Journal: Nature Communications

Article Title: TIGIT deficiency promotes autoreactive CD4 + T-cell responses through a metabolic‒epigenetic mechanism in autoimmune myositis

doi: 10.1038/s41467-025-59786-z

Figure Lengend Snippet: PBMCs were collected from polymyositis (PM), dermatomyositis (DM), systemic lupus erythematosus (SLE), or healthy controls (HC). TIGIT, CD226, and CD96 expression in cells in PBMCs was measured by flow cytometry. a Schematic employed to visualize TIGIT, CD226, and CD96 on T cells binding to CD155 on antigen-presenting cells (APC). b – g CD4 + T cells from HC or PM, DM or SLE patients were stimulated with anti-CD3/CD28 beads (αCD3/CD28) for 3 d. The expression of TIGIT, CD226, and CD96 in pre- and post-activation of CD4 + T cells was measured by flow cytometry of biological replicates (HC = 27, PM = 27, DM = 23, SLE = 23). h Naive CD4 + T cells isolated from PBMCs of HC or patients with PM, were stimulated with αCD3/CD28. TIGIT expression was measured by flow cytometry. Data from four biologically independent replicates. h – l Total CD4 + T cells from patients with PM or HC were transfected with pcDNA3.1-hTIGIT or pcDNA3.1-Vector by electroporation. Cells were then stimulated by αCD3/CD28 for 3 d. h Overexpression of TIGIT was confirmed by Western blot. The experiment was repeated three times independently with similar results. i , j Overexpression of TIGIT in CD4 + T cells from HC or PM was confirmed by flow cytometry. Data from five biologically independent replicates. k , l IFNγ and IL-17A production by CD4 + T cells from PM patients or HC was measured by flow cytometry. Data from five biologically independent replicates. m – o CD4 + T cells from PM patients or HC were stimulated with αCD3/CD28 in the presence of CD155-Fc (10 μg/ml) or control IgG1 for 3 d. IFNγ, IL-17A, and FoxP3 expression in CD4 + T cells from PM patients or HC was measured by flow cytometry. Data from five biologically independent replicates. All data are mean ± SEM. Statistics were done by one-way ANOVA with adjustments for multiple comparisons in c , e , g , and two-way ANOVA with adjustments for multiple comparisons in j , l , n , and o .

Article Snippet: Briefly, CD4 + T cells were activated with anti-CD3/CD28 beads in RPMI containing 2 g/L 12 C-glucose for 3 d. T cells were then collected and labeled in modified RPMI 1640 containing 2 g/L [ 13 C6]-glucose (MCE, Cat# HY-B0389A) for 24 h. For GC‒MS, cells were plated at 2 × 10 6 cells/well in 24-well plates.

Techniques: Expressing, Flow Cytometry, Binding Assay, Activation Assay, Isolation, Transfection, Plasmid Preparation, Electroporation, Over Expression, Western Blot, Control

Journal: Cancer cell

Article Title: Eradication of triple negative breast cancer cells by targeting glycosylated PD-L1

doi: 10.1016/j.ccell.2018.01.009

Figure Lengend Snippet: (A) Interaction of PD-1 and PD-L1 proteins with or without PNGase F. Confocal image shows bound PD-1/Fc fusion proteins on the membrane of BT549-PD-L1 cells. (B) Flow cytometry measuring PD-1 binding on the membrane of BT549 cells expressing gPD-L1 with or without PNGase F. (C) Co-immunoprecipitation and Western blot analysis measuring the interaction of PD-1 and PD-L1 in BT549 cells expressing gPD-L1 or ngPD-L1. (D) Time-lapse microscopy quantification showing the dynamic interaction between PD-L1 and PD-1. Representative phase, red fluorescent (nuclear restricted RFP) and green fluorescent (green fluorescent labeled PD-1/Fc protein) merged images of gPD-L1- or ngPD-L1-expressing BT549 cells at 12 h (top). Scale bar, 100 μm. (E) Flow cytometry measuring the interaction of membrane bond PD-1 on gPD-L1 or ngPD-L1 expressing BT549 cells. Cells were pretreated with MG132 prior to experiment. (F) T cell-mediated tumor cell killing assay in gPD-L1- or ngPD-L1-expressing BT549 cells. Representative phase, red fluorescent (nuclear restricted RFP), and green fluorescent (NucView 488 Caspase 3/7 substrate) merged images (10× magnification) are shown. Green fluorescent cells were counted as dead cells. The quantitative ratio of dead cells showed in bar graph. (G) Tumor growth of 4T1 cells expressing gPD-L1 or ngPD-L1 in BALB/c or BALB/c SCID mice. n = 7 mice per group. (H) Quantification of intracellular cytokine stain of IFNγ in CD8+, CD3+ T cell populations in BALB/c mice. n = 8 mice per group. *p < 0.05, statistically significant by Student’s t-test. Error bars, mean ± S.D. of three independent experiments. See also Figure S2

Article Snippet: Briefly, to prime tumor cell-specific T cells, we co-cultured tumor cells with anti-CD3 antibody and IL-2–stimulated human peripheral blood mononuclear cells (Stemcell Technologies, Vancouver, BC, Canada) for 5 to 7 days and then isolated and expanded the T cell population using ImmunoCult Human CD3/CD28 T Cell Activator (Stemcell Technologies).

Techniques: Membrane, Flow Cytometry, Binding Assay, Expressing, Immunoprecipitation, Western Blot, Time-lapse Microscopy, Labeling, Staining

Journal: Cancer cell

Article Title: Eradication of triple negative breast cancer cells by targeting glycosylated PD-L1

doi: 10.1016/j.ccell.2018.01.009

Figure Lengend Snippet: (A) PD-1/PD-L1 blockade by glycosylated PD-L1 antibodies. Kinetic graph showing quantitative binding of PD-1/Fc protein on BT549 cells expressing PD-L1 at hourly time points after treatment with glycosylated PD-L1 antibodies. (B) Blockade of PD-L1 and PD-1 interaction by the glycosylated PD-L1 antibodies STM004 and STM108. (C) Schematic diagram of various PD-L1 NQ mutants used in this study. The numbers indicate amino acid positions of the PD-L1 protein. (D) Western blot analysis of wild-type and mutant PD-L1 using STM004 or STM108 antibody. (E) Epitope mapping of glycosylated PD-L1–binding antibodies by High-Mass MALDI mass spectrometry (CovalX service). (F) Interaction of human PD-1 (hPD-1) or mouse PD-1 (mPD-1) protein with human PD-L1 (hPD-L1) on BT549 cells or mouse PD-L1 (mPD-L1) or hPD-L1 on 4T1 cells, with or without STM108 antibody. (G) Tumor growth of 4T1 cells expressing human PD-L1 (4T1-hPD-L1) in BALB/c mice treated with STM004 or STM108 antibody. Tumors were measured at the indicated time points and dissected at the endpoint. n = 7 mice per group. (H) Intracellular cytokine stain of IFNγ in CD8+ CD3+ T cell populations. n = 7 mice per group. (I) Immunofluorescence staining of the protein expression pattern of PD-L1, CD8, and granzyme B (GB) in a 4T1-hPD-L1 tumor mass. Scale bar, 100 μm (20 μm in magnified sections). (J) Quantitative binding affinity of gPD-L1 antibody (STM108) to glycan 1 and 2. Glycan array 100 was probed with biotin-labeled gPD-L1 antibody. gPD-L1 antibody bound to two glycans (1 and 2), and the bindings were compromised by a mixture of B3GNT3 substrate or product, mixture of DiLacNAc and GlcNAcβ1,3-Gal. (K) Western blot analysis of glycosylation of PD-L1 protein in BT549 cells by STM108 (gPD-L1). BT549 control (CTRL) or B3GNT3−/− cells were treated with 25 ng/ml EGF or gefitinib overnight. *p < 0.05, statistically significant by Student’s t-test. Error bars, mean ± S.D. of three independent experiments. See also Figure S5, Table S3, and Table S4

Article Snippet: Briefly, to prime tumor cell-specific T cells, we co-cultured tumor cells with anti-CD3 antibody and IL-2–stimulated human peripheral blood mononuclear cells (Stemcell Technologies, Vancouver, BC, Canada) for 5 to 7 days and then isolated and expanded the T cell population using ImmunoCult Human CD3/CD28 T Cell Activator (Stemcell Technologies).

Techniques: Binding Assay, Expressing, Western Blot, Mutagenesis, Mass Spectrometry, Staining, Immunofluorescence, Glycoproteomics, Labeling, Control