Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: CCR7 + (c4) MoMFs drive MASLD progression through enhanced antigen presentation and are recruited via the CCL19/CCL21 axis. (A) Feature plots displaying the expression of H2-Eb2 , Ccr7 , Cd209a , and Ciita . (B) Flow cytometry analysis showing CCR7 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (C) Correlation between the proportion of CCR7 + MoMFs and the NAFLD activity score in the combined cohort of NCD-fed and MCD-fed mice. (D, E) Dot plots show the enriched GO (D) and KEGG (E) terms of DEGs in CCR7 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. (F) Dot plots show the average expression levels of selected DEGs. Dot size denotes the percentage of expressed cells, and the color bar denotes scaled average expression. (G) Flow cytometry analysis showing MHC-II expression levels in CCR7 + MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (H) Percentage of CCR7 + cells among total intrahepatic MoMFs in mice. (I, J) Plasma ALT and AST levels in each group. (K–M) Representative images (K) and quantification of H&E staining (L) and Sirius red staining (M) in liver paraffin sections. Scale bars, 200 μm. (N) Relative mRNA levels of Ccl19 (left) and Ccl21 (right) in liver tissues of NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice. (O) Feature plots displaying the expression of Top2a , Mki67 , and Stmn1 . (P) Flow cytometry analysis showing Ki67 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (Q) Dot plots show the enriched GO terms of DEGs in Ki67 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. (R) Feature plots displaying the expression of Cd3d , Gzma , and Gzmb . (S) Flow cytometry analysis showing CD3 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (T) Dot plots show the enriched GO terms of DEGs in CD3 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. Abbreviations: ALT, alanine transaminase; AST, aspartate aminotransferase; CDHFD, choline-deficient high-fat diet; HFD, high-fat diet; H&E, Hematoxylin and Eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; MCD, methionine/choline-deficient diet; MoMFs, monocyte-derived macrophages; NAFLD, nonalcoholic fatty liver disease; NCD, normal control diet; UMAP, uniform manifold approximation and projection.

Article Snippet: To further determine the functional contribution of CCR7 + MoMFs, we administered a neutralizing antibody against CCR7 (MAB3477, R&D Systems) or an IgG2A isotype (clone 4B12, R&D Systems) to MCD-fed mice.

Techniques: Immunopeptidomics, Expressing, Flow Cytometry, Activity Assay, Clinical Proteomics, Staining, Derivative Assay, Control

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: The basal cluster 1 (c1) is depleted during MASLD progression and acquires CD14 + and CCR7 + phenotypes under inflammatory stimulation in vitro. (A) Feature plots displaying the expression of Uba52 , Rpl27 , Rpl15 , and Ifi27l2a . (B) The typical flow cytometry gating strategy and statistical analysis of cluster 1 relative to total MoMFs in NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice. (C) Correlation between the proportion of cluster 1 and the NAFLD activity score in the combined cohort of NCD-fed and MCD-fed mice. (D) UMAP plots displaying the major cellular subsets identified in the NCD and MCD groups. (E) Trajectory analysis of clusters 0, 1, 4, and 5 using Monocle 2. (F) Heatmap displaying gene expression dynamics across pseudotime for selected marker genes in clusters 0, 1, 4, and 5. (G–I) Flow cytometry analysis of CD14 (G), CCR7 (H), and CCR3 (I) expression in cluster 1 cells isolated from NCD-fed mouse liver, with or without LPS stimulation. (J) Dot plots depicting the expression of genes in liver macrophages derived from healthy and MASLD samples ( GSE212837 ). Abbreviations: CDHFD, choline-deficient high-fat diet; HFD, high-fat diet; LPS, lipopolysaccharide; MASLD, metabolic dysfunction–associated steatotic liver disease; MCD, methionine/choline-deficient diet; MoMFs, monocyte-derived macrophages; NCD, normal control diet; UMAP, uniform manifold approximation and projection.

Article Snippet: To further determine the functional contribution of CCR7 + MoMFs, we administered a neutralizing antibody against CCR7 (MAB3477, R&D Systems) or an IgG2A isotype (clone 4B12, R&D Systems) to MCD-fed mice.

Techniques: In Vitro, Expressing, Flow Cytometry, Activity Assay, Gene Expression, Marker, Isolation, Derivative Assay, Control

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: Heterogeneity and dynamics of intrahepatic MoMFs during MASLD progression. This scRNA-seq analysis delineated 7 distinct MoMFs clusters (c0–c6) in the liver with dynamic changes during steatohepatitis progression. CD14 + (c0) MoMFs, the predominant CCR2 + population, exhibited multifaceted functions beyond inflammation, including roles in fibrosis, lipid uptake, and antigen presentation. The basal cluster (c1) is depleted during MASLD progression and acquires CD14 + and CCR7⁺ phenotypes under inflammatory stimulation in vitro. Lipid overload drives mitochondrial dysfunction and apoptosis in CD206 + (c2) MoMFs in MASLD. CCR3 + (c3) MoMFs drive MASLD progression through lipid accumulation, oxidative stress, and CCL5-mediated recruitment. CCL19/CCL21-recruited CCR7 + (c4) MoMFs exacerbate MASLD pathogenesis through enhanced antigen presentation. In addition, Ki67 + (c5) proliferative and CD3 + (c6) TCR-related MoMFs maintained stable proportions but exhibited functional alterations during MASLD. Abbreviations: MASLD, metabolic dysfunction–associated steatotic liver disease; MoMFs, monocyte-derived macrophages; scRNA-seq, single-cell RNA sequencing.

Article Snippet: To further determine the functional contribution of CCR7 + MoMFs, we administered a neutralizing antibody against CCR7 (MAB3477, R&D Systems) or an IgG2A isotype (clone 4B12, R&D Systems) to MCD-fed mice.

Techniques: Immunopeptidomics, In Vitro, Functional Assay, Derivative Assay, Single Cell, RNA Sequencing

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: CCR7 + (c4) MoMFs drive MASLD progression through enhanced antigen presentation and are recruited via the CCL19/CCL21 axis. (A) Feature plots displaying the expression of H2-Eb2 , Ccr7 , Cd209a , and Ciita . (B) Flow cytometry analysis showing CCR7 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (C) Correlation between the proportion of CCR7 + MoMFs and the NAFLD activity score in the combined cohort of NCD-fed and MCD-fed mice. (D, E) Dot plots show the enriched GO (D) and KEGG (E) terms of DEGs in CCR7 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. (F) Dot plots show the average expression levels of selected DEGs. Dot size denotes the percentage of expressed cells, and the color bar denotes scaled average expression. (G) Flow cytometry analysis showing MHC-II expression levels in CCR7 + MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (H) Percentage of CCR7 + cells among total intrahepatic MoMFs in mice. (I, J) Plasma ALT and AST levels in each group. (K–M) Representative images (K) and quantification of H&E staining (L) and Sirius red staining (M) in liver paraffin sections. Scale bars, 200 μm. (N) Relative mRNA levels of Ccl19 (left) and Ccl21 (right) in liver tissues of NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice. (O) Feature plots displaying the expression of Top2a , Mki67 , and Stmn1 . (P) Flow cytometry analysis showing Ki67 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (Q) Dot plots show the enriched GO terms of DEGs in Ki67 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. (R) Feature plots displaying the expression of Cd3d , Gzma , and Gzmb . (S) Flow cytometry analysis showing CD3 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (T) Dot plots show the enriched GO terms of DEGs in CD3 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. Abbreviations: ALT, alanine transaminase; AST, aspartate aminotransferase; CDHFD, choline-deficient high-fat diet; HFD, high-fat diet; H&E, Hematoxylin and Eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; MCD, methionine/choline-deficient diet; MoMFs, monocyte-derived macrophages; NAFLD, nonalcoholic fatty liver disease; NCD, normal control diet; UMAP, uniform manifold approximation and projection.

Article Snippet: To further determine the functional contribution of CCR7 + MoMFs, we administered a neutralizing antibody against CCR7 (MAB3477, R&D Systems) or an IgG2A isotype (clone 4B12, R&D Systems) to MCD-fed mice.

Techniques: Immunopeptidomics, Expressing, Flow Cytometry, Activity Assay, Clinical Proteomics, Staining, Derivative Assay, Control

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: The basal cluster 1 (c1) is depleted during MASLD progression and acquires CD14 + and CCR7 + phenotypes under inflammatory stimulation in vitro. (A) Feature plots displaying the expression of Uba52 , Rpl27 , Rpl15 , and Ifi27l2a . (B) The typical flow cytometry gating strategy and statistical analysis of cluster 1 relative to total MoMFs in NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice. (C) Correlation between the proportion of cluster 1 and the NAFLD activity score in the combined cohort of NCD-fed and MCD-fed mice. (D) UMAP plots displaying the major cellular subsets identified in the NCD and MCD groups. (E) Trajectory analysis of clusters 0, 1, 4, and 5 using Monocle 2. (F) Heatmap displaying gene expression dynamics across pseudotime for selected marker genes in clusters 0, 1, 4, and 5. (G–I) Flow cytometry analysis of CD14 (G), CCR7 (H), and CCR3 (I) expression in cluster 1 cells isolated from NCD-fed mouse liver, with or without LPS stimulation. (J) Dot plots depicting the expression of genes in liver macrophages derived from healthy and MASLD samples ( GSE212837 ). Abbreviations: CDHFD, choline-deficient high-fat diet; HFD, high-fat diet; LPS, lipopolysaccharide; MASLD, metabolic dysfunction–associated steatotic liver disease; MCD, methionine/choline-deficient diet; MoMFs, monocyte-derived macrophages; NCD, normal control diet; UMAP, uniform manifold approximation and projection.

Article Snippet: To further determine the functional contribution of CCR7 + MoMFs, we administered a neutralizing antibody against CCR7 (MAB3477, R&D Systems) or an IgG2A isotype (clone 4B12, R&D Systems) to MCD-fed mice.

Techniques: In Vitro, Expressing, Flow Cytometry, Activity Assay, Gene Expression, Marker, Isolation, Derivative Assay, Control

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: Heterogeneity and dynamics of intrahepatic MoMFs during MASLD progression. This scRNA-seq analysis delineated 7 distinct MoMFs clusters (c0–c6) in the liver with dynamic changes during steatohepatitis progression. CD14 + (c0) MoMFs, the predominant CCR2 + population, exhibited multifaceted functions beyond inflammation, including roles in fibrosis, lipid uptake, and antigen presentation. The basal cluster (c1) is depleted during MASLD progression and acquires CD14 + and CCR7⁺ phenotypes under inflammatory stimulation in vitro. Lipid overload drives mitochondrial dysfunction and apoptosis in CD206 + (c2) MoMFs in MASLD. CCR3 + (c3) MoMFs drive MASLD progression through lipid accumulation, oxidative stress, and CCL5-mediated recruitment. CCL19/CCL21-recruited CCR7 + (c4) MoMFs exacerbate MASLD pathogenesis through enhanced antigen presentation. In addition, Ki67 + (c5) proliferative and CD3 + (c6) TCR-related MoMFs maintained stable proportions but exhibited functional alterations during MASLD. Abbreviations: MASLD, metabolic dysfunction–associated steatotic liver disease; MoMFs, monocyte-derived macrophages; scRNA-seq, single-cell RNA sequencing.

Article Snippet: To further determine the functional contribution of CCR7 + MoMFs, we administered a neutralizing antibody against CCR7 (MAB3477, R&D Systems) or an IgG2A isotype (clone 4B12, R&D Systems) to MCD-fed mice.

Techniques: Immunopeptidomics, In Vitro, Functional Assay, Derivative Assay, Single Cell, RNA Sequencing

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: CCR7 + (c4) MoMFs drive MASLD progression through enhanced antigen presentation and are recruited via the CCL19/CCL21 axis. (A) Feature plots displaying the expression of H2-Eb2 , Ccr7 , Cd209a , and Ciita . (B) Flow cytometry analysis showing CCR7 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (C) Correlation between the proportion of CCR7 + MoMFs and the NAFLD activity score in the combined cohort of NCD-fed and MCD-fed mice. (D, E) Dot plots show the enriched GO (D) and KEGG (E) terms of DEGs in CCR7 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. (F) Dot plots show the average expression levels of selected DEGs. Dot size denotes the percentage of expressed cells, and the color bar denotes scaled average expression. (G) Flow cytometry analysis showing MHC-II expression levels in CCR7 + MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (H) Percentage of CCR7 + cells among total intrahepatic MoMFs in mice. (I, J) Plasma ALT and AST levels in each group. (K–M) Representative images (K) and quantification of H&E staining (L) and Sirius red staining (M) in liver paraffin sections. Scale bars, 200 μm. (N) Relative mRNA levels of Ccl19 (left) and Ccl21 (right) in liver tissues of NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice. (O) Feature plots displaying the expression of Top2a , Mki67 , and Stmn1 . (P) Flow cytometry analysis showing Ki67 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (Q) Dot plots show the enriched GO terms of DEGs in Ki67 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. (R) Feature plots displaying the expression of Cd3d , Gzma , and Gzmb . (S) Flow cytometry analysis showing CD3 + MoMFs proportions among total MoMFs across NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice, with representative plots (left) and quantitative data (right). (T) Dot plots show the enriched GO terms of DEGs in CD3 + MoMFs between the NCD and MCD groups. Dot size denotes the enriched gene counts. The color bar denotes the adjusted p -value of enrichment. Abbreviations: ALT, alanine transaminase; AST, aspartate aminotransferase; CDHFD, choline-deficient high-fat diet; HFD, high-fat diet; H&E, Hematoxylin and Eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; MCD, methionine/choline-deficient diet; MoMFs, monocyte-derived macrophages; NAFLD, nonalcoholic fatty liver disease; NCD, normal control diet; UMAP, uniform manifold approximation and projection.

Article Snippet: To target CCR7 + MoMFs, mice received twice-weekly intravenous (i.v.) injections of an anti-mouse CCR7 neutralizing antibody (MAB3477, R&D Systems) at 10 μg per mouse or an IgG2A isotype control (clone 4B12, R&D Systems)., To target CCR2 + MoMFs, mice were fed an MCD diet for 4 weeks.

Techniques: Immunopeptidomics, Expressing, Flow Cytometry, Activity Assay, Clinical Proteomics, Staining, Derivative Assay, Control

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: The basal cluster 1 (c1) is depleted during MASLD progression and acquires CD14 + and CCR7 + phenotypes under inflammatory stimulation in vitro. (A) Feature plots displaying the expression of Uba52 , Rpl27 , Rpl15 , and Ifi27l2a . (B) The typical flow cytometry gating strategy and statistical analysis of cluster 1 relative to total MoMFs in NCD-fed, CDHFD-fed, MCD-fed, and HFD-fed mice. (C) Correlation between the proportion of cluster 1 and the NAFLD activity score in the combined cohort of NCD-fed and MCD-fed mice. (D) UMAP plots displaying the major cellular subsets identified in the NCD and MCD groups. (E) Trajectory analysis of clusters 0, 1, 4, and 5 using Monocle 2. (F) Heatmap displaying gene expression dynamics across pseudotime for selected marker genes in clusters 0, 1, 4, and 5. (G–I) Flow cytometry analysis of CD14 (G), CCR7 (H), and CCR3 (I) expression in cluster 1 cells isolated from NCD-fed mouse liver, with or without LPS stimulation. (J) Dot plots depicting the expression of genes in liver macrophages derived from healthy and MASLD samples ( GSE212837 ). Abbreviations: CDHFD, choline-deficient high-fat diet; HFD, high-fat diet; LPS, lipopolysaccharide; MASLD, metabolic dysfunction–associated steatotic liver disease; MCD, methionine/choline-deficient diet; MoMFs, monocyte-derived macrophages; NCD, normal control diet; UMAP, uniform manifold approximation and projection.

Article Snippet: To target CCR7 + MoMFs, mice received twice-weekly intravenous (i.v.) injections of an anti-mouse CCR7 neutralizing antibody (MAB3477, R&D Systems) at 10 μg per mouse or an IgG2A isotype control (clone 4B12, R&D Systems)., To target CCR2 + MoMFs, mice were fed an MCD diet for 4 weeks.

Techniques: In Vitro, Expressing, Flow Cytometry, Activity Assay, Gene Expression, Marker, Isolation, Derivative Assay, Control

Journal: Hepatology Communications

Article Title: Single-cell profiling reveals hepatic monocyte-derived macrophages heterogeneity during steatotic liver disease

doi: 10.1097/HC9.0000000000000928

Figure Lengend Snippet: Heterogeneity and dynamics of intrahepatic MoMFs during MASLD progression. This scRNA-seq analysis delineated 7 distinct MoMFs clusters (c0–c6) in the liver with dynamic changes during steatohepatitis progression. CD14 + (c0) MoMFs, the predominant CCR2 + population, exhibited multifaceted functions beyond inflammation, including roles in fibrosis, lipid uptake, and antigen presentation. The basal cluster (c1) is depleted during MASLD progression and acquires CD14 + and CCR7⁺ phenotypes under inflammatory stimulation in vitro. Lipid overload drives mitochondrial dysfunction and apoptosis in CD206 + (c2) MoMFs in MASLD. CCR3 + (c3) MoMFs drive MASLD progression through lipid accumulation, oxidative stress, and CCL5-mediated recruitment. CCL19/CCL21-recruited CCR7 + (c4) MoMFs exacerbate MASLD pathogenesis through enhanced antigen presentation. In addition, Ki67 + (c5) proliferative and CD3 + (c6) TCR-related MoMFs maintained stable proportions but exhibited functional alterations during MASLD. Abbreviations: MASLD, metabolic dysfunction–associated steatotic liver disease; MoMFs, monocyte-derived macrophages; scRNA-seq, single-cell RNA sequencing.

Article Snippet: To target CCR7 + MoMFs, mice received twice-weekly intravenous (i.v.) injections of an anti-mouse CCR7 neutralizing antibody (MAB3477, R&D Systems) at 10 μg per mouse or an IgG2A isotype control (clone 4B12, R&D Systems)., To target CCR2 + MoMFs, mice were fed an MCD diet for 4 weeks.

Techniques: Immunopeptidomics, In Vitro, Functional Assay, Derivative Assay, Single Cell, RNA Sequencing

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 CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

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 box plots showing flow cytometry analysis of the percentage of human CD4 and CD8 Tem (CD45RA-CCR7-) Tem cells from PBMCs isolated from control individuals ( n = 10) or individuals with Alstrom syndrome ( n = 10). Data are presented as mean +/− SD. Unpaired nonparametric Mann–Whitney T test; * P < 0.05. ( B , C ) Flow cytometry analysis of subcutaneous human abdominal adipose tissue ( B ) and lysed whole blood ( C ), including CD8 + T cells, and the CD8 + T cell sub-populations; naive (CD3 + CD8 + CD45RA + CD27 + ), central memory (CD3 + CD8 + CD45RA − CD27 + ), effector memory (CD3 + CD8 + CD45RA − CD27 − ). In adipose, CD8 + T cells are expressed as a percentage of CD3+ events, and CD8 + T cell sub-populations are expressed as a percentage of CD8+ 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 CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

Techniques: Flow Cytometry, Isolation, Control, MANN-WHITNEY

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 CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

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 CCR7 (clone: REA108) , Miltenyi Biotech , Cat #130-120-463.

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

Journal: Scientific Reports

Article Title: CCR7 immune cell receptor expression in inflammatory breast cancer

doi: 10.1038/s41598-026-43437-4

Figure Lengend Snippet: CCR7 protein expression in IBC and non-IBC cell lines. ( A ) Immunoblot analysis of protein lysates from IBC (A3250, SUM149, IBC-3, SUM190) and non-IBC cell lines (MDA-MB-468, MCF-7, TF7D, SK-Br-3) expressed high levels of CCR7 in vitro. ( B ) Ratio of CCR7 compared to β-tubulin demonstrated similar levels of overall CCR7 protein expression between IBC and non-IBC cell lines.

Article Snippet: Immunohistochemical (IHC) staining for CCR7 (R&D systems, catalog no. MAB197-100) was performed using Leica Bond RX autostainer.

Techniques: Expressing, Western Blot, In Vitro

Journal: Scientific Reports

Article Title: CCR7 immune cell receptor expression in inflammatory breast cancer

doi: 10.1038/s41598-026-43437-4

Figure Lengend Snippet: CCR7 and CCL21 expression in IBC versus non-IBC patient samples. ( A – D ) CCR7 and CCL21 expression were significantly higher among IBC patients ( n = 137) compared to non-IBC patients ( n = 252), ( p = 0.0007 and 0.0018, respectively). ( B ) Among IBC patients, HER2-positive and basal subtypes had significantly higher levels of CCR7 compared to luminal subtypes ( p = 0.0002 and 0.0161, respectively) and ( C ) ER-negative status exhibited higher CCR7 expression compared to ER-positive status ( p = 0.0098). ( E ) Similarly, HER2-positive subtypes demonstrated higher CCL21 expression compared to luminal subtypes ( p = 0.0423 and 0.0366), while ( F ) ER status did not impact CCL21 expression. ( G ) CCR7 positively correlated with expression of its ligand, CCL21 ( r = 0.39, p < 0.0001) and ( H ) CCL21 was correlated with LYVE-1, a lymphangiogenic marker in breast cancer ( r = 0.24, p < 0.0001).

Article Snippet: Immunohistochemical (IHC) staining for CCR7 (R&D systems, catalog no. MAB197-100) was performed using Leica Bond RX autostainer.

Techniques: Expressing, Marker

Journal: Scientific Reports

Article Title: CCR7 immune cell receptor expression in inflammatory breast cancer

doi: 10.1038/s41598-026-43437-4

Figure Lengend Snippet: CCR7 TMA scoring scales. Representative TMA images of CCR7 scoring scales by intensity, ( A ) 1 + intensity, ( B ) 2 + intensity, ( C ) 3 + intensity. Note that the example for 1 + staining intensity is not from this sample cohort as no patients in this study had 1 + CCR7 intensity.

Article Snippet: Immunohistochemical (IHC) staining for CCR7 (R&D systems, catalog no. MAB197-100) was performed using Leica Bond RX autostainer.

Techniques: Staining