Fig 2: Shielding and inflammatory regulation effects of DPP-ADSCs targeted to injured arteries at 24 h post-surgery. a) The leukocyte adhesion shielding effect of ADSCs bound to injured vessels was observed by en face immunostaining using anti-CD45 and anti-CD90. b) The adhesion number of leukocytes was calculated based on en face staining. c) The platelet adhesion-shielding effect of ADSCs bound to injured vessels was observed by en face immunostaining using mepacrine and CD90. d) The adhesion number of platelets was calculated based on en face staining. e) The relative expression of inflammatory genes (Selp, Il6, Il1b, and Il10) and pro-regeneration genes (Hgf and Vegfa) of injured arteries across the different treatments. All data are representative of (n = 5) animals per group.

Fig 3: MMF inhibits effector CD8+ T cell differentiation and TA/Ac partially rectifies the defective effector differentiation of C1qbp-deficient CD8+ T cells.(A) Schematic of MMF-treated P14 experiments. CD90.1/90.1 or CD90.1/90.2 P14 cells were cultured in vitro with dimethyl sulfoxide (DMSO) or MMF respectively for 1 day, and then 1:1 mixed and transferred into CD45.1/45.1 recipient mice, followed by infection with the LCMV Armstrong strain. Flow cytometry analysis was done at day 7 after infection. (B to D) Flow cytometry of splenic CD8+ T cells (B) and the donor P14 CD8+ T cells (C and D) as in (A). Frequency of the donor P14 cells (among splenic CD8+ T cells) (B), KLRG1+ P14 or CD127+ P14 subsets (among donor P14 cells) (C), and CD62L+ P14 subsets (among donor P14 cells) (D) as in (A) (n = 4 per group). (E) Schematic of TA/Ac-treated P14 experiments. CD90.1/90.1 or CD90.1/90.2 WT or C1qbp-deficient P14 cells were activated in vitro with plate-bound anti-CD3e (1 µg/ml) and anti-CD28 (1 µg/ml) for 1 day and cultured with DMSO or TA/AC in the presence of anti-CD3e and anti-CD28 for another day, and then were 1:1 mixed and transferred into CD45.1/45.1 recipient mice, followed by infection with LCMV Armstrong strain. Flow cytometry analysis was done at day 7 after infection. (F to H) Flow cytometry of splenic CD8+ T cells (F) and the donor P14 CD8+ T cells (G and H) as in (E). Frequency of the donor P14 cells (among splenic CD8+ T cells) (F), KLRG1+ P14 subsets (among donor P14 cells) (G), and CD62L+ P14 subsets (among donor P14 cells) (H) as in (E) (n = 3 per group). Each symbol represents an individual mouse; small horizontal lines indicate the means (±SD). *P < 0.05, **P < 0.01, and ***P < 0.001, ****P < 0.0001 (two-tailed paired Student’s t test). Data are representative of three independent experiments (error bars, SD).

Fig 4: Characterization of ADSCs. (a) Flow cytometric analysis of surface markers of ADSCs. ADSCs were positive for MSC markers including CD29 (99.71%) and CD90 (98.64%) and were negative for CD45 (0.43%) and CD11b/c (0.37%). (b) Representative images of adipogenic differentiation and osteogenic differentiation. The visual field was filled with red lipid droplets stained with Oil Red O solution in images of adipogenic differentiation. Calcium nodules with burrs were formed in images of osteogenic differentiation, which were stained red by Alizarin Red S solution (scale bar: 100 µm). CTRL, Control group; osteogenesis, osteogenic induction group; adipogenesis, adipogenic induction group; ADSCs, Adipose-derived mesenchymal stem cells.

Fig 5: Expression of CD4, CD90.1, CD3, and CD28 and TCR Vß usage in SLP-76OST–edited and control CD4+ T cells. (A) After gating on live singlets, SLP-76OST–edited and control CD4+ T cells that were expanded for 34 d were analyzed by flow cytometry for the expression of CD4, CD90.1, CD3, and CD28. Numbers in dot plots and histograms indicate the percentage of cells within the specified gates and the percentage of positive cells, respectively. Data are representative of three independent experiments. (B) SLP-76OST–edited and control CD4+ T cells were expanded for AP-MS and analyzed using a panel of 24 TCR Vß–specific antibodies. The clonogram shows that both populations have preserved a rather similar polyclonal Vß repertoire. Data are representative of two independent experiments. FSC, forward scatter; SSC, side scatter.