Fit or senescent MSCs (± IFNγ-activated) were lysed and total RNA was extracted using an RNeasy plus mini kit (QIAGEN). Normalized RNA was used to convert cDNA using Quantitect reverse transcription kit (QIAGEN). Sybr green (Perfecta Sybr green fast mix, Quanta Biosciences) real-time PCR was performed with IDO primer pairs as described previously.22 (link) IDO protein were detected using primary rabbit anti-human IDO1 (1:1,000; EMD Millipore Corporation, Billerica, MA) or rabbit anti-human β-actin (1:1000; Cell Signaling Technology, Inc, Danvers, MA), and secondary horseradish peroxide–coupled goat anti-rabbit IgG h+l (1:10 000; Bethyl Laboratories, Inc, Montgomery, TX). ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ) was used to detect immunoreactive blots. IDO activity was blocked using 1-methyl-DL-tryptophan (1 mM concentration) (Sigma-Aldrich) in MSC and T-cell coculture.
1 methyl dl tryptophan
Manufactured by Merck Group
Sourced in United States
1-methyl-DL-tryptophan is a chemical compound that functions as an indoleamine 2,3-dioxygenase (IDO) inhibitor. It is used as a laboratory reagent for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection system, by Cytiva (2 mentions)
Primary rabbit anti human ido1, by Merck Group (2 mentions)
Secondary horseradish peroxide coupled goat anti rabbit igg h l, by Fortis Life Sciences (2 mentions)
Anti tgf β, by R&D Systems (2 mentions)
Anti il 6 antibody, by R&D Systems (2 mentions)
Anti il10 antibody, by R&D Systems (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Transwell system, by Corning (2 mentions)
Rabbit anti human β actin, by Cell Signaling Technology (1 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Ro 61 8048, by Bio-Techne (1 mentions)
Complete freund s adjuvant cfa, by Merck Group (1 mentions)
Veritas microplate luminometer, by Promega (1 mentions)
1 methyl d tryptophan, by Merck Group (1 mentions)
1 methyl l tryptophan, by Merck Group (1 mentions)
Recombinant human ifn γ, by Thermo Fisher Scientific (1 mentions)
5 6 carboxyfluorescein diacetate succinimidyl ester cfse, by Thermo Fisher Scientific (1 mentions)
Celecoxib, by Abcam (1 mentions)
10 protocols using 1 methyl dl tryptophan
1
Quantifying IDO Expression and Activity
2
Induction of Experimental Autoimmune Encephalomyelitis
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Desiccated, killed mycobacterium tuberculosis H37Ra was obtained from Bioscientific Pty Ltd. Complete Freund’s adjuvant (CFA) was purchased from Sigma-Aldrich (MO, USA). Myelin oligodendrocyte glycoprotein (MOG35-55; amino acid [10 ] sequence: H-Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys-OH) and pertussis toxin were obtained from Sapphire Biosciences. Interchangeable syringes were obtained from Cadence Inc (VA, USA). 1-Methyl-DL-tryptophan (Sigma-Aldrich, MO, USA) was diluted in 5N HCl and adjusted to pH 7 before injection [11 (link)]. Ro 61-8048 (Tocris, Bristol, UK) was dissolved in DMSO and diluted in sterile 0.9% saline to a concentration of 10 mg/ml, and the pH was adjusted to 7.5 [12 (link)].
3
Assessing PBMC-Mediated Cytotoxicity on HepG2 Cells
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Resting or IFNγ (20 ng/ml) or TNFα (20 ng/ml) activated MSCs or HepG2-Luciferase cells were seeded in 96 well plates at a concentration of 25,000–50,000 cells per well. Subsequently, 1000 ng/ml Staphylococcal enterotoxin B (SEB) (Toxin Technologies, United States) activated peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals using an IRB approved protocol at the University of Wisconsin-Madison were added in different ratios. Two days later, 50 μl of 3 mg/ml D-Luciferin (Chem-Impex Inc., United States) was added into each well and the relative light units (RLU) were measured using the VeritasTM Microplate luminometer (Turner BioSystems, United States). For co-culture with MSCs, 50,000 HepG2-Luciferase cells and 250,000 SEB activated PBMCs were added into a 24 well plate with appropriate numbers of resting or IFNγ (20 ng/ml) or TNFα (20 ng/ml) activated MSCs in the presence or absence of 1-methyl-DL-tryptophan (1 mM) (Sigma-Aldrich, United States). Two days later, total cells were subjected to the luciferase assay as described above.
4
Quantification of IDO1 and AhR in MSCs
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Approximately 1 million MSCs were harvested from a single 75-cm2 flask at 80% confluency. Cells had been treated for 12h with 50 ng/ml recombinant human IFN-γ (Invitrogen, Carlsbad, CA), and/or 1-methyl-DL-tryptophan, 1-methyl-D-tryptophan, or 1-methyl-L-tryptophan (Sigma-Aldrich, St. Louis, MO). Whole-cell protein lysates were run in a 4-20% polyacrylamide gel electrophoresis apparatus and then transferred to PVDF membrane, which was blocked in 5% non-fat milk in Tris-buffered saline + 0.05% Tween-20. Protein was detected using primary rabbit anti-human IDO1 (1:1000; EMD Millipore Corporation, Billerica, MA), primary mouse anti-human AHR (1:1000; ThermoFisher, Waltham, MA) or primary rabbit anti-human β-actin (1:1000; Cell Signaling Technology, Danvers, MA), and secondary horseradish peroxide-coupled goat anti-rabbit IgG h + l (1:10,000; Bethyl Laboratories, Montgomery, TX). ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ) was used to detect immunoreactive blots.
5
Measuring Kynurenine in Breast Cancer Coculture
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Cell culture supernatant were collected, and the concentration of kynurenine was measured with ELISA kits in accordance with the manufacturer’s instructions (Bioswamp). In brief, Vγ9Vδ2 T cells (effector) were co-incubated with breast cancer cells (target) at different effector:target (E:T) ratios (0:1, 1:1, 5:1, and 15:1) at 37°C for 12 hours (tumor cells number = 2.5×105). In some experiments, the breast cancer and Vγ9Vδ2 T cells were treated with 0.5 mM 1-methyl-DL-tryptophan (Sigma-Aldrich) for 12 hours. Co-incubation meant that residual 1-MT in the medium was not washed out and remained in the subsequent experiment. Lastly, the cell culture mixture was centrifuged, and the culture supernatant was collected to clean tubes for standard kynurenine measurement.
6
Investigating MenSCs' Immunomodulatory Effects
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To assess the effects of MenSCs on CD4 + T cells proliferation, CD4 + T cells were co-cultured in different settings as mentioned above. Before co-culturing, CD4 + T cells were washed and labeled with 5 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, USA).
To investigate the effects of MenSCs secretome on CD4 + T cell proliferation, a transwell system (Corning, USA) was utilized. Briefly, IFN-γ/IL-1β-treated MenSCs were seeded in the lower chamber of a 24-well transwell plate. Later, CD4 + T cells were added into the upper chamber and plates were incubated for five days in incubator. In some settings, 1 mM 1-Methyl-DL-tryptophan (Sigma, USA) as IDO blocker, 20 mM Indomethacin (Sigma, USA) as PGE2 inhibitor, 10 µg/mL anti-IL-6 antibody (R&D systems, USA), 10 µg/mL anti-IL10 antibody (R&D systems, USA) or 10 µg/mL anti-TGF-β (R&D systems, USA) were added to the co-cultures in order to investigate the involvement of aforesaid molecules on modulatory effects of MenSCs on T cell proliferation or Treg generation. After 5 days, proliferation of CD4 + T cells and frequency of Tregs were assessed by flow cytometry.
To investigate the effects of MenSCs secretome on CD4 + T cell proliferation, a transwell system (Corning, USA) was utilized. Briefly, IFN-γ/IL-1β-treated MenSCs were seeded in the lower chamber of a 24-well transwell plate. Later, CD4 + T cells were added into the upper chamber and plates were incubated for five days in incubator. In some settings, 1 mM 1-Methyl-DL-tryptophan (Sigma, USA) as IDO blocker, 20 mM Indomethacin (Sigma, USA) as PGE2 inhibitor, 10 µg/mL anti-IL-6 antibody (R&D systems, USA), 10 µg/mL anti-IL10 antibody (R&D systems, USA) or 10 µg/mL anti-TGF-β (R&D systems, USA) were added to the co-cultures in order to investigate the involvement of aforesaid molecules on modulatory effects of MenSCs on T cell proliferation or Treg generation. After 5 days, proliferation of CD4 + T cells and frequency of Tregs were assessed by flow cytometry.
7
Vγ9Vδ2 T Cell-Mediated Breast Cancer Killing
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Breast cancer cells were first labeled with 0.5 μM green fluorescence dye CFSE (Thermo Fisher), followed by incubation with Vγ9Vδ2 T cells according to the designated E (effector, Vγ9Vδ2 T): T (target, cancer cell) ratios (0:1, 5:1, and 15:1) at 37°C in a humidified atmosphere with 5% CO2. After 6 hours, cells were harvested and stained with PI (SUNGENE BIOTECH) for 10 minutes at room temperature. The percentage of dead target cells (CFSE+ PI+) out of the total target cells was identified via flow cytometry. In some experiments, breast cancer cells (MCF-7 and MDA-MB-231) and Vγ9Vδ2 T cells were treated with 0.5 mM 1-Methyl-DL-tryptophan (Sigma-Aldrich), 100 nM Linrodostat (Selleck) or with 10 μg ml−1 PD-L1 (CD274) antibody (Selleck) for 6 or 12 hours. Co-incubation meant that residual 1-MT or anti-PD-L1 in the medium was not washed out and remained in the subsequent tumor killing assays.
8
Modulating CD8+ T cell activation
9
Macrophage Phagocytosis of Paracoccidioides
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Thioglycollate-induced peritoneal macrophages were isolated by adherence (2 h at 37°C in 5% CO2) to plastic-bottom tissue-culture plates (1×106 cells/well in 24 well plates). Macrophages were washed to remove nonadherent cells and cultivated overnight with fresh complete medium (DMEM, Dulbecco's Modified Eagle's Medium, Sigma, containing 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin) in the presence or absence of recombinant IFN-γ (20 ng/ml in culture medium, BD-Pharmingen) and/or 1MT (1-methyl-d,l-tryptophan, Sigma-Aldrich). 1MT was used in the concentration of 1 mM that was previously shown to efficiently inhibit the IDO activity of macrophages [6] (link). Macrophage cultures were infected or not with P.brasiliensis yeasts in a macrophage∶yeast ratio of 25∶1 and cocultivated for 4 h. This ratio was previously determined and was shown to be non-deleterious to macrophage cultures and adequate for killing assays [38] (link). The monolayers were then washed to remove nonadherent cells and incubated for an additional 48 h period in the presence or absence of IFN-γ (20 ng/ml in culture medium, BD Biosciences) and/or 1MT.
10
Immunomodulatory Effects of MenSCs
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To assess the effects of MenSCs on CD4+ T cells proliferation, CD4+ T cells were co-cultured in different settings as mentioned above. Before co-culturing, CD4+ T cells were washed and labeled with 5 μM 5,6carboxy uorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, USA).
To investigate the effects of MenSCs secretome on CD4+ T cell proliferation, a transwell system (Corning, USA) was utilized. Brie y, IFN-γ/IL-1β-treated MenSCs were seeded in the lower chamber of a 24-well transwell plate. Later, CD4+ T cells were added into the upper chamber and plates were incubated for ve days in incubator. In some settings, 1 mM 1-Methyl-DL-tryptophan (Sigma, USA) as IDO blocker, 20 mM Indomethacin (Sigma, USA) as PGE2 inhibitor, 10 µg/ml anti-IL-6 antibody (R&D, USA), 10 µg/ml anti-IL10 antibody (R&D, USA) or 10 µg/ml anti-TGF-β (R&D, USA) were added to the co-cultures in order to investigate the involvement of aforesaid molecules on modulatory effects of MenSCs on T cell proliferation or Treg generation. After 5 days, proliferation of CD4+ T cells and frequency of Tregs was assesses by ow cytometry.
To investigate the effects of MenSCs secretome on CD4+ T cell proliferation, a transwell system (Corning, USA) was utilized. Brie y, IFN-γ/IL-1β-treated MenSCs were seeded in the lower chamber of a 24-well transwell plate. Later, CD4+ T cells were added into the upper chamber and plates were incubated for ve days in incubator. In some settings, 1 mM 1-Methyl-DL-tryptophan (Sigma, USA) as IDO blocker, 20 mM Indomethacin (Sigma, USA) as PGE2 inhibitor, 10 µg/ml anti-IL-6 antibody (R&D, USA), 10 µg/ml anti-IL10 antibody (R&D, USA) or 10 µg/ml anti-TGF-β (R&D, USA) were added to the co-cultures in order to investigate the involvement of aforesaid molecules on modulatory effects of MenSCs on T cell proliferation or Treg generation. After 5 days, proliferation of CD4+ T cells and frequency of Tregs was assesses by ow cytometry.
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