Journal: Advanced Science

Article Title: Oral Astilbin Nanogels Modulate Senescence‐Associated CD4 + T Cells and Promote T reg Induction in Aged Mice with Colitis

doi: 10.1002/advs.78123

Figure Lengend Snippet: Mechanistic validation of Ast‐mediated regulation of senescence‐associated CD4 + T cells in aged colitis. (A) Experimental flowchart of NG treatment in aged colitis mice ( n = 6). (B) Representative fecal samples following occult blood testing. (C) Curve chart analysis of DAI ( n = 6). (D) Representative images of colons and corresponding quantification of colon length ( n = 6). (E) Quantification of PD‐1 + CD4 T cells in colonic lamina propria cells ( n = 6). (F) Representative FC plots and quantitative analysis of CD4 + CD27 − CD28 − T cells in colonic lamina propria cells ( n = 6). (G) Schematic illustration of the isolation and sorting strategy for senescence‐associated (CD28 − CD27 − ) and non‐senescence‐associated (remaining) CD4 + T cells. Created with BioRender. (H) MFI of PD‐1 expression on senescence‐associated and non‐senescence‐associated CD4 + T cells after treatment with PBS or Ast for 2 days ( n = 3). (I) Cell viability of senescence‐associated and non‐senescence‐associated CD4 + T cells ( n = 3). (J) Foxp3 + T reg differentiation capacity of senescence‐associated and non‐senescence‐associated CD4 + T cells in the presence of PBS or Ast for 2 days ( n = 3). (K) Experimental groups for therapeutic evaluation in aged colitis mice. (L) Curve chart analysis of DAI ( n = 6). (M) Quantification of colon length ( n = 6). Quantitative analysis of (N) CD4 + PD‐1 + T cells and (O) CD4 + CD27 − CD28 − T cells in colonic lamina propria cells ( n = 6). (P) Schematic illustration of CD4 + T cells cultured in the presence of plate‐bound agonistic aPD‐1 or Ast. Created with BioRender. (Q) Quantitative analysis of CD4 + CD27 − CD28 − T cells following treatment with plate‐bound agonistic aPD‐1 or Ast ( n = 3). Data are presented as mean ± SD and analyzed by Two‐way ANOVA with Tukey's post‐test (C, L) or One‐way ANOVA with Tukey's post‐test (D‐F, H‐J, M‐O, Q). ns represents not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Purified blocking anti‐mouse PD‐1 (Clone RMP1‐14), agonistic anti‐mouse PD‐1 (Clone RMP1‐30), anti‐mouse CD3 (Clone 17A2), and anti‐mouse CD28 (Clone 37.51) were purchased from BioXcell (USA).

Techniques: Biomarker Discovery, Fecal/Stool Samples, Analysis, Isolation, Expressing, Cell Culture, FACS

Journal: PLOS One

Article Title: Identification of Galectin-9 (Gal-9) as a B7-H4 binding partner and characterization of their glycosylation-dependent interaction that modulates T cell signaling within a multi-ligand/receptor network

doi: 10.1371/journal.pone.0355964

Figure Lengend Snippet: (A) SPR analysis of Gal-9 binding kinetics to PD-1, TIM-3, CD28, and B7-H4. Human PD-1, TIM-3, and CD28 were fused to the same mouse IgG2a Fc tags. 1.2 µg/mL Gal-9 was injected over a Protein A/G-coated Biacore chip to assess binding kinetics to each immobilized Fc-fusion protein. (B) Flow cytometry analysis of the binding between the four proteins (shown in Panel A) and 293T cells transiently overexpressing either WT Gal-9 or the Gal-9 R65A mutant. ( C ) Cell-surface binding of CD28 to Gal-9 analyzed by Co-IP-Western blotting. Cell-surface expression of CD28 on Jurkat T cells was examined by flow cytometry (left panel); CD28 expression was detected using an anti-CD28 antibody (red), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel); 400 µL of whole cell lysates (WCL) from Jurkat T cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-CD28 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-CD28 antibody from that used for IP, together with an anti-Gal-9 antibody, as indicated. ( D ) Cell-surface binding of B7-H4 to Gal-9 was analyzed by Co-IP-Western blotting. Cell-surface expression of B7-H4 on 293T cells transiently transfected with B7-H4 (293T-B7-H4) was examined by flow cytometry (left panel); B7-H4 expression was detected with an anti-B7-H4 antibody (blue), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel), 400 µL of WCL from 293T-B7-H4 cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-B7-H4 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-B7-H4 antibody from that used for IP, and an anti-Gal-9 antibody, as indicated. Anti-GAPDH antibody was used as an internal loading control for Western blotting of WCL samples in (C) and (D). (E) Gal-9-mediated stimulation of pCD28 signaling activity in Jurkat T cells. Jurkat T cells were treated with or without plate-bound B7.1, and Gal-9’s dose-dependent effects on pCD28 were analyzed by western blotting. Fold change (FC) in pCD28 band intensity relative to the CD28 loading control was quantified using ImageJ and GraphPad Prism.

Article Snippet: Antibodies and reagents used for western blotting were primarily obtained from ABclonal unless otherwise indicated: Phospho-CD28 (pCD28; Y191) Rabbit mAb (Cell Signaling Technology, 16399), CD28 Rabbit mAb (A20346), Phospho-Akt (pAKT; S473) Rabbit mAb (AP1208), Pan-Akt Rabbit mAb (A18675), HRP-conjugated β-Actin Rabbit mAb (AC028), Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, 5872), and Lactose (LABLEAD BIOTECH, 5989-81-1).

Techniques: Binding Assay, Injection, Flow Cytometry, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, Incubation, Transfection, Activity Assay

Journal: PLOS One

Article Title: Identification of Galectin-9 (Gal-9) as a B7-H4 binding partner and characterization of their glycosylation-dependent interaction that modulates T cell signaling within a multi-ligand/receptor network

doi: 10.1371/journal.pone.0355964

Figure Lengend Snippet: (A-C) B7-H4 modulates pCD28 and pAKT signaling in T cells in a glycosylation-dependent manner. Jurkat (A), MOLT-4 (B), and primary T cells (C) were treated with 8 µg/mL Gal-9 in the presence or absence of 25 µg/mL hB7-H4-Fc, hB7-H4 IgV-Fc, or de-glycosylated B7-H4 MGAT1- . Western blotting was performed to detect pCD28 and pAKT signaling activity (top panels). Fold changes (FC) in pCD28 and pAKT band intensity relative to the corresponding CD28 and AKT loading controls were quantified using Image J and GraphPad Prism (middle and bottom panels, respectively). Gal-9-mediated stimulation of pCD28 and pAKT signaling, and its regulation by hB7-H4-Fc, were independently replicated in Jurkat (Panel A and ), MOLT-4 T cells (Panel B and ), and primary T cells (Panel C and ) in two or three experiments. (D) Gal-9 stimulates NFAT signaling. Jurkat-NFAT-Luc reporter cells showed dose-dependent luciferase induction by Gal-9, but not hB7-H4-Fc. (E) B7-H4 attenuated Gal-9-induced CD28 signaling. Jurkat-NFAT-Luc reporter cells were treated with 3.3 µg/mL Gal-9, 25 µg/mL hB7-H4-Fc, or both. (F) Flow cytometry analysis of B7-H4 expression on 293F cells. B7-H4 expression on 293F cells (293F-B7-H4) was confirmed, the 293F-B7-H4 cells were used in panel G. (G) Cell-surface B7-H4 attenuated Gal-9-induced CD28 signaling. Jurkat-NFAT-Luc reporter cells were co-cultured with 293F or 293F-B7-H4 cells in the presence or absence of 3.3 µg/mL Gal-9. One-way ANOVA was used to statistically analyze the mean of NFAT-mediated luminescence signals among groups in Panels E and G. All data are presented as mean ± s.e.m.

Article Snippet: Antibodies and reagents used for western blotting were primarily obtained from ABclonal unless otherwise indicated: Phospho-CD28 (pCD28; Y191) Rabbit mAb (Cell Signaling Technology, 16399), CD28 Rabbit mAb (A20346), Phospho-Akt (pAKT; S473) Rabbit mAb (AP1208), Pan-Akt Rabbit mAb (A18675), HRP-conjugated β-Actin Rabbit mAb (AC028), Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, 5872), and Lactose (LABLEAD BIOTECH, 5989-81-1).

Techniques: Glycoproteomics, Western Blot, Activity Assay, Luciferase, Flow Cytometry, Expressing, Cell Culture