Journal: EMBO Reports

Article Title: DNA methylation-mediated memory of obesity in CD4 T lymphocytes perpetuates immune dysregulation

doi: 10.1038/s44319-026-00765-w

Figure Lengend Snippet: ( A ) Representative flow cytometry plot (left) showing human CD4 T cell subsets: naive CD4 T cells (CCR7 + CD45RO-), Tcm (CCR7 + CD45RO + ), Tem (CCR7-CD45RO + ), and TEMRA (CCR7-CD45RO-). Quantification (right) displays the frequencies of Tem (CD45RO + CCR7-) within live CD4 + T cells. Isolated CD4 T cells were treated with 50 μM of oleic acid, palmitate, or stearic acid overnight (right) followed by activation with plate-bound anti-CD3 (2.5 μg/mL) and anti-CD28 (1.5 μg/mL) for 48 h. The untreated control group received the ethanol:BSA (1:4) solution. Each point represents a technical replicate ( n = 4). Kruskal–Wallis with Dunn’s correction; data are presented as mean ± SD, * P < 0.05. ( B ) Representative flow cytometry plot (left) showing the expression of human CD4 Treg (CD4+Foxp3 + ). Quantification (right) shows the frequency of CD4 Treg (CD4+Foxp3 + ) pre-treated overnight with 50 μM oleic acid, palmitate, or stearic acid, then activated for 48 h as described above. The untreated control group received the vehicle control solution. Each point represents a technical replicate from n = 3 donors. Kruskal–Wallis with Dunn’s correction; data are presented as mean ± SD, * P < 0.05. ( C ) Scatter box plots showing relative STK26 mRNA expression in human CD4 T cells isolated from PBMCs from healthy volunteers and pre-treated overnight with 50 μM palmitate without activation, followed by either no activation or 48 h activation with plate-bound anti-CD3/CD28. Untreated controls received the vehicle control solution. Expression was normalized to the housekeeper gene 18S. Each point represents a technical replicate from n = 4 biological replicates. Data are presented as mean +/− SD; Unpaired nonparametric T test (Mann–Whitney), *** P < 0.001. ( D ) Representative western blot images (left) and densitometric quantification (right) of Stk26 and β-actin protein levels in human CD4 T cells pre-treated overnight with 50 μM palmitate or vehicle control, followed by either no activation or activation with plate-bound anti-CD3/CD28 for 48 h. Data are presented as mean +/− SD ( n = 3 donors). Two-tailed Student’s T test with Shapiro–Wilk normality test; * P < 0.05. ( E ) Representative western blot images (left) and densitometric quantification (right) showing LC3II and β-actin protein levels in human CD4 T cells pre-treated overnight with 50 μM palmitate or vehicle control and activated with CD3/CD28 beads for 48 h. To inhibit autophagy, palmitate or vehicle-control-treated CD4 T cells were treated with 25 μM chloroquine (CQ) or vehicle-control overnight. Data are presented as mean +/− SD ( n = 4 donors). Unpaired nonparametric T test (Mann–Whitney); * P < 0.05. ( F ) Scatter box plots of STK26 mRNA expression in human CD4 T cells cultured overnight with adipose-conditioned media from healthy range BMI or BMI > 30 osteoarthritis patients, followed by activation with CD3/CD28 beads for 48 h. Gene expression was normalized to β-actin. Data are presented as mean +/− SD ( n = 6 donors of adipose-conditioned media). Unpaired nonparametric T test (Mann–Whitney); * P < 0.05. ( G ) Scatter box plots showing relative CDKN1C mRNA expression in human CD4 T cells pre-treated overnight with 50 μM palmitate without activation, followed by either no activation or 48 h activation with plate-bound anti-CD3/CD28. Controls received the vehicle control solution. Expression was normalized to the housekeeper 18S. Each point represents a technical replicate ( n = 3–4 donors). Data are presented as mean +/− SD; unpaired nonparametric T test (Mann–Whitney); ** P < 0.01, *** P < 0.001. ( H ) Scatter plots showing the percentages of human CD57+ and IL-4 + CD4 TEMRA cells (CD4 + CD45RO-CCR7-CD57+ and CD4 + CD45RO-CCR7-IL-4 + ) pre-treated overnight with 50 μM oleic acid, palmitate, or stearic acid without activation. Cells were then either left non-activated or activated with plate-bound anti-CD3/CD28 for 48 h. Controls received the vehicle control solution. Each point represents a technical replicate from n = 3 donors. Data are presented as mean +/− SD; Kruskal–Wallis with Dunn’s correction; * P < 0.05, ** P < 0.01, *** P < 0.001. .

Article Snippet: Human CD57 (clone: REA769) , Miltenyi Biotech , Cat #130-111-811.

Techniques: Flow Cytometry, Isolation, Activation Assay, Control, Expressing, MANN-WHITNEY, Western Blot, Two Tailed Test, Cell Culture, Gene Expression

Journal: Cells

Article Title: Empowerment of CAR-T Cells by IL-7 and IL-15 Boosts Their Efficacy Against HER2-Positive Tumors with Enhanced Expansion and Persistence

doi: 10.3390/cells15060547

Figure Lengend Snippet: Treatment with IL-15 induced a population of CD57 + CAR-T cells, whose CD57 expression was downregulated upon tumor challenge. ( A , B ) CD57 expression level of CD4 + and CD8 + CAR-T cells with different interleukin treatments during CAR-T cell expansion measured by flow cytometry. ( A ) Representative histogram of CD57 expression on day 15 after initial anti-CD3/CD28 activation of CD4 + and CD8 + CAR-T cells. ( B ) Statistical evaluation of CD57 expression on day 8 and day 15 by matched ANOVA by donors. *: p < 0.05. ( C ) Nine days after the initial anti-CD3/28 activation (start), CAR-T cells were used for the repetitive tumor killing assay. Before each round of tumor killing, the CD57 level of CD4+ and CD8 + CAR-T cells was measured by flow cytometry. Group comparison of CD57 expressions was conducted between HER2-CAR-T cells with different treatments. ( D ) IL-2+7+15 HER2-CAR-T cells were sorted by anti-CD57 magnetic beads on day 15 after activation. On day 16, the positively sorted CD57 hi CAR-T cells, the remaining CD57 lo cells, and bulk CAR-T cells were proceeded to a repetitive tumor killing assay for three rounds. Before each round of tumor killing, the CD57 level of CD8 + cells was measured. ( E ) On day 16, CD57 hi , CD57 lo and bulk CAR-T cells were used to kill tumor cells at different effector–target (E:T) ratios. CAR-T cells were co-incubated with tumor cells for 24 h. Remaining live tumor cells and HER2 expression were identified by flow cytometry.

Article Snippet: The magnetic separation of CD57 hi and CD57 lo cells was conducted using anti-CD57 magnetic beads (Miltenyi Biotec, 130-092-073) and a QuadroMACSTM separator (Miltenyi Biotec, 130-090-976) according to the manufacturer’s instruction.

Techniques: Expressing, Flow Cytometry, Activation Assay, Comparison, Magnetic Beads, Incubation

Journal: Journal of Translational Medicine

Article Title: Exercise-mobilized lymphocytes enhance antibody-based immunotherapy in multiple myeloma through CD16 + NK cell–mediated cytotoxicity

doi: 10.1186/s12967-026-07888-7

Figure Lengend Snippet: Exercise-mobilized NK cells display enhanced cytotoxic phenotypes and differential responses to DRd vs MRd. ( A ) Baseline CD38% positive and MFI) and CD47% positive) on NK cells measured in peripheral blood mononuclear cells (PBMCs) collected at rest (REST; red) or during acute exercise at 80% VO2max (EX; blue). ( B ) NK-cell frequency (% of CD45+ lymphocytes) after overnight IL-15 culture under control, DRd (daratumumab + lenalidomide + dexamethasone), or MRd (magrolimab + lenalidomide + dexamethasone) conditions. Representative CD3 vs. CD56 dot plots are shown on the right for REST and EX across regimens, with the NK-cell gate (CD3−CD56+) indicated. ( C ) Expression of monoclonal-antibody–associated markers after overnight culture: CD38% positive and MFI) under control vs. MRd conditions, and CD47% positive and MFI) under control vs. DRd conditions. ( D ) Fc-gamma receptor (FcγR) profiles after overnight culture, showing the expression of CD16 and CD32% positive and MFI). ( E ) Expression of activating and cytotoxic markers after overnight culture: NKG2C, CD57, NKG2D, and DNAM-1% positive and MFI). In all bar graphs, red bars represent REST and blue bars represent EX. Bars show the mean ± SEM with paired donor values overlaid and connected by dotted lines. PBMCs from N = 10 healthy donors were used. Each condition was run in duplicate, and the results were averaged per donor. NK cells were defined as CD3−CD56+ lymphocytes. Statistical tests were two-sided. Panel A: paired t-test (EX vs. REST). Panel B: one-way repeated-measures ANOVA within each exercise state (REST or EX), followed by Dunnett’s multiple comparisons test vs. The control group. Panels C–E: two-way repeated-measures ANOVA with factors for exercise (REST vs. EX) and regimen (e.g., control vs. DRd), followed by Tukey’s multiple comparisons test.Significance is indicated by asterisks or exact p -values where appropriate. Thresholds were set at * p < 0.05, ** p < 0.01, and *** p < 0.001. “ns” denotes not significant. Abbreviations: PBMC, peripheral blood mononuclear cell; NK, natural killer; MFI, mean fluorescence intensity; DRd, daratumumab/lenalidomide/dexamethasone; MRd, magrolimab/lenalidomide/dexamethasone

Article Snippet: Cells were then stained for 15–20 min at room temperature with fluorochrome-conjugated antibodies: CD8-VioBlue, CD57-VioBlue, CD3-VioGreen, CD47-FITC, NKG2C-FITC, CD4-PE, NKG2D-PE, CD45-PE-Vio615, CD16-PerCP-Vio700, CD32-PE-Vio770, CD38-APC, DNAM-1-APC, and CD56-APC-Vio770 (Miltenyi Biotec, Germany).

Techniques: Control, Expressing, Fluorescence