Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) Flow cytometric analysis (FCA) of CD25 (left) and CD69 (right) staining and quantification in CTL, n = 6/11 individual patients. ( B ) FCA of lymphocyte-activation gene 3 (LAG3), programmed cell death protein 1 (PD1), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) staining and quantification as indicated, n = 6/4 (not activated/+CD3/CD28) individual patients. P values (left to right): 0.2842, 0.2188, 0.6875, 0.5494, 0.1250, and >0.9999. ( C ) Quantification of relative cytotoxicity using a flow cytometric killing assay, n = 5 individual patients. ( D ) Quantification of caspase-3 activity reporter using a CTL-K562 killing assay, n = 4 individual patients. Data were represented as mean ± SEM with dots indicating individual values ( A , B ) or as individual data points ( C , D ). P values: as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test, as appropriate.

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Staining, Activation Assay, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in isolated mitochondria from CTL, n = 7 individual patients. ( B ) FCA of JC1 green and quantification of red/green fluorescence in isolated mitochondria from CTL, n = 4 individual patients. ( C ) Immunoblot of OXPHOS complexes using isolated mitochondria of CTL and normalized densitometric quantification of complex V, n = 5 individual patients. I–V complex I–V; H heat shock protein 60 (HSP60). Subunits detected by the antibody cocktail: complex I: subunit NDUFB8; complex II: subunit 30 kDa (SDHB); complex III: subunit Core 2 (UQCRC2); complex IV: subunit II (COXII); complex V: ATP synthase subunit alpha (ATP5). ( D , E ) Representative immunoblot of mitochondrial fusion and fission proteins of isolated mitochondria of CTL ( D ) and normalized densitometric quantification ( E ) of the respective proteins, n = 5/4/3 individual patients. ( F ) Representative projections of 3D image stacks of CTL stained with MitoTracker (MT, green, left) and the respective skeleton images (white, right), generated using mitochondrial analyzer, time points as indicated, representative of six individual patients. ( G ) Quantification of mitochondrial count per cell, n = 29 cells from three individual patients. ( H ) Quantification of mitochondrial length (left) and mean mitochondrial area using mitochondrial analyzer (right), n = 180/170 mitochondria from 14/22 cells from three individual patients (length) and n = 50 cells from three individual patients (area). ( I ) Representative projection of 3D image stacks (two per time point) of translocated mitochondria to the IS in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, and quantification of mitochondrial translocation to the CTL IS, time points as indicated, n = 14 from 5 individual patients. If not stated otherwise, data were represented as mean ± SEM. P values as indicated, paired t -test or Wilcoxon matched-pairs signed-rank test ( A – E ), and unpaired t -test or Mann–Whitney test ( G – I ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Isolation, Fluorescence, Western Blot, Staining, Generated, Translocation Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Mitochondrial damage drives T-cell immunometabolic paralysis after major surgery

doi: 10.1038/s44321-025-00324-1

Figure Lengend Snippet: ( A ) FCA of MitoTracker green and quantification in CD8 + CTL, n = 7 individual patients. ( B ) Representative Seahorse OCR plot. ( C ) Fold change of basal OCR, maximum OCR, spare respiratory capacity (SRC) and ATP production, n = 6 individual patients. ( D ) Quantification of mitochondrial length, n = 92 mitochondria from 28 cells from three individual patients. ( E ) Quantification of mitochondrial count per cell, n = 30/26 cells from three individual patients. ( F ) Representative confocal microscopy image of mitochondria translocated to the TCR in CTL as indicated by MitoTracker (MT) Deep Red and CD3 FITC staining, T2 image as depicted in Fig. , upper right image. ( G ) Quantification of mitochondrial translocation in proximity to the CTL IS, n = 12 from four individual patients. ( H ) Representative impedance plot and ( I ) quantification of relative CTL cytotoxicity using ECIS, n = 6 individual patients. If not stated otherwise, data are represented as mean ± SEM. P values as indicated, one-sample t -test ( C ), paired t -test or Wilcoxon matched-pairs signed-rank test ( A – C, H , I ), and unpaired t -test or Mann–Whitney test ( D – G ), as appropriate. .

Article Snippet: CD69 FITC , Miltenyi , 130-112-801.

Techniques: Confocal Microscopy, Staining, Translocation Assay, MANN-WHITNEY

Journal: iScience

Article Title: Impact of 3D cell culture hydrogels derived from basement membrane extracts or nanofibrillar cellulose on CAR-T cell activation

doi: 10.1016/j.isci.2025.113234

Figure Lengend Snippet: CAR-T cell cytotoxicity in short term cultures is comparable between NFC, Matrigel, and BME CD20 CAR-T cells were co-cultured with Daudi (Burkitt lymphoma cell line) cells labeled with CellTrace Violet (CTV) for 24 h at the different effector to target (E:T) ratios specified, in standard 2D suspension (control) or embedded in the specified hydrogels. (A) Representative gating strategy for analyzing the viability of Daudi and CD20 CAR-T cells by flow cytometry. (B) Number of viable CAR-T cells recovered after culture in the 1:1 E:T condition (50,000 Daudi +50,000 CAR-T cells/well) as measured by flow cytometry using counting beads (Flow-Count fluorospheres). Data were analyzed using a one-way ANOVA with Tukey’s multiple comparison test. (C) Specific lysis (%) of Daudi cells induced by CD20 CAR-T cells at the specified E:T ratios. Specific apoptosis was calculated by applying the following formula: [(%viable untreated − %viable treated)/%viable untreated] × 100. (D and E) Bar plot showing median fluorescence intensity (MFI) of (D) IFNγ and (E) CD69 in CAR-T cells cultured for 24 h in the described conditions. Data from two or three independent experiments are presented as means ± SD. Data were analyzed using a two-way ANOVA with Tukey’s multiple comparison test (∗ p < 0.05, ∗∗ p < 0.005). NFC, nanofibrillar cellulose; MG, Matrigel; BME, basement membrane extract.

Article Snippet: Anti-Human CD69 FITC , Miltenyi , Cat# 130-113-523; RRID: AB_2733656.

Techniques: Cell Culture, Labeling, Suspension, Control, Flow Cytometry, Comparison, Lysis, Fluorescence, Membrane

Journal: Frontiers in Immunology

Article Title: A conserved element in the first intron of Cd4 has a lineage specific, TCR signal-responsive, canonical enhancer function that matches the timing of cell surface CD4 upregulation required to prevent lineage choice error

doi: 10.3389/fimmu.2024.1469402

Figure Lengend Snippet: Deletion of coreNCE/E4m reduces CD4 cell surface expression in a developmental stage-specific manner and significantly reduces TCR responsiveness in vitro. (A) CD4 cell surface expression levels on RLM11 and AKR1G1 cells with CRISPR-Cas9 deletion of the coreNCE/E4m on none (blue), one (green), or both (red) of the alleles of Cd4 compared to the corresponding parental cell lines (black). The numbers in the histograms represent the CD4 MFI of individual clones. Shaded histogram is the isotype control stain. (B) The bar graph represents the relative CD4 expression normalized to that of the parental cell line; n = at least 2 and up to 5 different clones. The Turkey-Kramer HSD multiple comparison test was used to show that coreNCE/E4m- unmodified, heterozygous, and double-deleted clones are significantly different from each other in RLM11, but not AKR1G1 cells ns = not significant; *p<0.05; **p<0.01; ***p<0.001. (C) The ratio of the CD4 MFI of ConA-stimulated (CD69 + ) to that in unstimulated (CD69 - ) RLM11 and coreNCE/E4m-deleted RLM11 (ΔNCE) cells as well as in ConA-stimulated (CD5 + ) to that in unstimulated (CD5 - ) AKR1G1 and coreNCE/E4m-deleted AKR1G1 cells as a measure of responsiveness to TCR stimulation. A two-tailed t test was used to determine whether this ratio was significantly different between NCE-sufficient and NCE-deficient cells.n = 6 for RLM11 and n = 3 for AKR1G1; **p<0.01; ns = not significant (D–F) Kinetics of CD4 re-expression in unmodified and coreNCE/E4m-deleted RLM11 (D) and AKR1G1 (E) cells stripped of cell surface CD4 using pronase. Linear regression was performed to determine the rate of CD4 re-expression, and the ratio of CD4 expression rates (ΔNCE: NCE) was calculated for each cell line (F) . A two-tailed t test was performed to determine whether this ratio was significantly different between RLM and AKR cells. The error bars represent +/- 1 standard deviation. n = 3; **p<0.01. *p<0.05; ***p<0.001. ns = not significant.

Article Snippet: Then, the cells were stained for 40 minutes at 4°C with a combination of fluorophore-conjugated antibodies, such as anti-mouse CD4-PE Clone GK1.5 (BD Biosciences Cat# 557308, RRID: AB_396634), anti-human CD3 isotype control [PE-conjugated (BioLegend Cat# 300408, RRID: AB_314062); FITC-conjugated (BioLegend Cat# 300440, RRID: AB_2562046); APC-conjugated (BioLegend Cat# 300439, RRID: AB_2562045)], anti-mouse CD8α-APC (Clone 53-6.7 17-0081-83 eBioscience), anti-mouse TCRβ-Alexa647 (HM3621 Invitrogen), anti-mouse CD69-FITC (Clone H1.2F3 14-0691-81 eBioscience), anti-mouse CD5-FITC (clone 53-7.3), and anti-mouse CD24-PE (Biolegend 101803).

Techniques: Expressing, In Vitro, CRISPR, Clone Assay, Control, Staining, Comparison, Two Tailed Test, Standard Deviation