Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: ( A ) Workflow of in vitro SATB1 KO experiments in human Treg and Tconv cells. Treg and Tconv cells were isolated from the blood of healthy donors, ex vivo expanded, CRISPR-edited, and challenged with or without IL-12. Cells were cultured with IL-2 throughout the experiment (Treg: 600 IU/ml; Tconv: 200 IU/ml). KO cells were phenotypically characterized by flow cytometry, bulk RNA-seq, and ATAC-seq analysis. ( B ) Heatmap displaying fold changes of pro- and anti-inflammatory flow cytometry markers in SATB1 KO Treg cells and SATB1 KO Tconv cells, normalized to their respective AAVS1 KO controls. The fold change was calculated based on the percentage of the individual marker pregated on living cells. n = 12–18 (biological replicates), ratio paired t test. ( C , D ) opt-SNE contour plot of IL-12-treated AAVS1 KO and SATB1 KO Treg ( C ) and Tconv cells ( D ). Expression levels (MFI) of tested flow cytometry markers plotted on opt-SNE plot, n = 12–18 (biological replicates). ( E ) Absolute percentages of intracellular cytokine flow cytometry stainings of IL-12-treated AAVS1 KO and SATB1 KO Treg cells. n = 12–17 (biological replicates), paired t test. ( F ) Extracellular IL-10 levels determined by LEGENDplex TM assay of IL-12-treated AAVS1 KO and SATB1 KO Treg cells. Absolute values (left), normalized to AAVS1 KO controls (right), n = 10, paired t test (left), ratio paired t test (right). ( G ) Absolute percentages of intracellular cytokine flow cytometry stainings of IL-12-treated AAVS1 KO and SATB1 KO Tconv cells. n = 12–18 (biological replicates), paired t test. ( H ) Extracellular IL-10 levels determined by LEGENDplex TM assay of IL-12 or control-treated AAVS1 KO and SATB1 KO Tconv. Absolute values (left), normalized to AAVS1 KO controls (right), n = 7 (biological replicates), paired t test (left), ratio paired t test (right). .

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques: In Vitro, Isolation, Ex Vivo, CRISPR, Cell Culture, Flow Cytometry, RNA Sequencing, Marker, Expressing, Control

Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: Human Treg and Tconv cells were isolated, expanded, activated, and nucleofected with Cas9 RNPs targeting AAVS1 , FOXP3 , and SATB1 . The cells were cultured with or without the pro-inflammatory cytokine IL-12. ( A ) Scatter dot plot displaying KO efficiencies with median. KO efficiencies were determined by amplicon NGS sequencing and TIDE analysis. n = 2–9 (biological replicates). ( B ) Bar graphs indicate the mean of relative mRNA expression (ΔCt) of SATB1 and FOXP3 in AAVS 1 and SATB1 KO Treg cells normalized to 18S rRNA levels. RNA of FACS-sorted living cells was isolated, and qPCR was performed in technical duplicates. n = 4–5 (biological replicates), paired t test. ( C ) Flow cytometry analysis of canonical pro- and anti-inflammatory markers in FOXP3 KO Treg cells stimulated with or without IL-12. Percentages of marker-positive cells were normalized to the respective AAVS1 KO Treg cells with or without IL-12 stimulation. n = 6–7 (biological replicates), ratio paired t test. ( D ) Flow cytometry gating strategy of AAVS1 KO control Treg and Tconv cells without IL-12 conditioning. ( E ) Bar graph plots quantifying flow cytometry marker expressions of AAVS1 , FOXP3 , and SATB1 KO Treg cells treated with or without IL-12. Data are also partially shown in Fig. . n = 6–17 (biological replicates), paired t test. ( F ) Bar graph plots quantifying flow cytometry marker expressions of AAVS1 and SATB1 KO Tconv cells treated with or without IL-12. Data are also partially shown in Fig. . n = 12–18 (biological replicates), paired t test.

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques: Isolation, Cell Culture, Amplification, Sequencing, Expressing, Flow Cytometry, Marker, Control

Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: ( A , B ) opt-SNE plot of untreated or IL-12-treated AAVS1 KO Treg ( C ) and Tconv cells ( D ). ( C , D ) Expression levels (MFI) of tested flow cytometry markers of untreated AAVS1 KO and SATB1 KO Treg ( A ) and Tconv cells ( B ) plotted on opt-SNE density plot. n (Treg) = 12–17 (biological replicates), n (Tconv) = 12–18 (biological replicates).

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques: Expressing, Flow Cytometry

Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: ( A ) Venn diagram of differentially accessible chromatin regions in SATB1 KO Treg and Tconv cells normalized to AAVS1 KO control cells ( P value < 0.05, Wald test using DESeq2). ( A , B ) n = 3 (biological replicates). ( B ) PCA plot of AAVS1 and SATB1 KO Treg and Tconv cells treated with IL-12, analyzed by ATAC-seq. ( C ) Venn diagram of differentially expressed genes in SATB1 KO Treg and Tconv cells normalized to AAVS1 KO control cells ( P value < 0.05, Wald test using DESeq2). ( D ) PCA plot of AAVS1 and SATB1 KO Treg and Tconv cells analyzed by RNA-seq after IL-12 conditioning. ( E ) Heatmaps indicating log2 fold change of overlapping RNA-seq data of SATB1 KO Treg and Tconv cells treated with IL-12. Human transcription factors were indicated in bold. ( C – E ) n = 3 (biological replicates). .

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques: Control, RNA Sequencing

Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: ( A ) Mean fluorescence intensity (MFI) of GATA3 expression of SATB1 KO and AAVS1 control KO Treg and Tconv cells treated with or without IL-12. n = 6–9 (biological replicates), paired t test. ( B ) Mean fluorescence intensity (MFI) of CD86 and FOXP3 expression of SATB1 KO and AAVS1 control KO Treg and Tconv cells treated with or without IL-12. n = 8–9 (biological replicates), paired t test. ( C , D ) AAVS1 and SATB1 KO Tconv cell expansion rate at day 14, 15, 16, and 18 of culture with or without the addition of IL-12. ( C ) Absolute cell counts with mean and SD, ( D ) cell counts of SATB1 KO Tconv cells normalized to respective AAVS1 KO control cells. n = 6 (biological replicates). ( E , F ) Extracellular IL-6 levels determined by LEGENDplex TM assay of IL-12-treated AAVS1 KO and SATB1 KO Treg ( E ) or Tconv cells ( F ). Absolute values (left), normalized to AAVS1 KO controls (right), n = 7–10 (biological replicates), paired t test.

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques: Fluorescence, Expressing, Control

Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: ( A , B ) Volcano plot of differentially accessible chromatin regions of SATB1 KO Treg versus AAVS1 KO Treg cells ( A ) and SATB1 KO Tconv versus AAVS1 KO Tconv cells ( B ), n = 3 (biological replicates). Genes with a P value < 0.05 are highlighted, calculated by a Wald test using DESeq2. ( C , D ) Volcano plot of differentially expressed genes of SATB1 KO Treg versus AAVS1 KO Treg cells ( C ) and SATB1 KO Tconv versus AAVS1 KO Tconv cells ( D ), n = 3 (biological replicates). Genes with a P value < 0.05 are highlighted, calculated by a Wald test using DESeq2. ( E ) Heatmaps displaying z-scores of equally regulated chromatin and gene expression changes in SATB1 KO Treg and Tconv cells treated with IL-12. Differentially regulated TFs are highlighted in purple. Top hits of Reactome (REAC) and biological pathway (BP) analysis and associated adjusted P values ( P adj) if significant are highlighted. n = 3 (biological replicates). .

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques: Gene Expression

Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: Heatmaps display z-scores of RNA- and ATAC-seq data of SATB1 KO and AAVS1 KO Treg and Tconv cells treated with IL-12. TFs differently regulated in RNA- and ATAC-seq data after SATB1 KO are highlighted in purple. Genes associated with “Interleukin-10 signaling”, “Positive regulation of leukocyte proliferation”, or “cytokine-mediated signaling” are highlighted. REAC Reactome, BP biological pathway. n = 3 (biological replicates).

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques:

Journal: EMBO Reports

Article Title: SATB1 is a targetable modulator of JAK-STAT signaling and cytokines in human Treg and Tconv cells

doi: 10.1038/s44319-026-00812-6

Figure Lengend Snippet: Differently expressed genes (DEGs) of the cytokine–cytokine receptor pathway ( A ) and the JAK-STAT signaling pathway ( B ) in SATB1 KO Treg and Tconv cells treated with IL-12, normalized to the respective IL-12 AAVS1 KO conditions based on RNA-seq data. Color scheme indicates log2 fold changes of gene expression and the size of the dots the -log10 of the P value calculated by a Wald test using DESeq2, n = 3 (biological replicates). .

Article Snippet: Recombinant human IL-12 , Miltenyi , Cat#: 130-096-704.

Techniques: RNA Sequencing, Gene Expression