Journal: Cell Reports Medicine

Article Title: Circulating natural killer cells are phenotypically and functionally altered in age-related macular degeneration

doi: 10.1016/j.xcrm.2026.102792

Figure Lengend Snippet: NK cell proliferation, biogenesis, and metabolic respiration are heightened during active resolution of neovascular lesions (A) scRNA-seq experimental design for 12- to 16-week-old wild-type male mice treated intraperitoneally (i.p.) with PBS or IL-18 and subjected to laser-induced CNV ( n = 4 mice/group, ≥3 lesions/eye). Scale bar: 50 μm. (B) Isolectin B4 analysis of CNV size in RPE/choroid flatmounts ( n = 11 PBS; n = 17 IL-18 [lesions]). (C) UMAP (Scanpy) of CNV tissue from PBS- and IL-18-treated mice ( n = 8 eyes/condition). Bar chart shows treatment-specific cell-type proportions; see also A. (D) Dot plot of known marker genes across cell types. Dot size indicates proportion of cells in cluster expressing a gene; color indicates relative level of expression. (E) UMAP showing predicted cell-cycle phase with summary bar chart of phase distribution per cell type. (F) Top 20 DEGs in NK cells (IL-18 vs. PBS), ranked by Wilcoxon significance; see also B. (G) Dot plot of NK-related genes in NK1 and NK2 populations under PBS or IL-18 treatment. (H) Ncr1 and CD3ε expression in cells from RPE/choroid of mice ( n = 8 eyes). (I) Pou2f2 , Klrg1, Klrk1 , and Klrb1c gene expression in PBS- versus IL-18-treated mice. (J) Functional enrichment analysis of genes differentially upregulated in NK2 vs. NK1 cells in IL-18-treated samples only; see also A. (K and L) Flow cytometry analysis of CNV tissue from 8- to 10-week-old C57Bl/6J mice treated with PBS or IL-18 ( n = 3 mice/group); see B for gating. (M and N) Flow cytometry of CD69 and IFN-γ expression in splenocytes from naive C57Bl/6J mice (8–10 week old) treated ex vivo with PBS or rIL-18 (three mice per group); see C. (O) Seahorse analysis of energetic phenotype of NK cells isolated from C57Bl/6J mouse splenocytes unstimulated vs. IL-2/IL-12 pair vs. IL-18 alone. Bar charts presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01. Following normality testing, unpaired t tests or one-way ANOVA with Dunnett’s multiple comparison tests were used to compare groups.

Article Snippet: Human IL-12 , Miltenyi Biotec , Cat# 130-096-705.

Techniques: Marker, Expressing, Gene Expression, Functional Assay, Flow Cytometry, Ex Vivo, Isolation, Comparison

Journal: Cell Reports Medicine

Article Title: Circulating natural killer cells are phenotypically and functionally altered in age-related macular degeneration

doi: 10.1016/j.xcrm.2026.102792

Figure Lengend Snippet: NK cells are found localized adjacent to neovessels in nAMD (A) HRMEC cells were treated for 24 h with 100 μM etoposide and analyzed for expression of markers of cellular senescence ( n = 3). (B) Representative image of β-galactosidase expression in control versus etoposide-treated HRMECs ( n = 3). Scale bar: 200 μm. (C) NKG2DL expression on HRMECs treated with etoposide ( n = 3); see primer list in A. (D–H) NK92MI cells were treated for 18 h with IL-15/18 and incubated with or without HRMECs for 4 h, scale bar: 400 μm, (E) expression of CD107a measured by flow cytometry ( n = 4), (F) cell viability measured by MTS assay ( n = 3), (G) lytic cell death measured by LDH assay ( n = 4), and (H) target cell apoptosis measured by caspase-3/-7 assay ( n = 4). (I) PBMCs were isolated from healthy donors, treated with NK-activating cytokines (IL-12/15 or IL-15/18) for 18 h, and incubated with or without HRMECs for 4 h. Expression of CD107a was measured by flow cytometry ( n = 3–5); see also B. (J and K) Representative and control immunostaining of NCR1 (red) and UEA-1 (green) in cross-sections from non-disease donor controls ( n = 3); see donor information in D. (L) H&E staining in human donor eye with CNV. Orange autofluorescence due to RPE lipofuscin. ∗denotes area of CNV. (M–P) Representative immunostaining of NCR1 (red) and UEA-1 (green) in cross-sections of patients with nAMD ( n = 3); higher magnifications of lesional tissue of one donor shown in Na&b. High-magnification images of two further donors shown in (O) and (M). Closed white arrows denote areas of NCR1 + staining at or adjacent to blood vessels. Open white arrows denote areas of NCR1 + staining in choroidal tissue. Yellow arrows denote areas of NCR1 + staining at the neovascular membrane. Graphical data presented as mean ± SEM. ∗ p < 0.05 ∗∗ p < 0.01, ∗∗∗ p < 0.005, ∗∗∗∗ p < 0.001. Following normality testing, unpaired t tests or one-way ANOVA with Dunnett’s multiple comparison tests were used to compare groups.

Article Snippet: Human IL-12 , Miltenyi Biotec , Cat# 130-096-705.

Techniques: Expressing, Control, Incubation, Flow Cytometry, MTS Assay, Lactate Dehydrogenase Assay, Isolation, Immunostaining, Staining, Membrane, Comparison

Journal: Cell Reports Medicine

Article Title: Circulating natural killer cells are phenotypically and functionally altered in age-related macular degeneration

doi: 10.1016/j.xcrm.2026.102792

Figure Lengend Snippet: NK cell immunotherapy reduces neovascular lesion volume and repairs neovessel barrier integrity in models of retinal neovascularization (A) Schematic representation of therapeutic administration of isolated, activated NCR1-GFP − or NCR1-GFP + NK cells in liCNV model in C57Bl/6J mice. (B) RPE/choroid flatmounts from wild-type mice receiving adoptive transfer of IL-12/-15/-18-activated NCR1-GFP − NK cells (top panel) or NCR1-GFP + NK cells were stained for isolectin B4 (red) and DAPI (blue) at 3 days post-liCNV ( n = 4 eyes, 3–4 lesions/eye). Scale bar: 58 μm. (C and D) Schematic representation of therapeutic administration of isolated, activated NK cells in the liCNV model. (D) RPE/choroid flatmounts were stained for isolectin B4 (green) and CNV volume was analyzed by confocal microscopy and IMARIS. n = 5 mice/group, 3 CNVs/eye. Scale bar: 115 μm. (E) Schematic representation of adoptive transfer of isolated, activated C57Bl/6J NK cells in a JR5558 model of spontaneous retinal vascularization ( n = 3–4 mice/group). (E) Fundus fluorescein angiography (FFA) was performed prior to (top), and 5 days following (bottom), NK cell administration. (F) Quantification of leakage area from retinal neovascular lesions. Graphical data presented as mean ± SEM; statistical analysis by unpaired t test or two-way ANOVA with Fisher’s LSD test where ∗ p ≤ 0.05, ∗∗ p < 0.01.

Article Snippet: Human IL-12 , Miltenyi Biotec , Cat# 130-096-705.

Techniques: Isolation, Adoptive Transfer Assay, Staining, Confocal Microscopy

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

Journal: bioRxiv

Article Title: Tissue-adapted NK cells shape pathogenic cDC1 niches in early arthritis

doi: 10.64898/2026.05.01.716870

Figure Lengend Snippet: a , Violin plots detailing normalised XCL1 , XCL2, and CCL5 expression across annotated immune cell types in paired blood and synovial fluid (CITE-seq, n = 11). b , Frequencies (left) and geometric mean fluorescence intensities (geoMFI; right) of XCL1 + NK cells, calculated as a percentage of total NK cells (gated as live, Lin - CD3 - CD7 + events) in blood ( n = 8) and synovial fluid ( n = 7; 7 paired, 1 unpaired) following a 6-hour unstimulated culture containing brefeldin A and monensin. c , Representative flow cytometry of intracellular XCL1 in total NK cells stimulated for up to 4 hours with PMA and ionomycin, with brefeldin A and monensin. d , e , Enrichment scores for transcriptional modules associated with cytokine-stimulated NK cells ( d ) and antigen-receptor T cell activation ( e ) within NK subpopulations. f , Absolute change in XCL1 + NK cell frequency relative to unstimulated controls in paired blood and synovial fluid ( n = 8) following ex vivo stimulation with cytokines (IL-12, IL-15, and IL-18) or equimolar bead-conjugated activating receptor antibodies (actR; targeting CD2, CD226, CD244, NKG2D, NKp30, and NKp46). g , Quantification of XCL1 protein in paired serum and synovial fluid from patients with JIA, measured by ELISA ( n = 7). Bars indicate the mean ± s.d. Statistical significance was determined using paired two-tailed t -tests (restricted to the 7 matched pairs for b ), paired two-tailed t -tests with Benjamini-Hochberg correction ( d ), and a Wilcoxon signed-rank test following four-parameter logistic regression ( g ).

Article Snippet: We performed cytokine stimulations with IL-12 (10 ng/mL, Miltenyi, 130-096-704), IL-15 (10 ng/mL, Miltenyi, 130-095-760), or IL-18 (100 ng/mL, MBL International, B001-5).

Techniques: Expressing, Fluorescence, Flow Cytometry, Activation Assay, Ex Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test