Sample Collection
Fresh whole blood and bone marrow aspirates were collected longitudinally at diagnosis, through induction, and up to two years post-transplant. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were isolated using a Ficoll-density gradient approach. Cells were frozen in Cryo-storage media (90% FBS, 10% DMSO) using pre-chilled containers before transfer to long-term liquid nitrogen storage. Matched plasma and bone marrow interstitial fluid (BMIF) were isolated by centrifugation and frozen at -80°C.
Single-Cell Sequencing and Pipeline Flow Cytometry
Frozen BMMC and PBMC aliquots were thawed and processed in 9-24 batches of 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 for PBMC and two panels for BMMC. These samples were also processed using multiplexed scRNA-seq using 10x Genomics 3' scRNA-seq with Cell Hashing for PBMC as well FACS sorting to remove dead cells and CITE-seq with a custom antibody panel for BMMC. 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).
Proteomics and Serology
Protein expression profiling was performed using the Olink Explore 1536 platform for plasma and the Olink Explore 3072 platform for BMIF. Pathogen-specific IgG responses for Influenza and SARS-CoV-2 were quantified using Meso Scale Discovery (MSD) multiplex serology assays.
Flow Cytometry
High-dimensional spectral flow cytometry utilized four panels for PBMCs and two panels for BMMCs, with gating and data analysis performed in CellEngine.