Sample collection
All collection sites used a common lab manual, common protocols, on-site training prior to the start of work, and regular coordination meetings to ensure consistency in sample collection across locations. All samples were assayed at the Allen Institute, except for plasma proteomics, which was outsourced (Olink). Blood was drawn into BD sodium heparin (NaHeparin) vacutainer tubes for PBMC or K2-EDTA vacutainer tubes for plasma. PBMC isolation was performed using Ficoll-Paque gradient separation, which was started within 2 hours after blood draw to avoid effects of sample degradation (Savage, et al. 2021). PBMCs were cryopreserved in 90% FBS (ThermoFisher) / 10% DMSO (Fisher Scientific) at a target of 5 x 106 cells/mL by slow freezing in a Coolcell LX (VWR) overnight in a -80°C freezer followed by transfer to liquid nitrogen.
Autoantibody and inflammatory marker testing
Anti-citrullinated protein antibodies (ACPA) was measured using the anti-CCP3 IgG assay on an ELISA platform (QuantaLite, Werfen). Samples were considered positive for anti-CCP3 if this measurement was ≥20 units, as suggested by the manufacturer. Rheumatoid factor (RF) immunoglobulin (Ig) A (RFIgA) and IgM (RFIgM) were also tested by ELISA with the following clinical laboratory-established cutoffs: >1.7 International Units for RFIgA, and >6.7 International Units for RFIgM. Nephelometry was utilized to measure High sensitivity C-reactive protein (hsCRP) in milligrams per liter (mg/L). Erythrocyte sedimentation rates (ESR) were performed using clinical lab methodologies with results in millimeters per hour (mm/hr). Anti-CCP3, RFIgA and RFIgM tests were performed on all ALTRA participants and all testing for these autoantibodies was performed at the University of Colorado in the Exsera Biolabs, a College of American Pathologists and Clinical Laboratory Improvement Amendments (CAP/CLIA) certified laboratory. hsCRP and ESR testing were performed using local clinical laboratories at UCSD and CU.
HCMV serology
Human Cytomegalovirus (HCMV) serology tests were performed at the University of Washington’s Clinical Virology Laboratory in the Department of Laboratory Medicine (https://depts.washington.edu/uwviro/). Plasma or serum samples (200 µL) collected in tandem with blood samples were analyzed using the FDA-approved LIAISON® CMV IgG Assay to detect CMV IgG class antibodies. Results consisted of a CMV Ab Screen Index Value ranging from <0.20 to >10.00, as well as a ‘Positive’ or ‘Negative’ call for anti-CMV IgG detection.
Flow cytometry, scRNA-seq, and TEA-seq single-cell assays
Frozen PBMC aliquots were thawed and processed in batches of 12-23 samples, plus a uniform batch control sample, as described in Genge, et al. (2021). After thawing, cells were washed, resuspended, counted, and divided for parallel processing using four panels for high dimensionality spectral flow cytometry (described in Heubeck, et al. (2022)) as well as multiplexed scRNA-seq using 10x Genomics 3' scRNA-seq with Cell Hashing. Pipelines batches contained samples from multiple cohorts collected across projects for the Allen Institute for Immunology. For additional details of our paired experimental pipeline, see our Multiplexed scRNA-seq Pipeline documentation and Genge, et al. (2022).
For TEA-seq, samples were thawed and stained with sample specific cell hashing antibodies and processed in the same batch. Cells were sorted to remove dead cells, debris, and neutrophils. A panel of 166 target-specific barcoded oligonucleotide-conjugated antibodies (ADT) (BioLegend TotalSeq™-A Human Universal Cocktail, V1.0 and 3 additional antibodies) were used. TEA-seq library preparation was performed as described previously (Swanson, et al. (2021); Thomson, et al. (2023)).
Plasma proteomics
K2-EDTA source tubes were gently inverted, centrifuged, and 80–90% of the plasma supernatant was removed for immediate freezing at −80°C. Plasma was assayed after the first freeze/thaw. The Olink Explore 1536 platform was used to assay relative protein abundance of 1,472 target proteins per sample. Positions of samples were randomized across plates to balance distribution of age and sex. Multiple samples from the same participant were run on the same plate. Seven batches in total were used to measure all samples, each of which included the same set of 12 bridging controls that were used to normalize expression data across batches.
References
Genge PC, Roll CR, Heubeck AT, Swanson E, Kondza N, Lord C, et al. Optimized workflow for human PBMC multiomic immunosurveillance studies. STAR Protoc. 2021;2: 100900.
doi:10.1016/j.xpro.2021.100900
Heubeck A, Savage A, Henderson K, Roll C, Hernandez V, Torgerson T, et al. Cross-platform immunophenotyping of human peripheral blood mononuclear cells with four high-dimensional flow cytometry panels. Cytometry A. 2023;103: 500–517.
doi:10.1002/cyto.a.24715
Savage AK, Gutschow MV, Chiang T, Henderson K, Green R, Chaudhari M, et al. Multimodal analysis for human ex vivo studies shows extensive molecular changes from delays in blood processing. iScience. 2021;24: 102404.
doi:10.1016/j.isci.2021.102404
Swanson E, Lord C, Reading J, Heubeck AT, Genge PC, Thomson Z, et al. Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq. Elife. 2021;10.
doi:10.7554/eLife.63632
Thomson Z, He Z, Swanson E, Henderson K, Phalen C, Zaim SR, et al. Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment. Nat Immunol. 2023;24: 1947–1959.
doi:10.1038/s41590-023-01641-8