Cd45ra beads

PBMCs were isolated from reduction chambers obtained from Vitalant Research Institute or the NMCP. All blood products were first diluted at a 1:1 ratio using FACS buffer (PBS supplemented with 2% FBS and 2 mM EDTA). Ficoll (Stemcell Technologies) was then slowly added at the bottom of the tube at a ratio of 2:1. The samples were then centrifuged at 931 g for 30 min. After centrifugation, the PBMC layer underlying the top supernatant was transferred to a new tube and cells were washed 3x in FACS buffer. Where indicated, PBMCs were activated by stimulation for 3 days with 5 μg/ml PHA (Sigma-Aldrich) in the presence of 100 IU/ml IL-2 (Life Technologies). Activated PBMCs were always maintained in R10 media containing 20 IU/ml IL-2. For sorting experiments, memory CD4+ T cells (Tm cells) were purified using the EasySep CD4 negative selection enrichment kit (Stem cell Technologies) followed by depletion of naïve T cells using CD45RA beads (Miltenyi Biotec) prior to antibody staining.

PBMCs were isolated from reduction chambers obtained from Vitalant Research Institute and Stanford Blood Bank using Ficoll-Hypaque density gradients, and then cultured in R10. For sorting experiments, CD4+ T cells were purified by negative selection using the EasySep CD4 enrichment kit (Stem Cell Technologies), and further enriched for memory cells by depletion of naïve T cells using CD45RA beads (Miltenyi Biotec), prior to lectin staining and sorting as described further below. Where indicated, PBMCs were first stimulated for 3 days with 5 µg/ml PHA in the presence of 10 IU/ml IL-2 prior to CyTOF-Lec analysis.

For cell sorting, 10 mL of peripheral blood was collected from either HDs or patients, and CD4⁺T, CD4⁺CD45RA⁺T, CD4⁺CD45RA⁻T, or CD4⁺CD25⁻T cells were obtained from PBL using the human CD4⁺T Cell Isolation Kit II and CD45RA beads (Miltenyi Biotec). The purity of the cells was 90% or greater as determined by reanalysis. Typically, the cells were incubated with 1,25(OH)2D at final concentration 20 nM or DMSO as control under the stimulation with precoated 5 μg/mL αCD3, soluble 5 μg/mL αCD28, and 200 U/mL rhIL-2 at 1 × 10⁵ per well in 96-well U-bottom plates, and three replicate wells were set up. The cells were cultured in a humidified CO₂-containing atmosphere at 37°C for 5~7 days in complete RPMI-10 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.5 mM sodium pyruvate, 0.05 mM nonessential amino acids, 2 mM L-glutamine, and 10 mM HEPES (all from GIBCO). For cell proliferation, purified T cell was stained with CFSE and detected cell division by flow cytometry after 5 days of incubation.