Cd45ra apc hi100

The following conjugated antibodies were used to characterize CD45RC T cell subpopulations: CD3-VioGreen (REA613), CD4-PerCP-Vio700 (REA623), CD8-PE-Vio770 (REA734), from Milteny Biotec, Bergisch-Gladbach, Germany; CD45RA-APC (HI100) from BD Biosciences, San Jose, CA, USA; and CD45RC-FITC (MT2) from IQ Product, Houston, TX, USA. Cell viability was systematically assessed (LIVE/DEAD Fixable Near-IR Dead Cell Stain kit; Fischer Scientific, Pittsburgh, PA, USA). Briefly, 10⁶ cells were incubated with the viability dye according to the manufacturer’s recommendations before incubation with the antibodies. Data were collected using a FACS-Canto II (BD Biosciences) cytometer and analyzed using the FlowJo software, Ashland, OR, USA. The expression of CD45RC is bimodal on CD4⁺ T cells, some cells expressing low levels of CD45RC (CD45RC^low), and others expressing high levels (CD45RC^high). On CD8⁺ T cells, expression of CD45RC is trimodal, the first fraction of cells expressing low levels (CD45RC^low), the second fraction expressing intermediate levels (CD45RC^Int), and the last fraction expressing high levels of CD45RC (CD45RC^high). Figure S1 illustrates the gating strategy.

Large-scale manufacturing of CAR-T cells on CliniMACs Prodigy was carried out under GMP-like conditions into Gene-Cell Therapy clean rooms of the Cell Therapy Unit of Hospital Universitario Reina Sofía (Córdoba, Spain). Two different aphereses from a healthy donor were thawed, and around 100 × 10⁶ (link) T cells were inoculated into the CliniMACs prodigy bioreactor (Miltenyi Biotec). CD4 and CD8 cells were selected with CD8 and CD4 Reagent (Miltenyi Biotec), cultured with IL-7 and IL-15 (Miltenyi Biotec), and activated with αCD3 andαCD28 GMP T cell TransAct (Miltenyi Biotec). On day 2 of the process, these cells were transduced with AWARI-LVs (MOI = 5). Cells were cultured in TexMACs GMP medium containing GMP-grade IL-15 and IL-7 (Miltenyi Biotec) for 9 or 10 days. Final product was collected with 100 mL of NaCl 0.9% + 0.5% human serum albumin (HSA).
Cells were stained with CD3-APC, CD4-FITC, CD8-APC Vio770, CD14-PE Vio770, and CD45-Vioblue (Miltenyi Biotec). To assess the efficiency of transduction, CAR-T cells were stained with CD-19 Biotin and anti-Biotin PE (Miltenyi Biotec). Viability was tested by using 7-AAD (Miltenyi Biotec). Phenotype was determined with CD45RA-APC (HI100) and CCR7-BV421 (2-L1-A RUO) from BD Pharmingen. Cells were acquired on a MACsQuant cytometer (Miltenyi Biotec) and analyzed with MACsQuantify Analysis Software (Miltenyi Biotec).