Ghost 780

Intracellular cytokine staining was conducted on day 3, after the T cells were cultured for 72 h. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 h. Brefeldin A 1000X (eBioscience) was added for the final 4.5 h. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4 °C. Cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17A (FITC; eBioscience), IFNγ (eFluor450; eBioscience), and/or FOXP3 (eFluor450, eFluor660; eBioscience). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD4⁺TCRβ⁺ Live cells.

PB was collected from transplanted mice via retro-orbital puncture at 6 weeks. After euthanasia at 15 weeks, BM was isolated by crushing femurs and tibias with a mortar and pestle. PB and BM engraftment (expressed as the percentage of hCD45⁺ cells of total CD45⁺ [hCD45⁺mCD45⁺]) were determined by staining for mCD45-PE (30-F11), hCD45-APC (HI30), and DAPI. For VCN and lineage distribution analysis, hCD45⁺ cells were enriched from BM using a bead-based selection kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Human CD45-enriched cells were stained with Ghost 780 (viability dye; Tonbo Biosciences, San Diego, CA, USA) and the following antibodies: hCD45-FITC (HI30), mCD45-PE (30F11), hCD3-PerCp-Cy5.5 (UCHT1), hCD33-BV421 (WM53), hCD34-APC (581), and hCD19-PE-Cy7 (SJ25C1). All antibodies were purchased from BD Biosciences (San Jose, CA, USA). Genomic DNA was extracted from hCD45-enriched cells using the Purelink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) and analyzed for VCN as described above.