Journal: Breast cancer research and treatment

Article Title: Eribulin promotes proliferation of CD8 + T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells.

doi: 10.1007/s10549-023-07111-x

Figure Lengend Snippet: Fig. 3 Phenotypic change of CD3/CD28-stimulated T cells in human PBMC by eribulin treatment. a Frequency of CD45RA+ (left), CD45RO+ (middle), and CCR7+ (right) cells in CD8+ T cells (CD3+ CD8+ cells) on day 6 that was standardized by the value of control cells from six (CD45RA), four (CD45RO), and five (CCR7) donors, respectively. b, c Representative flow cytometric plots show- ing CD127 and KLRG1 (b), and TCF-1 (c) expression in CD8+ T cells (CD3+ CD8+ cells) treated with DMSO (control) (left), eribulin

Article Snippet: A 24-well plate was coated with 1 μg/ml anti-CD3 antibody and 1 μg/ml anti-CD28 antibody (Miltenyi Biotec, Burlington, MA, USA).

Techniques: Control, Expressing

Journal: Breast cancer research and treatment

Article Title: Eribulin promotes proliferation of CD8 + T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells.

doi: 10.1007/s10549-023-07111-x

Figure Lengend Snippet: Fig. 4 Effect of eribulin on cell proliferation and phenotypic change of CD3/CD28-stimulated mouse T cells. a Experimental set-up. Sple- nocytes from female BALB/c − AnNCr mice were stimulated with CD3/CD28 antibody and IL-7 in the presence of DMSO (control) or eribulin for 6 days. b Representative flow cytometric plots show- ing CD62L and CD44 expression in mouse CD8+ T cells (CD3+ CD8+ cells) treated with DMSO (control) (left) or eribulin (right). Data panel shows average frequency of CD44+ CD62L− cells (left) and CD44+ CD62L+ cells (right) in CD8+ T cells. Numbers, percent CD44+ CD62L− cells (black square) and CD44+ CD62L+ cells (red square). The error bars represent the standard deviations of the val- ues obtained. The experiments were performed in triplicate in each sample. The experiments were repeated independently at three times, and one representative result is provided in the figures. *p < 0.05 by two-tailed t test

Article Snippet: A 24-well plate was coated with 1 μg/ml anti-CD3 antibody and 1 μg/ml anti-CD28 antibody (Miltenyi Biotec, Burlington, MA, USA).

Techniques: Control, Expressing, Two Tailed Test

Journal: Breast cancer research and treatment

Article Title: Eribulin promotes proliferation of CD8 + T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells.

doi: 10.1007/s10549-023-07111-x

Figure Lengend Snippet: Fig. 5 Alteration of CD4/CD8 ratio in CD3/CD28-stimulated T cells when co-cultured with TNBC cell lines. a, b Representative flow cytometric plots showing CD4 and CD8 expression on T cells (CD3+ cells) that were co-cultured with none (control) (left), MDA-MB-231 (a) or Hs578T (b) (middle) and MDA-MB-231 (a) or Hs578T (b) + 1 nM of eribulin (right). Numbers, percent CD4+ or CD8+ cells. Data panel shows average CD4/CD8 ratio. 6 days after CD3/

Article Snippet: A 24-well plate was coated with 1 μg/ml anti-CD3 antibody and 1 μg/ml anti-CD28 antibody (Miltenyi Biotec, Burlington, MA, USA).

Techniques: Cell Culture, Expressing, Control

Journal: Breast cancer research and treatment

Article Title: Eribulin promotes proliferation of CD8 + T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells.

doi: 10.1007/s10549-023-07111-x

Figure Lengend Snippet: Fig. 6 Anti-tumor activity of CD3/CD28-stimulated T cells to TNBC cells with eribulin treatment. a, b Cell numbers of MDA-MB-231 (upper), Hs578T (middle), MDA-MB-157 (lower) cells when after co-culture with or without CD3/CD28-stimulated PBMCs in the pres- ence or absence of eribulin for 24 h. PBMCs were collected from two different donors (Donor A; a, Donor B; b). The error bars represent the standard deviations of the values obtained. The experiments were performed in triplicate in each donor. The experiments were repeated

Article Snippet: A 24-well plate was coated with 1 μg/ml anti-CD3 antibody and 1 μg/ml anti-CD28 antibody (Miltenyi Biotec, Burlington, MA, USA).

Techniques: Activity Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A) Schematic of the SEC-coupled AS-MS workflow. A total of 6,336 compounds were screened in pooled format using CD28 protein, followed by SEC and LC-MS to detect protein-bound ligands. (B) Representative SEC/UV chromatograms from the primary screen using 2.5 μM CD28 and 1 μM compound concentration. A distinct protein peak is observed in CD28 + compound samples (P1+, P2+) but absent in the compound-only control (P−), confirming successful protein separation. (C) Hit selection was based on mass error ≤5 ppm, absence in P− control, P+/P− intensity ratio ≥3, consistent retention time, clear isotopic pattern, and signal >1000 counts. A total of 56 compounds were initially selected, and 25 were confirmed as CD28 binders. (D) Fraction bound (%) of selected compounds from the retest screen using 5 μM CD28. Compounds 5MS, 7MS, 12MS, 13MS, 17MS, 19MS , and 20MS showed the highest Fb% values (>30%), indicating strong binding to CD28.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Control, Selection, Binding Assay

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A) Single-dose binding responses of 25 hits at 100 μM using the Dianthus platform in PBST buffer (PBS + 0.05% Tween-20) with 2% DMSO. Compounds higher or lower three times the SD of CD28 only (Blank) were selected for dose dependent analysis. (B–D) Dose–response MST binding curves for compounds 5MS, 19MS , and 20MS , respectively. Each compound was prepared at an initial concentration of 500 μM and subjected to a 14-point 1:1 serial dilution (final range: 500 to 0.06 μM). Data represent mean ± standard error mean (SEM) from n = 5 independent measurements.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Binding Assay, Concentration Assay, Serial Dilution

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: Each compound was serially diluted from 500 to 0.06 μM in PBST with 2% DMSO, while CD28 protein concentration was held constant. KD values were determined by nonlinear regression using GraphPad Prism, fitted to a 1:1 binding model. Data are presented as mean ± SEM from n = 5 independent experiments.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Protein Concentration, Binding Assay

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: The CD28 Blockade Bioassay (Promega, Cat. #JA6101) was employed to assess the ability of 19MS-5 to inhibit CD28–B7 interactions in a co-culture system of CD28 Effector Cells (Jurkat) and aAPC/Raji Cells. The compound was evaluated in a 10-point dose–response format, and luminescence was quantified using the Bio-Glo™ Luciferase Assay System. Dose–response curve was fitted using nonlinear regression (fourparameter logistic model) in GraphPad Prism. Data are presented as mean ± SEM from n = 5 independent experiments.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Bioassay, Co-Culture Assay, Luciferase

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A-B) Compound 5MS docked into CD28, showing surface view (A) and detailed interaction diagram (B). (C-D) Compound 19MS-5 binding mode, displaying surface representation (C) and interaction network (D). (E-F) Compound 5MS-5 docked configuration, illustrated through surface view (E) and binding interactions (F). (G-H) Compound 19MS binding analysis, showing surface representation (G) and detailed interactions (H) . In all panels, the protein surface is colored according to electrostatic potential (blue: positive, red: negative, white: neutral), and green dashed lines indicate hydrogen bonds or favorable electrostatic interactions. The ligands are represented as gray stick models, and interacting residues are labeled with their three-let-ter amino acid codes and residue numbers. Favorable interactions are color-coded as follows: green, hydrogen bonds; light green, carbon–hydrogen interactions; dark pink, π–π stacking interactions; purple, π–sigma interactions; light pink, hydrophobic interactions. All docking studies were performed using Maestro Schrödinger (version 2023.2) with induced fit protocols to account for protein flexibility during ligand binding.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Binding Assay, Labeling, Residue, Ligand Binding Assay

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: RMSD analysis of CD28 protein-ligand complexes during 50 ns molecular dynamics simulations.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques:

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A) Schematic overview of the experimental setup. Human PBMCs were overlaid onto fully differentiated Mu-cilAir™ tissue (Epithelix) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 48 h in the presence of vehicle (0.1% DMSO), 19MS-5 (10, 25, or 50 μM), or FR104 (10 μg/mL). (B–D) Quantification of IFN-γ, IL-2, and TNF-α secretion (pg/mL) in apical supernatants by ELISA. 19MS-5 suppressed CD28-induced cytokine production in a dose-dependent manner, with levels at 50 μM comparable to FR104. Statistical comparisons to the “CD3/CD28 + Vehicle” group were performed using one-way ANOVA followed by Dunnett’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 relative to “CD3/CD28 + Vehicle” group.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Janus Kinase Inhibitor Baricitinib Modulates Human Innate and Adaptive Immune System.

doi: 10.3389/fimmu.2018.01510

Figure Lengend Snippet: Figure 5 | Baricitinib inhibits T cell proliferation and differentiation of Th1 and Th17 cells. Human naive CD4+ T cells were purified and cultured with anti-CD3 antibody, anti-CD28 antibody, and various cytokines [interleukin (IL)-12, TGF-β, IL-6, IL-1β, and IL-23]. (A) T cell proliferation assessed by [3H] thymidine incorporation. (B) Percentage of interferon (IFN)-γ-producing cells in T cells in the presence of baricitinib and other inhibitors. (C) Representative flow cytometry plots showing IFN-γ production in T cells. (D) Percentage of IL-17-producing cells in T cells in the presence of baricitinib and other inhibitors. (E) Representative flow cytometry plots showing IL-17 production in T cells. Data are mean ± SD of three different donors per group. *p < 0.05 (by Mann–Whitney U test).

Article Snippet: The obtained cells were cultured in flat-bottomed 96-well plates (2 × 105 cells/well) coated with anti-CD3 antibody (2 μg/ml; R&D systems, Minneapolis, MN, USA) and supplemented with soluble anti-CD28 antibody (0.5 μg/ml; R&D systems) and cytokines [IL-12 (10 ng/ml), TGF-β (1 ng/ml), IL-6 (10 ng/ml), IL-1β (5 ng/ml), and IL-23 (10 ng/ml)].

Techniques: Purification, Cell Culture, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: CD38 expression by neonatal human naive CD4 + T cells shapes their distinct metabolic and tolerogenic properties

doi: 10.1172/JCI200062

Figure Lengend Snippet: ( A and B ) CD25 + percentages from CB and AB naive CD4 + T cells after 96 hours of anti-CD3/CD28 stimulation under No Cytokine or IL-2 + TGF-β conditions. ( C – E ) Marker expression on CD25 + fraction by flow cytometry. ( F and G ) ELISA of cytokines in supernatant. ( H ) Allogeneic MLR measuring suppressive capacity of CB and AB No Cytokine and IL-2 + TGF-β-stimulated cells. ( I ) Allogeneic MLR assay for CD25 + versus CD25 neg iTreg-stimulated cells. ( J – L ) FOXP3 CNS2 methylation was assessed by bisulfide sequencing in CD4 + CD25 + CD127 lo cells from iTreg cultures and endogenous blood Tregs (CD4 + FOXP3 + CD25 + CD127 lo ). Two separate experiments were analyzed together with adjustment for batch effects. ( J ) Heatmap, ( K ) violin plot, and ( L ) graphs of CpG island methylation ratios. ( M ) FOXP3 stability assessed through 2 rounds of stimulation and rest. CD25 + and FOXP3 MFI on CD25 + cells after second rest. Data points represent individual donors. ( H and I ) N = 5/group. ( A – E ) One of 3 representative experiments. ( H ) One of 2 representative experiments. ( F , G , I , and M ) Performed once. ( J – L ) Combined data from 2 experiments. ( B – G and M ) Two-way ANOVA with multiple comparisons used. ( I ) One-way ANOVA with multiple comparisons used. ( L ) Linear mixed model (included batch correction). * P ≤ 0.05, ** P ≤ 0.02, *** P ≤ 0.001, **** P < 0.0001. Horizontal bars and column heights in panels B – G and M depict mean values. In H and I , mean value plus standard deviation is shown.

Article Snippet: In , other protocols for cell activation were used: Human T-Activator CD3/CD28 Dynabeads and plate-coating with anti-CD3/CD28 antibodies (UCSF Antibody Core clone: OKT3 2 μg/mL; Miltenyi Biotec 130-093-375, clone: 15E8, 4 μg/mL) overnight at 4°C or for 4 hours at 37°C.

Techniques: Marker, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mlr Assay, Methylation, Sequencing, Standard Deviation