Journal: iScience

Article Title: Akkermansia muciniphila ameliorates fatty liver through microbiota-derived α-ketoisovaleric acid metabolism and hepatic PI3K/Akt signaling

doi: 10.1016/j.isci.2025.112458

Figure Lengend Snippet: Akk improves gut immune responses in obese mice (A) PHATE of T cells clustering in mice intestinal lymph nodes. (B–E) Percentages of CD3 + CD4 + Tregs, CD3 + CD8 + Tregs, CD4 + CD25 + Tregs, and CD4 + CD25 + Foxp3 + Tregs. Data are expressed as mean ± SD ( n = 6), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the Mod group, and p value was calculated using Dunnet’s post hoc test in one-way ANOVA.

Article Snippet: Anti-Mouse CD25 + PE , BioGems , Cat# 07312-60.

Techniques:

Journal: iScience

Article Title: Akkermansia muciniphila ameliorates fatty liver through microbiota-derived α-ketoisovaleric acid metabolism and hepatic PI3K/Akt signaling

doi: 10.1016/j.isci.2025.112458

Figure Lengend Snippet: Gut fungi is the key in Akk improving gut immune responses in organoids (A) Co-culture of colonic organoids, fecal microbiota and Akk. (B) Fluorescent images of colonic organoids with DAPI-labeled cells, and number of organoid crypts. Scale bars: 100 μm. (C) Relative abundance of fungal in co-culture system. (D) Percentages of CD3 + CD4 + , CD3 + CD8 + , CD4 + CD25 + , and CD4 + CD25 + Foxp3 + Tregs in organoids. (E) Relative content of metabolites in co-culture system. (F) Co-culture of colonic organoids, fluconazole, fecal microbiota and Akk. (G) Fluorescent images of colonic organoids with DAPI-labeled cells, and number of organoid crypts. Scale bars: 50 μm. (H) Percentages of CD3 + CD4 + , CD3 + CD8 + , CD4 + CD25 + , and CD4 + CD25 + Foxp3 + Tregs in organoids. (I and J) Relative content of metabolites (α-ketoisovaleric acid and glycylleucine) in co-culture system. Figures 7 A–7E and Figures 7 F–7J represent experiments from different batches, respectively. Data are expressed as mean ± SD (n = 6–8), ns p >0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the Mod group, and p value was calculated using Dunnet’s post hoc test in one-way ANOVA.

Article Snippet: Anti-Mouse CD25 + PE , BioGems , Cat# 07312-60.

Techniques: Co-Culture Assay, Labeling

Journal: Autoimmunity

Article Title: N6-methyladenosine modification of THBS1 induced by affluent WTAP promotes Graves' ophthalmopathy progression through glycolysis to affect Th17/Treg balance.

doi: 10.1080/08916934.2024.2433628

Figure Lengend Snippet: Figure 2. RNA sequencing (RNA-seq) was performed to capture differentially expressed genes in CD4+ T cells. (A) Pearson correlation between NS and GO group samples. NS: healthy control group. (B) Reads per kilobase per million mapped reads (RPKM) distribution of NS and GO group. (C) RPKM density distribution indicated the accurate results of RNA-seq. (D) a volcano plot was presented to narrate differentially expressed genes between the NS group and the GO group (total: 679, up: 308, down: 371). (E) Gene ontology analysis was presented to demonstrate the TOP 20 signaling pathway enriched by differentially expressed genes through a dot plot. (F) Kyoto Encyclopedia of genes and Genomes (KEGG) enrichment analysis of differentially expressed genes in CD4+ T cells.

Article Snippet: For the Treg cells, FITC plus anti-human CD4 (Proteintech, FITC-65134), PE anti-human CD25 antibody (Biolegend, 302605), Alexa Fluor® 647 anti-mouse/rat/human FOXP3 antibody (Biolegend, 320013) were applied.

Techniques: RNA Sequencing, Control

Journal: Autoimmunity

Article Title: N6-methyladenosine modification of THBS1 induced by affluent WTAP promotes Graves' ophthalmopathy progression through glycolysis to affect Th17/Treg balance.

doi: 10.1080/08916934.2024.2433628

Figure Lengend Snippet: Figure 3. Analysis of methylation differences in CD4+ T cells between NS group and GO group. (A) The distribution of m6A peak density from GO along a meta gene. (B) Presentation of differences in CDS, stop C, 5’UTR, 3’UTR, and start C occupancy between the two groups. (C) Motif sequences for GO group and NS group. (D) A total of 3277 m6A sites (hypermethylated _ sites 1302, hypomethylated _ sites, 1975) were identified by MeRIP-seq. (E) 1302 hypermethylated _ sites corresponded to 1143 genes and 1975 hypomethylated _ sites corresponded to 1601 genes. (F) The chromosomal distribution of corresponding m6A peaks. Hype _ sites: hypermethylated _ sites; hypo _ sites: Hypomethylated _ sites.

Article Snippet: For the Treg cells, FITC plus anti-human CD4 (Proteintech, FITC-65134), PE anti-human CD25 antibody (Biolegend, 302605), Alexa Fluor® 647 anti-mouse/rat/human FOXP3 antibody (Biolegend, 320013) were applied.

Techniques: Methylation

Journal: Autoimmunity

Article Title: N6-methyladenosine modification of THBS1 induced by affluent WTAP promotes Graves' ophthalmopathy progression through glycolysis to affect Th17/Treg balance.

doi: 10.1080/08916934.2024.2433628

Figure Lengend Snippet: Figure 4. Functional analysis of hypermethylated and hypomethylated genes between NS group and GO group. (A) Gene ontology enrichment analysis of hyper methylated genes in CD4+ T cells of GO group. (B) KEGG enrichment analysis of hypermethylated genes in GO group. (C) Gene ontology enrichment analysis of hypomethylated genes in GO group. (D) KEGG enrichment analysis of hypomethylated genes in GO group.

Article Snippet: For the Treg cells, FITC plus anti-human CD4 (Proteintech, FITC-65134), PE anti-human CD25 antibody (Biolegend, 302605), Alexa Fluor® 647 anti-mouse/rat/human FOXP3 antibody (Biolegend, 320013) were applied.

Techniques: Functional Assay, Methylation

Journal: Autoimmunity

Article Title: N6-methyladenosine modification of THBS1 induced by affluent WTAP promotes Graves' ophthalmopathy progression through glycolysis to affect Th17/Treg balance.

doi: 10.1080/08916934.2024.2433628

Figure Lengend Snippet: Figure 5. MeRIP-seq combined with RNA-seq reveals THBS1 as a target gene in CD4+ T cells of GO. (A) Veen diagram presented the number of intersecting genes according to MeRIP-seq and RNA-seq. Hypermethylated and upregulated genes: 81; hypermethylated and downregulated genes: 4; hypomethylated and upregu lated genes: 6; hypomethylated and downregulated genes: 121. (B) TOP 6 mRNA genes (FGF11, PRKCZ, GNG4, PDGFB, THBS1, and FN1) which were characterized with hypermethylation and up-regulated were lying in the PI3K-Akt signaling pathway. (C) The expression maps of 6 candidate genes (FGF11, PRKCZ, GNG4, PDGFB, THBS1, and FN1) were detected by RT-qPCR. (D) The knockdown efficiency of si-THBS1-1 (P = 0.008), si-THBS1-2 (P = 0.006), and si-THBS1-3 (P = 0.001) was presented by RT-qPCR results. The knock-down efficiency of si-THBS1-3 (p = 0.001) is the most desirable. (E) Western blot verified the knockdown effect of si-THBS1-3. (F) The lactic acid content in CD4+T cells was significantly reduced after the silence of THBS1. (G) Glucose uptake in CD4+T cells was subsequently reduced by THBS1 knockdown. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For the Treg cells, FITC plus anti-human CD4 (Proteintech, FITC-65134), PE anti-human CD25 antibody (Biolegend, 302605), Alexa Fluor® 647 anti-mouse/rat/human FOXP3 antibody (Biolegend, 320013) were applied.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Knockdown, Western Blot

Journal: Autoimmunity

Article Title: N6-methyladenosine modification of THBS1 induced by affluent WTAP promotes Graves' ophthalmopathy progression through glycolysis to affect Th17/Treg balance.

doi: 10.1080/08916934.2024.2433628

Figure Lengend Snippet: Figure 6. WTAP is abnormal in CD4+ T cells of GO and regulates the m6A level and expression of THBS1. (A) Potential m6A loci on THBS1 sequence between NS group and GO group. (B) The expression of four common m6A modifying enzymes (METTL3, METTL14, WTAP, KIAA1429) was verified by RT-qPCR, only WTAP was significantly highly expressed. (C) The interfered effect of si-WTAP-1 (P = 0.001), si-WTAP-2 (P = 0.001), and si-WTAP-3 (P = 0) on WTAP was confirmed by RT-qPCR, and si-WTAP-3 was selected for the subsequent experiment. (D) The sequence motif (GGACA) of WTAP was in accord with “RRACH”. (E) Silencing of WTAP signifi cantly reduced the expression level of THBS1 compared with the control group. (F) MeRIP-PCR showed that the knockdown of WTAP decreased the m6A abun dance of THBS1. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For the Treg cells, FITC plus anti-human CD4 (Proteintech, FITC-65134), PE anti-human CD25 antibody (Biolegend, 302605), Alexa Fluor® 647 anti-mouse/rat/human FOXP3 antibody (Biolegend, 320013) were applied.

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Control, Knockdown

Journal: Clinical & Translational Immunology

Article Title: Anti‐C1q autoantibodies from systemic lupus erythematosus patients enhance CD40–CD154‐mediated inflammation in peripheral blood mononuclear cells in vitro

doi: 10.1002/cti2.1408

Figure Lengend Snippet: T cell proliferation, activation and IL‐10 secretion in activated T cells are not affected by bound C1q, bound C1q/anti‐C1q and soluble C1q, respectively, whereas TNF secretion is decreased in the presence of soluble C1q. T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and cultured on bound HSA, bound C1q, bound C1q preincubated with anti‐C1q‐positive systemic lupus erythematosus serum (bound C1q/anti‐C1q), or together with soluble C1q without coating. T cells were activated by tetrameric anti‐CD3/CD28. Cytokines (a) TNF and (b) IL‐10 as well as activation markers (c) CD25 and (d) CD69 were analysed after 24 h by ELISA and flow cytometry, respectively. For proliferation assessment, cells were stained with CFSE prior to the experiment and (e) per cent dividing cells and (f) proliferation index were analysed by flow cytometry after 96 h. Data points represent six different healthy donors used to obtain PBMCs analysed in independent experiments with connecting lines linking data points of a single individual. Median values are shown as solid horizontal lines. The Friedman test with Dunn's posttest correction (all vs bound HSA), * P < 0.05. (c, d) Relative intensity is calculated by normalising MFI of bound C1q, bound C1q/anti‐C1q and soluble C1q to bound HSA. The horizontal dashed line marks the relative change in intensity of 1.0 (Supplementary figure a depicts the gating strategy).

Article Snippet: Staining for surface marker expression was performed for 30 min at 4°C and included the following antibodies: mouse anti‐human CD25 APC and CD69 PE (both from Immunotools) (antibody panel 1), or CD11c APC (Biolegend), CD40 FITC, CD80 FITC and CD86 FITC (all from BD) (antibody panel 2).

Techniques: Activation Assay, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Ustekinumab Improves Psoriasis without Altering T Cell Cytokine Production, Differentiation, and T Cell Receptor Repertoire Diversity

doi: 10.1371/journal.pone.0051819

Figure Lengend Snippet: The percentage of nTreg (FoxP3 + CD127 low CD25 high CD4 + T cells/CD4 + T cells) was similar among the seven volunteers. Flow cytometry data from four patients and three healthy controls are shown.

Article Snippet: For identification of nTreg (FoxP3 + CD127 low CD25 high CD4 + T cells), magnetically collected CD4 + T cells during the third blood sampling were directly stained with monoclonal antibodies to CD127-FITC and CD25-PE in cell surface staining buffer containing 0.1M PBS and 2% FCS (Biowest, Nuaillé, France).

Techniques: Flow Cytometry