Anti cd15

Cells were labeled with anti-human LIN cocktail (anti-CD3/CD14/ CD16/CD19/CD20/CD56), anti-CD15, anti-CD34, anti-CD38, anti-CD45RA (Biolegend) and anti-CD90 (BD Biosciences), antibodies specification described above, to perform purification of Primitive subpopulations (Lin- CD34+CD38- CD45RA- cells). The stained samples were FACS purified with the BD Aria cell sorter (BD Biosciences), achieving purity ranging between 92% and 99%.
Sorted primitive (HSC+MPP) populations were transduced as described above in “In vitro transduction of HD and WAS patients’ derived BM or MPB CD34+ cells” section. After transduction, cells were collected, washed, and transplanted in NSGW41 mice. An aliquot of cells was cultured in Iscove’s modified Dulbecco’s medium (IMDM), 10% fetal bovine serum (Cambrex, East Rutherford, NJ, USA) with stem cell factor (SCF), FLT3-L thrombopoietin (TPO) and IL-3 (all from Preprotech) at 20 ng/ml concentration (liquid culture) and harvested after 15 days to perform VCN estimation. An additional aliquot was used to perform Colony Forming Cell (CFC) assay according to the manufacturer’s procedure in Methocult medium (Stem Cell Technologies, Vancouver, Canada). At day 14, colonies were scored, singly picked and analyzed to evaluate the percentage of transduction.

Human KCs and immune cell subsets were obtained from healthy human volunteers using a published protocol [89 (link)]. Lesional, uninvolved and healthy control skin was obtained and processed as described [90 (link)]. RNA was extracted using RNeasy columns (Qiagen, Cat No. 74104) and evaluated using the Agilent 2100 Bioanalyzer. A commercial kit was used for reverse transcription (Applied Biosystems, Cat No. 4368814). QRT-PCR was done with primers for TNFRSF9 (Applied Biosystems, Cat No. Hs00155512_m1) and RPLP0 (Applied Biosystems, Cat No. Hs99999902_m1) using the 7990HT Fast Real-Time PCR system (Applied Biosystems). Whole blood was obtained from healthy donors and analyzed by flow cytometry for cell surface TNFRSF9 (Biolegend, Cat. No. 309810), anti-CD15 (BioLegend, Cat No. 323022), anti-CD3 (BioLegend, Cat No. 344808) and anti-CD14 (BD Pharmingen, Cat. No. 555397). Samples were analyzed using the LSR II flow cytometer (BD Biosciences).