Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Immunoblot analysis of Ripk1 in FACS-sorted Tcon (CD4 + YFP – ) and Treg (CD4 + YFP + ) cells from Ripk1 Δ Foxp3 and Foxp3 Cre (control) mice. Pictures of cervical ( B ) and inguinal ( C ) area of 9-week-old Ripk1 Δ Foxp3 and Foxp3 Cre mice. Arrows indicate lymph nodes. D Representative histological sections of paraffin-embedded tissue of the indicated organs of Ripk1 Δ Foxp3 and Foxp3 Cre mice stained with hematoxylin and eosin. E Heat map of histological score of the indicated organs of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 5, each group). F Heat map of bone marrow erythropoiesis and granulopoiesis of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 5, each group).

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Western Blot, Control, Staining

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Representative dot plots of Foxp3-expressing cells within CD4 + cells of Ripk1 Δ Foxp3 and Foxp3 Cre mice. Percentages (left) and absolute cell number (right) of CD4 + Foxp3 + cells ( B ), CD4 + cells ( C ), CD8 + cells ( D ), and B cells ( E ) of spleen and lymph nodes (LN) from Ripk1 Δ Foxp3 and Foxp3 Cre mice. F Concentration of the indicated cytokines in sera from Ripk1 Δ Foxp3 and Foxp3 Cre mice determined by Luminex assay ( Ripk1 Δ Foxp3 n = 13 , Foxp3 Cre n = 5). Bar graphs represent the mean ± SEM. Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant. G Representative zebra plots (left) and frequencies (right) of Treg cell activation markers CD62L and CD44 in CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice. B – E , G Mean ± SEM are given. Each symbol represents a single mouse in the scatter plots. H Representative histograms and geometric mean fluorescence intensity (gMFI) bar graphs of ICOS (upper) and TIGIT (lower) expression in CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 2, each). I Representative histograms and gMFI bar graphs of Ki67 expression in CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 2, each). J Representative histograms (top) and summary bar graph (bottom) of active caspase-3/7 in CD4 + YFP + cells from spleen and lymph nodes (LN) of Ripk1 Δ Foxp3 and Foxp3 Cre mice ( n = 5, each). B – G , J Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Expressing, Concentration Assay, Luminex, Two Tailed Test, MANN-WHITNEY, Activation Assay, Fluorescence

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Immunoblot analysis of Ripk1 in purified Treg cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 (control) mice treated with tamoxifen or vehicle for the indicated time points. B Representative dot plots, C Foxp3 + hCD2 + percentages, and D Foxp3 geometric mean fluorescence intensity (gMFI) of events within live CD4 + cells of MACS-sorted naïve CD4 + T cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 ( Ripk1 fl/fl ; n = 4) and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 ( Ripk1 wt/wt control; n = 4) mice treated with tamoxifen for 72 h and subsequently with Treg cell polarizing conditions. Data was obtained from four independent experiments. E Representative dot plots (left) and bar graphs (right) of viability analysis determined by CellEvent Caspase-3/7 and LIVE/DEAD Fixable Blue staining of MACS-sorted CD4 + hCD2 + cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 ( Ripk1 fl/fl ; n = 5) and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 ( Ripk1 wt/wt control; n = 5) mice and treated in vitro with Tamoxifen for 72 h. Data was obtained from five independent experiments. F Frequencies of viable cells determined by LIVE/DEAD Fixable Blue staining of MACS-sorted CD4 + hCD2 + cells from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 ( Ripk1 fl/fl ; n = 8) and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 ( Ripk1 wt/wt control; n = 8) mice and treated in vitro with Tamoxifen for 72 h and, subsequently with TNF (50 ng/ml), anti-CD3 and anti-CD28, combination of anti-CD3, anti-CD28 and TNF (50 ng/ml) or left untreated for 16 additional hours. C – F Data in bar graphs are represented as mean ± SEM. Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, n.s. not significant.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Western Blot, Purification, Control, Fluorescence, Staining, In Vitro, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Distribution of the percentages of YFP - and YFP + cells within the CD4 + Foxp3 + population of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 11). B Representative dot plots of Foxp3-expressing cells within CD4 + cells (upper) and YFP - expressing cells within Foxp3 + cells (lower) from spleen, lymph nodes (LN) and thymus of Ripk1 fl/fl Foxp3 Cre/wt mice. C Ki67 positive events in YFP - and YFP + cells within CD4 + CD25 + cells from spleen and lymph nodes (LN) of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 11). D Proliferation represented by inverse geometric mean fluorescence intensity (gMFI) of CTV dilution from FACS sorted CD4 + CD25 + YFP – and CD4 + CD25 + YFP + cells of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 4) stimulated with anti-CD3 (1 µg/ml), anti-CD28 (2 µg/ml) and IL-2 (10 ng/ml) for 72 h. E Active caspase-3/7 positive events in YFP - and YFP + cells within CD4 + CD25 + cells from spleen and lymph nodes (LN) of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 11). F Viable cell frequency of FACS sorted CD4 + CD25 + YFP - and CD4 + CD25 + YFP + cells of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 6) stimulated with anti-CD3 (1 µg/ml), anti-CD28 (2 g/ml) and IL-2 (10 ng/ml) for 16 h. G Representative zebra plots (left) and distribution in frequencies (right) of resting (rTreg; CD62L + CD44 - ) and effector (eTreg; CD62L - CD44 + ) Treg cells within CD4 + Foxp3 + cells from spleen and lymph nodes (LN) of Ripk1 wt/wt Foxp3 Cre/wt mice ( n = 6) and Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 7). Data in bar graphs are represented as mean ± SEM. Statistical analyses were performed by Kruskal-Wallis test followed by Dunn´s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, otherwise not significant. H Geometric mean fluorescence intensity (gMFI) of Foxp3 in CD4 + CD25 + cells from spleen and lymph nodes (LN) of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 6) and Ripk1 wt/wt Foxp3 Cre/wt mice ( n = 4). I Fold change of average percentages of YFP + vs. YFP - events within eTreg cells from spleen and lymph nodes (LN) of Ripk1 wt/wt Foxp3 Cre/wt mice and Ripk1 fl/fl Foxp3 Cre/wt mice. J Frequency of Foxp3 + cells in CD4 + cells in lamina propria (LP) and in the intraepithelial lymphocyte compartment (IEL) of small intestine of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 4). K Distribution in frequency of YFP - and YFP + within CD4 + Foxp3 + cells and their respective Helios ( L ) and RORγt expression ( M ) in lamina propria (LP) and in the intraepithelial lymphocyte (IEL) compartment of small intestine of Ripk1 fl/fl Foxp3 Cre/wt mice ( n = 4). ( A , C – F , H and J – M ) Data in bar graphs are represented as mean ± SEM. Statistical analyses were performed by two-tailed Mann–Whitney tests. * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Expressing, Fluorescence, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death and Differentiation

Article Title: Ripk1 is critical for preserving effector regulatory T cells and the suppressive transcriptional program in regulatory T cells

doi: 10.1038/s41418-025-01550-3

Figure Lengend Snippet: A Chord plot of differentially expressed genes in FACS sorted CD4 + CD25 + YFP - (Ripk1 +/+ ) and CD4 + CD25 + YFP + (Ripk1 –/– ) cells from Ripk1 fl/fl Foxp3 Cre/wt mice. Upregulated genes in CD4 + CD25 + YFP + (Ripk1 –/– ) cells are labeled in red (positive logFC) and downregulated genes are labeled in blue (negative logFC). Chord plot shows a detailed relationship between the log-fold change of differentially expressed genes (DEG) (left semicircle) and their enriched gene ontology (GO) terms (right semicircle). B Heat map analysis of Treg gene signature in FACS sorted CD4 + CD25 + YFP – (Treg Ripk1 +/+ ) and CD4 + CD25 + YFP + (Treg Ripk1 –/– ) cells from Ripk1 fl/fl Foxp3 Cre/wt mice. C Representative CNS2/TSDR methylation patterns of FACS sorted CD4 + CD25 + YFP – and CD4 + CD25 + YFP + cells from Ripk1 fl/fl Foxp3 Cre/wt mice. Amplicons are vertically arranged, each representing a single CpG-motif. Color code indicates the percentage of CNS2/TSDR methylation in the purified populations ( n = 4). Data was obtained from two independent experiments. D Seurat clustering of scRNA-seq of FACS-sorted CD4 + CD25 hi cells from spleen and lymph nodes of Ripk1 fl/fl Foxp3 Cre/wt mice. UMAP projections represent clusters of pre-labeled YFP - (left) and YFP + (right) cells. E scRNASeq volcano plot showing changes in gene expression of CD4 + CD25 hi YFP + (Treg Ripk1 –/– ) cells vs. CD4 + CD25 hi YFP - (Treg Ripk1 +/+ ) cells. Heat map analysis of ( F ) Bcl-2 family members, ( G ) NF-κB pathway components, ( H ) Top20 downregulated NF-κB target genes in YFP + cells and ( I ) NF-κB target genes of cytokines/chemokines in FACS sorted CD4 + CD25 + YFP - (Treg Ripk1 +/+ ) and CD4 + CD25 + YFP + (Treg Ripk1 –/– ) cells from Ripk1 fl/fl Foxp3 Cre/wt mice.

Article Snippet: Naïve CD4 + T cells were isolated from pooled spleen and lymph node single-cell suspensions from Ripk1 fl/fl Cre-ER T2 Foxp3 hCD2 and Ripk1 wt/wt Cre-ER T2 Foxp3 hCD2 mice using Naive CD4 + T Cell Isolation kit (130-104-453, Miltenyi Biotec) according to manufacturer ́s instructions.

Techniques: Labeling, Methylation, Purification, Gene Expression

Journal: Science Advances

Article Title: Galectin-3 exacerbates autoimmune diabetes by limiting regulatory T cell differentiation and function

doi: 10.1126/sciadv.adz7916

Figure Lengend Snippet: Pancreatic islets from 12-week-old nondiabetic Galectin-3 +/+ NOD and Galectin-3 −/− NOD mice were harvested and subject to flow cytometry analysis ( n = 5). Frequencies of CD4 + and CD8 + T cells ( A ) and T reg cells (CD4 + CD25 + FOXP3 + ) ( B ) are shown as representative fluorescence-activated cell sorting plots. ( C to E ) Frequencies of T H 1 (CD4 + IFN-γ + ) (C), T H 2 (CD4 + IL-4 + ) (D), and T H 17 (CD4 + IL-17 + ) (E) subsets among CD45 + cells in islets of 12-week-old Galectin-3 +/+ NOD and Galectin-3 −/− NOD mice. ( F to J ) Frequencies of perforin + CD8 + (F), Tc1 (CD8 + IFN-γ + ) (G), Tc2 (CD8 + IL-4 + ) (H), Tc17 (CD8 + IL-17 + ) (I), and granzyme B + CD8 + (J) subsets among CD45 + cells in islets of 12-week-old Galectin-3 +/+ NOD and Galectin-3 −/− NOD mice. Data are expressed as mean ± SEM. *P < 0.05.

Article Snippet: The administration of Galectin-3 consistently significantly decreased the frequency of CD4 + CD25 + FOXP3 + T reg cells, while the proportions of T H 1, T H 2, and T H 17 subsets remained unaffected (fig. S5).

Techniques: Flow Cytometry, Fluorescence, FACS

Journal: Science Advances

Article Title: Galectin-3 exacerbates autoimmune diabetes by limiting regulatory T cell differentiation and function

doi: 10.1126/sciadv.adz7916

Figure Lengend Snippet: ( A ) Frequency of CD4 + CD25 + FOXP3 + T reg cells after exposure of naïve CD4 + T cells to T reg cell–inducing conditions with or without Galectin-3 recombinant protein at different concentrations ( n = 6). ( B ) Frequency of Ki67 + cells after T reg cell induction ( n = 6). ( C ) Apoptosis of T reg cells was determined by annexin V staining ( n = 3). ( D ) The percentage of proliferated T reg cells was defined by carboxyfluorescein diacetate succinimidyl ester assay after 72 hours of culture ( n = 4 to 6). ( E and F ) Cytokine profile of culture supernatants of isolated T reg cells ( n = 4). ( G ) CD8 + T cells were stimulated with plate-coated anti-CD3/CD28 and cultured either alone or with Galectin-3–pretreated or vehicle-pretreated T reg cells at the indicated ratios. Perforin expression in CD8 + T cells was measured by flow cytometry after 72 hours. ( H ) CD8 + T cells were cocultured with T reg cells in a Transwell system in the presence or absence of Galectin-3 protein, IL-10, or TGF-β neutralizing antibodies. Perforin expression was measured by flow cytometry after 72 hours. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The administration of Galectin-3 consistently significantly decreased the frequency of CD4 + CD25 + FOXP3 + T reg cells, while the proportions of T H 1, T H 2, and T H 17 subsets remained unaffected (fig. S5).

Techniques: Recombinant, Staining, CFSE Assay, Isolation, Cell Culture, Expressing, Flow Cytometry

Journal: Science Advances

Article Title: Galectin-3 exacerbates autoimmune diabetes by limiting regulatory T cell differentiation and function

doi: 10.1126/sciadv.adz7916

Figure Lengend Snippet: ( A ) Expression levels of candidate Galectin-3–binding receptors in T reg cells ( n = 3). ( B ) Expression of LAG3 was examined on T reg cells after induction in different groups as indicated ( n = 3 to 6). ( C and D ) Naïve CD4 + T cells purified from WT C57BL/6J splenocytes were cultured under T reg cell–polarizing conditions in vitro, treated with Galectin-3 protein alone or in combination with LAG3 neutralizing antibody for 72 hours. The frequencies of CD4 + CD25 + FOXP3 + T reg cells (C) as well as Ki67 + cells (D) were assessed by flow cytometry ( n = 7). ( E ) Heatmap showing the expression of T reg cell function–associated genes in control T reg cells versus Galectin-3–treated T reg cells ( n = 3). ( F ) Volcano plot of DEGs between groups. Log 2 FC, log 2 fold change. ( G ) GSEA plot showing the enrichment of the MAPK signaling pathway in Galectin-3–treated T reg cells compared to control T reg cells. ( H and I ) Immunoblot analysis of MEK/ERK phosphorylation in T reg cells stimulated with Galectin-3, with or without LAG3 blockade. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NES, normalized enrichment score; FDR, false discovery rate.

Article Snippet: The administration of Galectin-3 consistently significantly decreased the frequency of CD4 + CD25 + FOXP3 + T reg cells, while the proportions of T H 1, T H 2, and T H 17 subsets remained unaffected (fig. S5).

Techniques: Expressing, Binding Assay, Purification, Cell Culture, In Vitro, Flow Cytometry, Cell Function Assay, Control, Western Blot, Phospho-proteomics

Journal: Science Advances

Article Title: Galectin-3 exacerbates autoimmune diabetes by limiting regulatory T cell differentiation and function

doi: 10.1126/sciadv.adz7916

Figure Lengend Snippet: ( A ) Schematic diagram showing the protocol of TD139 treatment. ( B ) Incidence of diabetes expressed as percentage of diabetic mice at different ages ( n = 20). ( C ) Glucose excursion curve after receiving IPGTT ( n = 8). ( D ) Serum insulin concentration at 0 and 15 min during the IPGTT in 12-week-old TD139 or vehicle-treated NOD mice ( n = 7 to 8). ( E ) Representative H&E staining of pancreas from 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). ( F ) Insulitis scores calculated on the basis of histological evaluation of pancreatic section as in (E). ( G ) Frequencies of CD4 + and CD8 + T cells in total CD3 + T cells from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). ( H ) Frequencies of T reg cells (CD4 + CD25 + FOXP3 + ) from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). ( I to K ) Frequencies of perforin + CD8 + T, Tc1 (CD8 + IFN-γ + ), Tc2 (CD8 + IL-4 + ), Tc17 (CD8 + IL-17 + ), and granzyme B + CD8 + T subsets from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 3 to 4). ( L ) Frequencies of T H 1 (CD4 + IFN-γ + ), T H 2 (CD4 + IL-4 + ), and T H 17 (CD4 + IL-17 + ) from islets of 12-week-old NOD mice treated with TD139 or vehicle ( n = 4). Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01.

Article Snippet: The administration of Galectin-3 consistently significantly decreased the frequency of CD4 + CD25 + FOXP3 + T reg cells, while the proportions of T H 1, T H 2, and T H 17 subsets remained unaffected (fig. S5).

Techniques: Concentration Assay, Staining