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 western blots (top) and densitometric quantification (bottom) of Stk26 protein levels within peripheral LNs CD4 T cells isolated from female C57BL/6J mice on different diet groups for 14 weeks (groups 1–3 in Fig. ). Data are presented as mean +/− SD ( n = 3 mice per group). Unpaired nonparametric Mann–Whitney T test; n.s. ( B ) Representative flow cytometry histograms (left) showing LC3II expression in splenocytes from the three 14-week mouse groups (groups 1–3 in Fig. ). Splenocytes were cultured ex vivo in the presence of 100 μM of chloroquine within RPMI for 4 h. Scatter plots (right) show autophagy flux, calculated as the difference in mean fluorescence intensity (MFI) of LC3II between chloroquine-treated and vehicle control groups. Analysis was gated on live (near IR-) CD4 + CD44 + CXCR3+ or CD4 + CD44 + LFA1 + T cell populations. Data are from one experiment ( n = 6 female C57BL/6J mice per group). Results are shown as mean +/− SD. Unpaired nonparametric T test (Mann–Whitney), ** P < 0.01. ( C ) Representative flow cytometry plots (left) of CD4 + KLRG1+ cells gated within the live (near IR-) CD4 T cell population from peripheral LNs of female C57BL/6J mice on a 14-week CD or HFD-RE. Scatter plots (right) show the percentage of CD4 + KLRG1+ cells within the CD4 + CD44+ or CD4 + LFA1+ populations. Data is from one experiment ( n = 5–6 mice per group) and is presented as mean +/− SD. Unpaired nonparametric T test (Mann–Whitney), * P < 0.05. ( D ) Scatter plots showing CD4 and CD8 (CD45RA + CCR7-) human TEMRA from PBMCs of control ( n = 10) and individuals with Alstrom syndrome ( n = 10). Data are presented as mean +/− SD. Paired two-tailed Wilcoxon T test; * P < 0.05. ( E ) Scatter plots showing the percentage of human CD4 TEMRA (CCR7-CD45RO-) cells from PBMCs from individuals ( n = 13) before and after semaglutide treatment. All samples were gated on live (near IR-) populations. Paired nonparametric Wilcoxon T test; n.s. ( F ) Flow cytometry analysis of human CD4 + TEMRA cells (CD3 + CD4 + CD45RA + CD27 − ) in subcutaneous abdominal adipose tissue and lysed whole blood. In adipose, data are expressed as a percentage of CD4 + T cells. Samples are from participants of a 10-week randomized controlled trial of exercise training ( n = 14) compared to control ( n = 13). Pre-intervention = day one of the intervention (before exercise). Post-intervention= 36 h after the 10-week intervention/control period. Repeated measures analyses of variance (ANOVAs); n.s. ( G ) Heatmap displaying transcriptomic data of selected genes with previous implications with autophagy and/or senescence from murine CD44+ memory T cells isolated from spleen of female C57BL/6J mice on different diet groups for 14-week ( n = 3 female C57BL/6J mice per group; groups 1–3 in Fig. ). The analysis was performed using the Nanostring Mouse PanCancer Immune Profiling panel, covering 770 genes including genes related to autophagy and/or senescence. .

Article Snippet: Human CD45RO (clone: REA611) , Miltenyi Biotech , Cat #130-119-620.

Techniques: Western Blot, Isolation, MANN-WHITNEY, Flow Cytometry, Expressing, Cell Culture, Ex Vivo, Fluorescence, Control, Two Tailed Test

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 ) Scatter plots showing flow cytometry analysis of the percentage of human CD4 T cells, CD4 Tem (CCR7-CD45RO + ) and inflammatory CD4 T cells (CXCR3 + ) from PBMCs isolated from individuals ( n = 13) before and after semaglutide treatment. All samples were gated on live populations. Paired nonparametric Wilcoxon T test; n.s. ( B , C ) Flow cytometry analysis of human subcutaneous abdominal adipose tissue ( B ) and lysed whole blood ( C ), including CD3 + T cells, CD4 + T cells, and the CD4 + T cell sub-populations; naive (CD3 + CD4 + CD45RA + CD27 + ), central memory (CD3 + CD4 + CD45RA-CD27 + ), effector memory (CD3 + CD4 + CD45RA − CD27 − ). In adipose, CD3 + T cells are expressed as a percentage of CD45+ events, CD4 + T cells are expressed as a percentage of CD3+ events, and CD4 + T cell sub-populations are expressed as a percentage of CD4+ events. Samples are from participants of a 10-week randomized controlled trial of exercise training ( n = 14) compared to a control ( n = 13). Pre-intervention = day one of the intervention (before exercise). Post-intervention = 36 h after the 10-week intervention/control period. Repeated measures analyses of variance (ANOVAs); n.s. .

Article Snippet: Human CD45RO (clone: REA611) , Miltenyi Biotech , Cat #130-119-620.

Techniques: Flow Cytometry, Isolation, Control

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 CD45RO (clone: REA611) , Miltenyi Biotech , Cat #130-119-620.

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