Fig 1: Expression dynamics of adhesion molecules on CAR T cells and the effect on CARIS avidity.(A) Hypothetical schematic of mechanisms of LFA-1 activation. (B to D) Flow cytometry for CD11a (B) and CD18 (C) expression on HER2-CAR T cells at baseline and for aLFA-1 (D) in response to plate bound recombinant HER2, using an LFA-1 extended-open conformation-specific antibody (m24). MFI, mean fluorescence intensity. (E) WB for membrane fractions from HER2-CAR T cells at baseline (0 min) and in conjugation with LN229 (15 and 60 min) probed for CAR (CD3ζ), LFA-1 (CD18), LAT, and CD71. (F) Percentage LFA-1 (CD18) recruited to the LR fractions from (E). (G) ImageStream flow cytometry for HER2-CAR T cells conjugated with LN229 (30 min). CARs (emerald GFP), actin (phalloidin), nuclei (DAPI), and aLFA-1 (m24). (H to J) Fluorescence intensity of activated aLFA-1 (H) and its correlation with actin intensity (I) or CAR intensity (J) at the CARIS of mature synapses. N = 3; n = 9007. (K and L) Flow cytometry for ICAM-1 expression at baseline on CD4 and CD8 (K) and product (L) of HER2-CAR T cells. (M) Schematic representation of z-Movi acoustic force microscopy for testing CARIS avidity. A force ramp of 0 to 1000 pN was applied to pull HER2-CAR T cells off an LN229 monolayer toward acoustic nodes. (N) z-Movi ultrasound microscopy measuring the percentage of HER2-CAR T cells bound to LN229 at 500 pN after 2, 5, 20, or 40 min of incubation. (O) Detachment forces for individual cells after 5 or 40 min of incubation. Median represents the force needed to detach 50% of CAR T cells (EF50). Mean of three donors + SD [(B) to (D), (F), (K), and (L)], data pooled from three donors [(H) to (J)] or three to five avidity runs [(N) and (O)] compared using one-way [(B), (C), and (O)] or RM two-way Holm-Sidak corrected ANOVA [(D) and (N), ratio paired t test (F), Mann-Whitney U test (H), or Spearman correlation (non-Gaussian distribution) [(I) and (J)]. *P < 0.05, **P < 0.01, ****P < 0.0001. (A) and (M) created using Biorender.com.
Fig 2: Schematic illustration of the mechanism of MSC-exos for the treatment of ischemic AKI. In ischemic AKI, the injuries of TECs could lead to cell cycle arrest in G2/M phase and apoptosis. MSC-exos targeted injured kidney especially the proximal tubules due to VLA-4 and LFA-1 mediated adhesive interactions. Moreover, miR-125b-5p was enriched in MSC-exos and exerted tubular repair effect via suppressing the expression of p53, which not only up-regulated CDK1 and Cyclin B1 to rescue tubular G2/M arrest, but modulated Bcl-2 and Bax to inhibit TECs apoptosis.
Fig 3: Integrin VLA-4 and LFA-1 guide the homing of MSC-exos to injured kidney. (A) Western blotting analysis of VLA-4 (integrin α4, integrin β1) and LFA-1 on MSC-exos. (B) Western blotting analysis of VCAM-1 and ICAM-1 in control or cells with H/R injury (n = 4). (C) Western blotting analysis of VCAM-1 and ICAM-1 in sham or I/R injured mice tissues (n = 4). (D) Flow cytometry detected the effect of siICAM-1 and siVCAM-1 transfection on cellular uptake of DiI-labeled MSC-exos in H/R-injured HK-2 cells. The red peak represented NC siRNA treated and the purple one represented siICAM-1 and siVCAM-1 transfected cells. (E) Flow cytometry detected the effect of VLA-4 and LFA-1 blockade on cellular uptake of DiI-labeled MSC-exos in H/R-injured HK-2 cells. The red peak represented isotype control treated MSC-exos and the orange one represented anti-VLA-4 and anti-LFA-1 antibodies treated MSC-exos. (F) Imaging of fluorescence intensity in isotype control or blocking antibodies treated group (n = 6). (G) Fluorescent images showed the accumulation of isotype control or blocking antibodies treated DiD-labeled MSC-exos in I/R mice (n = 6). Scale bars, 25 μm. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student's t-test.
Fig 4: Interferon-stimulated gene 15 (ISG15)-induced CCL18 secretion by macrophages was dependent on the ISG15 receptor, leukocyte function-associated antigen-1 (LFA-1), and SRC family kinase (SFK) signaling. (A) Immunofluorescence demonstrated that the ISG15 receptor CD11a/CD18, also called LFA-1, was expressed on the membranes of human macrophages. Macrophages were treated with 295nM rISG15 for 24 h. Scale bars, 10 μm. (B) ELISA results showed small molecular inhibitors of LFA-1, A286982, inhibited CCL18 secretion by macrophages. (C) Small molecular inhibitors of LFA-1 and SRC, A286982 and PP2, hindered the ISG15-induced activation of SFK signaling. (D) PP2 reduced the CCL18 secretion by ISG15-treated macrophages in a dose-dependent manner, as determined by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 5: Exploration of the cellular uptake efficiency, ROS scavenging ability, macrophage polarization, anti-inflammatory and antisenescence activities of USPB@SeDMSN@NM in vitro. a Western blots of integrin β2 and LFA-1 on USPB@SeDMSN, neutrophils, NMs, and USPB@SeDMSN@NM at the same protein amount loading. b In vitro binding to activated endothelial cells. Confocal microscopy images of USPB@SeDMSN@NM/Dil compared with those of USPB@SeDMSN@Lip/Dil and free Dil after 4 h of incubation. HUVECs were pretreated with TNF-α (25 ng/mL) for 6 h. Scale bar, 20 μm. c ICAM-1 expression in normal HUVECs and inflamed HUVECs induced by TNF-α. d Quantitative analysis of ICAM-1 protein expression from c normalized to that of GAPDH (n = 3). e Fluorescence microscopy was used to assess the attenuation of the inflammatory response in LPS-induced macrophages treated with various formulations, including USPB, SeDMSN, USPB@SeDMSN, and USPB@SeDMSN@NM. Green fluorescence indicates the presence of ROS, as detected by the DCFH-DA probe. f Quantitative analysis of the average optical density of DCF (green fluorescence) in RAW264.7 cells. The results are expressed as the means ± SD (n = 4). The related typical inflammatory factors g IL-6, h IL-1β, and i TNF-α were detected via an ELISA kit. The results are presented as the means ± SD (n = 3). j The mRNA levels of IL-10 after treatment with LPS alone or in combination with different formulations for 24 h (n = 3). All the data were normalized to that of β-actin. Quantitative analysis of k HIF-1α, l iNOS, m Arg1, n CD86, and o CD206 protein expression from panel (p), normalized to that of β-actin (n = 3). p Hypoxia-inducible factor (HIF-1α) and macrophage polarization-related proteins (M1: iNOS, CD86; M2: Arg1, CD206) were measured via western blotting in RAW264.7 cells treated with LPS or LPS plus different nanozymes for 24 h. q Representative optical microscopy images of the attenuation effects of USPB@SeDMSN@NM on the formation of foam cells induced by both LPS and oxLDL. Scale bar: 200 μm. r DNA damage in HUVECs was assessed by determining the colocalization of γ-H2AX (green) and DAPI (blue) across different treatment groups. Scale bar: 200 μm. s Quantification of Oil Red O-stained area size in RAW264.7 cells, as calculated from panel (q); n = 4. t Quantitative analysis of the average optical density of γ-H2AX, derived from (r). The data are presented as the means ± SD (n = 4). u Quantitative results of β-gal (blue) expression in HUVECs under different treatments, as shown in (v). The data are expressed as the means ± SD (n = 4). v Antisenescence activity of USPB@SeDMSN@NM verified by SA-β-gal staining. The model of cellular senescence was induced by 100 μM H2O2. Scale bar: 200 μm. Statistical significance was determined by one-way ANOVA and two-tailed Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001
Supplier Page from Abcam for Anti-CD11+CD18 antibody [24]