytometer Atom per cell (25
Cryopreserved 145,000 Mass Spectrometer Targeted multiple reaction monitoring mass spectrometry (26
Fresh 51,000 Flow cytometer QuantiBrite (28
Fresh 39,000 Flow cytometer QIFI Kit (28
Fresh 173,000 Flow cytometer QSC (28
Fresh 98,000 Flow cytometer QuantiBrite (29
Cryopreserved, fixed 140,677 Mass cytometer QSC Current study 
CD4+ monocytes Fresh 3,600 Flow cytometer QuantiBrite (28
Fresh 4,200 Flow cytometer QIFI Kit (28
Fresh 24,000 Flow cytometer QSC (28
Cryopreserved, fixed 26,004 Mass cytometer QSC Current study 
TargetSpecificationsMedian ABCInstrumentAssayReference
CD4+ T lymphocytes Cryopreserved 47,000 Flow cytometer QuantiBrite (25
Cryopreserved, fixed 42,000 Flow cytometer QuantiBrite (25
Cryopreserved, fixed 46,000 Mass cytometer Atom per cell (25
Cryopreserved 145,000 Mass Spectrometer Targeted multiple reaction monitoring mass spectrometry (26
Fresh 51,000 Flow cytometer QuantiBrite (28
Fresh 39,000 Flow cytometer QIFI Kit (28
Fresh 173,000 Flow cytometer QSC (28
Fresh 98,000 Flow cytometer QuantiBrite (29
Cryopreserved, fixed 140,677 Mass cytometer QSC Current study 
CD4+ monocytes Fresh 3,600 Flow cytometer QuantiBrite (28
Fresh 4,200 Flow cytometer QIFI Kit (28
Fresh 24,000 Flow cytometer QSC (28
Cryopreserved, fixed 26,004 Mass cytometer QSC Current study 

Because all clusters with reduced CD4 ABC were Th cells, we exemplarily performed manual gating and CD4 ABC analysis of CD4+ T cells and their major subsets, confirming the result of the systematic analysis (reduction of CD4 ABC by 14%, p = 0.0156, Supplemental Fig. 3B; data of manual subsetting not shown). Interestingly, low HLA-DR ABC on mDC were strongly associated with low CD4 ABC of certain Th cell clusters (Supplemental Fig. 3E).

Combining unsupervised clustering of high-dimensional mass cytometry data with receptor expression level analysis sensitively identified known and novel immune cell aberrations in active SLE, illustrating the utility of bead-based assays in biomarker identification studies. In summary, osmium-labeled beads facilitated the integration of ABC assays in high-dimensional immune phenotyping studies by mass cytometry, extending its application range and providing a means to report standardized receptor expression data in mass cytometry.

In this study, we present a simple and stable bead-labeling approach for mass cytometry using OsO4, a highly reactive compound used before for cell membrane and interior staining in electron microscopy and mass cytometry (3033). The approach enables osmium labeling of common, commercially available functionalized polystyrene beads and facilitates their unambiguous detection by mass cytometry by introducing a stable and high-intensity signal independent of the readout of the bead functionality. Corresponding to the labeling stability observed in this study, OsO4 has been shown to covalently bind to unsaturated bonds, such as present in benzyl groups, which are abundant in polystyrene (3234). Beads were likewise successfully labeled with ruthenium tetroxide (data not shown). Notwithstanding the potential use of OsO4 containing isotopically enriched Os for a higher degree multiplexing of bead-based assays, ruthenium labeling of beads directly offers a second, independent detection channel for functionalized beads.

The acquisition of osmium-labeled beads did not interfere with the detection of lanthanides, palladium, and platinum (i.e., metals used in analytical reagents, sample barcoding, etc.), and osmium-labeled beads successfully captured metal conjugates irrespective of which conjugation strategy was employed to generate these conjugates. Mixtures of osmium-labeled beads with cells and normalization beads were routinely acquired on the mass cytometer, and their data were deconvoluted according to osmium, iridium, and cerium signals. Although not formally tested, osmium-based cell size measurements or sample barcoding (30, 31) are likely to be compatible with the use of beads if the beads are prepared to elicit an osmium signal intensity distinct from that of cells or labeled with ruthenium tetroxide being detectable in atomic mass channels distinct from osmium or by the potential use of different osmium tetroxides holding isotopically purified Os isotopes, all of which should allow for a clear discrimination of beads from cells. The osmium label also permitted exclusion of bead doublets according to high versus average osmium signal intensity, and bead labeling with OsO4 did not interfere with Ab capture. Taken together with their storability, either with or without captured Ab conjugates, alone or pooled with other beads, osmium-labeled beads proved a highly versatile and compatible tool for various bead-based assays in mass cytometry.

Consistently, we successfully employed osmium-labeled beads for compensation of signal spillover using Catalyst. In this study and, likewise, in Ab quality control experiments, osmium labeling secures that Ab conjugate signal intensity readouts are based on representative bead populations and that beads with no or low signal intensity of the captured Ab are reliably detected. Notably, for the purpose of compensation, osmium-labeled beads can be spiked into the cell sample for spillover correction on a per sample basis.

The new possibility to assess low bead-associated Ab conjugate signal intensities also facilitated bead-assisted quantification of cellular receptors in ABC assays modified for mass cytometry, requiring a standard based on beads that capture many, few, or no Ab conjugates. We obtained ABC values for CD4 on Th cells largely agreeing with published data obtained by flow cytometry and mass spectrometry (3, 2529) (Table II). Remaining variance in CD4 ABC could be because of deviations in the cell preparation protocol, in which cell fixation is mandatory for mass but not flow cytometry, differences in Ab conjugation for mass versus flow cytometry, or different production lots of QSC beads. We recommend preparing and running the matched cell and bead samples in pairs to achieve ideal conditions for the quantification of cellular ABC.

By pairing the power of high-dimensional immune phenotyping by mass cytometry to quantify large numbers of immune cells subsets from a single sample with the ability of ABC assays to quantify the expression levels for individual receptors, we analyzed the ABC of four receptors in 60 cell clusters in a pilot study of five SLE patients and controls. Although the accuracy of this analysis largely benefits from sample barcoding, the conversion of marker expression signal intensities to ABC values enabled by osmium-labeled QSC beads establishes comparability of cellular receptor expression data across different mass cytometry platforms. Our data confirmed the increase of Siglec-1 on monocytes and mDC clusters in SLE consistent with an IFN-α signature in these patients (9, 35, 36) and, in accordance with data of in vitro–generated dendritic cells in SLE, downregulated HLA-DR on ex vivo mDC (37). We further identified reductions of CD4 ABC in SLE Th cells, affecting memory rather than naive CD4+ T cell clusters. All Th clusters with significantly reduced CD4 ABC expressed CD25 or CCR4, increased levels of CD95, and CCR7, CXCR3, CD39, and CD127 to various extents, indicating that Th1, Th2, and regulatory T cell clusters were affected. By contrast, CD4 ABC of activated CD38+HLA-DRhigh CD4+ T cells (cluster 30) were comparable between patients and controls. Notably, both CD4 expressed by Th cells as a coreceptor of the TCR, and HLA-DR expressed by APCs, such as mDC, directly interact in the immunological synapse (38). Consistently our results may indicate defective cross-talk between Th/regulatory T cells and mDC and related limitations in cellular immunity and immune regulation in SLE.

By pairing the power of high-dimensional immune phenotyping by mass cytometry with the ability of ABC assays to quantify individual receptors, we circumvent the high efforts of previous approaches (25) and introduce the reporting of receptor expression data that are comparable across different CyTOF instruments and other platforms, facilitating standardization in biomedical research. By converting conventional flow cytometry beads (e.g., those used for spillover correction and quantification) into osmium-labeled beads suitable for respective assays in mass cytometry, our approach paves the way to a broad application of bead-based assays in mass cytometry.

We are grateful to Vera Bockhorn for revising the implementation of the FlowSOM script.

This work was supported by German Research Foundation Grant ME 3644/5-1 and TRR130 TP24, the German Rheumatism Research Centre Berlin, European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357, the Else Kröner-Fresenius-Foundation, German Federal Ministry of Education and Research e:Med sysINFLAME Program Grant 01ZX1306B, and the Leibniz Science Campus for Chronic Inflammation (http://www.chronische-entzuendung.org).

The online version of this article contains supplemental material.

Abbreviations used in this article:

ABC

Ab-binding capacity

B2M

β2-microglobulin

CSM

CyTOF staining medium

mDC

myeloid dendritic cells

PFA

paraformaldehyde

QSC

Quantum Simply Cellular

RT

room temperature

SLE

systemic lupus erythematosus

t-SNE

t-distributed stochastic neighbor embedding.

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L.B., A.R.S., and H.E.M. are listed inventors on a related patent application that was submitted by their employer, the German Rheumatism Research Centre Berlin (EP 18169878.8). The other authors have no financial conflicts of interest.

Supplementary data