Journal: Frontiers in Immunology

Article Title: Tumor cells express and maintain HMGB1 in the reduced isoform to enhance CXCR4-mediated migration

doi: 10.3389/fimmu.2024.1358800

Figure Lengend Snippet: CXCL12 and HMGB1 are expressed in MCF-7, MDA-MB-231, and PC-3 cancer cell lines. (A, B) qRT-PCR analysis (normalized on 18S ) is shown for CXCL12 and HMGB1 mRNA in cancer cells. Data are shown as mean ± SEM of 5 independent experiments. Statistical analysis of the differences among the cell lines was performed using one way ANOVA, followed by Tukey’s multiple comparison test: **p < 0.01. (C) CXCL12 and HMGB1 expression assessed by confocal microscopy. In the merged images, CXCL12 is shown in red and HMGB1 in green. Nuclei and cell membranes were counterstained with DAPI (blue) and MemBrite Fix Dye (cyan), respectively. Representative images, of at least 3 independent experiments, are shown. Scale bar represents 20 µm. (D, E) CXCL12 (D) and HMGB1 (E) expression was evaluated in at least 40 cells and expressed as fluorescence intensity. Horizontal lines represent means. Statistical analysis of the differences among the cell lines was performed using one way ANOVA, followed by Tukey’s multiple comparison test: *p < 0.05, ****p < 0.0001. (F) Confocal microscope z-stack overlays showing the intracellular localization of CXCL12 in MCF-7 cells (scale bar 20 μm). CXCL12 is shown in red, while nuclei were counterstained with DAPI (blue) and cell membranes with the MemBrite Fix Dye (cyan).

Article Snippet: Nuclei were counterstained with 2 mM DAPI (62248, Thermo Fisher Scientific), and the cell membrane was stained using the MemBrite Fix cell surface staining kit from Biotium, following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Comparison, Expressing, Confocal Microscopy, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Tumor cells express and maintain HMGB1 in the reduced isoform to enhance CXCR4-mediated migration

doi: 10.3389/fimmu.2024.1358800

Figure Lengend Snippet: CXCL12 uptake by cancer cells. (A) qRT-PCR analysis (normalized on 18S ) is shown for CXCR4 mRNA expression in cancer cells. Data are shown as mean ± SEM of 5 independent experiments. (B) CXCR4 expression analyzed by flow cytometry in MCF-7, MDA-MB-231, and PC-3 cells. Data are shown as relative MFI (rMFI, mean ± SEM) of 3 independent experiments. (C) Uptake of CXCL12-Atto647 by cancer cells analyzed by flow cytometry. Left: A representative histogram showing fluorescence intensity of unstimulated (blue) and CXCL12-Atto647 stimulated cells (red). Right: CXCL12-Atto647 uptake shown as rMFI (mean ± SEM) of 5 independent experiments. (D) Uptake of CXCL12-Atto674 by cancer cells treated with the CXCR4 antagonist AMD3100. Chemokine uptake shown as rMFI (mean ± SEM) of 6 independent experiments. Statistical analysis of the differences among the cell lines was performed using two-way ANOVA, followed by Šídák’s multiple comparisons test: ****p < 0.0001. (E) CXCL12-Atto647 uptake assessed by confocal microscopy in MCF-7, MDA-MB-231, and PC-3 cells. Left: In the merged images, CXCL12 is shown in red. Nuclei and cell membranes were counterstained with DAPI (blue) and MemBrite Fix Dye (cyan), respectively (scale bars: 20 μm). Right: CXCL12-Atto647 uptake was evaluated in at least 10 cells and expressed as fluorescence intensity. Horizontal lines represent the mean values. Statistical analysis of the differences among the cell lines was performed using one way ANOVA, followed by Tukey’s multiple comparison test: *p < 0.05, ****p < 0.0001.

Article Snippet: Nuclei were counterstained with 2 mM DAPI (62248, Thermo Fisher Scientific), and the cell membrane was stained using the MemBrite Fix cell surface staining kit from Biotium, following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Confocal Microscopy, Comparison

Journal: The EMBO Journal

Article Title: Photoreceptor nanotubes mediate the in vivo exchange of intracellular material

doi: 10.15252/embj.2020107264

Figure Lengend Snippet: A, B Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin + (SirActin) and GFP + (A) and tubulin + (ViaFluor) and MitoTracker Green + (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy. C Quantification of photoreceptor protrusions in Nrl::GFP retinal cultures (culture wells, n = 8, images, n = 24, protrusions, n = 868). Data are presented as mean ± SEM. D Images from FRAP experiments carried out on protrusion connected and isolated Nrl::GFP cultures pre‐ and post‐photobleaching. Area of photobleaching (white dashed circles). Analysed regions (yellow, orange and red dashed circles). Photoreceptor FRAP results shown from multiple experiments (isolated cells, n = 14; connected to recipient, n = 21; connected donor, n = 21). All data presented as percentage, mean ± SEM and fitted to the curve in a non‐linear regression with two‐phase association. E Time lapse imaging showing movement of MitoTracker Green + puncta in photoreceptors connected by a protrusion. White arrows point to MTG + puncta inside of the protrusion during the time lapse imaging. Data information: n.s. not statistically significant, **** P ≤ 0.0001 and *** P < 0.001; unpaired t ‐test and extra‐sum‐of‐squares F ‐test (Y0 constrained to 0.0). Scale bars: 5 μm. Source data are available online for this figure.

Article Snippet: To live‐image actin or microtubules, cultures were supplemented overnight with SiR‐Actin (100 μM) (CY‐SC001, Cytoskeleton Inc. Denver, CO, USA) or ViaFluor ® (50 μM) (70062, Biotium, Fremont, CA, USA).

Techniques: Microscopy, Isolation, Imaging

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet: C57BL/6J mice were dosed with three 30 μg intraperitoneal injections of mouse serum albumin (MSA)-conjugated IL-2 or muteins diluted in PBS on day 0, 3, and 6. On day 7, spleen and lymph nodes were harvested for immune cell profiling. ( A-D ) IL-2 treatment decreases the frequency of T and B cells while increasing the frequency of NK cells and granulocytes in spleen. T cells were gated as CD3+, B cells were defined as CD3-CD19+, NK cells were analyzed as CD3-NK1.1+, and granulocytes were defined as CD3-Ly6G+SSC hi . All populations were expressed as a percentage of live CD45+ cells. For absolute numbers and gating, see . ( E ) IL-2-REH increases the frequency of regulatory T cells in spleen. Tregs were defined as IL-2Rα+Foxp3+ and expressed as a percentage of liveCD3+CD4+. Similar results were observed in lymph nodes. For absolute numbers, see . For gating, see . ( G-J ) IL-2 increases the frequency of effector memory CD8+ T cells with a decrease in naïve CD8+ T cells while IL-2-REH increases the frequency of central memory CD8+ T cells. ( G ) Gating scheme to identify naïve, central memory (CM), and effector memory (EM) CD8+ T cells. ( H-J ) Quantification of memory cell frequency in spleen expressed as a percent of liveCD3+CD8+ T cells. For absolute numbers, see . For gating, see . ( K-L ) IL-2, IL-2-REH, and IL-2-REK enhance IFNγ producing CD8+ T cells in proportion to their agonist activity. For cytokine recall, lymph node cells were restimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of Monensin (GolgiStop) for 4 hr. For gating, see . Bar graphs show mean ± standard deviation of n = 3 mice per group. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons and are representative of two or more independent experiments.

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Activity Assay, Standard Deviation

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet: ( A ) Quantification of absolute Treg numbers in spleen. ( B ) Gating scheme to identify regulatory T cells. ( C–E ) IL-2 administration increases the frequency and number of IL-2Rα+ conventional T cells in spleen. ( C ) Representative flow cytometry plots showing IL-2Rα expression in CD4+FoxP3- T cells. ( D ) Quantification of data in C. ( E ) Absolute numbers of CD4+FoxP3-IL-2Rα+ T cells in spleen. Data are expressed as mean ± standard deviation and were analyzed by one-way ANOVA with Tukey’s multiple comparison relative to PBS, n = 3 mice per group.

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Flow Cytometry, Expressing, Standard Deviation

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet: ( A–C ) Increased frequency of Tregs in IL-2-REH-treated mice is not the result of differentiation from conventional CD4+ T cells. ( A ) Schematic of experimental design. Conventional CD4+ T cells from B6-Foxp3 EGFP mice (CD45.2) were transferred to congenic recipients (CD45.1). Mice were treated with three 30 μg injections of IL-2-REH or PBS control on day 0, 3, and 6 prior to analysis on day 7. Flow cytometry plots ( B ) and quantification ( C ) of Treg frequencies. Tregs were defined as IL-2Rα+Foxp3+ and expressed as a percent of CD3+CD4+ T cells. For gating, see . ( D–F ) IL-2-REH induces proliferation of Tregs with reduced activity on CD8+ T cells relative to IL-2. ( D ) Schematic of proliferation tracking experiment. C57BL/6J mice were treated with a single 30 μg injection of cytokine on day 0 followed by EdU injections on day 0, 1, and 2 prior to analysis on day 3. Flow cytometry plots ( E ) and quantification ( F ) of EdU incorporation in Tregs (CD3+CD4+Foxp3+) and CD8+ T cells (CD3+CD8+). Dashed line indicates EdU incorporation in PBS-treated mice. For gating, see . Bar graphs show mean ± standard deviation of n = 3 mice per group. Groups were compared by unpaired t-test and data are representative of two independent experiments.

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Flow Cytometry, Activity Assay, Injection, Standard Deviation

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet: Analysis of IL-2 receptor expression on NK cells and T cells. Histograms of IL-2Rα (CD25), IL-2Rβ (CD122), and γ c (CD132) expression in red compared to fluorescence minus one (FMO) control shown in gray. For receptor staining, a single cell suspension of spleen and lymph nodes from B6-Foxp3 EGFP mice was stained with a cell surface panel to identify NK cells (CD3-NK1.1+), CD4+ T cells (CD3+CD4+Foxp3-), CD8+ T cells (CD3+CD8+), and Tregs (CD3+CD4+Foxp3+). For blasts, a single cell suspension of spleen and lymph nodes from B6-Foxp3 EGFP mice was stimulated with 2.5 μg/mL plate bound αCD3, 5 μg/mL αCD28, and 100 IU/mL IL-2 for 2 days and rested overnight in complete media without IL-2. For NK cell signaling, magnetic-activated cell sorting (MACS) isolated NK cells from C57BL/6J mice were combined with CellTrace Violet (CTV) loaded carrier cells, stimulated for 20 min with cytokine prior to fixation, permeabilization, and staining for pSTAT5. NK cells were gated as CTV–. For T cell signaling, spleen and lymph nodes from B6-Foxp3 EGFP mice were pre-stained with a T cell phenotyping panel. Cells were stimulated for 20 min before fixation, permeabilization, and staining for pSTAT5. T cells were gated as CD4+ (CD3+CD4+Foxp3–), CD8+ (CD3+CD8+), or Treg (CD3+CD4+Foxp3+) based on cell surface markers and GFP expression. Data are representative of two or more independent experiments.

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Expressing, Fluorescence, Staining, FACS, Isolation

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet: ( A–C ) IL-2 partial agonist activity depends on IL-2Rα and γ c expression. Correlation plots showing STAT5 phosphorylation and receptor expression in NK cells, CD4+ T cells, CD8+ T cells, and Tregs. Dashed lines represent receptor expression normalized to the highest and lowest expressing cell types. pSTAT5 MFI at saturating cytokine concentration was normalized to stimulation with IL-2. See also . ( D–E ) IL-2Rβ affinity regulates cell type selectivity of IL-2-REH. C57BL/6J mice were dosed with three 30 μg intraperitoneal injections of IL-2-REH or H9-REH diluted in PBS on day 0, 3, and 6 followed by analysis on day 7. Flow cytometry plots ( D ) and quantification ( E ) of Treg frequency expressed as a percent of CD3+CD4+ T cells. Bar graphs show mean ± standard deviation of n = 3 mice per group. Dashed line represents Treg frequency in PBS-treated mice. Groups were compared using an unpaired t-test. ( F–G ) IL-2Rα expression controls proliferation in response to IL-2-REH. Wild-type and IL-2Rα -/- CTLL-2 cells were mixed at 1:1 ratio and cultured in the presence of 200 nM IL-2 or IL-2-REH for three days prior to quantification of IL-2Rα expression by flow cytometry. Bar graph shows mean ± standard deviation of duplicate wells. Groups were compared using an unpaired t-test. ( H–I ) IL-2-REH signaling is more sensitive to negative regulation by suppressor of cytokine signaling 1 (SOCS1). SOCS1-p2a-eGFP expressing CTLL-2s were mixed with wild-type CTLL-2 cells, rested overnight, and stimulated with 1 μM IL-2 or IL-2-REH for 30 min prior to fixation, permeabilization, and detection of pSTAT5 by phosphoflow cytometry. Wild-type and SOCS1 overexpressing cells were gated on the basis of GFP expression. ( H ) Histograms showing pSTAT5 staining in wild-type (blue) and SOCS1 expressing (green) CTLL-2 cells. ( I ) pSTAT5 staining in SOCS1 expressing cells expressed as a ratio to pSTAT5 staining in wild-type CTLL-2s. Bar graph shows mean ± standard deviation of duplicate wells. Groups were compared using an unpaired t-test. MFI, mean fluorescence intensity. Results are representative of at least two independent experiments.

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Activity Assay, Expressing, Concentration Assay, Flow Cytometry, Standard Deviation, Cell Culture, Cytometry, Staining, Fluorescence

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet: (A-B) Suppression of conventional CD4+ T cell proliferation by Tregs from cytokine-treated mice. Tregs from PBS, IL-2, or IL-2-REH-treated B6-Foxp3 EGFP mice were isolated by fluorescence-activated cell sorting (FACS) and co-cultured with CellTrace Violet (CTV) labeled CD4+ conventional T cells from PBS-treated mice in the presence of αCD3αCD28 coated beads for 3 days. Proliferation of conventional cells was assessed by CTV dilution. ( A ) Contour plots of Treg sorting scheme. Histograms of CTV dilution in Tcons cultured with varying ratios of Tregs as indicated. ( B ) Quantification of Tcon proliferation. The dashed line represents the frequency of divided cells in the absence of Tregs. Data are shown as mean ± standard deviation of triplicate wells. Data are representative of two independent experiments. ( C ) Kinetics of Treg expansion in response to cytokine treatment. B6-Foxp3 EGFP mice were administered 30 μg of MSA-IL-2 or MSA-IL-2-REH on day 0, 3, and 6. The frequency of Foxp3 EGFP+ cells was tracked every three days in blood. Dashed line represents Foxp3 EGFP+ frequency in untreated mice. Data were analyzed by unpaired t-test with multiple corrections using the Holm-Šidák method. Data are expressed as mean ± standard deviation of n = 5 mice per group and are representative of a single experiment.

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Isolation, Fluorescence, FACS, Cell Culture, Labeling, Standard Deviation

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet: ( A ) Schematic of T cell transfer colitis. On day 0, 3 × 10 5 naïve CD4+CD45 RB hi T cells were transferred into C.B-17 scid mice with or without 1 × 10 4 Tregs. Starting on day 24 at the onset of weight loss, mice were administered 5 μg IL-2-REH twice weekly for 3 weeks. Treg frequency and activation status was monitored in blood on day 42 and a final week of cytokine treatment was begun on day 57. ( B ) Body weight measurements from T cell transfer recipients. Body weight was monitored weekly and expressed as mean ± standard error for n = 8 (CD45RB hi and CD45RB hi +Treg), n = 7 (CD45RB hi +Treg+IL-2-REH), and n = 5 (negative control) mice, respectively. ( C ) IL-2-REH expands Tregs in the context of T cell colitis. ( C ) Frequency of Foxp3+Tregs in blood on day 42. Data are shown as mean ± standard deviation and were compared by one-way ANVOA with Tukey’s post hoc test. ( D–F ) IL-2-REH enhances activation of Tregs during T cell colitis. Expression of IL-2Rα ( D ), GzmB ( E ), and ICOS ( F ) in Tregs was monitored by flow cytometry in the blood on day 42. Data are shown as mean ± standard deviation and were compared by unpaired t-test.

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Activation Assay, Negative Control, Standard Deviation, Expressing, Flow Cytometry

Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet:

Article Snippet: CD4+ T cells were isolated from the spleens of BALB/c mice (Taconic) by MACS followed by FACS sorting for CD45RB hi CD25- (naïve) and CD45RB lo CD25+ (Treg).

Techniques: Expressing, Recombinant, Modification, Sequencing, Cell Isolation, Selection, SYBR Green Assay, Flow Cytometry, Staining, Activation Assay, Software

Journal: PLoS ONE

Article Title: Jun Is Required in Isl1 -Expressing Progenitor Cells for Cardiovascular Development

doi: 10.1371/journal.pone.0057032

Figure Lengend Snippet: Sagittal sections of E10.5 mutant ( B, D, G ) and control ( A, C, F ) embryos analyzed by pHH3 and NFATc1 immunostaining and by TUNEL assay. pHH3 immunostaining (pink) reveals similar proliferation rates in mutant and control OFT cells ( A, B ). Dotted lines show representative areas used for cell counting with ImageJ software. TUNEL assay reveals similar numbers of TUNEL-positive (green) OFT cells in mutant and control embryos ( C, D ). ( E ) Quantitative analysis of the percentage of pHH3- and TUNEL-positive OFT cells in serial sections showing a statistically insignificant trend toward less proliferation and more apoptosis in conditionally deleted embryos at E10.5. Results are expressed as mean ± SEM of the percent positive nuclei. The statistical significance of differences between groups was analyzed by the Student’s t-test. ( F, G ) Expression of the endocardial marker NFATc1 (pink), is not significantly different between conditional mutant and control embryos. Saggital sections of anti-Tfap2a immunostained Jun flox/− ( H ) and Isl1 Cre/+ ; Jun flox/− ( I ) embryos showing no significant difference in Tfap2a-expressing neural crest cells (arrowheads) at E10.5. Sections were co-stained with DAPI to illustrate nuclei. A, atrium; AVC, atrioventricular cushion; LV, left ventricle; OFT, outflow tract; OFTC, outflow tract cushion; SMA, smooth muscle actin.

Article Snippet: Immunofluorescence (IF) and horseradish peroxidase (HRP) immunostaining were performed as described previously using rabbit monoclonal anti-Jun 60A8 (1∶100 IF; Cell Signaling Technology, Danvers, MA), rat monoclonal anti-CD31 MEC13.3 (Pecam1; 1∶100; BD Pharmingen, Franklin Lakes, NJ), mouse monoclonal anti-Isl-1 39.4D5 (1∶25 IF; Developmental Studies Hybridoma Bank, Iowa City, IA), rabbit polyclonal anti-GFP (1∶200 IF, Life Technologies), mouse monoclonal anti-SMA 1A4 (1∶200 IF, Life Technologies), rabbit polyclonal phospho-histone H3 Ser10 (1∶200 IF, Cell Signaling Technology), mouse monoclonal AP-2 alpha (Tfap2a) 3B5 (1∶4 IF, Developmental Studies Hybridoma Bank) and mouse monoclonal NFATc1 7A6 (1∶25 IF, Developmental Studies Hybridoma Bank).

Techniques: Mutagenesis, Control, Immunostaining, TUNEL Assay, Cell Counting, Software, Expressing, Marker, Staining