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: Translational Lung Cancer Research

Article Title: Nanoassemblies loaded with low-dose paclitaxel can enhance the response of lung cancer immunotherapy by activating dendritic cells

doi: 10.21037/tlcr-2025-180

Figure Lengend Snippet: Detection of the immune state of the TIME. (A) Schematic diagram of the administration and detection schedule. The proportion of (B,C) CD4 + T cells and (D,E) CD8 + T cells of tumors from mice treated with NS, FA NAs, and FA-PTX NAs containing different doses of PTX at different time points as detected by flow cytometry. The numbers in the right area indicate the percentages of (B) CD4 + or (D) CD8 + T cells. (F) The distribution of CD8 + T cells in the TIME as detected by IHC. Scale bar =100 μm. (G) The concentration of TGF-β detected in tumors via ELISA in the different treatment groups at different time points. (H) The killing rate of splenic T cells as measured by lactate dehydrogenase release assay. FA-PTX NA, folic acid-modified nanoassemblies loaded paclitaxel; FA NA, folic acid-modified nanoassemblies; IHC, immunohistochemical; NS, normal saline; TIME, tumor immune microenvironment; TGF-β, transforming growth factor-beta.

Article Snippet: The cell suspensions were then stained with fluorochrome-labeled antibody-targeting murine CD4-FITC (eBioscience, San Diego, CA, USA), CD8-APC (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), or an appropriate isotype control antibody and then analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA).

Techniques: Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Release Assay, Modification, Immunohistochemical staining, Saline

Journal: Translational Lung Cancer Research

Article Title: Nanoassemblies loaded with low-dose paclitaxel can enhance the response of lung cancer immunotherapy by activating dendritic cells

doi: 10.21037/tlcr-2025-180

Figure Lengend Snippet: Antitumor mechanism analysis. (A) HE staining of tumor tissues. Scale bar =100 μm. (B) The apoptosis induction as determined by TUNEL assay. Scale bar =50 μm. (C) The number of CD4 + and (D) CD8 + T cells in tumors detected by IHC. Scale bar =50 μm. (E) The distribution of CD8 + T cells in tumors detected by IHC. Scale bar =100 μm. (F-H) Tumor cell apoptosis and the abundance of CD4 + T cells and CD8 + T cells were assessed by counting the rate of TUNEL-positive cells and the number of CD4 + and CD8 + T cells, respectively. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance. FA NA, folic acid-modified nanoassemblies; FA-PTX NA, folic acid-modified nanoassemblies loaded with paclitaxel; HE, hematoxylin and eosin; IHC, immunohistochemistry; NS, normal saline; PD-1, programmed cell death protein-1; TUNEL, terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling.

Article Snippet: The cell suspensions were then stained with fluorochrome-labeled antibody-targeting murine CD4-FITC (eBioscience, San Diego, CA, USA), CD8-APC (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), or an appropriate isotype control antibody and then analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA).

Techniques: Staining, TUNEL Assay, Modification, Immunohistochemistry, Saline, End Labeling