Journal: Nature Communications

Article Title: Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint blockade

doi: 10.1038/s41467-024-51496-2

Figure Lengend Snippet: Single-cell RNA-seq analysis of a type I interferon-α/β receptor (IFNAR) and type II interferon-γ receptor (IFNGR) subunit gene expression shown in units of transcript per million (TPM)/10 (black bars; mean ± SEM) in patient melanoma cells (left) versus tumor-infiltrating T-cells (right). Gene expression of additional cytokine and growth factor receptor subunits is also shown (gray bars). b Violin plots of IFNAR1 and IFNAR2 gene expression (TPM/10; median, bold white line; top and bottom quartiles, thin white lines) in patient melanoma (MM) versus tumor-infiltrating T-cells generated from the single-cell RNA-seq dataset above. c IFNAR1 and IFNAR2 surface protein expression (percent positivity, mean ± SEM, left) by patient tumor cells and tumor-infiltrating lymphocytes (TILs), with representative flow cytometric histograms shown (right). d Relative IFNAR1 and IFNAR2 gene expression (fold change, mean ± SEM) in human melanoma cell lines (black bars) versus human activated CD3 + T-cells (gray bars) as determined by RT-qPCR. e IFNAR1 and IFNAR2 surface protein expression (percent positivity, mean ± SEM, left) by human A2058 and A375 melanoma cells, with representative flow cytometric histograms shown for A2058 melanoma cells (right). f Relative Ifnar1 and Ifnar2 gene expression (fold change, mean ± SEM) in murine melanoma cell lines (black bars) versus murine activated CD3 + T-cells (gray bars) as determined by RT-qPCR. g IFNAR1 and IFNAR2 surface protein expression (percent positivity, mean ± SEM, left) by murine melanoma cells, with representative flow cytometric histograms shown for B16-F10 and YUMM1.7 melanoma cells (right). IFNAR1 (left) and IFNAR2 (right) surface protein expression (percent positivity, mean ± SEM) by PD-1 + versus PD-1 - h , human A2058 and A375 and i , murine melanoma cell subsets, with representative flow cytometric histograms shown for A2058 cells. Results represent biologically independent samples of a , b n = 1252 MM cells (left panel) and 2040 T-cells (right panel), c n = 4, and biologically independent experiments of d , f , i n = 3, e n = 12, g n = 6, and h n = 7 (A2058) and n = 6 (A375). Statistical analyses included b Mann-Whitney test, two-sided, c paired t -test, one-sided, and h , i unpaired t -test, two-sided. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant. See also Supplementary Figs. – . Source data, including exact p -values, are provided as a Source Data file.

Article Snippet: The following antibodies (abs) were used for flow cytometric analyses of human cells: Alexa Fluor 647-conjugated (BD Biosciences) or PerCP-eFluor 710-conjugated anti-human PD-1 (clone MIH4, 20 μg/mL, Thermo Fisher Scientific, Waltham, MA) and Alexa Fluor 647-conjugated or PerCP-eFluor 710-conjugated mouse IgG1 isotype controls (clone MOPC-31C, 20 μg/mL, BD Biosciences; clone P3.6.2.8.1, 20 μg/mL, Thermo Fisher Scientific), unconjugated anti-human PD-1 clinical ab, nivolumab (100 μg/mL, Bristol Myers Squibb, Cambridge, MA) was obtained from the BWH Pharmacy, and Ultra-LEAF unconjugated human IgG4 isotype control (clone QA16A15, 100 μg/mL, BioLegend), FITC-conjugated or PE-conjugated anti-human IgG4 (clone HP6023, 1:50, Abcam, Waltham, MA), PE-conjugated anti-human PD-L1 (clone 29E.2A3, 15 μg/mL, BioLegend) and PE-conjugated mouse IgG2b isotype control (clone MPC-11, 15 μg/mL, BioLegend), APC-conjugated anti-human PD-L2 (clone 24 F.10C12, 15 μg/mL, BioLegend) and APC-conjugated mouse IgG2a isotype control (clone MOPC-173, 15 μg/mL, BioLegend), PE-conjugated anti-human IFNAR1 (clone 85228, 20 μg/mL, Thermo Fisher Scientific) and PE-conjugated mouse IgG1 isotype control (clone P3.6.2.8.1, 20 μg/mL, Thermo Fisher Scientific) or PE-conjugated mouse IgG1 isotype control (clone MOPC-21, 20 μg/mL, BioLegend), Alexa Fluor 750-conjugated anti-human IFNAR1 (clone 85228, 20 μg/mL, R&D Systems, Minneapolis, MN) and Alexa Fluor 750-conjugated mouse IgG1 isotype control (clone 11711, 20 μg/mL, R&D Systems), APC-conjugated or APC-Vio770-conjugated anti-human IFNAR2 (clone REA124, 20 μg/mL, Miltenyi Biotec, Gaithersburg, MD) and APC-conjugated or APC-Vio770-conjugated human IgG1 isotype control (clone REA293, 20 μg/mL, Miltenyi Biotec), FITC-conjugated anti-human IFN-β (clone A1(IFNb)), 5 μg/mL, and FITC-conjugated mouse IgG1 (clone MOPC-21, 5 μg/mL, Thermo Fisher Scientific), PE-conjugated anti-human IFN-α[2b] (clone 7N4-1, 1.5 μg/mL) and PE-conjugated mouse IgG1 isotype control (clone MOPC-21, 1.5 μg/mL, BD Biosciences), FITC-conjugated anti-mouse Interferon alpha (clone RMMA-1, 50 μg/mL, PBL Assay Science, Piscataway, NJ) and FITC-conjugated rat IgG1 (clone eBRG1, 50 μg/mL, Thermo Fisher Scientific), anti-mouse Interferon-beta 1 (clone D2J1D, 75 ng/mL), rabbit IgG isotype control (clone DA1E, 75 ng/mL Cell Signaling Technology, Danvers, MA), and PE-conjugated donkey anti-rabbit IgG (clone Poly4064, 1.25 μg/mL BioLegend).

Techniques: RNA Sequencing, Gene Expression, Generated, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The cells were then resuspended in wash buffer and incubated in MACS human FcR blocking solution (20μl/sample, cat no . 130–059-901, Miltenyi Biotec) and primary antibodies against CD45-FITC (12μl/sample, Cat no . 130–114-567, Clone 5B1, Miltenyi Biotec), CD31-FITC (4μl/sample, Cat no . 130–110-668, Clone REA730, Miltenyi Biotec), CD90-PE (3.2μl/sample, Clone 5E10, eBioscience, Thermo Fisher Scientific), CD56-BV421 (1:100, Cat no . 562752, BD Bioscience, San Jose, CA, USA), CD82-PE-vio770 (Cat no . 130–101-302 10μl/sample, Miltenyi Biotec) and CD34-APC (20μl/sample, clone 581, BD Bioscience) in darkness at 4°C for 30 min. Propidium iodide (PI, 10μl/sample, cat 556463, BD Bioscience) immediately before sorting to exclude non-viable cells.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing, Quantitative Proteomics

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: A-i, Sorting strategy (CD45−CD31−) identifying CD34+CD56− or CD34−; A-ii, CD34+CD90− and CD34+CD90+ cells; A-iii, CD56+CD82+ cells; A-iv, CD90+ cells; B, Work-flow; C-E, adipogenic, fibrogenic and myogenic differentiation of CD34+CD90−CD56− (C), CD34+CD90+CD56− (D) and CD56+CD82+CD34− (E) populations by IHC; F, % mononuclear cells in human skeletal muscle (n=17, study 2, non-T2DM); G, PDGFRα in FAPs (scalebar 50 μm); H, COL1A1 expressing CD90+CD56− (FAPs), CD56+ (MuSCs) and CD90−CD56− (Unidentified) cells (n=6, study 2, non-T2DM) after 3–9 days in vitro; I, Clonal experiment; J, Colony formation (%) of single sorted FAPs and MuSCs (n=12, study 2, non-T2DM); K, Limiting dilution assay on FAPs and MuSCs (n=3, study 2, non-T2DM), Solid line is non-linear fit; L, MyHC+/Desmin+ myotubes from single sorted MuSCs; M, Perilipin1+/Collagen1+ cells from single sorted FAPs; N, Biological processes in FAPs (n=3, study 2, non-T2DM); O, % of EdU+ MuSCs and FAPs (n=10–16, study 2, non-T2DM); P, EdU+ MuSCs (i) and FAPs (ii) 96h post sort. Cells obtained from of mm. Rectus Abdominus/Gastrocnemius. *p<0.05, **p<0.01 and ***p<0.001.

Article Snippet: The cells were then resuspended in wash buffer and incubated in MACS human FcR blocking solution (20μl/sample, cat no . 130–059-901, Miltenyi Biotec) and primary antibodies against CD45-FITC (12μl/sample, Cat no . 130–114-567, Clone 5B1, Miltenyi Biotec), CD31-FITC (4μl/sample, Cat no . 130–110-668, Clone REA730, Miltenyi Biotec), CD90-PE (3.2μl/sample, Clone 5E10, eBioscience, Thermo Fisher Scientific), CD56-BV421 (1:100, Cat no . 562752, BD Bioscience, San Jose, CA, USA), CD82-PE-vio770 (Cat no . 130–101-302 10μl/sample, Miltenyi Biotec) and CD34-APC (20μl/sample, clone 581, BD Bioscience) in darkness at 4°C for 30 min. Propidium iodide (PI, 10μl/sample, cat 556463, BD Bioscience) immediately before sorting to exclude non-viable cells.

Techniques: Expressing, In Vitro, Limiting Dilution Assay

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The cells were then resuspended in wash buffer and incubated in MACS human FcR blocking solution (20μl/sample, cat no . 130–059-901, Miltenyi Biotec) and primary antibodies against CD45-FITC (12μl/sample, Cat no . 130–114-567, Clone 5B1, Miltenyi Biotec), CD31-FITC (4μl/sample, Cat no . 130–110-668, Clone REA730, Miltenyi Biotec), CD90-PE (3.2μl/sample, Clone 5E10, eBioscience, Thermo Fisher Scientific), CD56-BV421 (1:100, Cat no . 562752, BD Bioscience, San Jose, CA, USA), CD82-PE-vio770 (Cat no . 130–101-302 10μl/sample, Miltenyi Biotec) and CD34-APC (20μl/sample, clone 581, BD Bioscience) in darkness at 4°C for 30 min. Propidium iodide (PI, 10μl/sample, cat 556463, BD Bioscience) immediately before sorting to exclude non-viable cells.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing, Quantitative Proteomics

Journal: Science translational medicine

Article Title: Role of radiation-induced circulating myeloid-derived suppressor cells on systemic lymphopenia after chemoradiotherapy for glioblastoma

doi: 10.1126/scitranslmed.abn6758

Figure Lengend Snippet: A. Illustration depicting GL261 cell implantation, five fractionated 2 Gy radiation treatment (RT) to head along with CB1158 and Tadalafil treatments, MDSC and T cell profiling in peripheral blood, spleen, and tumor. Quantification of G and M-MDSC (B and C), CD4 and CD8 T cells (D and E) in peripheral blood, spleen, and tumor. Data are shown as mean ± S.D. (n=5 mice/group). P-values were determined by one-way ANOVA with post hoc Tukey’s test.

Article Snippet: The following fluorophore-conjugated antibodies were used for different T cell subpopulation staining: CD3 (Order no. 130-113-129, Clone: BW264/56), CD4 (Order no. 130-121-336, Clone: M-T321), CD8 (Order no. 130-110-683, Clone: REA734), CD45RO (Order no. 130-113-561, Clone: REA611), CCR7 (Order no. 130-120-468, Clone: REA546), and CD127 (Order no. 130-113-413, Clone: REA614), all obtained from Miltenyi Biotech.

Techniques:

Journal: Science translational medicine

Article Title: Role of radiation-induced circulating myeloid-derived suppressor cells on systemic lymphopenia after chemoradiotherapy for glioblastoma

doi: 10.1126/scitranslmed.abn6758

Figure Lengend Snippet: A. To study the mechanism of radiation-induced lymphopenia, the C57BL6 mice were injected with GBM tumor cells (GL261), and treated with five fractions of 2 Gy cranial irradiation (RT). The PBMC and MDSC were analyzed on various days as indicated in the following groups; no tumor, no tumor+RT, tumor, and tumor+RT. B-E. The absolute counts of peripheral blood CD4, CD8, G-MDSC, and M-MDSC cells. F & G. The quantitative radiance value and their representative images over time. Data are shown as mean ± S.D. (n=5 mice/group). P-values were determined by two-way ANOVA with post hoc Tukey’s test. The indicated p-values are between the tumor and tumor+RT cohorts at 14 and 21 days post tumor implantation.

Article Snippet: The following fluorophore-conjugated antibodies were used for different T cell subpopulation staining: CD3 (Order no. 130-113-129, Clone: BW264/56), CD4 (Order no. 130-121-336, Clone: M-T321), CD8 (Order no. 130-110-683, Clone: REA734), CD45RO (Order no. 130-113-561, Clone: REA611), CCR7 (Order no. 130-120-468, Clone: REA546), and CD127 (Order no. 130-113-413, Clone: REA614), all obtained from Miltenyi Biotech.

Techniques: Injection, Irradiation, Tumor Implantation

Journal: Science translational medicine

Article Title: Role of radiation-induced circulating myeloid-derived suppressor cells on systemic lymphopenia after chemoradiotherapy for glioblastoma

doi: 10.1126/scitranslmed.abn6758

Figure Lengend Snippet: A. Illustration depicting the patient (NLP and LP) sorted MDSC were co-cultured with healthy pan T cell and the (B) T cell proliferation (Ki67+) and (C) T cell activation (TCR zeta+) were evaluated. D. Illustration depicting infusion of sorted G-MDSC (CD11b+Ly6G+Ly6C−) from GBM-bearing mice treated with or without RT sorted G-MDSC infused into non-tumor recipient mice and evaluated peripheral T cells 24 hours post MDSC infusion in recipient naïve mice. The MDSC from irradiated mice significantly reduced (E) CD4 and (F) CD8 T cells when compared to the unirradiated control. G. Illustration depicting GBM-bearing mice treated with anti-Ly6G antibody (α Ly6G) or isotype control during and after RT. The peripheral T cell and MDSC were analyzed at time points indicated in the graph. I. Depletion of G-MDSC, and (J) No change in M-MDSC during α Ly6G treatment. Depleting of G-MDSC led to rescuing (K) CD4 and (L) CD8 and (H) enhanced survival. Data are shown as mean ± S.D. (n=3 patients/group and n=5 mice/group). P-values were determined by two-tailed with unpaired student’s t-test in B and C, two-way ANOVA with post hoc Tukey’s test in I-L, and generalized Wilcoxon test in H. The indicated p-values in H-L are a comparison between isotype+RT and Ly6G+RT cohorts.

Article Snippet: The following fluorophore-conjugated antibodies were used for different T cell subpopulation staining: CD3 (Order no. 130-113-129, Clone: BW264/56), CD4 (Order no. 130-121-336, Clone: M-T321), CD8 (Order no. 130-110-683, Clone: REA734), CD45RO (Order no. 130-113-561, Clone: REA611), CCR7 (Order no. 130-120-468, Clone: REA546), and CD127 (Order no. 130-113-413, Clone: REA614), all obtained from Miltenyi Biotech.

Techniques: Cell Culture, Activation Assay, Irradiation, Control, Two Tailed Test, Comparison

Journal: Science translational medicine

Article Title: Role of radiation-induced circulating myeloid-derived suppressor cells on systemic lymphopenia after chemoradiotherapy for glioblastoma

doi: 10.1126/scitranslmed.abn6758

Figure Lengend Snippet: Heat map showing differential expression MDSC, T cells with other myeloid and lymphoid cells specific genes in NLP and LP at baseline and week 6 (A). t-SNE representation of single-cell gene expression of the distribution of different T cell subtypes with markers expression (B). TN - naive, TCM - central memory, TEM - effector memory, TEMRA - terminal effector, TREG - regulatory T cells. The size and color of each circle represents the proportion of cells within the group expressing each transcript (C). Flow cytometric analysis of NLP and LP PBMC revealed the changes in CD4 (D), and CD8 T cells (E). Flow cytometric gating strategy demonstrating for CD4 and CD8 T cells on CD3 cells at week 6 between NLP and LP (F). Data are shown as mean ± S.D. P-values were determined by two-way ANOVA with post hoc Tukey’s test.

Article Snippet: The following fluorophore-conjugated antibodies were used for different T cell subpopulation staining: CD3 (Order no. 130-113-129, Clone: BW264/56), CD4 (Order no. 130-121-336, Clone: M-T321), CD8 (Order no. 130-110-683, Clone: REA734), CD45RO (Order no. 130-113-561, Clone: REA611), CCR7 (Order no. 130-120-468, Clone: REA546), and CD127 (Order no. 130-113-413, Clone: REA614), all obtained from Miltenyi Biotech.

Techniques: Quantitative Proteomics, Gene Expression, Expressing

Journal: Nature Communications

Article Title: GPSM1 restricts CD73 + CD103 + T reg cells in adipose tissue, critical for promoting obesity-related metabolic deterioration

doi: 10.1038/s41467-026-73349-w

Figure Lengend Snippet: a Representative western blot of GPSM1 expression in blood CD4 + T cells from lean and obese human donors ( n = 5 per group). b – e Correlation between GPSM1 mRNA expression in peripheral blood CD4 + T cells with metabolic quantitative traits, including BMI ( b , n = 38 biologically independent individuals), Fasting plasma glucose ( c , n = 38 biologically independent individuals), HbA 1c ( d , n = 38 biologically independent individuals), and C-peptide ( e n = 38 biologically independent individuals) from human subjects. GPSM1 mRNA level was normalized to ACTB mRNA. f Representative immunofluorescence images and quantification of staining with antibodies against GPSM1 (red) and CD4 (green) in visceral fat from individuals without/with overweight or obesity. Scale bars, 100 µm. ( n = 3 per group). g – k Correlation between GPSM1 mRNA expression in adipose CD4 + T cells with clinical metabolic traits, including BMI ( g , n = 30 biologically independent individuals), waist circumference ( h , n = 30 biologically independent individuals), insulin ( i , n = 30 biologically independent individuals), HOMA-IR ( j , n = 30 biologically independent individuals), and HbA 1c ( k , n = 29 biologically independent individuals) from visceral fat of human with overweight or obesity. GPSM1 mRNA level was normalized to ACTB mRNA. l , m Representative western blot of GPSM1 expression in CD4 + T cells from subcutaneous white adipose tissue (scWAT, l n = 3 per group) and epididymal white adipose tissue (eWAT, m n = 3 per group) from male mice fed a normal chow diet (NCD) or high-fat diet (HFD). n RT-qPCR analysis of GPSM1 mRNA expression in T h0 and T reg cells induced from naïve mouse T cells ( n = 4 per group). o Representative western blot of GPSM1 expression in T h0 and T reg cells ( n = 3 per group). p , q Naïve T cells were isolated from murine spleens and infected with Lv-shCON or Lv-shGPSM1 and then induced into T regs . Representative flow cytometry and quantification of T reg proportion ( p n = 3 per group). Representative western blot of GPSM1 and FOXP3 expression ( q n = 3 per group). r – u Naïve CD4 + T cells were isolated from murine spleens or human blood and activated for two days using plate-bound anti-CD3 and soluble anti-CD28 antibodies. Naïve CD4 + T cells were then transduced with either Adv-CON or Adv-GPSM1 and subsequently polarized under TGFβ and IL-2 treatment for four days to T regs differentiation. r , t Representative flow cytometry and quantification of murine ( r ) and human ( t ) T reg proportion ( n = 3 per group). s , u Representative western blot of GPSM1 and FOXP3 expression in mouse ( s , n = 3 per group) and human ( u n = 3 per group). All data are shown as means ± SEM. P values are determined by unpaired two-tailed Student’s T-test in ( f , n , p , r , t ) Pearson’s correlation analysis showing R values, and two-tailed P values in ( b – e , g – k ). Source data are provided as a Source Data file.

Article Snippet: The human naïve CD4 + T Cell Isolation Kit II (Cat. No. 130-094-131, Miltenyi Biotec) was used to obtain human naïve CD4 + T cells.

Techniques: Western Blot, Expressing, Clinical Proteomics, Immunofluorescence, Staining, Quantitative RT-PCR, Isolation, Infection, Flow Cytometry, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: GPSM1 restricts CD73 + CD103 + T reg cells in adipose tissue, critical for promoting obesity-related metabolic deterioration

doi: 10.1038/s41467-026-73349-w

Figure Lengend Snippet: a , b Representative flow cytometry picture and quantification of T reg proportion in scWAT and eWAT from CD4 + T cell GPSM1 -knockout ( GPSM1 ΔT ) male mice and male mice bearing a conditional LoxP-flanked allele of GPSM1 ( GPSM1 f/f ) under NCD ( a n = 6 per group) or HFD ( b n = 9 per group) feeding. c , d RT-qPCR analysis of mRNA expression of anti-inflammatory and pro-inflammatory genes in scWAT ( c ) and eWAT ( d ) from HFD-fed male GPSM1 f/f or GPSM1 ΔT mice ( n = 7 per group). e , Serum cytokine profiling of anti-inflammatory, pro-inflammatory, and chemotactic cytokines in HFD-fed male GPSM1 f/f and GPSM1 ΔT mice ( n = 6 per group). f , g Representative flow cytometry and quantification of T reg proportion in the scWAT and eWAT of male FOXP3-YFP -Cre mice and T reg -specific GPSM1 -knockout ( GPSM1 ΔTreg ) mice under NCD ( f , n = 6 per group) and HFD ( g , n = 6 per group) feeding. h, i , RT-qPCR analysis of mRNA expression of anti-inflammatory and pro-inflammatory anti-inflammatory cytokines genes in scWAT ( h ) and eWAT ( i ) from HFD-fed male GPSM1 f/f or GPSM1 ΔTreg mice ( n = 7 per group). All data are presented as means ± SEM. P values are determined by unpaired two-tailed Student’s T-test in ( a, b, f and g ) or two-way ANOVA with Sidak’s multiple-comparisons test in ( c , d , h and i ). Source data are provided as a Source Data file.

Article Snippet: The human naïve CD4 + T Cell Isolation Kit II (Cat. No. 130-094-131, Miltenyi Biotec) was used to obtain human naïve CD4 + T cells.

Techniques: Flow Cytometry, Knock-Out, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: GPSM1 restricts CD73 + CD103 + T reg cells in adipose tissue, critical for promoting obesity-related metabolic deterioration

doi: 10.1038/s41467-026-73349-w

Figure Lengend Snippet: The single nucleus RNA-sequencing (snRNA-seq) of scWAT from male GPSM1 f/f and GPSM1 ΔTreg male mice was performed ( n = 3 per group). a Unsupervised clustering and dimensionality reduction of the CD4 + T cells data in a Uniform Manifold Approximation and Projection (UMAP) plot showing various cells classes. b The relative proportions of cells assigned to each cell class across different experimental conditions or sample groups. c The UMAP dimensionality reduction visualization of T reg cells. d Heat map of marker gene expression of T reg subpopulations. e Biological process (BP) analysis of differentially expressed genes for cluster 0. f quantification of the frequencies of CD73 + CD103 + T regs in scWAT from male FOXP3-YFP -Cre and GPSM1 ΔTreg mice under HFD condition ( n = 6 per group) ( g , h ). The UMAP dimensionality reduction visualization ( g ) and relative proportions ( h ) of four adipocyte subsets. i BP analysis for each subset of adipocytes. j–n Male GPSM1 f/f and GPSM1 ΔTreg mice were fed on NCD and peritoneally administered with CL-316243 for 7 days. j Oxygen consumption (VO2) was monitored ( n = 7 per group). k Regression-based analysis of absolute VO 2 against body weight ( n = 7 per group). l Serum adenosine levels ( n = 7 per group). m Mitochondrial Respiration rates were determined from scWAT of indicated genotypes. Oligomycin (Oligo)-stimulated, FCCP-induced, and Rotenone& antimycin A (R&A)- stimulated respiration is shown ( n = 6 per group). n Representative western blot of UCP1 expression in scWAT from male GPSM1 f/f and GPSM1 ΔTreg mice ( n = 3 per group). o , p Representative western blot of UCP1 expression ( o , n = 3 per group) and average oxygen consumption rate (OCR, p , n = 12 per group) of differentiated primary beige adipocytes treated with collecting conditioned media (CM) from sorted CD73 + CD103 + T regs versus CD73 + CD103 - T regs . q , r The UMAP dimensionality reduction visualization ( q ) and relative proportions ( r ) of four adipose tissue macrophages subsets. s , Quantification of the frequencies of TREM2 + F4/80 + macrophages in scWAT and eWAT from male GPSM1 f/f and GPSM1 ΔTreg mice under HFD condition ( n = 8 per group) t , BP analysis for each subset of macrophages. u RT-qPCR analysis of IL-10 mRNA in CD73 + CD103 + T regs and CD73 + CD103 - T regs ( n = 4 per group). v Bone marrow-derived macrophages were induced polarization into M1 macrophage and treated with CM from sorted CD73 + CD103 + T regs versus CD73 + CD103 - T regs for 24 h. RT-qPCR analysis of mRNA expression of anti-inflammatory and pro-inflammatory genes in macrophages ( n = 4 per group). All data are shown as means ± SEM. P values are determined by unpaired two-tailed Student’s T-test in ( f , l , m , p , s , and u ) or two-way ANOVA with Sidak’s multiple-comparisons test in ( j , v ), and two-sided analysis of covariance (ANCOVA) in ( k ), and hypergeometric test in ( e , i and t ). Source data are provided as a Source Data file.

Article Snippet: The human naïve CD4 + T Cell Isolation Kit II (Cat. No. 130-094-131, Miltenyi Biotec) was used to obtain human naïve CD4 + T cells.

Techniques: RNA Sequencing, Marker, Gene Expression, Western Blot, Expressing, Quantitative RT-PCR, Derivative Assay, Two Tailed Test

Journal: Biomolecules

Article Title: Efficacy of HDAC Inhibitors in Driving Peroxisomal β-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders

doi: 10.3390/biom13121696

Figure Lengend Snippet: Entinostat most effectively stimulates phagocytosis of myelin debris and upregulates expression of genes involved in lipid export. ( A ) Scheme showing the receptors involved in the uptake of myelin debris ( CD36 , MSR - 1 , and MERTK ), the proteins responsible for the export of lipids ( ABCA1 , ABCG1 , and APOE ) and the transcription factor, liver X receptor (LXR), responsible for their synthesis. ( B ) RT-qPCR testing of the phagocytic receptors CD36 , MSR-1 , and MERTK in human primary macrophages derived from healthy donors pre-treated with entinostat, vorinostat, tefinostat (2 µM), or the solvent control DMSO for 24 h ( n = 4–5). Data were normalized to HPRT1 . ( C ) RNA-seq transcriptional levels of CES1 in human healthy control macrophages ( n = 8) differentiated in the presence of murine myelin debris (GSE245235). ( D ) IncuCyte live imaging of myelin debris phagocytosis in HDACi or solvent-treated human primary macrophages ( n = 6). Fluorescence intensity was measured in the IncuCyte imaging platform with 30 min intervals for up to 20 h and ( E ) the phagocytic capacity was compared between entinostat, vorinostat, tefinostat, or DMSO solvent-treated macrophages 3 h, 6 h, and 9 h after myelin debris uptake ( n = 6). ( F ) RT-qPCR tests of the lipid transporters ABCG1, ABCA1 , and APOE in human primary macrophages derived from healthy donors pre-treated with entinostat, vorinostat, tefinostat (2 µM), or the DMSO solvent control for 24 h ( n = 4–5). To make interindividual variants more comparable for the statistical analysis, the RT-qPCR data was log transformed. A mixed model followed by Dunnett’s post-hoc test was used for statistical analysis (**** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05; ns = not significant). Bar graphs in ( B , E , F ) indicate means ± S.D. Symbols were used to visualize data derived from same donors. Illustration in ( A ) was created with BioRender.com, accessed on 5 September 2023.

Article Snippet: For analysis of CD36 protein levels, macrophages were treated with vorinostat, entinostat, tefinostat (2 μM), or the DMSO solvent control for 24 h. Harvested cells were washed and blocked with 4% Beriglobin P for 30 min at 4 °C and then stained with APC-conjugated mouse anti-human CD36 (Cat.No.130-110-741, Miltenyi Biotec) or APC-conjugated mouse anti-human isotype control (Cat.No.130-113-434, Miltenyi Biotec) antibodies.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Solvent, Control, RNA Sequencing, Imaging, Fluorescence, Transformation Assay

Journal: Frontiers in immunology

Article Title: Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity.

doi: 10.3389/fimmu.2022.819929

Figure Lengend Snippet: FIGURE 1 (A) Family tree illustrates the individuals with KLF2 c.951dup p.(Glu318Argfs*87) mutation. Black arrows point at the index patient IV:2 diagnosed with idiopathic juvenile arthritis and the index patient III:2 with lymphopenia of unknown origin. The variant positive family members suffer from lymphopenia, arthritis, lymphoma, and malignancies marked with small black boxes in four alternative corners, each indicating a different condition. Summary and timeframe (years) of clinical events in patients II:1 (B), II:2 (C), III:2 (D) and IV:2 (E) is shown. The major health events and medications are indicated by arrows (a to x). The corresponding health events are explained in detail in chapter 2.1. CLL, Chronic lymphocytic leukemia; MTX, methotrexate; SSZ, sulfasalazine; CSA, cyclosporine; PL, prednisolone; ATM, sodium aurothiomalate; CP, cyclophosphamide; CMB, chlorambucil; VS, vincristine sulfate; H, hydroxydaunorubicin; CVID, common variable immunodeficiency; HCQS, hydroxychloroquine; ETN, etanercept.

Article Snippet: After permeabilization the nuclei were washed and stained for APC conjugated antibody against human KLF2 (Miltenyi Biotec; 130-111-040) along with isotype control (Miltenyi Biotec; 130- 120-709) for 30 minutes at 4°C.

Techniques: Mutagenesis, Variant Assay, Medications

Journal: Frontiers in immunology

Article Title: Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity.

doi: 10.3389/fimmu.2022.819929

Figure Lengend Snippet: FIGURE 2 (A) KLF2 protein structure. The conserved C2H2-type zinc finger structure at the C-terminus consists of three loops that are crucial for DNA- binding. The KLF2 c.951dup p.(Glu318Argfs*87) mutation is located in the middle of the second zinc finger loop. The duplication of Glu318 causes a shift in the reading frame which leads to a stop codon 87 amino acids later. (B) Comparison of the sequence coding the human mutant to human, mouse, and zebrafish with KLF2 zinc fingers. The mutated part of the KLF2 sequence [c.951dup p.(Glu318Argfs*87)] is typed with red color. The sequence shown begins with the nuclear localization signal (NLS: amino acids (aa) 254 - 274), followed by the first zinc- finger (Zf1: aa277 - 297), the second zinc-finger (Zf2: aa304 - 327) and the third zinc finger (Zf3: aa334 - 254). Within the zinc finger sequences, there are three conserved DNA-binding domains (marked with blue boxes). The black arrows point at important amino acids that bind directly to DNA. The KLF2-PE/APC antibody binding sequences (A) are intact both in the wildtype and the variant alleles. KLF2 was detected by western blot in patient (III:2) skin fibroblasts (C) and neutrophils (D) as 37 kDa and 60 kDa molecular weight forms correspondingly. 30µg of protein was separated on a 12% gel and transferred into PVDF membrane. The KLF2 and GAPDH antibody binding was detected by anti-mouse or anti- rabbit secondary antibodies (IRDye 680RD or 800CW). The membranes were imaged by Odyssey infrared scanner and band intensities quantified from three different sample sets. (E) KLF2 expression in CD4+ and CD8+ lymphocytes. The KLF2 expression of isolated PBMCs from mutation carriers (III:1 and III:2) and sex-matched controls was determined by flow cytometry. (F) Localization of KLF2 protein was studied in control PBMCs with flow cytometry using three different permeabilization strategies: first without any permeabilization/fixation (used when staining cell membrane targets), second with Cytoperm buffer (used when staining cytoplasmic targets) and finally, a Fixation/permeabilization buffer (used when staining nuclear transcription factors). Quantitation of MFI values is shown in bar chart below. (G) Nuclei from patient and control PBMCs were isolated, fixed, permeabilized and stained with KLF-APC and isotype control antibodies. Quantitation of MFI values is shown in bar chart below.

Article Snippet: After permeabilization the nuclei were washed and stained for APC conjugated antibody against human KLF2 (Miltenyi Biotec; 130-111-040) along with isotype control (Miltenyi Biotec; 130- 120-709) for 30 minutes at 4°C.

Techniques: Binding Assay, Mutagenesis, Comparison, Sequencing, Variant Assay, Western Blot, Molecular Weight, Membrane, Expressing, Isolation, Cytometry, Control, Staining, Quantitation Assay

Journal: Frontiers in immunology

Article Title: Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity.

doi: 10.3389/fimmu.2022.819929

Figure Lengend Snippet: FIGURE 3 KLF2 interactome analysis. (A) Schematic workflow overview of the MAC-tagged based BioID purification coupled with mass spectrometry. (B) Dot-plot visualization (BFDR ≤0.05) of the KLF2-WT and KLF2-MUT interactors (prohits-viz.org). Each node corresponds to the abundance of the average spectral count for each prey. (C, D) The polar plot shows the molecular level localization of KLF2-WT and KLF2-MUT obtained by MS-microscopy respectively (proteomics.fi). ACTF, Actin filament; JUNC, Cell junction; CEN, Centrosome; CHR, Chromatin; ER, Endoplasmic reticulum; END, Endosome; EXO, Exosome; FOCA, Focal adhesion; GOL, Golgi; INTF, Intermediate filament; LYS, Lysosome; MIC, Microtubule; MT, Mitochondrion; NE, Nuclear envelope; NUC, Nucleolus; NUCPL, Nucleoplasm; PER, Peroxisome; PM, Plasmamembrane; PRO, Proteasome. (E, F) Significantly enriched (q < 0.05, calculated with Fisher exact test and Benjamini–Hochberg multiple-testing correction) Reactome pathway annotations from the KLF2-WT and KLF2-MUT interactors, respectively. Y-axis corresponds to the percentage of the interactors found in the given pathway.

Article Snippet: After permeabilization the nuclei were washed and stained for APC conjugated antibody against human KLF2 (Miltenyi Biotec; 130-111-040) along with isotype control (Miltenyi Biotec; 130- 120-709) for 30 minutes at 4°C.

Techniques: Mass Spectrometry, Microscopy

Journal: Frontiers in immunology

Article Title: Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity.

doi: 10.3389/fimmu.2022.819929

Figure Lengend Snippet: FIGURE 5 (A) CD3+CD4+ T cell subgroups in KLF2 c.951dup p.(Glu318Argfs*87) variant positive family members compared to healthy controls. Mutation carriers´ and controls´ unstimulated PBMCs were stained for regulatory T helper cell markers CD4, CD25, CD127 and FOXP3 and analyzed by flow cytometry. Th1 and Th 17 subpopulations were stained ex vivo (whole blood) with CD4, CXCR3, CCR6, CD45RA and CD10 antibodies and analyzed by flow cytometry. (B) Representative results of flow cytometry analysis of CD25+CD127lowFOXP3+ regulatory T cells (Treg). The gating strategy is presented along with the data. Regulatory T cells of patient (III:2) and sex-matched controls´ were studied for the expression of CD45RA, CCR6, CCR7 and CXCR3 with flow cytometry. (C) The Treg populations I-V were studied for the expression of CCR6, CCR7 and CXCR3. Representative results of patient and control Treg populations are shown. The regulatory T cell populations I, II, III, IV and V are presented as the number of cells and percentages. The percentage of CXCR3, CCR6 and CCR7 positive cells.

Article Snippet: After permeabilization the nuclei were washed and stained for APC conjugated antibody against human KLF2 (Miltenyi Biotec; 130-111-040) along with isotype control (Miltenyi Biotec; 130- 120-709) for 30 minutes at 4°C.

Techniques: Variant Assay, Mutagenesis, Staining, Cytometry, Ex Vivo, Expressing, Control

Journal: Cell Reports Methods

Article Title: Dynamic stimulation promotes functional tissue-like organization of a 3D human lymphoid microenvironment model in vitro

doi: 10.1016/j.crmeth.2025.101105

Figure Lengend Snippet: Static vs. dynamic: The lymph node (A) Expression comparison of specific functional markers between static and dynamic scaffolds. White arrow indicates the direction through which the cells organize following the direction of the flow. Yellow circles indicate the material pores around which endothelial cells position and organize. z Stacks were acquired using a 30× (a, c) and a 60× (b, d) objective lens. (B) RT-qPCR expression analysis of PDPN and CD31 in the LN model. The analysis was made on static 3D culture, dynamic 3D culture, and 2D culture. The data have been normalized on the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene. All data are represented by mean ± SEM. Tukey’s multiple comparison test was performed; p < 0.05; n = 6 for 3D static and dynamic conditions, n = 2 for 2D condition. (C) Flow cytometry analysis of the ratio between HLF and HUVEC cells after 15 days of maturation, showing the contribution in terms of percentage of the two cell types in the LN model. HLF cells are CD73 + /CD31 − , and HUVEC cells are CD73 + /CD31 + . Data are represented by mean ± SEM. (D) Comparison of ECM protein deposition between static and dynamic scaffolds of the LN model. z Stacks were acquired using a 20× (a, c, d, f) and a 60× (b, e) objective lens. Scale bars, 50 μm.

Article Snippet: CD73 Antibody, anti-human, REAfinityTM , Miltenyi , CAT#130-111-909 RRID: AB_2659167.

Techniques: Expressing, Comparison, Functional Assay, Quantitative RT-PCR, Flow Cytometry

Journal: PLoS ONE

Article Title: Comparative mRNA and miRNA transcriptome analysis of a mouse model of IGFIR-driven lung cancer

doi: 10.1371/journal.pone.0206948

Figure Lengend Snippet: (A) Schematic of doxycycline inducible expression of IGFIR in the mouse lung via the SPC promoter. (B) Unsupervised hierarchical clustering dendrogram of RNA-Seq data from tumor (T, red) and non-transgenic normal lung (N, grey) samples. (C) Volcano plot of log2 fold changes and differential expression p values between tumor and normal lung tissue. (D) Pie chart illustrating percentage of genes up and down-regulated in IGFIR-driven tumors. (E) Dot plots of endogenous murine Igf1r (padj = 5.71E-11) and human IGFIR transgene (padj = 5.65E-249) mRNA expression following mapping to a hybrid genome. (F) Heatmap showing differential expression of markers of AT2 and Club cells as well as subtypes of non-small cell lung cancer. ADC, adenocarcinoma, SCC, squamous cell carcinoma, mADC, mucinous adenocarcinoma. Adjusted p-values for (C) and (E) were obtained from DESeq2.

Article Snippet: To further enrich for tumor cells, a biotinylated anti-human IGF1R antibody (1:10, clone REA271, Miltenyi Biotech) was added to the epithelial enriched fraction for 30 min on ice followed by positive selection and separation as above.

Techniques: Expressing, RNA Sequencing, Transgenic Assay, Quantitative Proteomics

Journal: Cell reports

Article Title: Overcoming CD226-related immune evasion in acute myeloid leukemia with CD38 CAR-engineered NK cells

doi: 10.1016/j.celrep.2024.115122

Figure Lengend Snippet:

Article Snippet: CD25 Antibody, anti-human REAfinity – Clone REA570 , Miltenyi Biotec , Cat# 130-122-302; RRID: AB_2801883.

Techniques: Purification, Functional Assay, Virus, Recombinant, Binding Assay, Staining, Western Blot, Modification, Transfection, Fluorescence, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Cell Isolation, Plasmid Preparation, Generated, Sequencing, Software