Journal: bioRxiv

Article Title: Clinically relevant SMAC mimetics do not enhance human T cell proliferation or cytokine production

doi: 10.1101/2021.11.09.466489

Figure Lengend Snippet: SMAC mimetics activate non-canonical NF-kB in human T cells at concentrations that kill cancer cells. A, T cells were magnetically purified from PBMC and stimulated with plate-bound anti-CD3 or anti-CD3/CD28 in the presence of the indicated concentrations of birinapant for 24 or 48 hours. Cells were harvested and total cellular protein was extracted, run on an SDS-PAGE gel, and blotted with an antibody to NF-kB2 that detects both inactive full-length (p100) and active cleaved (p52) isoforms. B, T cells were magnetically purified and stimulated with plate-bound anti-CD3/CD28 in the presence of the indicated concentrations of birinapant for 48 hours, then assessed for viability via Annexin/PI staining. C, MDA-MB231 breast cancer cell lines were cultured in the indicated concentrations of SMAC mimetic for 48 hours, then assessed for viability via Annexin/PI staining.

Article Snippet: Isolated T cells (0.5-1 × 10 5 per well) or unseparated PBMCs (1 × 105 per well) were stimulated for 48-72 hours either with Dynabeads™ Human T Activator CD3/CD28 antibody coated beads (Thermo Fisher) at 2:1 or 4:1 bead:cells ratios or with plate-bound anti-CD3 and soluble anti-CD28, each at 2 ug/ml in the presence of varying concentrations of IAP antagonists.

Techniques: Purification, SDS Page, Staining, Cell Culture

Journal: bioRxiv

Article Title: Clinically relevant SMAC mimetics do not enhance human T cell proliferation or cytokine production

doi: 10.1101/2021.11.09.466489

Figure Lengend Snippet: SMAC mimetics induce no change in T Cell or PBMC proliferation. A, Flow cytometry gating scheme. Cells were gated on CD25+ and CTe450 followed by CTe450 histograms for percent of cells divided. B-C , T cells were magnetically separated from whole PBMC samples, stained with CTe450 to detect cell division, and stimulated with Dynabeads at a concentration of 1:4 beads to cells in the presence of the indicated concentration of SMAC mimetic for 48 hours. Cells were then stained for surface antigens and analyzed via flow cytometry for percent of cells divided ( B ) and CD25 MFI ( C ). D, PBMCs were stained with CTe450 to detect cell division and stimulated with Dynabeads at a concentration of 1:4 beads to cells in the presence of the indicated SMAC mimetic at various concentration for 48 hours. Cells were then stained for surface antigens and analyzed via flow cytometry for percent of cells divided.

Article Snippet: Isolated T cells (0.5-1 × 10 5 per well) or unseparated PBMCs (1 × 105 per well) were stimulated for 48-72 hours either with Dynabeads™ Human T Activator CD3/CD28 antibody coated beads (Thermo Fisher) at 2:1 or 4:1 bead:cells ratios or with plate-bound anti-CD3 and soluble anti-CD28, each at 2 ug/ml in the presence of varying concentrations of IAP antagonists.

Techniques: Flow Cytometry, Staining, Concentration Assay

Journal: bioRxiv

Article Title: Clinically relevant SMAC mimetics do not enhance human T cell proliferation or cytokine production

doi: 10.1101/2021.11.09.466489

Figure Lengend Snippet: Birinapant does not affect IFNγ or IL-2 secretion. Unseparated PBMC or magnetically purified T cells were stimulated with Dynabeads at a 1:4 ratio beads to cells in the presence of 0 uM or 1 uM Birinapant. Supernatant was collected after 48 hours (IL-2) or 72 hours (IFNγ) and analyzed via ELISA. A, Average cytokine concentration in supernatants from 5 subjects +/− SD. B, Cytokine concentrations were normalized to each subject’s no SMAC mimetic control and plotted individually.

Article Snippet: Isolated T cells (0.5-1 × 10 5 per well) or unseparated PBMCs (1 × 105 per well) were stimulated for 48-72 hours either with Dynabeads™ Human T Activator CD3/CD28 antibody coated beads (Thermo Fisher) at 2:1 or 4:1 bead:cells ratios or with plate-bound anti-CD3 and soluble anti-CD28, each at 2 ug/ml in the presence of varying concentrations of IAP antagonists.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: bioRxiv

Article Title: Clinically relevant SMAC mimetics do not enhance human T cell proliferation or cytokine production

doi: 10.1101/2021.11.09.466489

Figure Lengend Snippet: Birinapant does not affect the proportion of T cells producing cytokines. PBMC were stimulated with plate-bound anti-CD3/CD28 with or without 1 uM Birinapant for 48 hours, treated with BFA for 5 hours (without restimulation), stained for surface antigens and intracellular cytokines, and assessed via flow cytometry. A, Gating scheme for flow cytometry analysis. B, The percent of CD4 or CD8 T cells that were producing the indicated cytokine(s) was normalized to each subject’s no SMAC mimetic control and plotted individually. Top , total proportion of cells producing IFNγ. Bottom , proportion of cells producing both IFNγ and TNFα.

Article Snippet: Isolated T cells (0.5-1 × 10 5 per well) or unseparated PBMCs (1 × 105 per well) were stimulated for 48-72 hours either with Dynabeads™ Human T Activator CD3/CD28 antibody coated beads (Thermo Fisher) at 2:1 or 4:1 bead:cells ratios or with plate-bound anti-CD3 and soluble anti-CD28, each at 2 ug/ml in the presence of varying concentrations of IAP antagonists.

Techniques: Staining, Flow Cytometry

Journal: JCI Insight

Article Title: Regulation of bile duct epithelial injury by hepatic CD71 + erythroid cells

doi: 10.1172/jci.insight.135751

Figure Lengend Snippet: (A–C) Representative plots and quantification of CD71+Ter119+ cells (A), CD4+ T cells and CD8+ T cells (B), and activation marker NKp46 in CD3–CD49b+ cells and total NK cell counts (C) in livers of RRV-infected mice pretreated with either rat IgG or anti-CD71 antibody. Differences in mean values analyzed by 2-tailed Student’s t test; **P < 0.01; ****P < 0.0001; n = 4/group.

Article Snippet: In these assays, 4 × 10 5 of responder cells were seeded into 96-well round-bottom plates individually or together with suppressor cells (neonatal hepatic mononuclear cells) at a 1:1 ratio and then stimulated for 72 hours with 0.125 μg/mL anti–mouse CD3 antibody (clone 145-2C11, BioXcell).

Techniques: Activation Assay, Marker, Infection

Journal: JCI Insight

Article Title: Regulation of bile duct epithelial injury by hepatic CD71 + erythroid cells

doi: 10.1172/jci.insight.135751

Figure Lengend Snippet: Adult hepatic mononuclear cells (MNCs) can produce TNF-α upon anti-CD3 stimulation; therefore, they were used as responder cells. Neonatal liver harvested at 24 hours of life, or 3 days after antibody depletion followed by RRV infection, was used as erythroblast pool to function as effector cells. TNF-α levels were measured in supernatant 72 hours after coculture. “+” denotes presence. “–” denotes absence. One-way ANOVA test was performed. *P < 0.05, ***P < 0.001. n = 3/group.

Article Snippet: In these assays, 4 × 10 5 of responder cells were seeded into 96-well round-bottom plates individually or together with suppressor cells (neonatal hepatic mononuclear cells) at a 1:1 ratio and then stimulated for 72 hours with 0.125 μg/mL anti–mouse CD3 antibody (clone 145-2C11, BioXcell).

Techniques: Infection

Journal: Aging (Albany NY)

Article Title: p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

doi: 10.18632/aging.101268

Figure Lengend Snippet: Gene expression analysis of macrophage polarization markers (M1, Nos2 and Il1b ; M2, Arg1 ) of alginate bead model (AB model)-elicited peritoneal macrophages from wild type mice via qPCR. ( A ) mRNA expression of Nos2 and Arg1 in AB-elicited macrophages adherence-selected from CD11b-enriched peritoneal lavage, as compared to expression in naïve bone marrow-derived macrophages (M0) or following polarization to M1 (IFN-γ for 24 hrs; M1 ctrl) or M2 (IL-4 for 24 hours; M2 ctrl) states. Gapdh expression was used an internal reference gene control. Data shows mean ± standard deviation (n=3). *** p-value < 0.001 compared to M0 control. ( B ) Peritoneal macrophages elicited by the alginate bead model were treated ex vivo with immunomodulatory agents. qPCR analysis of mRNA expression of indicated genes was normalized to β2-microglobulin ( B2m ) expression was determined following 72 hour incubation with M1-inducing stimuli (LPS at 1 ng/mL + IFN-γ at 10 ng/mL) or M2-inducing cytokines (IL-4 at 20 ng/mL + IL-13 at 10 ng/mL). Fold change in gene expression following treatment is depicted as mean ± standard deviation relative to non-treated controls; ***, p-value < 0.001. Results are representative of 3 independent experiments with peritoneal lavage cells pooled from at least 3 mice.

Article Snippet: Following elicitation of macrophages via the alginate bead model (2-3 weeks post-injection), or 1 mL of 3% proteose peptone (3 days post-injection) (BD Biosciences; Franklin Lakes, NJ), peritoneal lavage cells were collected and 2.5×10 5 to 5.0×10 5 cells per well were plated overnight in a 24-well plate in complete medium, followed by ≤ 72 hour treatments of carrier-free recombinant mouse cytokines IL-4, IL-13, IFNγ (PeproTech; Rocky Hill, NJ) and IFNα (BioLegend; San Diego, CA), LPS (Sigma Aldrich; St Louis, MO), high molecular weight (HMW) Poly(I:C) (InvivoGen; San Diego, CA), JAK1/2 inhibitor Ruxolitinib (ApexBio; Boston, MA) and STAT6 inhibitor AS1517499 (Axon Medchem; Reston, VA).

Techniques: Expressing, Derivative Assay, Standard Deviation, Ex Vivo, Incubation

Journal: Aging (Albany NY)

Article Title: p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

doi: 10.18632/aging.101268

Figure Lengend Snippet: Peritoneal lavage cells elicited by alginate-encapsulated SCs from p16 Ink4a/Luc mice were treated ex vivo with immunomodulatory agents for 72 hours. ( A ) p16 Ink4a promoter-driven luciferase activity (black bars) and β-galactosidase activity (via 4-MUG hydrolysis) (gray bars) were measured following treatment with M1- and M2-polarizing stimuli: LPS at 1 ng/mL, IFN-γ at 10 ng/mL, LPS/IFN-γ co-treatment, Poly(I:C) at 10 μg/mL, IFN-α at 100 U/mL, IL-4 at 20 ng/mL, IL-13 at 10 ng/ml, and IL-4/IL-13 co-treatment. Results are shown as the mean ± standard deviation for at least 3 independent experiments with statistical significance between treated and non-treated samples depicted. ( B ) Microphotograph of SAβG-stained adherence-selected macrophages with or without stimulation with LPS (1 ng/mL) for 72 hours (10x objective). ( C ) mRNA expression of p16 Ink4a and β-galactosidase ( Glb1 ) (relative to B2m expression) in macrophages from wild type mice with or without LPS stimulation for 72 hours analyzed via qPCR, as normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). ( D&E ) Kinetics of p16 Ink4a promoter-driven luciferase activity per cell with or without LPS stimulation ( D ) or IL-4 stimulation ( E ), normalized to activity from non-treated cells at time zero. Results are shown as the mean ± standard deviation (n=3). Statistical significance with respect to non-treated control at time zero is indicated. ( F ) Luciferase activity and β-galactosidase activity (via 4-MUG hydrolysis) from proteose peptone-elicited lavage cells following stimulation with IL-4 (20 ng/mL) for 72 hours, normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). ( G-J ) Dose-dependent response of JAK1/2 inhibitor Ruxolitinib ( G&H ) and STAT6 inhibitor AS1517499 ( I&J ) on luciferase activity ( G&I ) and viability via CyQuant Direct assay ( H&J ) following 72 hours treatment of AB-elicited macrophages in the presence (gray bars) or absence (black bars) of IL-4 (10 ng/mL) stimulation. Results of luciferase activity and viability are representative of two independent experiments, depicted as mean ± standard deviation of data normalized to respective controls lacking inhibitors (with or without IL-4). Luciferase activity and viability are depicted as the percent signal relative to non-treated (NT) controls. Statistical significance between IL-4 stimulated and non-stimulated cells at each concentration of inhibitor is shown. Results are representative of three independent experiments, depicted as mean ± standard deviation. ( K ) Relative luciferase activity per cell following repolarization of AB-elicited macrophages (via adherence-enriched peritoneal lavage) with M1- and M2-inducing agents. Macrophages were left non-treated (NT) or treated with either LPS (1 ng/ml) or IL-4 (20 ng/ml) for 72 hours (days 1-3), as indicated. For each treatment set, samples were collected at 72 hours (no further treatment; days 4-6 = ×). Alternatively, cells were washed and placed in fresh medium (-), medium containing LPS (1 ng/mL) and IFN-γ (10 ng/mL), or medium containing IL-4 (20 ng/mL) and IL-13 (10 ng/mL) and incubated for an additional 72 hours prior to sample collection (as indicated for days 4-6). Luciferase activity is expressed as the percent activity per cell relative to non-treated (NT) controls after the first 72 hours. Results are representative of two independent experiments. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.

Article Snippet: Following elicitation of macrophages via the alginate bead model (2-3 weeks post-injection), or 1 mL of 3% proteose peptone (3 days post-injection) (BD Biosciences; Franklin Lakes, NJ), peritoneal lavage cells were collected and 2.5×10 5 to 5.0×10 5 cells per well were plated overnight in a 24-well plate in complete medium, followed by ≤ 72 hour treatments of carrier-free recombinant mouse cytokines IL-4, IL-13, IFNγ (PeproTech; Rocky Hill, NJ) and IFNα (BioLegend; San Diego, CA), LPS (Sigma Aldrich; St Louis, MO), high molecular weight (HMW) Poly(I:C) (InvivoGen; San Diego, CA), JAK1/2 inhibitor Ruxolitinib (ApexBio; Boston, MA) and STAT6 inhibitor AS1517499 (Axon Medchem; Reston, VA).

Techniques: Ex Vivo, Luciferase, Activity Assay, Standard Deviation, Staining, Expressing, CyQUANT Assay, Concentration Assay, Incubation

Journal: Aging (Albany NY)

Article Title: p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

doi: 10.18632/aging.101268

Figure Lengend Snippet: Primary cultures of adipose-derived mesenchymal stromal cells (AdMSC) isolated from p16 Ink4a/Luc mice were irradiated (20Gy) and cultured for 10 days for senescence induction. Mock irradiated cells were passaged and used as a proliferating cell control. Response of senescent and proliferating AdMSCs to immunomodulatory agents were compared to that of peritoneal lavage cells elicited by the alginate bead model. ( A-C ) Characterization of senescent and proliferating AdMSCs. Microphotographs of SAβG-stained cells depicts positive staining of senescent cells, as well as an enlarged and flattened morphology, compared to that of proliferating cell control ( A ). p16Ink4a promoter-driven luciferase activity ( B ) and β-galactosidase activity measured via 4-MUG hydrolysis ( C ) were measured in senescent and proliferating AdMSCs, confirming senescent phenotypes. ( D-K ) Dose-response curves of LPS ( D&E ), Poly(I:C) ( F&G ), IFN-α ( H&I ), and IFNγ (10 ng/mL), IL-4 (20 ng/mL) and IL-13 (10 ng/mL) ( J&K ) on p16 Ink4a promoter-driven luciferase activity (left panels: D,F,H&J ) and β-galactosidase activity measured via 4-MUG hydrolysis (right panels: E,G,I&K) after 72hr treatment. No effect on viability was observed via CyQuant Direct assay (>80% viability). Results are shown as the mean ± standard deviation for at least 3 experiments, with statistical comparison to non-treated controls; *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001. nd, not determined.

Article Snippet: Following elicitation of macrophages via the alginate bead model (2-3 weeks post-injection), or 1 mL of 3% proteose peptone (3 days post-injection) (BD Biosciences; Franklin Lakes, NJ), peritoneal lavage cells were collected and 2.5×10 5 to 5.0×10 5 cells per well were plated overnight in a 24-well plate in complete medium, followed by ≤ 72 hour treatments of carrier-free recombinant mouse cytokines IL-4, IL-13, IFNγ (PeproTech; Rocky Hill, NJ) and IFNα (BioLegend; San Diego, CA), LPS (Sigma Aldrich; St Louis, MO), high molecular weight (HMW) Poly(I:C) (InvivoGen; San Diego, CA), JAK1/2 inhibitor Ruxolitinib (ApexBio; Boston, MA) and STAT6 inhibitor AS1517499 (Axon Medchem; Reston, VA).

Techniques: Derivative Assay, Isolation, Irradiation, Cell Culture, Staining, Luciferase, Activity Assay, CyQUANT Assay, Standard Deviation