Journal: Journal of Cardiovascular Pharmacology

Article Title: Evidence of Cardiotoxic Immune Activation by Triple Immune Checkpoint Blockade: A Translational Alert for Clinical Surveillance in Patients With Cancer

doi: 10.1097/FJC.0000000000001798

Figure Lengend Snippet: Effects of nivolumab–relatlimab combined with ipilimumab or atezolizumab on secretion of cytokines in the supernatant of cocultures of hPBMCs with HFC cells. HFC cells were cocultured with lymphocytes (effector–target ratio 5:1) and treated for 48 hours at 37°C with the combination of nivolumab + relatlimab (light gray bars), ipilimumab or atezolizumab (dark gray bars) used alone, or in combinations (black bars) at the indicated concentration. IL-2 (A) and granzyme B (B) levels (pg/mL) were obtained by using the duoset ELISA kit from R & D Systems performed on cell supernatants. Cells untreated or treated with an unrelated hIgG (with bars) were used as negative controls. Error bars depict means ± SD. P -values for the combinations relative to respective single agents are *** P < 0.001; ** P < 0.01.

Article Snippet: Supernatants were collected after 48 hours of coculture and analyzed for IL-2 (DY202) and granzyme B (DY2906) concentrations using DuoSet ELISA kits (R&D Systems, Minneapolis, MN) endowed with a sensitivity of 50 pg/mL.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiovascular Pharmacology

Article Title: Evidence of Cardiotoxic Immune Activation by Triple Immune Checkpoint Blockade: A Translational Alert for Clinical Surveillance in Patients With Cancer

doi: 10.1097/FJC.0000000000001798

Figure Lengend Snippet: Proposed mechanism of immune checkpoint inhibitor (ICI)-driven disruption of immune tolerance and cardiomyocyte injury. Under physiologic conditions, regulatory T cells (Tregs) and inhibitory checkpoint pathways—including cytotoxic T-lymphocyte–associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1)—cooperate to maintain peripheral tolerance and protect cardiac tissue from autoreactive immune responses. ICI therapy blocks these inhibitory signals at multiple levels: (I) anti-CTLA-4 antibodies prevent CTLA-4 engagement on both activated T cells and Tregs, thereby dampening Treg-mediated suppression and enhancing effector T-cell activation; (ii) anti-LAG-3 antibodies abrogate an additional inhibitory checkpoint on activated T cells, further potentiating T-cell cytotoxicity; and (iii) antibodies targeting PD-1 or PD-L1 disrupt the PD-1/PD-L1 axis, which normally delivers inhibitory signals on engagement of PD-1 on activated T cells with PD-L1 expressed on cardiomyocytes. Cumulative blockade of these checkpoints removes critical layers of peripheral tolerance and unleashes effector T cells that recognize cardiac antigens as targets, leading to immune attack against cardiomyocytes. This loss of tolerance contributes to proinflammatory cytokine secretion (eg, IL-2, IL-1β, IL-6, IL-8, CCL2, IL-17), enhanced cytotoxic mediator release such as granzyme B, and activation of the cardiomyocyte NLRP3–MyD88–NF-κB axis, ultimately driving myocardial inflammation and injury. The figure summarizes the central paradigm supported by our coculture experiments, wherein triplet checkpoint blockade (anti-CTLA-4 + anti-PD-1 + anti-LAG-3 or anti-PD-L1) produced the strongest disruption of immune tolerance and the most pronounced cardiotoxic effects.

Article Snippet: Supernatants were collected after 48 hours of coculture and analyzed for IL-2 (DY202) and granzyme B (DY2906) concentrations using DuoSet ELISA kits (R&D Systems, Minneapolis, MN) endowed with a sensitivity of 50 pg/mL.

Techniques: Disruption, Activation Assay, Produced

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a Schematic illustrating the generation of the high-throughput screening platform for Foxp3-mediated Il-2 inhibition and the inhibitor screening workflow. b Treg functional inhibitor screening identified Timosaponin AIII (TAIII) as a “hit”. c Human PBMCs were activated through TCR stimulation and treated with 1 μM of the indicated compounds for 48 h. IL-2 secretion levels were measured by ELISA (upper panel). Cell viability was measured using the CellTiter-Glo assay (lower panel). d Chemical structure of TAIII. e FoxP3a-IL-2-Fluc-reporter were generated by introducing IL-2-promoter-Fluc-reporter that containing a FoxP3 binding site, into Jurkat T cells, the monoclonal cells were picked out post-puromycin selection, followed delivery of a FoxP3a over-expression lentiviral construct containing a BSD resistance cassette. Cells were treated with or without (w/o) TAIII, the target effect towards Foxp3a-mediated IL-2 suppression were determined by luciferase assay. Data are presented as mean ± SD ( n = 3–4 biological replicates); ordinary one-way ANOVA with Dunnett’s test. f TAIII impairs Treg differentiation and functional marker expression in a dose-dependent manner. Human CD4 + Naïve T cells were isolated from umbilical cord blood of healthy donors, and cultured under iTreg polarization conditions for 5 days. iTreg cells were then subjected to TAIII treatment, mRNA levels of indicated genes were assessed post-16 h by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test. g – h Naïve human CD4 + T cells were obtained and differentiated into the iTreg lineage as in e . Expression levels of Foxp3 were assessed post-72 h treatment w/o TAIII by flow cytometry. Data are presented as mean ± SD Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. Source data are provided as a Source Data file.

Article Snippet: Human T or CAR-T cells were cultured in X-VIVO (Lonza, 04-418Q) with 10% FBS (Gibco, 10100-147), 1% GlutaMAX (Gibco, 35050061), 1% NEAA (Gibco, 11140050), 1% Antibiotic-Antimycotic (Gibco, 15240062), 100 IU IL-2 (R&D Systems, 202-IL) and Dynabeads Human T-activator CD3/CD28 (Gibco, 1132D).

Techniques: High Throughput Screening Assay, Inhibition, Functional Assay, Enzyme-linked Immunosorbent Assay, Glo Assay, Generated, Binding Assay, Selection, Over Expression, Construct, Luciferase, Marker, Expressing, Isolation, Cell Culture, Quantitative RT-PCR, Flow Cytometry

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a CD19 CAR-T cells were generated from human T cells as described in the Methods. After expansion, CAR-T cells were treated with DMSO or TAIII for 72 h. The proportion of FoxP3 + Treg in CD4 + T was then analyzed by flow cytometry. Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. b – c CAR-T and Untransduced T cells (UNT) cells were treated with TAIII (0, 0.5, 1, 2 µM) for 24 and 72 h under non-stimulatory conditions (without tumor cells). T cell proliferation and cell viability/apoptosis were assessed using CellTiter-Glo assays and flow cytometry (PI/Annexin V/7-AAD staining). Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t-test or ordinary one-way ANOVA with Dunnett’s test. CAR-T cells were co-cultured with Raji-Luc ( d ) or Nalm6-Luc ( e ) cells at effector-to-target (E:T) ratios of 1:2 or 1:4 under indicated treatments. Cytotoxicity was measured by residual luciferase activity, and IFN-γ, IL-2, and TNF-α levels were determined by ELISA. UNT cells were included as negative controls; Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Šídák’s multiple comparisons test. f Real-time cytotoxicity of CAR-T cells co-cultured with Raji cells was monitored over 36 h using Incucyte® cytolight red and annexin V staining; Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Šídák’s multiple comparisons test. g IFN-γ and TNF-α protein levels in CAR-T–Raji co-cultures after 16 h were measured by ELISA; Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. h FoxP3a (FoxP3) mRNA levels in CAR-T–Raji co-cultures were determined by qRT-PCR. Data are presented as mean ± SD from independent experiments ( n = 3–4 biological replicates); two-sided unpaired t -test.

Article Snippet: Human T or CAR-T cells were cultured in X-VIVO (Lonza, 04-418Q) with 10% FBS (Gibco, 10100-147), 1% GlutaMAX (Gibco, 35050061), 1% NEAA (Gibco, 11140050), 1% Antibiotic-Antimycotic (Gibco, 15240062), 100 IU IL-2 (R&D Systems, 202-IL) and Dynabeads Human T-activator CD3/CD28 (Gibco, 1132D).

Techniques: Generated, Flow Cytometry, Staining, Cell Culture, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a Human PBMCs (1 × 10⁶) were activated with anti-CD3/CD28 beads for 4 days, then treated with NECA ± TAIII for 24 h. Bulk RNA sequencing was performed, and a heatmap displays selected mRNAs and surface markers of Treg and Teff subsets. b PBMCs were stimulated with CD3/CD28 beads and treated with indicated compounds for 48 h. IFN-γ and IL-2 in supernatants were measured by ELISA. Data represent mean ± SEM of three independent experiments, each in triplicate; analyzed by ordinary one-way ANOVA with Dunnett’s test. c T cells were generated as in ( a ). IFN-γ, IL-2, and FoxP3 transcription ( c ) and FoxP3 expression during 10-day induced Treg (iTreg) differentiation ( d ) were determined by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. e – f Human CD4⁺ naive T cells were skewed to Tregs for 7 days. Suppressive function was assessed by co-culture with CFSE-labeled responder Teff cells; CFSE dilution in CD4⁺CFSE⁺ Teff cells was analyzed by flow cytometry to determine the percentage of undivided Teff cells. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. g – h iTreg cells (4-day differentiation) were transduced with shCK or shA2AR constructs (shA2AR-2, -3), and mRNA levels of indicated genes, including FoxP3, were measured by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); One-way ANOVA with Dunnett’s test. i iTreg cells were generated and the recruitment change of CREB on FoxP3a (FoxP3) promoter w/o TAIII treatment was confirmed by CUT & RUN assay. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test.

Article Snippet: Human T or CAR-T cells were cultured in X-VIVO (Lonza, 04-418Q) with 10% FBS (Gibco, 10100-147), 1% GlutaMAX (Gibco, 35050061), 1% NEAA (Gibco, 11140050), 1% Antibiotic-Antimycotic (Gibco, 15240062), 100 IU IL-2 (R&D Systems, 202-IL) and Dynabeads Human T-activator CD3/CD28 (Gibco, 1132D).

Techniques: RNA Sequencing, Enzyme-linked Immunosorbent Assay, Generated, Expressing, Quantitative RT-PCR, Co-Culture Assay, Labeling, Flow Cytometry, Transduction, Construct

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a CD19 CAR-T cells were generated as in (Fig. ) and co-cultured with Raji-Luc or Nalm6-Luc cells under indicated conditions for 24 h; the CD19 levels were assessed by measuring the luciferase signal. Data represent pooled biological replicates; Data are presented as SD (n = 3 biological replicates); two-way ANOVA with Tukey’s test. b – c CAR-T cells from different donors were co-cultured with Raji-Luc or Nalm6-Luc cells under indicated treatments. IFN-γ, IL-2, and TNF-α in supernatants were measured by ELISA. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA or two-way ANOVA with Dunnett’s test. d CD19 CAR-T cells were co-cultured with Raji cells; FoxP3 mRNA levels were determined by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. e Effects of TAIII on central memory T cells (CD62L⁺CD45RO⁺, Tcm) were assessed after 48 h co-culture with Raji cells at E:T ratios of 1:1, 1:2, or 1:4 by flow cytometry. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test. f A2AR knockdown (KD) CD19-CAR-T cells were generated via shRNA transduction (GFP⁺), and knockdown efficiency was confirmed by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. g Intracellular cAMP levels in A2AR-KD CAR-T cells were measured to assess TAIII-mediated inhibition of A2AR signaling; Two-way ANOVA with Šídák’s multiple comparisons test. h – i A2AR-KD, parental CD19-CAR-T, and UNT cells were co-cultured with Nalm6-Luc cells for 24 h. Cytotoxicity was measured by residual luciferase activity at E:T ratios of 1:1 and 1:2 (normalized to Nalm6-only controls), and IL-2 secretion was quantified by ELISA at E:T = 1:1. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA or ordinary one-way ANOVA with Tukey’s test.

Article Snippet: Human T or CAR-T cells were cultured in X-VIVO (Lonza, 04-418Q) with 10% FBS (Gibco, 10100-147), 1% GlutaMAX (Gibco, 35050061), 1% NEAA (Gibco, 11140050), 1% Antibiotic-Antimycotic (Gibco, 15240062), 100 IU IL-2 (R&D Systems, 202-IL) and Dynabeads Human T-activator CD3/CD28 (Gibco, 1132D).

Techniques: Generated, Cell Culture, Luciferase, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Co-Culture Assay, Flow Cytometry, Knockdown, shRNA, Transduction, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: The Glucose Transporter GLUT3 Controls Regulatory T Cell Function

doi: 10.64898/2026.03.26.714439

Figure Lengend Snippet: (A and B) Relative body weight changes (A) and experimental autoimmune encephalomyelitis (EAE) clinical scores (B) in WT, GLUT1-deficient ( Slc2a1 fl/fl Cd4 Cre ) and GLUT3-deficient ( Slc2a3 fl/fl Cd4 Cre ) mice following immunization with MOG 35-55 peptide emulsified in CFA; means ± SEM of 6-11 mice per cohort. (C) RT-qPCR analysis of Slc2a1 (GLUT1) and Slc2a3 (GLUT3) expression in isolated splenic CD4 + T cells from WT, GLUT1-deficient and GLUT3-deficient mice used in EAE experiments; data are shown as means ± SEM of 4-11 mice. (D and E) Representative flow cytometric analysis of Foxp3 + Treg cells in the central nervous system (CNS) of WT, GLUT1- and GLUT3-deficient mice 20 days after MOG 35-55 immunization (D) , with quantification of Treg cell frequencies and absolute cell numbers (E) ; means ± SEM of 6-11 mice. (F and G) Frequencies of GM-CSF and IL-2-producing CD4 + T cells in the CNS of WT, GLUT1-and GLUT3-deficient mice 20 days after immunization with MOG 35-55 peptide and restimulation with PMA/ionomycin for 5 h (F) , with quantification of total cell numbers of GM-CSF and IL-2 producing CD4 + T cells in the CNS (G) ; means ± SEM of 6-11 mice. Statistical analyses shown in (A, B, C, E, G) were performed using two-way ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001; ns, non-significant.

Article Snippet: For Th1 differentiation, cells were cultured with 2.5 μg/mL anti-IL-4 (clone 11B11, Bio X Cell), 10 ng/mL recombinant human IL-2 and 10 ng/mL recombinant murine IL-12 (both PeproTech).

Techniques: Quantitative RT-PCR, Expressing, Isolation