Cd4 fitc

Flow cytometric analyses were conducted using an LSR II flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Version 7.6.5; Flowjo). All antibodies against human or mouse cells were used at appropriate dilutions, as determined by previous titration. Doublet discrimination was carried out and non-viable cells were excluded by 4,6 diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich). Mouse cells were characterized using the antibodies as follows: CD3-V500, CD4-V450, CD11c-APC Cy7, SiglecF-AF647, Ly6G-FITC, CD11b-V500, B7-1-V450, B7-2-PE Cy7, CD62L-FITC (BD Biosciences); mPDCA1-APC, B220-PE (Miltenyi Biotec); CD8-PE Cy7, F4/80-PerCP, SiglecH-PE, TLR4-AF488, TLR9-FITC, MHC-I-FITC, MHC-II-V450, PD-L1-PerCP, ICOS-L-PE, OX40L-APC (eBioscience); TLR7-PE (Abcam); p75NTR-AF488 (Advanced Targeting Systems), CD45-Pacific Blue (Biolegend), and panTrk-FITC (Cell Signaling Technology). Human cells were characterized using the antibodies as follows: TrkA-PE (R&D Systems); BDCA-2-FITC, BDCA-4-PE, p75NTR-APC, p75NTR-FITC, p75NTR-PE (Miltenyi Biotec); CD45-V500, CD3-PE, CD4-FITC, CD8-PerCP, FcεRIα-FITC, IL-3R-PE Cy7, CD184-PE Cy7, MHC-I-PE Cy7, MHC-II-PE, CD80-V450, CD86-PE, CD83-V500, OX40L-V500, PD-L1-PE Cy7, CCR7-V450, CCR9-APC (BD Biosciences), CD3-APC eFluor780, CD4-APC, CD8-PE Cy7, CD25-PE (eBioscience), and CD69-PerCP Cy5.5 (BioLegend).

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).

PBMCs from patients with SS and healthy controls were washed with FACS buffer (PBS–Mg–Ca, 2% FBS) and suspended in FACS buffer with Fc receptor (FCR) blocker and TANDEM enhancer (Miltenyi Biotec, Woking, UK) on ice for 10 min. Cells were stained with either recombinant mAb14 at 5 µg/mL or biotinylated mAb14 at 5 µg/mL, or matched isotype-control mouse IgG1ĸ at 5 µg/mL for 2 h. Cells were washed and stained with either APC AffiniPure F (ab’) 2 fragment goat anti-mouse IgG or streptavidin-APC for 45 min on ice. Cells were then washed and suspended in FACS buffer with CD4-FITC (Miltenyi Biotec, Woking, UK) and CD26-PE (Miltenyi Biotec, Woking, UK) for 30 min on ice, washed in FACS buffer, and suspended in FACS reader buffer. DAPI viability staining solution was used to distinguish between live and dead cells. ∆ positive staining for mAb14 = (%mAb14 + cells) − (%isotype control + cells).

CD4 depletion was performed using human CD4 MicroBeads (Miltenyi Biotec, cat. no. 130-045-101; >99% depletion efficiency) according to manufacturer’s instructions. Magnetic separation was performed using the autoMACS Pro Separator (Miltenyi Biotec). Purity of the isolated subsets was checked using flow cytometry after staining with a cocktail of antibodies containing CD14 PE-Vio770 (Miltenyi Biotec, cat. no. 130-098-074), CD3 APC (BioLegend, cat. no. 3004399) and CD4 FITC (Miltenyi Biotec, cat. no 130-114-722). Absolute number of CD14⁺ cells used for subsequent incubations was determined using BD Trucount Tubes (BD, cat. no. 340334) and no-wash staining procedure with the above antibody cocktail.