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

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: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: Examination of tissue and cell expression of IL-32 by quantitative real-time PCR. (a) Tissue expression of IL-32. WBC, white blood cells. (b) Human peripheral blood mononuclear cells (PBMCs) expressed IL-32. PBMCs were cultured with or without concanavalin A. PBMCs were also stimulated by immobilized anti-human CD3 and anti-human CD28 antibodies. Cont, control. (c) IL-32 expression of monocytes and B cells after the depletion of CD3 + cells. (d) Peripheral CD4 + T cells were cultured with the indicated inflammatory cytokines for 24 hours. (e) Human monocyte-derived dendritic cells (MoDCs) were cultured with lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNFα) for 24 hours to induce maturation. The data are representative of at least three independent studies.

Article Snippet: PBMCs were stimulated with Con A or plate-coated anti-human CD3 antibodies and anti-human CD28 antibodies (R&D Systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Control, Derivative 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: 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