murine t cells  (MedChemExpress)

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A , B ) Cytotoxic CD4 + T cells were generated from OT-II mice and treated with the Gli inhibitor GANT61 at 5 µM or carrier control 24 h prior to a killing assay of Ova-pulsed B cell targets on day 7. ( A ) Pooled killing assays including anti-MHC Class II negative control. n = 2–3 biological replicates from 2–3 independent experiments. Bars represent the mean; error bars shown are SD. p values were calculated using a two-way ANOVA with Tukey’s multiple comparison test (10:1 p = 0.008, 5:1 p = 0.0135). ( B ) IFN-γ and Granzyme B levels were assessed by flow cytometry at day 8 in unstimulated (top panel) or restimulated CD4 + T cells (bottom panel). Quantification of steady-state Granzyme B levels and IFN-γ levels upon restimulation are shown on the right. Data shown is pooled from n = 3 biological replicates from 3 independent experiments. Bars represent the mean; error bars shown are SD. Statistical significance was assessed by a paired two-tailed Student’s t test. ( C ) On day 7, CD4 + T cells were co-cultured with P815 target cells at indicated effector to target ratios and subjected to a flow-cytometry-based cytotoxicity assay in the presence of 10 µM FPL 64176 or carrier control at 20 h. Top panel shows a representative killing assay (Data points represent mean of two technical replicates; error bars indicate SD). Bottom panel shows quantification of n = 4 biological replicates from 2 independent experiments normalised to killing of carrier-treated cells at a 2.5:1 effector:target ratio. Bars represent the mean; error bars shown are SD. p values were calculated using a two-way ANOVA with Šídák’s multiple comparisons test (2.5:1 p = 0.007, 1.25:1 p = 0.0007, 0.625:1 p = 0.0017). ( D ) On day 19, human Vγ9Vδ2 + T cells were co-cultured with zoledronate-pulsed K562 and Raji target cells at the indicated effector to target ratios and subjected to an LDH cytotoxicity assay in the presence of the indicated concentrations of GANT61 or carrier control at 5 h. Representative killing assays from n = 2 biological replicates from 2 independent experiments. Data points represent mean of two technical replicates; error bars indicate SD. ( E ) On day 23–24, human Vγ9Vδ2 + T cells were co-cultured with P815 target cells at indicated effector to target ratios and subjected to a flow-cytometry-based cytotoxicity assay in the presence of 10 µM FPL 64176 or carrier control at 4.5 h. Top panel shows a representative killing assay. Data points represent mean of two technical replicates; error bars indicate SD. Bottom panel shows quantification of n = 3 biological replicates from 2 independent experiments normalised to killing of carrier-treated cells at a 10:1 effector:target ratio. Bars represent the mean; error bars shown are SD. p values were calculated using a two-way ANOVA with Šídák’s multiple comparisons test (10:1 p = 0.0005, 5:1 p = 0.0006, 2.5:1 p = 0.0028, 1.25:1 p = 0.001). Symbols indicate biological replicates or individual human donors. * p < 0.05, ** p < 0.01, *** p < 0.001. ns = not significant. .

Article Snippet: Alternatively, naive murine CD4 + T cells were isolated using negative selection (Naive CD4 + T Cell Isolation Kit, Cat no. 130-104-453, Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Generated, Control, Negative Control, Comparison, Flow Cytometry, Two Tailed Test, Cell Culture, Cytotoxicity Assay, LDH Cytotoxicity Assay

Journal: Frontiers in Immunology

Article Title: CD4+FoxP3+ T regulatory cells subsets release small extracellular vesicles containing cell death-related proteins as potential mechanism of T cell suppression

doi: 10.3389/fimmu.2026.1777669

Figure Lengend Snippet: nTregs, iTregs and RATregs produce sEV. nTregs were obtained from spleens of FoxP3 GFP mice using the CD4+CD25+ Miltenyi kit. 10 6 /mL of nTregs were cultured in the presence of polyclonal activation (αCD3/αCD28) for 72h. To produce iTregs, the CD4+CD25- T cells fraction obtained from the nTregs purification was used. iTregs were obtained after 5 days in culture in the presence of polyclonal activation, IL-2 and TGF-β, whereas RATregs had retinoic acid (RA) added in addition to IL-2 and TGF-β. Tregs characterization included FoxP3, CD25 and CD73 expression analysis by flow cytometry (A) Representative dot plots displaying the phenotype of nTregs (black), iTregs (blue) and RATregs (pink). (B) Bar plots depicting the MFI for FoxP3, CD25 and CD73 from all Tregs subsets. (C) Graph showing the number of sEV per 10 6 cells obtained from nTregs, iTregs and RATregs. (D) Size distribution of sEV obtained from nTregs, iTregs and RATregs determined by NTA. (E) Abundance of sEV by size range also determined by NTA. (F) Western blot showing Alix as marker for sEV and β-actin as a marker for (Tregs) cells. A-E. Each experiment was performed 4–8 times (each dot corresponds to a single cell culture). (F) Western blot was performed 2 times. *p < 0.05 according to One-way ANOVA test; ns, not significant. Bar lines represent Mean ± SEM.

Article Snippet: In brief, nTregs were obtained using the Murine CD4+CD25+ T regulatory Cell Isolation Kit (Cat n°130-091-041, Miltenyi Biotec, CA, USA) following the provider ́s instructions.

Techniques: Cell Culture, Activation Assay, Purification, Expressing, Flow Cytometry, Western Blot, Marker, Single Cell

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Kaplan-Meier curve of GBM patients on standard of care (SOC; red, median=12.4 months) or combinations of SCO with metformin (blue, media=13 months) or DPP4 inhibitors (DPP4i, median=15.9 months) based on the SEER dataset. (B) DPP4 expression from immune lineages as determined by the scRNA-seq of 13 brain tumour patients (3 Grade II; 1 Grade III-IV; 9 GBM). Mean fluorescence intensity (MFI) of DPP-4 from (C) tumor-infiltrating and (D) splenic immune populations. n=5; *** p<0.001 by two-way ANOVA. (E) Representative gating strategy used to define effector, progenitor exhausted (PEX), or terminally exhausted (TEX) populations. (F) DPP-4 MFI from effector, PEX, and TEX populations. n=8; ** p<0.01 by two-way ANOVA. (G) Exhausted CD8 + T cells from GBM and tumor-adjacent tissue were defined as: CD45 + CD11b - CD3 + PD1 + TIGIT + . DPP-4 MFI from TOX + and TOX - population. n=4; * p<0.05 by paired t-test.

Article Snippet: CD3 + murine T cells were then collected using the Pan T-Cell Isolation Kit II (Miltenyi Biotec #130-095-130) as per the manufacturer’s protocol.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Representative histogram depicting CFSE dilution of mouse CD8 + T cells activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin, saxagliptin, or vehicle control for 4 days. (B) The percentage of proliferating mouse CD8 + T cells treated with sitagliptin vs. control. n=4; ** p<0.01 by paired t-test. (C) Representative zebra plots depicting Granzyme B and IFNγ production from OT-I T cells on Day 5. (D) The percentage of OT-I T cells expressing high levels of Granzyme B or IFNγ. n=4; * p<0.05 by paired t-test. (E) Representative zebra plot showing 7-AAD levels in SB28 and ovalbumin-expressing SB28 (SB28-OVA) cells co-cultured with OT-I CD8 + T cells at a 1:1 ratio. (F) The frequency of lysed wild-type SB28 or SB28-OVA cells cultured with sitagliptin- or vehicle-treated OT-I T cells. n=5; * p<0.05, ns=not significant by paired t-test.

Article Snippet: CD3 + murine T cells were then collected using the Pan T-Cell Isolation Kit II (Miltenyi Biotec #130-095-130) as per the manufacturer’s protocol.

Techniques: Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Mouse CD8 + T cells activated with anti-CD3/CD28 plus 100IU IL-2 in the presence of 100 μM sitagliptin or vehicle control for 4 days were intracellularly stained with Ki67 to determine proliferative capacity. n=4; *** p<0.001 by paired t-test. (B) Human CD8 + T cells isolated from healthy PBMCs were expanded with anti-CD3/CD28 and 100IU IL-2 for 6 days, and CFSE dilution determined by flow cytometry. n=5; * p<0.05 by paired t-test . (C) Schematics of the killing assay. Splenic CD8 + T cells from transgenic OT-I mice were isolated and activated with 10 ng OVA for 2 days, co-cultured at 1:1 E:T with either OVA- or empty vector-expressing cancer cells for six hours and then stained with apoptotic marker 7-AAD to determine cancer cell death by flow cytometry. (D) Mouse CD8 + T cells activated with anti-CD3/CD28 plus 100 IU IL-2 in the presence of 100 μM sitagliptin or vehicle control for 4 days were stained with exhaustion marker, anti-PD-1 and anti-2B4, to determine exhaustion post treatment. n=5; * p<0.05 by paired t-test. (E) Schematics of the LCMV experiment. Naïve CD45.1 + p14 CD8 + T cells were adoptively transferred into recipient wild-type CD45.2 + mice, and then recipient mice were infected with 4 x 10 6 PFU LCMV clone 13 intravenously (i.v.) the day after. Recipient mice were treated intraperitoneally with 20 mg/kg sitagliptin daily for 5 continues doses, 2-day break, and 5 more doses for a total of 10 doses. Mice were then observed daily for indications of endpoint. (F) Splenic CD8 + T cells isolated from (E) at endpoint and stained with exhaustion marker, anti-PD-1 and anti-2B4, to determine sitagliptin induced effect in LCMV Clone 13 model. n=4; * p<0.05 by t-test. (G) Frequency of grandparents for TEX, PEX, and effector populations was determined for OT-I CD8 + T cells repetitively stimulated with 10 ng OVA for 5 days. n=7; * p<0.05, ns=not significant by paired t-test.

Article Snippet: CD3 + murine T cells were then collected using the Pan T-Cell Isolation Kit II (Miltenyi Biotec #130-095-130) as per the manufacturer’s protocol.

Techniques: Control, Staining, Isolation, Flow Cytometry, Transgenic Assay, Cell Culture, Plasmid Preparation, Expressing, Marker, Infection

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin, or vehicle control for 4 days. Top differentially up-regulated pathways in sitagliptin-treated CD8 + T cells based on genes expressed >2-fold with p<0.05. n=6. Mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin, or vehicle control for 4 days and changes in cellular bioenergetics were determined with a Seahorse assay. (B) Oxygen consumption rate (OCR) and (C) Maximal respiration (top) and reserve capacity (bottom) of sitagliptin- vs. vehicle-treated CD8 + T cells. n=6; *** p<0.001, **** p<0.0001 by t-test. (D) Mouse CD8 + T cells were activated for 2 days and stained with the mitochondrial membrane potential sensitive dye TMRM at 100 nM for 30 minutes. TMRM MFI of vehicle- vs sitagliptin-treated CD8 + T cells. n=5; **p<0.01 by paired t-test. (E) Proton efflux rate (PER) and (F) extracellular acidification rate (ECAR) of CD8 + T cells from B, C. n=6. (G) CD8 + T cells activated for two days were incubated with the glucose mimetic, 2-NBDG (50 μM) for 30 min. The percentage of T cells with 2-NBDG uptake was determined by flow cytometry. n=4; ** p<0.01 by paired t-test.

Article Snippet: CD3 + murine T cells were then collected using the Pan T-Cell Isolation Kit II (Miltenyi Biotec #130-095-130) as per the manufacturer’s protocol.

Techniques: Control, Staining, Membrane, Incubation, Flow Cytometry

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Volcano plot of differentially regulated genes (1799 down and 762 up) upon sitagliptin treatment for 4 days. (B) Top differentially down-regulated pathways in sitagliptin-treated CD8 + T cells based on genes expressed >2-fold with p<0.05. n=6. (C) Splenic mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin or vehicle control for 2 days and then stained with 20 nM of the mitochondrial permeable stain, Mitotracker green, for 30 minutes to quantify mitochondria mass. n=4; ns=not significant by paired t-test. (D) Splenic mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin or vehicle control overnight. Cells were collected and lysed with RIPA buffer containing phosphatase/protease inhibitors, and 20 μM of protein were loaded on an SDS-Page for protein resolution, and western blotting analysis. (E) Splenic mouse CD8 + T cells were activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of 100 μM sitagliptin or vehicle control for 2 days, and then ATP levels were determined with CellTiter-Glo Luminescent Cell Viability Assay. n=4; ** p<0.01 by paired t-test. (F) CD8 + T cells activated for two days were incubated with the superoxide indicator, MitoSOX Deep Red, at 500 nM for 60 minutes. The percentage of T cells positive for superoxide accumulation was determined by flow cytometry. n=4; * p<0.05 by paired t-test. (G) CD8 + T cells from OT-I mice were isolated and stimulated with 10 ng OVA repetitively for 5 days to elicit exhaustion. On Day 6, cells were incubated with Mitotracker and TMRM. TMRM MFI was normalized to mitochondrial mass. n=4; * p<0.05 by paired t-test.

Article Snippet: CD3 + murine T cells were then collected using the Pan T-Cell Isolation Kit II (Miltenyi Biotec #130-095-130) as per the manufacturer’s protocol.

Techniques: Control, Staining, SDS Page, Western Blot, Cell Viability Assay, Incubation, Flow Cytometry, Isolation

Journal: bioRxiv

Article Title: Pharmacologic DPP-4 inhibition promotes CD8⁺ T cell metabolic fitness to enhance anti-tumor activity

doi: 10.64898/2026.03.31.715681

Figure Lengend Snippet: (A) Top differentially altered pathways in DPP4 high vs. DPP4 low human GBM-infiltrating CD8 + T cells. Pathways with an adjusted p-value < 0.05 were considered significantly enriched. (B) Phospho-proteomic array fluorescent signal from splenic CD8 + T cells pooled from 4 biological replicates and activated overnight with anti-CD3/CD28 plus 100 IU/mL IL-2 with 100 μM sitagliptin or vehicle control. (C) Differentially regulated pathways from (B) . (D) Targeted metabolomics was performed with mouse CD8 + T cells activated for two days with 100 μM sitagliptin or vehicle control. Metabolite quantification for GABA shunt pathway intermediates was determined. n=4; * p<0.05, ** p<0.01, **** p<0.0001, ns=not significant by paired t-test. (E) Human CD8 + T cells from healthy human donors were isolated form PBMCs and activated with anti-CD3/CD28 plus 100 IU/mL IL-2 in the presence of sitagliptin (100 μM), the GAD1 inhibitor, L-Allylglycine (100 μM), or combination treatment for 6 days. Cells were then collected, stained with trypan blue, and quantified using an automatic cell counter. n=4; * p<0.05, *** p<0.001 by mixed effect analysis. Human CD8 + T cells from healthy human donors were isolated from PBMCs and expanded with anti-CD3/CD28 plus 100 IU/mL IL-2 for 6 days to reduce donor variability. Cells were then further activated in the presence of sitagliptin (100 μM), L-Allylglycine (200 μM), or combination treatment for four days. Seahorse T-Cell Fitness Assay was performed to determine (F) OCR and (G) reserve capacity. n=6; * p<0.05 by Friedman test.

Article Snippet: CD3 + murine T cells were then collected using the Pan T-Cell Isolation Kit II (Miltenyi Biotec #130-095-130) as per the manufacturer’s protocol.

Techniques: Control, Isolation, Staining

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: To isolate primary murine T cells, C57BL/6J mice (Janvier Labs) were sacrificed by cervical dislocation and the spleen and lymph nodes were removed using aseptic technique.

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based CAR constructs lead to regression of syngeneic GB tumors in vivo (A) Schematic representation of the syngeneic model in vivo experiment. (B) Evaluation of mCAR expression on transduced murine T cells on treatment day by flow cytometry. A non-transduced sample was used to determine gating. Data gated on single live mCD3 + cells. (C) Weekly tumor volume evaluation in C57BL/6J mice orthotopically implanted with GL261/CD70 cells and subsequently treated ICT with anti-mCD70 CAR-T cells by MRI. A one-way ANOVA with a Dunnett’s multiple comparisons test was used to assess significance. (D) Indicative T2-weighed MRI sections (coronal plane) from N = 3 mice of each treatment group from (C), before and after treatment with anti-mCD70 CAR-T cells. (E) Comparison of tumor volumes among treatment groups from (C). For day-14, a one-way ANOVA followed by a Tukey’s multiple comparisons test was used. For day-21, a Welch’s ANOVA followed by a Dunnett’s T3 multiple comparisons test was used. For day-28, a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used. Data presented as mean (SD). (F) Overall survival of treated mice from (C). The log rank (Mantel-Cox) test was used to assess statistical significance. Bonferroni-adjusted significance threshold: p = 0.0166. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: To isolate primary murine T cells, C57BL/6J mice (Janvier Labs) were sacrificed by cervical dislocation and the spleen and lymph nodes were removed using aseptic technique.

Techniques: Construct, In Vivo, Expressing, Flow Cytometry, Comparison