Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: HDCA modulates Treg migration and atherosclerotic plaque composition via FXR signaling. ApoE−/− mice (C57BL/6J background, male, 8 weeks old) were fed a high-fat diet for 28 days to induce AS and subsequently received adoptive transfer of control or FXR-knockout (FXR KO) Treg cells generated by CRISPR/Cas9-mediated lentiviral transduction, with HDCA (30 μM) or vehicle treatment as indicated. (A) Representative Western blot analysis of FXR, PD-1, SHP-2, p-Raptor, RAC and IL-10R expression in isolated Treg cells from each group. (B) Oil Red O staining was performed to assess lipid accumulation in the aorta. (C) Masson's trichrome staining of aortic sections from FXR KO mice reveals comparable plaque area and collagen deposition in both HDCA-treated and untreated groups (magnification, 5 × ; scale bar, 1 mm). (D) Representative H&E images of aortic sections show the difference between untreated and HDCA-treated mice in the FXR KO groups (magnification, 5 × ; scale bar, 500 μm). Relative bar graphs show quantification of lesion area and lesion/media area ratio. (E) Confocal immunofluorescence was used to evaluate Foxp3+ Treg infiltration within atherosclerotic plaques (scale bar, 25 μm). (F) Immunohistochemistry images show Treg accumulation in the plaque area across all groups (magnification, 40 × ; scale bar, 100 μm). (G) Flow cytometry analysis of Treg proportions in aortic plaques and spleen from control and FXR KO mice, with or without HDCA treatment. (H) Western blot analysis of matrix remodeling-related proteins, including calpain 1 and matrix metalloproteinase 2, and the anti-inflammatory factor IL-10. Data are presented as mean ± SD (n = 5 biological replicates). Data with four groups were analyzed by one-way ANOVA with Tukey's post hoc test. Comparisons between two groups were performed using the non-parametric Mann-Whitney U test. ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Proteins were detected using the following antibodies: anti-CPT1a antibody (ab234111, abcam), anti-beta actin antibody (ab8226, abcam), anti-PERK antibody (ab229912, abcam), anti-ERK1+ERK2 antibody (ab184699, abcam), anti-S6K1 antibody (ab14708, abcam), anti-S6K1 (phospho T229) antibody (ab5231, abcam), Rac1/2/3 antibody (G-2) (sc-514583, Santa Cruz), anti-Calpain 1 antibody (ab108400, abcam), anti-MMP2 antibody (ab92536, abcam), anti-IL-10 antibody (ab310329, abcam), anti-ZNF671 antibody (HPA046099, Sigma-Aldrich), anti-MAPK6/ERK3 antibody (ab53277, abcam), SIAH1 recombinant rabbit monoclonal antibody (PSH01-80) (MA5-51926, Thermo Fisher), p-Stat1 antibody (pY701.4A) (sc-136229, Santa Cruz), Stat1 antibody (C-136) (sc-464, Santa Cruz), anti-FXR1 antibody (ab155124, abcam), phospho-Raptor (Ser792) polyclonal antibody (PA5-118730, Thermo Fisher), anti-PD1 antibody (ab214421, abcam), SHP-2 antibody (3752S, Cell Signaling Technology), IL-10R antibody (3F9) (sc-53654, Santa Cruz), GAPDH antibody (6C5) (sc-32233, Santa Cruz), rabbit anti-mouse IgG H&L (HRP) (ab6728, abcam), goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa FluorTM Plus 488 (A32731, Thermo Fisher).

Techniques: Migration, Adoptive Transfer Assay, Control, Knock-Out, Generated, CRISPR, Transduction, Western Blot, Expressing, Isolation, Staining, Immunofluorescence, Immunohistochemistry, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Bivalent bispecific CD28 antibodies reinforce T-cell responsiveness and revert anergy/quiescence in patients treated with bispecific CD3 antibodies

doi: 10.64898/2026.03.25.714198

Figure Lengend Snippet: A PBMCs were collected from patients before and after TCE treatment with CC-1 (day 1 and day 8, respectively), incubated ex vivo with CC-1 alone or in combination with BiCos, and subsequently subjected to functional analysis. B Patient-derived PBMC (n=5) were cultured with LNCaP-E tumor cells at an E:T ratio of 1:1 in the presence of 1 nM CC-1 with or without 0.5 nM of BiCo-1, BiCo-2 or isotype control (BiCo-iso). After 72 h, T-cell proliferation was quantified by 3 H-thymidine incorporation, tumor cell killing was determined by flow cytometry. C PBMCs from three patients obtained before and after treatment (day 1 and day 8 of subsequent cycles; see scheme in ) were incubated with CC-1 (5 nM) alone or together with BiCo-1 (5 nM). After 72 h, T-cell proliferation was assessed by 3 H-thymidine incorporation. D Leiden-based subclustering of scRNAseq data visualized by UMAP, depicting T cell populations within patient PBMC samples collected after therapy (day 8), and incubated for 3 days in vitro with LNCaP-E and 1 nM CC-1 with or without 0.5 nM BiCos annotated using canonical lineage markers. UMAP visualization of T-cell subclustering, resolving distinct functional T-cell states. E Dot plots summarizing temporal expression changes of selected genes involved in T-cell activation, cytotoxicity, proliferation and quiescence. Dot size indicates the fraction of cells expressing the respective gene; and color intensity reflects the mean expression. F Density plots illustrating shifts in patient T-cell (day 8) state distributions of T-cells treated in vitro with CC-1 alone or CC-1+BiCos, demonstrating a pronounced transition toward proliferative phenotypes. G PBMCs (n=4) were cultured with LNCaP-E cells at an E:T ratio of 1:2 in the presence of CC-1 (1 nM); medium, target cells, and constructs were replenished on day 4 to mimic chronic exposure. At day 7, PBMCs were re-stimulated with LNCaP-E cells at an E:T ratio of 1:1 in the presence of CC-1 (1 nM) alone or in combination with the indicated BiCo constructs (0.5 nM). On day 10, T-cell proliferation was accessed by 3 H-thymidine incorporation and tumor cell killing was evaluated by flow cytometry. H PBMCs (n = 4) were cultured with LNCaP-E cells at an E:T ratio of 1:2 in the presence or absence of CC-1 (1 nM) alone or in combination with BiCo constructs (0.5 nM). Medium, target cells, and constructs were replenished on day 4 and day 7 with (LNCaP-E cells E:T ratio of 1:1 on day 7). At day 10, T-cell proliferation was assessed by 3 H-thymidine incorporation and tumor cell killing was evaluated by flow cytometry. I PBMCs (n=3) were cultured with SHP-77 cells at an E:T ratio of 1:2 in the presence of tarlatamab (1 nM). Medium and target cells were replenished on day 4. After the exposure until day 7 mimicking induction of hyporesponsiveness, PBMCs were re-stimulated with SHP-77 cells at an E:T ratio of 1:1 in the presence of tarlatamab (1 nM) or in combination with BiCo-2 (0.5 nM). Proliferation was measured on day 10 by 3 H-thymidine incorporation, and tumor cell killing was assessed by flow cytometry.

Article Snippet: The human prostate cancer cell line LNCaP was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and SHP-77 cell line from American Type Culture Collection (ATCC).

Techniques: Incubation, Ex Vivo, Functional Assay, Derivative Assay, Cell Culture, Control, Flow Cytometry, In Vitro, Expressing, Activation Assay, Construct