Anti cd45ra

CD4+ T cells were enriched by magnetic activated cell sorting (MACS) using 20 µl of anti-CD4 MicroBeads (anti-CD4 MicroBeads, human, Miltenyi Biotec, Germany) and 80 µl MACS buffer (PBS with 0.5% BSA and 2 mM ethylenediaminetetraacetic acid) per 10⁷ cells. CD4+ T cells were stained for surface expression in MACS buffer at a concentration of 2 × 10⁸ cells/ml. Cells were washed and stained with 4′,6-diamidino-2-phenylindole (1:250) and then sorted using the flow cytometer BD FACSAria II (BD Biosciences, Germany) into the following CD4+ T-cell subpopulations: Treg (CD25highCD127low) and non-Treg: TN (CD45RA+ CCR7+), TCM (CD45RA-CCR7+), TEM (CD45RA− CCR7−), and TEMRA (CD45RA+ CCR7−).
Antibodies used for surface staining were anti-CCR7 (GO43H7, 1:50), anti-CD25 (M-A251, 1:100), anti-CD45RA (HI100, 1:50), anti-CD127 (A019D5, 1:100), anti-CD3 (UCHT1, 1:200), anti-CD4 (OKT4, 1:200), anti-PD-1 (EH12.2H7, 1:20), anti-TIGIT (A15153G, 1:20), anti-TIM-3 (F38–2E2, 1:20), and LAG-3 (11C3C65, 1:20) from BioLegend, and anti-KLRB1 (191B8, 1:10), anti-KLRF1 (4A4.D10, 1:50), and anti-KLRG1 (REA261, 1:10, 1:50 since 2018/06 due to more concentrated formulation) from Miltenyi Biotec. See also Supplementary Fig. 5 for the pre-gating strategy.

Basal expression of the different molecules was determined in peripheral blood obtained from seven RA patients, as described above. The effect of IL-15 on the expression of these molecules was studied in PBMCs obtained from 10 RA patients and cultured in medium alone or in the presence of IL-15 (50 ng/mL) (Peprotech INC, Rockyhill, NJ, USA) for 18 h. Overnight treatment with IL-15 has been seen to be the appropriate frame time for protein expression detection in previous studies [8 (link),16 (link)]. The surface stain in both cases was made with anti-CD4 (APC) or anti-CD4 (PECy7), anti CD45RA (APCFire), anti-CD8 (PB), anti-CD28 (BV), anti-CD44 (FITC), anti-CX3CR1 (PE), anti-CCR5 (PECy7), and anti-CD11a (FITC) (Biolegend, San Diego, CA, USA), as well as anti-CD3 (PerCP) and anti-CD49d (APC) (BD Bioscience). Cells from whole blood were stained as described above. PBMCs were stained for 30 min at 4 °C. Then, cells were washed and resuspended in PBS until they were acquired in a Navios flow cytometer and analysed with Kaluza software (Beckman Coulter Life Science, Brea, CA, USA). The cytometer compensation was carried out using the VersaComp Antibody Capture Bead Kit (Beckman Coulter). The marker settings for determining the negative/positive cell populations were established using the FMO strategy.

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.