Lsrii machine

For flow cytometry analysis, spleens were harvested from mice and processed as previously described (35 (link)). Extracted lungs were chopped in small pieces and incubated with a digestive mix containing RPMI with collagenase IV (50 μg/ml) and DNAseI (20 U/ml) for 30 min at 37°C. Spleens and lungs were mashed through a 70-μm strainer before red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer (Gibco). Cells were washed with PBS containing 1% FBS and surface receptors were stained using various antibodies. Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD44, CD38, ICOS, OX40, CD62L, Perforin, Granzyme B, and Tbet, CXCR5 were purchased from Biolegend. Fluorochrome-conjugated antibodies against mouse CD8a were purchased from BD Biosciences. Fluorochrome-conjugated antibodies against CXCR3 and Ki67 were purchased from Thermofisher. Stained cells were fixed with PBS containing 4% Formaldehyde. For intracellular staining, the forkhead box P3 (FOXP3) staining buffer set was used (eBioscience). Flow cytometry analysis was performed on a LSR II machine (BD Bioscience) and data were analyzed using FlowJo (Tree Star).

Mortality and morbidity were checked once a day each working day. Animals were observed individually once a week for the recording of clinical signs and body weight. Blood samples for hematology analyses and DNA analyses were collected 16 weeks after transplantation and at termination. Erythrocyte count, mean cell volume, packed cell volume, hemoglobin, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocyte count, leucocyte count, differential white cell count with cell morphology, and reticulocyte count were determined using the Scil Vet ABC Plus⁺ analyzer (Scil Animal Care, Treviglio, Italy).
Cytofluorimetric analyses of PB were performed on a Becton Dickinson LSR II machine with BD FACSDiva software. PB from mice was analyzed for hematopoietic reconstitution using the following antibodies: phycoerythrin (PE)-conjugated anti-mouse CD11b, anti-mouse CD3, and anti-mouse B220 (BD Biosciences, San Jose, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD45.1 and allophycocyanin (APC)-conjugated anti-mouse CD45.2 antibodies (BD Biosciences, San Jose, CA) were used to evaluate donor-host chimerism at 16 weeks and termination.
Genomic DNA was extracted from the blood for determination of VCN at 16 weeks using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany).

Growth curves were performed as previously described [41 (link), 46 (link)]; the number of viable cells was assessed on a TC-20 (Bio-Rad, Hercules, CA, USA). Cell cycle progression/proliferation was assessed by flow cytometry after BrdU/7AAD staining using the BrdU Flow Kit (BD Pharmingen, Franklin Lakes, New Jersey, USA) according to the manufacturer’s instructions and as described previously [41 (link), 46 (link), 50 (link), 51 (link)]. BrdU incubation was performed at a concentration of 50 µM for 1 h, and an LSR II machine (BD Biosciences) was used for flow cytometry. Apoptosis was assessed by Annexin-V/7AAD assays (BD Biosciences), as described earlier [41 (link), 46 (link), 50 (link), 51 (link)], using a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA).