Aria 3

The Hsp70 and RB1 promoters were cloned by PCR from the genome of PC12 cells, extracted with a Genomic DNA Kit (Tiangen Biotech., China). The plasmids Hsp70-pro(ΔpolyA⁺) and Hsp70-pro(ΔpolyA⁻) were constructed by replacing the CMV promoters of pEGFP-N1 and pEGFP-N1 (ΔpolyA⁻) with the Hsp70 promoter, using the restriction endonucleases AseI and EcoRI. The Hsp70 and RB1 ORFs were cloned from PC12 cDNA obtained by RT-PCR. The Hsp70 and RB1 ORFs were inserted into Hsp70-pro(ΔpolyA⁺) using BamHI to generate the Hsp70-pro-Hsp70(ΔpolyA⁺) and Hsp70-pro-Rb(ΔpolyA⁺) plasmids. RB1-pro(ΔpolyA⁺), RB1-pro(ΔpolyA⁻), RB1-pro-Hsp70(ΔpolyA⁺), RB1-pro-Rb(ΔpolyA⁺) and RB1-pro-Hsp70(ΔpolyA⁻) were constructed in a similar way. Cloning primers were as follows: Hsp70 promoter sense: 5′-TGGGAGAGGAGAGTGTGTCG-3′, antisense: 5′- GGGCGGAGAAGATCTCGAAG-3′; RB1 promoter sense: 5′-TCTTTGTAGCTGGACCTGGGCCT-3′; antisense: 5′-GGGAGCCAGCGAGCTGTGGAG-3′; Hsp70 ORF sense: 5′-ATGTCGGTGGTGGGCATAGAC-3′; antisense: 5′-ATCAATGTCCATCTCAGGAAGC-3′; RB1 ORF sense: 5′-ATGCCGCCCAAAACCCCCCGAAAAACGGCC-3′; antisense: 5′-AGCATGGATACCTCAAACAAGGAAGAGAAA-3′12 (link). 293T cells were transfected with all types of plasmids and sorted using a BD AriaIII (Becton Dickinson, USA).

The pMSCV Exp-mCcr4 NM_009916.2 (ns): P2A: EGFP (Vector ID: VB230314-1872tcs) vector was designed and created using VectorBuilder, Inc. (Guangzhou, China). The vector was then packaged into a recombinant MSCV retrovirus using VectorBuilder, Inc. (Guangzhou, China).
The general procedure for Treg cells was transduced as previously described^{24 (link)}. Transduction media was prepared with RPMI media with 100 mM HEPES and 10 μg/mL polybrene. We carefully pipetted 100 μL of the virus stock into each well containing 1 mL of the transduction media. The culture was spinoculated at 1,200 g for 90 min at 32 °C. Cells were incubated under standard culture conditions for 4 h, followed by a media change. Treg cells were sorted on days 5 or 6 using a BD Aria III (Becton Dickinson Company, Franklin Lakes, New Jersey).

Cellular phenotyping and sorting were performed on a BD ARIAIII cell sorter (Becton Dickinson, San Jose, CA, USA). The following fluorochrome-labelled mAbs conjugated to FITC, PE, PeCy7, PerCPCy5.5, APC, APC-Cy7, Brilliant Violet 510, Brilliant Violet 605, Pacific Blue and Alexa700 or appropriate isotype controls were used for cell surface staining: CD11b, CD11c, CD45 (clone 30-F1), CD86, CD103, CD140a (clone APA5), CD274 (PD-L1), MHC-class II (I-A^b), GR-1, F4/80, NK1.1, SCA-1 (clone D7), Siglec-F (BD Biosciences, Germany), TER119 and 120G8 (Dendritics, France). The surface staining time was 30 min on ice and cells were washed with staining buffer (1× HBSS, PAA, Germany) at 400 g for 5 min at room temperature (RT) before analysis. The number of acquired events was ≥ 500,000.
The viability of sorted cells was >90 % as indicated by Sytox blue (Life Technologies, Germany) staining. All mAbs were ordered from Biolegend, Germany unless indicated otherwise. Absolute cell counts were determined with AccuCount Fluorescent Particles 7.7 μm (Spherotech, Lake Forest, USA).

Cell labeling was performed on ice in 1X PBS with 5 mM EDTA and 2% serum. Samples were analyzed for donor chimerism (detectable by CD45.1 or GFP) on an LSRII or AriaIII (Becton Dickinson, San Jose, CA), as described previously [16 (link),17 (link),24 (link),27 (link),29 (link)–32 (link)]. BM HSC and multi-potent progenitor (MPP) populations and BM stromal populations were stained as previously described [16 (link),17 (link)]. Briefly, the steady state populations are characterized as peripheral blood (PB) HSCs: Lin^-/CD27⁺/cKit⁺/Sca1⁺/Flk2^- [33 (link)], PB MPP: Lin^-/CD27⁺/cKit⁺/Sca1⁺/Flk2⁺, BM HSC: Lin^-/cKit⁺/Sca1⁺/Flk2^-/CD34^-, BM MPP: Lin^-/cKit⁺/Sca1⁺/Flk2⁺/CD34⁺, Total VEC: CD45^-/Ter119^-/CD31⁺/Sca1⁺, Sinusoidal VEC (SECs): CD45^-/Ter119^-/CD31⁺/Sca1^mid/Tie2⁺; MSC: CD45^-/Ter119^-/CD31^-/Sca1⁺/CD51⁺ and OBL: CD45^-/Ter119^-/CD31^-/Sca1^-/CD51⁺. All antibodies were purchased from Biolegend, eBioscience, or BD Pharmingen.

Cells exposed to vaspin for 24 h were washed and collected by trypsinization. Cells were centrifuged (0.4 rcf, 3 min) and stained according to the Annexin-V apoptosis assay (Boncel et al., 2017). Cell pellets were dissolved in 50 μl cold Annexin-V labeling buffer and then 2.5 μl of FITC-labeled Annexin-V antibody was added (BioLegend) followed by 10 μl of propidium iodide (PI) solution (100 μg/ml; Sigma). After 20 min in darkness, 250 μl of Annexin-V labeling buffer was added and the samples were incubated on ice and in the dark for 15 min. Flow cytometric analysis (Aria III, Becton Dickinson) using the FITC configuration (488 nm excitation; emission: LP mirror 503, BP filter 530/30) and the PE configuration (547 nm excitation; emission: 585 nm) was performed immediately and at least 10,000 cells were counted. Cells were classified as necrotic (PI positive and Annexin-V negative; late apoptotic (PI positive and Annexin-V positive); early apoptotic (PI negative and Annexin-V positive); or normal (PI negative and Annexin-V negative) [11 (link)].