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

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Experimental method to assess resistance to CTL-mediated killing. (A) Co-culture setup: primary CD4 + T cells from PLWH are pulsed with mutant p53 peptide and cultured with autologous, pre-expanded CD8 + T cells and p53-specific scDb. IPDA is performed on the viable CD4 + T cell population isolated before and after the co-culture to assess for enrichment of infected cells, indicative of resistance. (B) scDb mechanism of action: scDbs bind to cognate peptide:MHC complexes on the target cell surface and also to the CD3ε subunit of the TCR complex to induce localized cytotoxicity. (C) Flow cytometry gating strategy to determine frequency of viable target cells. (D) Representative histograms of co-cultures with and without scDbs. The reduction in the viable CD4 + T cell count (marked region) was used to determine the amount of CTL-mediated lysis. I Representative viability data for study participant OM5418 for the experimental condition and seven control conditions.

Article Snippet: Effector cells for co-cultures were expanded through stimulation with soluble anti-CD3 mAb (15 ng/mL, clone OKT3, BioLegend, Cat. # 317302) in CD8 media (base media + 250 U/uL human IL-2 (R&D Systems, Cat. # 202-IL-050/CF) + 5 ng/mL recombinant human IL-7 (BioLegend, Cat. # 581906)) for 72 hours.

Techniques: Co-Culture Assay, Mutagenesis, Cell Culture, Isolation, Infection, Flow Cytometry, Cell Characterization, Lysis, Control

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Effect of active viral expression on cell death resistance. (A) Experimental setup: Healthy donor CD4 + T cells were activated for 72 hours with PHA and infected with different HIV-1 GFP-reporter constructs. Cells were then pulsed with exogenous p53 R175H peptide and co-cultured with pre-expanded autologous CD8 + T cells and p53-specific scDb or irrelevant control scDb. Viability and GFP-expression were analyzed through flow cytometry. (B) Genome maps of the HIV-1 reporter constructs used to infect target cells. Each row represents one reading frame. Striped segments indicate gene segments that are no longer translated due to insertion of a STOP-codon. (C) Representative flow plot showing viable uninfected (GFP-negative) and HIV-1-expressing (GFP-positive) cell populations. (D) Viability of uninfected target cells or cells infected with indicated HIV-1 reporter constructs after co-culture with the HLA-A2:p53 R175H -restricted scDb. Each dot shows the average of three technical replicates per condition. Symbols represent individual donors; assay was performed 2 independent times for each of the n=5 healthy donors, except for the full-length condition, where one donor was only tested once for technical reasons. A p-value less than 0.05 was considered significant. Abbreviations: PHA, phytohemagglutinin; LTR, long terminal repeat; IRES, internal ribosome entry site. ns: no significance (p > 0.05).

Article Snippet: Effector cells for co-cultures were expanded through stimulation with soluble anti-CD3 mAb (15 ng/mL, clone OKT3, BioLegend, Cat. # 317302) in CD8 media (base media + 250 U/uL human IL-2 (R&D Systems, Cat. # 202-IL-050/CF) + 5 ng/mL recombinant human IL-7 (BioLegend, Cat. # 581906)) for 72 hours.

Techniques: Expressing, Infection, Construct, Cell Culture, Control, Flow Cytometry, Co-Culture Assay