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
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