Journal: mBio

Article Title: Engineering of CD8 + T cells with an HIV-specific synthetic notch receptor to secrete broadly therapeutic antibodies for combining antiviral humoral and cellular immune responses

doi: 10.1128/mbio.03839-24

Figure Lengend Snippet: Engineered cells with a CD4-17b SNR can sense the Env-presenting cells and virions. ( A ) Schematic design of a CD4-17b-BFP cell. The CD4-17b molecule (Myc-tagged) targeting HIV-1 Env was used as the extracellular structural domain to bind to the Notch core and the cytoplasmic transcription factor Gal4-VP64 to generate the required synNotch receptor. Upon stimulation by the Env antigen, the detached transcription factor enters the nucleus and binds to the upstream activating sequence (UAS) of the regulated element, triggering the expression of BFP. The response element vector also carries a constitutive DsRed reporter for cell population analysis and purification. ( B ) The dual-positive rate of CD4-17b-BFP cells. Positive CD4-17b-BFP cells were measured by detecting both DsRed fluorescence and CD4-17b SNR stained with an anti-Myc antibody through flow cytometry, where the anti-Myc antibody was labeled by a secondary antibody conjugated to Alexa Fluor 488. ( C ) Expression of BFP in CD4-17b-BFP cells in response to 293T-gp160 cells. Left, schematic representation of CD4-17b-BFP cells reacting with the target cells. The immunofluorescence image (right) was taken after 48 h of co-culture of CD4-17b-BFP cells (red) with 293T-gp160 cells (green). An anti-Env 3B3 monoclonal antibody and the secondary antibody conjugated to Alexa Fluor 488 were used to detect 293T-gp160 cells. BFP and DsRed dual-positive cells are indicated by white arrows. The scale bar is 20 µm. ( D ) Flow cytometry analysis of the percentage of BFP-positive cells in the DsRed-positive CD4-17b-BFP cells described in panel C. ( E, F ) Flow cytometry analysis of the expression of BFP in CD4-17b-BFP cells (gated by DsRed + expression) after stimulation with gradient virus particles without ( E ) or with ( F ) the addition of 293T-gp160 cells for 24 h, reported as mean fluorescence intensity (MFI). HIV-1 NL4-3 particles were added at 0.05 µg (only in panel E), 0.25 µg, or 1.25 µg of p24. Mock, no virus or target cells added. In panels E and F, three independent experiments were performed and the error bars depict the standard deviation (SD). **, P < 0.01; ***, P < 0.001 (unpaired Student’s t -test).

Article Snippet: For detection of the expression of cell surface synNotch receptor, transduced cells were incubated with an anti-Myc polyclonal antibody (Proteintech, Rosemont, IL) for 1 h at 4°C, washed three times with PBS, then incubated with a secondary Alexa Fluor Plus 488-goat anti-rabbit IgG (H+L) cross-adsorbed antibody (Invitrogen, Carlsbad, CA) at a 1:500 dilution for 45 min at 4°C, and washed three times with PBS before analysis.

Techniques: Sequencing, Expressing, Plasmid Preparation, Purification, Fluorescence, Staining, Flow Cytometry, Labeling, Immunofluorescence, Co-Culture Assay, Virus, Standard Deviation

Journal: mBio

Article Title: Engineering of CD8 + T cells with an HIV-specific synthetic notch receptor to secrete broadly therapeutic antibodies for combining antiviral humoral and cellular immune responses

doi: 10.1128/mbio.03839-24

Figure Lengend Snippet: Functional anti-HIV-1 bNAb and BiTE can be produced after activation of the Jurkat T cells equipped with the synNotch circuits. ( A ) Schematic design of the response elements of the CD4-17b-Ab synNotch circuits. ( B ) The dual-positive rate of CD4-17b-Ab Jurkat cells, as determined by flow cytometry at 7 days after FACS purification, was analyzed through the detection of both Myc-tagged CD4-17b SNR and DsRed. Jurkat cells were engineered with CD4-17b SNR and the response element encoding for VN, VRC01, or N6-αCD3. UTD, untransduced. ( C ) The Ab expression of the CD4-17b-Ab cells in panel B after being stimulated with 293T-gp160 for 24 h was determined by RT-qPCR probed to VRC01, N6-αCD3, or both genes. Mock, stimulated with 293T cells. ( D ) Time course kinetics of VRC01 IgG secretion from CD4-17b-Ab or UTD Jurkat cells stimulated with 293T-gp160 (Env+, solid lines) or 293T (Env−, dashed lines) cells. ( E ) After 24 h of co-incubation of CD4-17b-Ab cells or UTD cells with pseudoviruses with the Env proteins from multiple HIV-1 strains, changes in the supernatant virus infectivity were detected by TZM-bl cells. ( F ) The p24 viral replication curves were measured with the co-culture supernatants of H9-HXB2 cells and CD4-17b-Ab cells or UTD cells. H9-HXB2 cells are H9 cells infected with HIV-1 HXB2 for 3 days. ( G ) The cell number of each group in panel F was monitored within the co-culture course. ( H, I ) Flow cytometry analysis of CD62L ( H ) and CD25 ( I ) T cell activation markers on CD4-17b-Ab Jurkat cells (gated by DsRed + expression) co-incubated with 293T-gp160 cells for 24 h, reported as mean fluorescence intensity (MFI). In panels C to I, three (C to G) or four ( H and I ) independent experiments were performed and the error bars depict SD. In panels C and E to I, statistical analysis was performed by unpaired Student’s t -test ( C and E ), two-way repeated measures analysis of variance ( F and G ), or unpaired Mann-Whitney U-test ( H and I ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: For detection of the expression of cell surface synNotch receptor, transduced cells were incubated with an anti-Myc polyclonal antibody (Proteintech, Rosemont, IL) for 1 h at 4°C, washed three times with PBS, then incubated with a secondary Alexa Fluor Plus 488-goat anti-rabbit IgG (H+L) cross-adsorbed antibody (Invitrogen, Carlsbad, CA) at a 1:500 dilution for 45 min at 4°C, and washed three times with PBS before analysis.

Techniques: Functional Assay, Produced, Activation Assay, Flow Cytometry, Purification, Expressing, Quantitative RT-PCR, Incubation, Virus, Infection, Co-Culture Assay, Fluorescence, MANN-WHITNEY

Journal: mBio

Article Title: Engineering of CD8 + T cells with an HIV-specific synthetic notch receptor to secrete broadly therapeutic antibodies for combining antiviral humoral and cellular immune responses

doi: 10.1128/mbio.03839-24

Figure Lengend Snippet: CD8 + T cells equipped with the bifunctional synNotch circuit can control viral replication and kill HIV-1 latency-reactivated cells. ( A ) Schematic diagram of the generation of CD8 + T cells equipped with the synNotch circuits. ( B ) The dual-positive rate of CD4-17b-Ab CD8 + T cells was determined by flow cytometry 7 days after FACS purification. ( C ) The p24 viral replication curves were measured with the co-culture supernatants of H9-HXB2 cells and CD4-17b-Ab CD8 + T cells or UTD cells. ( D ) The cell number of each group in panel C was monitored within the co-culture course. ( E ) Establishment of an HIV latent infection model using human primary CD4 + T cells. CD4 + T cells were first activated and expanded with anti-CD3 and anti-CD28 antibodies for 2 days. The cells were then infected with HIV-1 HXB2 and maintained with gradually decreasing concentrations of interleukin-2 (IL-2) for 5 days from day 2 post-infection to establish latency. (F to H) The survival of HIV-1 HXB2 latently-infected CD4 + T cells, without ( F ) or with 24 h of PMA ( G ) or JQ1 ( H ) pre-treatment, was analyzed by flow cytometry before and after 24 h of incubation with engineered or UTD CD8 + T cells. Anti-CD19 SNR-transduced cells severed as a non-targeting (NT) control. CD107a expression on the engineered or UTD CD8 + T cells was analyzed by flow cytometry following stimulation with the target cells (left). The relative percentage of survival cells (right) was calculated as described in the legend of . In panels C, D, and F to H, three ( C and D ) or four (F to H) independent experiments were performed and the error bars depict SD. Statistical analysis was performed by repeated measures analysis of variance ( C and D ) or Mann-Whitney U-test (F to H). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: For detection of the expression of cell surface synNotch receptor, transduced cells were incubated with an anti-Myc polyclonal antibody (Proteintech, Rosemont, IL) for 1 h at 4°C, washed three times with PBS, then incubated with a secondary Alexa Fluor Plus 488-goat anti-rabbit IgG (H+L) cross-adsorbed antibody (Invitrogen, Carlsbad, CA) at a 1:500 dilution for 45 min at 4°C, and washed three times with PBS before analysis.

Techniques: Control, Flow Cytometry, Purification, Co-Culture Assay, Infection, Incubation, Expressing, MANN-WHITNEY

Journal: Science Advances

Article Title: Cardiomyocyte-derived YOD1 promotes pathological cardiac hypertrophy by deubiquitinating and stabilizing STAT3

doi: 10.1126/sciadv.adu8422

Figure Lengend Snippet: ( A to F ) Representative Western blot analysis of YOD1 in primary cardiomyocytes (A), primary fibroblasts (C), and primary macrophages (E) subjected to Ang II stimulation at various time points and densitometric quantification (B, D, and F). n = 3. h, hours. ( G ) Single-cell mRNA sequencing was conducted on the hearts of mice subjected to sham operation and TAC treatment. For each group, single-cell suspensions from three to four hearts were pooled into one sample. The t-SNE distribution of clustering identified four distinct functional cardiomyocyte clusters: canonical cardiomyocyte, fibroblast-like cardiomyocyte, endothelial-like cardiomyocyte, and remodeling cardiomyocyte. CM, cardiomyocyte. ( H ) The dot plot illustrates the relative expression of Yod1 across various functional cardiomyocyte clusters. ( I to K ) Representative images of tetramethyl rhodamine isothiocyanate (TRITC)–labeled rhodamine-phalloidin staining in primary cardiomyocytes. Cardiomyocytes were transfected with plasmids containing either the empty vector (EV) or YOD1 overexpression (YOD1 oe ) and siRNAs targeting negative control (si-NC) or YOD1 (si-YOD1), followed by stimulation with Ang II (1 μM for 24 hours) or Vehicle (Veh). The surface area of the cardiomyocytes was assessed using TRITC-labeled rhodamine-phalloidin staining (I), accompanied by a corresponding quantitative analysis (J and K). n = 6. ( L to O ) Representative Western blot analysis of YOD1, MyHC, and ANP in HL-1 under Ang II stimulation (L) and densitometric quantification (M to O). n = 6. The cell processing procedure is consistent with that depicted in (J). n = 3. n.s., P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Subsequently, primary cardiomyocytes were incubated with TRITC-labeled phalloidin working solution (catalog no. CA1610, Solarbio, Beijing, China) at room temperature in the dark for 30 min. Last, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution (catalog no. C0065, Solarbio, Beijing, China), and fluorescence observation was conducted under a fluorescence microscope, followed by photography.

Techniques: Western Blot, Sequencing, Functional Assay, Expressing, Labeling, Staining, Transfection, Plasmid Preparation, Over Expression, Negative Control

Journal: Science Advances

Article Title: Cardiomyocyte-derived YOD1 promotes pathological cardiac hypertrophy by deubiquitinating and stabilizing STAT3

doi: 10.1126/sciadv.adu8422

Figure Lengend Snippet: Cardiomyocytes from (A) to (C) were transfected with plasmids containing either the empty vector or YOD1 (YOD1 oe ), followed by stimulation with Ang II (1 μM for 24 hours). Cardiomyocytes from (D) to (G) were transfected with plasmids containing either the empty vector or YOD1 (YOD1 oe ) and siRNAs targeting negative control or STAT3, followed by stimulation with Ang II (1 μM for 24 hours). ( A ) Representative Western blot analysis of STAT3 in total cell lysate, cytoplasmic lysate, and nuclear lysate. Glyceraldehyde phosphate dehydrogenase (GAPDH) and lamin B were used as loading controls. ( B and C ) Representative images of immunofluorescence staining illustrating STAT3 nuclear translocation in cardiomyocytes (B), along with the corresponding quantitative analysis (C). The staining results are presented as follows: Red represents STAT3, while blue indicates 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 50 μm. n = 6. ( D and E ) The surface area of the cardiomyocytes was assessed using TRITC-labeled rhodamine-phalloidin staining (D), accompanied by a corresponding quantitative analysis (E). n = 3. ( F and G ) Representative Western blot analysis of MyHC and ANP in cardiomyocytes (F) and densitometric quantification (G). n.s., P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Subsequently, primary cardiomyocytes were incubated with TRITC-labeled phalloidin working solution (catalog no. CA1610, Solarbio, Beijing, China) at room temperature in the dark for 30 min. Last, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution (catalog no. C0065, Solarbio, Beijing, China), and fluorescence observation was conducted under a fluorescence microscope, followed by photography.

Techniques: Transfection, Plasmid Preparation, Negative Control, Western Blot, Immunofluorescence, Staining, Translocation Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: γ-Secretase and Presenilin Mediate Cleavage and Phosphorylation of Vascular Endothelial Growth Factor Receptor-1 *

doi: 10.1074/jbc.M111.296590

Figure Lengend Snippet: VEGFR1-GFP-WT exhibits structural and functional characteristics of endogenous VEGFR1 receptors. PAECs were stably transfected with VEGFR1-GFP-WT and treated with VEGF (100 ng/ml) and/or PEDF (100 ng/ml) for 24 h. A , ELISA showing that VEGF induced VEGFR1 phosphorylation and that this was blocked by PEDF. Error bars = S.E. B , representative Western blot analysis of subcellular fractions using antibody against the C terminus of VEGFR1 confirmed that PEDF + VEGF induced the appearance of a VEGFR1 fragment in the cytosol ( boxed ), whereas PEDF alone induced translocation of full-length VEGFR1 to the nucleus ( boxed ). C , cells were stained for tubulin ( red ) and nuclei (DAPI; blue ). Cells were visualized by confocal fluorescence. In VEGFR1-GFP-WT cells, PEDF induced a nuclear translocation of VEGFR1. *, p < 0.05 versus VEGF. Error bars = S.E. IB , immunoblot.

Article Snippet: After treatment, the monolayers were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 for 5 min, and after blocking with 0.1% bovine serum albumin, cells were incubated for 1 h with anti-tubulin-TRITC antibody (Santa Cruz Biotechnology, Santa Cruz, CA) to illustrate the shape of the cells.

Techniques: Functional Assay, Stable Transfection, Transfection, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Translocation Assay, Staining, Fluorescence

Journal: Viruses

Article Title: The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner

doi: 10.3390/v12060629

Figure Lengend Snippet: Nafamostat mesylate potently inhibits transmembrane serine protease 2 (TMPRSS2)-dependent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into lung epithelium-derived Calu-3 cells. ( a ) Dual split protein (DSP)1-7 has the structure Renilla luciferase (RL)1–155-Ser-Gly-Gly-Gly-Gly-green fluorescent protein (GFP)1–156. DSP8-11 has the structure Met-RL156–311-Gly-Gly-Gly-Gly-Ser-GFP157–231. DSP1-7 and DSP8-11 reassociate efficiently, resulting in reconstitution of functional RL and GFP to generate luminescent and fluorescent signals, respectively. ( b ) Effector cells (293FT cells expressing DSP8-11 and S protein) and target cells (293FT or Calu-3 cells expressing DSP1-7, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2) were co-cultured. Both GFP (fluorescence) and RL (luminescence) signals were generated following DSP1-7 and DSP8-11 reassociation upon cell fusion. ( c ) Different combinations of the effector and target cells were cocultured, and the resulting RL activity was measured. Relative cell-fusion values were calculated by normalizing the RL activity of each co-culture to that of the co-culture of cells expressing S protein with those expressing both receptor and TMPRSS2, which was set to 100%. ( d ) Phase contrast images of SARS-CoV-2 S protein-mediated-cell fusion. Scale bars, 100 μm. ( e ) The fusion assay using wild type and ACE2-kockout Calu-3 cells. ( f ) Three clinically used pancreatitis and/or anticoagulant drugs were evaluated by the DSP assay for their effects on SARS-CoV-2 S-mediated membrane fusion. Relative cell-fusion value was calculated by normalizing the RL activity for each co-culture to that of the co-culture with dimethyl sulfoxide (DMSO) alone, which was set to 100%. gabe: gabexate mesylate, nafa: nafamostat mesylate, camo: camostat mesylate. ( g ) The DSP assay using Calu-3 (left) or H3255 (right) cells as target cells. DSP1-7 was constitutively expressed in Calu-3 and H3255 cells. ( h ) The DSP assay using Calu-3 cells was performed in the presence of various anticoagulants: edo, edoxaban; riva, rivaroxaban; dabi, dabigatran; api, apixaban; arga, argatroban; dare, darexaban. ( i ) In the “pretreatment” group, cells were pretreated with nafamostat mesylate (10-fold serial dilutions from 100 μM to 1 nM, 4 wells for each dose) for 1 h before infection. SARS-CoV-2 was then added and further incubated for 30 min. The culture medium was then changed to fresh medium with the same concentrations of nafamostat mesylate as those before infection. In the “no-pretreatment” group, cells were incubated with fresh medium for 1 h without nafamostat mesylate. SARS-CoV-2 was then added and further incubated for 30 min. The culture medium was then changed to fresh medium containing nafamostat mesylate as in the “pretreatment” group. Three (VeroE6/TMPRSS2) or 5 (Calu-3) days after infection, effective concentration (EC) 50 was determined using the Spearman–Karber formula based on the appearance of visually detectable cytopathic effect (CPE) in quadruplicate experiments.

Article Snippet: Nafamostat mesylate (Tokyo Chemical Industry, Tokyo, Japan), camostat mesylate (Wako, Tokyo, Japan), gabexate mesylate (Tokyo Chemical Industry, Tokyo, Japan), edoxaban, apixaban, rivaroxaban, dabigatran (Selleck Chemicals, Houston, TX, USA), argatroban (Tokyo Chemical Industry, Tokyo, Japan) and darexaban (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Derivative Assay, Luciferase, Ubiquitin Proteomics, Functional Assay, Expressing, Cell Culture, Fluorescence, Generated, Activity Assay, Co-Culture Assay, Single Vesicle Fusion Assay, Membrane, Infection, Incubation, Concentration Assay, Endpoint Dilution Assay

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Sorafenib suppress hepatic stellate cell viability and induce cellular death with cytoplasmic vacuolation depending on dose and duration of treatment. ( a ) Phase-contrast microscope showing the LX2 cells treated with different concentration of sorafenib (2.5 µM, 5 µM, 7.5 µM, 10 µM and 15 µM) in 7 h and 24 h in compare with untreated control cells. Cytoplasmic vacuoles were indicated by white arrow. Images were taken using × 20 objective, scale bar: 300 μm. ( b ) Cell death and viability of LX2 cells were shown with propidium iodide (PI) positive and negative populations of LX2 cells after treatment of different concentration of sorafenib for 24 h using flow cytometry. ( c ) The quantification of dose and time dependent percentage (%) cell death of LX2 cells with sorafenib treatment were measured. The bars represent mean ± s.d. from three independent experiments (ns > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001 One-way analysis of variance).

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Microscopy, Concentration Assay, Control, Flow Cytometry

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Involvement of non-apoptotic cell death in sorafenib induced LX2 cells depending on dose and time. ( a ) Phase-contrast microscope showing the LX2 cells exposed with 10 µM Sorafenib for 24 h after pre-treatment with protein synthesis inhibitor 25 μM cycloheximide (CHX), autophagy inhibitor, 25 μM chloroquine (CQ), pan-caspase inhibitor, 20 μM Z-VAD-FMK, anti-oxidants 5 μM N -acetylcysteine (NAC) and 5 μM humans serum albumin (ALB). Images were taken using × 20 objective, scale bar: 300 μm. ( b ) 10 µM Sorafenib induced percent cell death and viability of LX2 cells were shown with propidium iodide (PI) positive and negative populations of LX2 cells after pre-treatment with above mentioned inhibitors and anti-oxidants using flow cytometry. ( c ) The quantification of sorafenib induced % cell death of LX2 cells with sorafenib (10 µM) treatment after pre-treatment with above mentioned inhibitors were measured in compare with untreated control. The bars represent mean ± s.d. from three independent experiments (ns > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001 One-way analysis of variance).

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Microscopy, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Dose dependent sorafenib induced cytoplasmic vacuolation in LX2 cells are due to ER stress. ( a , b ) Phase-contrast and Transmission electron micrograph (TEM) of untreated control and 10 µM sorafenib treated LX2 cells for 24 h. TEM images were taken using × 12,000 magnification, scale bar: 1 μm. Cytoplasmic vacuoles (asterisk, *) were observed in 10 µM sorafenib treated LX2 cells. ( c ) The confocal microscopic images of 10 μM sorafenib treated LX2 cells showed alteration of ER tracker along with cytoplasmic vacuolation with respective to untreated control after 24 h. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). ( d ) The confocal microscopic images showing enhanced calreticulin expression LX2 cells after 10 μM sorafenib treatment after 12 h. Relative fluorescence intensities of ER tracker dye and calreticulin in LX2 cells after exposure with 10 µM sorafenib in compare with untreated control were represented in a graph. The bars represent mean ± s.d. from three independent experiments (ns > 0.05, **P < 0.01 Student's unpaired t test).

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Transmission Assay, Control, Staining, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Dose dependent sorafenib induced LC3 signalling. ( a ) The confocal microscopic images showed alteration of LC3B expression in LX2 cells after exposure with low (5 µM) and high (10 µM) concentration of sorafenib for 12 h along with stellate cells activation marker α-SMA. Nuclei were stained with DAPI. Merged pictures showing co-expression of LC3B and α-SMA to evaluate autophagic regulation in activated stellate cells after sorafenib exposure. Images were taken using × 40 objective, scale bar: 50 μm. ( b ) Fluorescence intensity of LC3B and α-SMA quantified and represented as relative fold change with respective to untreated control. The bars represent mean ± s.d. from three independent experiments. ( c , d ) Western blot analysis showing protein expression of LC3B conversion in 5 µM and 10 µM sorafenib treated LX2 cells for 12 h. As a negative control the LC3B conversion were also assessed after transfection with 100 nM siRNA against ATG5. Protein level of ATG5 in ATG5 siRNA transfected LX2 cells were shown. Protein expression were quantified using ImageJ software. αTubulin and GAPDH were used as loading controls. Data represent mean ± s.d. from three independent experiments (ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 two-way analysis of variance).

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Expressing, Concentration Assay, Activation Assay, Marker, Staining, Fluorescence, Control, Western Blot, Negative Control, Transfection, Software

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: ROS participate in sorafenib induced stress. ( a ) Sorafenib induced cellular reactive oxygen species (ROS) were measured by positive population of the fluorogenic dye, 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in LX2 cells with or without pre-treatment of different inhibitors and anti-oxidants N -acetylcysteine (NAC), human serum albumin (ALB), protein synthesis inhibitor cycloheximide (CHX), and pan-caspase inhibitor vZAD. ( b ) The quantification of percentage (%) H 2 DCFDA positive LX2 cells with sorafenib exposure after pre-treatment with above mentioned inhibitors. Statistical analysis performed with grouped data to show the significant difference among relative H 2 DCFDA positive cells. Data represent mean ± s.d. from three independent experiments (ns > 0.05, ***P < 0.001 One-way analysis of variance).

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques:

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Influence of Sorafenib on ROS and ER stress signalling. ( a ) qPCR showing different concentration of sorafenib induced fold changes of relative mRNA expressions of ROS generating enzyme gene such as NOX1, NOX4, NOXA1, CYBA, FMO2 for 24 h. ( b ) The relative mRNA expression of 10 µM sorafenib induced ROS generating enzyme genes after 24 h without and with pre-treatment of CHX, NAC and albumin (ALB). ( c ) qPCR showing different concentration of sorafenib induced fold changes of relative mRNA expression of ER stress or unfolded protein response (UPR) markers such as GPR78 (BiP), IRE1α, PERK, XBP1 and CHOP after 24 h. ( d ) Time dependent relative mRNA expression of 10 µM sorafenib induced ER stress markers were shown in different concentration and different time point up to 24 h. ( e ) The relative mRNA expression of 10 µM sorafenib induced ER stress markers without and with pre-treatment of CHX, NAC and albumin. Fold changes are normalised with expression of 18S rRNA. Data represent mean ± s.d. from three independent experiments (ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 One-way analysis of variance).

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Concentration Assay, Expressing

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Higher concentration of sorafenib induced UPR pathway associated with IRE1α-XBP1s axis. ( a , b ) Western blot analysis showing IRE1α, pIRE1α, GRP78 (BiP), calreticulin and XBP1s expression after treatment with 10 µM sorafenib treatment for 12 h and 24 h. αTubulin and GAPDH were used as a loading control. The immunoblots were depicted the influence of inhibitor of IRE1α 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) on 10 µM sorafenib induced protein level of IRE1α, pIRE1α, GRP78 (BiP), calreticulin and XBP1s. ( c , d ) Protein expression level were quantified using ImageJ software. GAPDH and αTubulin were used as a loading control. Relative protein ratios (normalized with loading control) were shown in a plot graph. Data represent mean ± s.d. from three independent experiments (ns > 0.05, *P < 0.05; **P < 0.01, ***P < 0.001 One-way analysis of variance).

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Concentration Assay, Western Blot, Expressing, Control, Software

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Inhibition of IRE1α through EDBS pre-treatment delayed sorafenib mediated cytoplasmic vacuolation and cell death. ( a ) Phase-contrast microscope showing the effect of pre-treated LX2 cells with EDBS on sorafenib (10 µM) induced cytoplasmic vacuolation at 12 h and 24 h. ( b ) The effect of EDBS on 10 µM sorafenib induced cell death at 12 h and 24 h were determined by Annexin V staining with propidium iodide exclusion using flow cytometry analysis. PI positive population were estimated to measure percent cell death.

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Inhibition, Microscopy, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Cytoplasmic vacuolation with endoplasmic reticulum stress directs sorafenib induced non-apoptotic cell death in hepatic stellate cells

doi: 10.1038/s41598-021-82381-3

Figure Lengend Snippet: Diagram showing dose dependent sorafenib induced cellular death through ER vacuolation. Therapeutic treatment with sorafenib leads to deactivation of HSCs by means of overlapping cellular processes such as autophagy, apoptosis, and non-apoptotic death. Depending on concentration and duration of sorafenib treatment the activated stellate cells can undergo autophagy. On the other hand, higher concentration of sorafenib lead activated stellate cells to undergo cytoplasmic vacuole mediated non-apoptotic cell death by suppressing apoptotic and autophagic pathway but with increasing reactive oxygen species and ER stress. ER vacuolation were mediated by functional activation of UPR pathway involving GPR78, IRE1α and XBP1s.

Article Snippet: Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N -acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).

Techniques: Concentration Assay, Functional Assay, Activation Assay

Journal: Gastroenterology

Article Title: Circulating and Tissue-Resident CD4 + T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

doi: 10.1053/j.gastro.2017.07.047

Figure Lengend Snippet: Expansion and detection of microbiota-specific memory CD4 + T cells (related to Figure 4 ). Total memory CD4 + T cells were isolated, labeled with CFSE, and cultured with autologous irradiated monocytes in the presence or absence of the indicated heat-inactivated bacteria and blocking antibodies to major histocompatibility complex II (MHCII). ( A ) Experimental setup. ( B ) Shown are the CFSE profiles and ICOS expression on days 3 and 6 of stimulation in a representative donor. ( C ) Frequencies (±SEM) of CFSE low proliferating cells after stimulation with S aureus and M tuberculosis (n = 5−21). Data representative of 5−10 independent experiments. Each dot represents an independent donor. ( D ) Frequencies (±SEM) of CFSE low proliferating CD4 + T cells in presence or absence of anti-MHCII. Data representative of 2 independent experiments. ( E, F ) CFSE profiles and expression of CD25 and OX40 on day 6 of stimulation in a representative donor. ( F ) Geometric mean and frequencies (±SEM) of ICOS, CD25, and OX40 expression on CFSE low CD4 + T cells on day 6. ( G ) Number of unique TCRβ clonotypes detected in bacteria-reactive CD4 + T cells. TCR Vβ sequencing was performed from 3 independent donors. Each circle represents an independent donor. TCR Vβ usage of the different reactivities of 3 independent donors is summarized in Supplementary Table 3 . ( H ) Heat map showing the frequency of shared clonotypes between different bacteria-reactive CD4 + T-cell responses. SEB and phytohemagglutinin (PHA) expanded memory T cells were used as controls. Data from 3 independent donors. The CDR3 sequences are summarized in Supplementary Table 4 . Statistics: ( C , D , F ) 1-way analysis of variance with Sidak’s multiple comparison test; ns, not significant; ∗ P ≤ .05; ∗∗ P ≤ .01; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.

Article Snippet: Memory CD4 + CD45RO + CD45RA − T cells were enriched from peripheral blood mononuclear cells (PBMCs) with untouched memory CD4 + T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany), sorted to >97% purity on a FACS ARIA III (BD, San Jose, CA) using CD45RA and CD45RO expression, and were labeled with carboxy-fluorescein succinimidyl ester (CFSE) or violet proliferation dye (Invitrogen, Carlsbad, CA).

Techniques: Isolation, Labeling, Cell Culture, Irradiation, Bacteria, Blocking Assay, Immunopeptidomics, Expressing, Sequencing, Comparison

Journal: Gastroenterology

Article Title: Circulating and Tissue-Resident CD4 + T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

doi: 10.1053/j.gastro.2017.07.047

Figure Lengend Snippet: Microbiota-reactive memory CD4 + T cells are clonally diverse and functionally heterogeneous. Memory CD4 + T cells were labeled with CFSE or violet proliferation dye (VPD)-450 and stimulated with heat-inactivated bacteria in the presence of autologous monocytes. ( A ) CFSE profiles and inducible T-cell costimulator (ICOS) expression on days 3 and 6 of stimulation in a representative donor. ( B ) Percentage of CFSE low CD4 + T cells of each individual donor (n = 18). ( C ) Pie charts showing TCR Vβ expression by proliferating VPD low cells measured by Vβ antibody staining on day 7 of stimulation. Average of 3 independent donors is depicted. TCR Vβ usage of the different reactivities of 3 independent donors is summarized in Supplementary Table 2 . ( D ) Mean (±SEM) cytokine production frequencies of proliferating CFSE low cells and non-activated CFSE high cells after phorbol myristate acetate/ionomycin stimulation (n = 12−13 independent donors). ( E ) Boolean gating analysis showing each possible combination of IL17A, IFN-gamma, and IL22 production by CFSE low proliferating cells. Data from 9 independent donors. ( F , G ) RAR-related orphan receptor γt (RORγt) and T-box expressed in T cells (T-bet) expression in proliferating CFSE low cells measured by intracellular staining on day 7 of stimulation. ( G ) Boolean gating analysis showing each possible combination of RORγt, T-bet, and GATA-binding factor-3 production by CFSE low proliferating cells. Data from 3 independent donors. Statistics: ( B ) 1-way analysis of variance with Sidak’s multiple comparison test; ( D ) 1-way analysis of variance with Bonferroni’s multiple comparison test.

Article Snippet: Memory CD4 + CD45RO + CD45RA − T cells were enriched from peripheral blood mononuclear cells (PBMCs) with untouched memory CD4 + T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany), sorted to >97% purity on a FACS ARIA III (BD, San Jose, CA) using CD45RA and CD45RO expression, and were labeled with carboxy-fluorescein succinimidyl ester (CFSE) or violet proliferation dye (Invitrogen, Carlsbad, CA).

Techniques: Labeling, Bacteria, Expressing, Staining, Binding Assay, Comparison

Journal: Gastroenterology

Article Title: Circulating and Tissue-Resident CD4 + T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

doi: 10.1053/j.gastro.2017.07.047

Figure Lengend Snippet: Cross-reactivity, cytokine production, and transcription factor expression by antigen-reactive memory CD4 + T cells (related to Figure 5 ). ( A ) CD154 + memory CD4 T cells from PBMCs were sorted after short-term stimulation with E coli and B animalis lysates and expanded them for 10−14 days with CD3/CD28 and IL2. The E coli Nissle - and B animalis− reactive T cell lines were CFSE-labeled and then co-incubated with autologous monocytes loaded with the various bacterial lysates. Frequencies (±SEM) of CFSE low proliferating cells after 5 days of stimulation with the indicated bacteria are shown. Data from 3−6 independent donors. ( B − F ) Total memory CD4 + T cells were isolated, labeled with CFSE and cultured with autologous irradiated monocytes in the presence of the indicated heat-inactivated bacteria or antigens. ( B , C ) Production of cytokines by proliferating CFSE low cells measured by intracellular staining after phorbol myristate acetate (PMA)/ionomycin stimulation on day 7 of primary stimulation with monocytes and heat-inactivated bacteria. Frequencies (±SEM) of 3−13 independent donors are depicted. Each dot represents an independent donor. ( D , E ) Related orphan receptor γt (RORγt), T-box expressed in T cells (T-bet), and GATA-binding factor-3 (GATA-3) expression on day 7 of primary stimulation with monocytes and influenza seasonal vaccination or heat-inactivated S aureus in a representative donor. ( E ) Boolean gating analysis showing each possible combination of RORγt, T-bet, and GATA-3 production by CFSE low proliferating cells. Data generated from 3 independent donors. ( F ) Production of IL10 by expanded CFSE low CD4 + cells after PMA/ionomycin stimulation. Frequencies (±SEM) and IFN-gamma/IL17A co-expression within IL10 + and IL10 − cells are depicted from 5−8 independent donors. Statistics: ( A, C ) 1-way analysis of variance with Sidak’s multiple comparison test; ( F ) Kruskal-Wallis test with Dunn’s multiple comparison; ns, not significant; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.

Article Snippet: Memory CD4 + CD45RO + CD45RA − T cells were enriched from peripheral blood mononuclear cells (PBMCs) with untouched memory CD4 + T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany), sorted to >97% purity on a FACS ARIA III (BD, San Jose, CA) using CD45RA and CD45RO expression, and were labeled with carboxy-fluorescein succinimidyl ester (CFSE) or violet proliferation dye (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Labeling, Incubation, Bacteria, Isolation, Cell Culture, Irradiation, Staining, Binding Assay, Generated, Comparison

Journal: Gastroenterology

Article Title: Circulating and Tissue-Resident CD4 + T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

doi: 10.1053/j.gastro.2017.07.047

Figure Lengend Snippet: Microbiota-reactive memory T cells promote intestinal stromal and epithelial cell activation. Healthy donor memory CD4 + T cells from peripheral blood were labeled with CFSE and stimulated with heat-inactivated bacteria in the presence of autologous monocytes. CD4 + CFSE low ICOS high cells were fluorescence-activated cell−sorted on day 7 and expanded for 10−14 days with anti-CD3/CD28 beads. Expanded cells were stimulated at equal numbers with phorbol myristate acetate (PMA)/ionomycin for 24 hours to produce conditioned supernatants. ( A, B ) Cell-free supernatants from different T-cell specificities were used to stimulate CCD18Co intestinal myofibroblasts and LIM1863 colon epithelial cells. Gene expression in stimulated cells was measured by quantitative polymerase chain reaction (qPCR) and normalized to control treatment (media containing PMA/ionomycin alone). Results of independent stimulations were pooled together into the following categories: Proteobacteria -reactive T cells ( S typhimurium − and E coli −reactive) ; Actinobacteria -reactive T cells ( B animalis- reactive); Firmicutes -reactive T cells ( F prausnitzii − and L acidophilus −reactive). Data are from 3 independent T-cell donors. ( C , D ) Supernatants from E coli −reactive CD4 + T cells were used to stimulate CCD18Co ( C ) or LIM1863 ( D ) cells. Supernatants were pretreated with 1 or more cytokine-neutralizing antibodies as indicated. Gene expression was median-normalized, log 2 transformed, and plotted as a heat map. Data representative of 2−3 independent experiments. ( E ) qPCR analysis of mucosal biopsies from the Oxford IBD cohort, categorized by endoscopic assessment of disease activity. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 5 . Statistics: ( A, B , E ) 1-way analysis of variance with Sidak’s multiple comparison test.

Article Snippet: Memory CD4 + CD45RO + CD45RA − T cells were enriched from peripheral blood mononuclear cells (PBMCs) with untouched memory CD4 + T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany), sorted to >97% purity on a FACS ARIA III (BD, San Jose, CA) using CD45RA and CD45RO expression, and were labeled with carboxy-fluorescein succinimidyl ester (CFSE) or violet proliferation dye (Invitrogen, Carlsbad, CA).

Techniques: Activation Assay, Labeling, Bacteria, Fluorescence, Gene Expression, Real-time Polymerase Chain Reaction, Control, Transformation Assay, Activity Assay, Comparison

Journal: Gastroenterology

Article Title: Circulating and Tissue-Resident CD4 + T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

doi: 10.1053/j.gastro.2017.07.047

Figure Lengend Snippet: Microbiota-reactive CD4 + T cells show a Th17-skewed phenotype in IBD patients. ( A, B ) PBMCs isolated from healthy donors and IBD patients were stimulated with heat-inactivated bacteria or SEB and analyzed for intracellular CD154 and cytokine expression. ( A ) Frequencies (±SEM) of IL17A, IFN-gamma, and IL22 expression in CD154 + TNF-α + memory CD4 + T cells (n = 23−33 independent donors). Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6 . ( B ) Frequencies (±SEM) of IL17A and IFN-gamma co-expression in CD154 + TNF-α + memory CD4 + T cells after short-term stimulation with heat-inactivated bacteria (n = 23−34 independent donors). ( C ) Lamina propria mononuclear cells (LPMCs) from inflamed IBD surgical specimens or non-inflamed and tumor-free surgical specimens from colorectal cancer patients were stimulated with heat-inactivated E coli. Boolean gating shows each possible combination of IL17A, IFN-gamma, and IL22 production by CD154 + TNF-α + memory CD4 + T cells (n = 6 and n = 7 independent donors for IBD and controls, respectively). ( D ) Quantitative polymerase chain reaction analysis of IL1B, IL6 , and IL23A in intestinal mucosal specimens categorized by endoscopic assessment of disease activity. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 5 . ( E ) CD4 + CD45RO + CD45RA − CD25 − CD8 − memory CD4 + T cells were isolated from healthy donor blood, labeled with CFSE, and stimulated with autologous monocytes pulsed with B animalis in the presence or absence of the indicated cytokines. Data represent mean (±SEM) fold-changes in IL17A or IFN-gamma expression frequencies relative to cells expanded without cytokines. Statistics: ( A, B , C ) Mann-Whitney test; ( D , E ) 1-way analysis of variance with Sidak’s multiple comparison test.

Article Snippet: Memory CD4 + CD45RO + CD45RA − T cells were enriched from peripheral blood mononuclear cells (PBMCs) with untouched memory CD4 + T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany), sorted to >97% purity on a FACS ARIA III (BD, San Jose, CA) using CD45RA and CD45RO expression, and were labeled with carboxy-fluorescein succinimidyl ester (CFSE) or violet proliferation dye (Invitrogen, Carlsbad, CA).

Techniques: Isolation, Bacteria, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Labeling, MANN-WHITNEY, Comparison

Journal: Gastroenterology

Article Title: Circulating and Tissue-Resident CD4 + T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

doi: 10.1053/j.gastro.2017.07.047

Figure Lengend Snippet: Functional characteristics of enteric bacteria-reactive CD4 + T cells in IBD (related to Figure 7 ). ( A ) PBMCs isolated from healthy donors or IBD patients were stimulated with the indicated heat-inactivated bacteria and analyzed for CD154 expression. Frequencies (±SEM) of reactive CD154 + cells among CD4 + T cells in peripheral blood are depicted (n = 30−38). Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6 . ( B ) Lamina propria mononuclear cell (LPMCs) were isolated from inflamed surgical specimens from IBD patients or non-inflamed and tumor-free surgical specimens from colorectal cancer patients. Isolated LPMCs were stimulated with the indicated heat-inactivated bacteria and analyzed for CD154 expression. Frequencies (±SEM) of reactive CD154 + TNF-α + cells among CD4 + T cells in peripheral blood are depicted (n = 10−20). ( C ) Microarray analysis of intestinal biopsies obtained at endoscopy (n = 6 controls, 24 ulcerative colitis (UC) and 37 Crohn’s disease (CD); Gene Expression Omnibus entry GSE16879). The relative abundance of memory CD4 + T cells in control and inflamed IBD tissue was estimated in silico using CIBERSORT (see for details). ( D−F ) PBMCs isolated from healthy and IBD patients were stimulated with the indicated heat-inactivated bacteria or SEB and analyzed for CD154 expression and intracellular cytokine expression. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6 . ( D ) IL2 expression in CD154 + TNF-α + memory CD4 + T cells after stimulation with the indicated heat-inactivated bacteria or SEB. Frequencies (±SEM) of 23-33 independent donors. Each dot represent an independent donor. ( E, F ) IL17A, IFN-gamma, IL22, and IL2 expression in CD154 + TNF-α + memory CD4 + T cells after stimulation with the indicated heat-inactivated bacteria. Each dot represents an independent donor. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6 . ( G ) IL17A expression in CD154 + TNF-α + memory CD4 + T cells after stimulation with S typhimurium . IBD patients were categorized by disease phenotype (UC, CD), disease activity according to clinical notes, and current treatment. ( H ) Total memory CD4 + T cells were isolated from healthy controls or IBD patients blood, labeled with CFSE and cultured with autologous irradiated monocytes in the presence of the indicated heat-inactivated bacteria or antigens. Production of IL10 by expanded CFSE low CD4 + cells after phorbol myristate acetate (PMA)/ionomycin stimulation on day 7 of stimulation. Frequencies (±SEM) are depicted from 9−11 independent donors. ( I ) CD4 + CD45RO + CD45RA − CD25 − CD8 − memory CD4 + T cells were isolated from healthy controls or IBD patients blood, labeled with CFSE, and stimulated with autologous monocytes pulsed with B animalis in the presence or absence of the indicated cytokines. Data represent mean (±SEM) fold changes in IL17A or IFN-gamma expression frequencies relative to cells expanded without cytokines. Statistics: ( A−E, G−H ) Mann-Whitney test; ( F ) 1-way analysis of variance with Sidak’s multiple comparison test; ns, not significant; ∗ P ≤ .05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.

Article Snippet: Memory CD4 + CD45RO + CD45RA − T cells were enriched from peripheral blood mononuclear cells (PBMCs) with untouched memory CD4 + T cell enrichment kit (Miltenyi Biotec, Bergisch Gladbach, Germany), sorted to >97% purity on a FACS ARIA III (BD, San Jose, CA) using CD45RA and CD45RO expression, and were labeled with carboxy-fluorescein succinimidyl ester (CFSE) or violet proliferation dye (Invitrogen, Carlsbad, CA).

Techniques: Functional Assay, Bacteria, Isolation, Expressing, Microarray, Gene Expression, Control, In Silico, Activity Assay, Labeling, Cell Culture, Irradiation, MANN-WHITNEY, Comparison