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Materials
Animals
Female DO11.10 OVA-specific TCR-transgenic (Tg), BALB/c, and C57BL/6 mice were bred and maintained in the Animal Center of the College of Medicine, National Taiwan University. All mice were used at 4-5 week of age throughout the studies. All procedures were approved by the institutional Animal Care and Use Committee.
Cell line EL4 cells
This cell line is a C57BL/6N mouse lymphoma. The appropriate medium used to maintain this cell line is complete Dulbecco’s modified Eagle’s medium with 10% FBS.
Cell culture reagents RPMI-1640 (10% FBS)
RPMI-1640 (Hyclone, USA) 1L
Fetal Bovine Serum (Hyclone, USA) 10%
L-glutamine (GIBCO BRI, MD, USA) 1%
PSA (GIBCO BRI, MD, USA) 1%
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HEPES (GIBCO BRI, MD, USA) 1%
Phosphate Buffered Saline (1L)
NaCl (Amersco, USA) 8g
Na2HPO4 (Sigma, USA) 2.871g
KCl (Merck, Germany) 0.2g
KH2PO4 (Ferak, Germany) 0.2g
ACK (RBC lysis buffer)
NH4Cl (Wako, Japan) 0.15M
KHCO3 (Wako, Japan) 10mM
EDTA (Sigma, USA) 0.1mM
Hank’s buffer
Hank’s Balanced Salts (Sigma, USA)
Dissolved the powder in 1L ddH2O and adjusted to pH 7.2~7.4 by NaHCO3
Cytokine
IL-2 (PeproTech Inc, USA) IL-4 (eBioscience, USA)
GM-CSF (PeproTech Inc, USA) TGF-β (R&D, USA)
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Others
Anti-mouse CD3ε (Biolegend, USA) Anti-mouse CD28 (Biolegend, USA)
Drugs
pentoxifylline (Sigma, USA)
Dissolved the powder in complete medium and stocked at -20℃
Triptolide (Sigma, USA)
Dissolved the powder in DMSO and stocked at -20℃
ELISA assay (R&D System, USA) Wash buffer (1L)
Tween 20 (Sigma, USA) 0.05%
added Tween 20 in PBS and adjusted to pH 7.2~7.4 Reagent diluents
IL-2 0.1% BSA, 0.05% Tween 20 in Tris-buffered saline, pH 7.2~7.4 IL-10 1% BSA in PBS, pH 7.2~7.4
IL-12p40 1% BSA in PBS, pH 7.2~7.4
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IL-12p70 1% BSA in PBS, pH 7.2~7.4 Blocking buffer
IL-2 1% BSA in PBS with 0.05% NaN3, pH 7.2~7.4 IL-10 1% BSA in PBS, pH 7.2~7.4
IL-12p40 1% BSA in PBS, pH 7.2~7.4
IL-12p70 1%BSA, 5% Sucrose in PBS, pH 7.2~7.4 Substrate solution
NelA-Blue tetramethylbenzidine substrate (TMB) (Clinical, USA) Stop solution
2N H2SO4 (Wako, Japan)
Flow cytometry
FACScan buffer (1L)
1X FACScan PBS 980ml
Fetal bovine serum (Hyclone, USA) 20ml
NaN3 1g
Foxp3 Fix/Perm Buffer Set (Biolegend)
(4X) Foxp3 Fix/Perm buffer (Biolegend, USA) (10X) Foxp3 Perm buffer (Biolegend, USA)
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Diluted the above buffer to 1x working solution with PBS
Magnetic cell sorting
CD4 (L3T4) MicroBeads, mouse (Miltenyi Biotec, Germany) autoMACS running buffer
Bovine serum albumin (Sigma, USA) 0.5%
EDTA (Sigma, USA) 2mM
Dissolved the above powder in 1X PBS then sterilized and filtrated with 0.2μm filter
autoMACS rinsing buffer
EDTA (Sigma, USA) 2mM
Dissolved powder in 1X PBS then sterilized
Methods
Isolation of naïve CD4T cells
Spleens were harvested from female BALB/c DO11.10 OVA-specific TCR-transgenic mice or C57BL/6 mice, and then grinded it into single-cell suspensions in Hank’s buffer. After centrifuging at 1500 rpm for 5 minutes, large tissues were removed and RBCs were depleted by adding 1 ml of ACK lysis buffer and washed these
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cells two to three times with Hank’s buffer. After determining the total cell number, spleen cells were prepared for the separation of CD4 T cell by CD4 T Cell Isolation Kit in Magnetic cell sorting machine. At first, spleen cells were re-suspended in 40μL per 1
× 107 cells of MACs running buffer and then add Biotin-Antibody Cocktail 10μL per 1
× 107 cellswas added, then mix well and incubate at 4℃~8℃ for 10 minutes. Cells were added with MACs running buffer 30μL per 1 × 107 cells and Anti-Biotin MicroBeads 20μL per 1 × 107 cellsand incubated at 4~8℃ for 15 minutes. Stained cells were washed with MACs running buffer and re-suspended with MACs running buffer in appropriate volume to get CD4T cells by auto-MACS.
Cell toxicity test
The EL-4 cell line was cultured in DMED with 10% FBS and in the concentration of 2 × 10 5 cells/ml; in addition, the cell line were treated with serial diluted dosages of PTX and TPT and used cell only as control. After cultured for 48 hours, the cells were collected from each condition, evaluated for cell viability using trypan blue staining.
The cell viability was shown in the percentage of alive cells in 500 cells per well.
Culture of bone marrow-derived dendritic cells (BMDCs)
The femurs and tibias were collected from mice, washed with 75% alcohol for 5
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seconds, and transferred to HBSS buffer. The bone marrow cells were flushed out with 5ml needle and red blood cells were lysed by ACK. After washed with HBSS, cells were re-suspended in RPMI-1640 medium and adjusted to the concentration of 1 × 106 cells/ml. Cells were cultured with 10% FBS-RPMI with recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, 500 U/ml) and interleukin-4 (IL-4, 1000 U/ml, Pepro Tech Inc., Rocky Hill, NJ ). On day 2, day 4, and day 6, the medium was removed by aspiration and lymphocytes were cultured in fresh medium containing GM-CSF and IL-4 in the same concentration as the first day. The percentages of bone marrow-derived DCs were evaluated by FACSort cell analyzer (Becton-Dickinson) on day 6.
Drug treatment on CD4T cells IL-2 secretion
Purified CD4 T cells were adjusted to the concentration of 1 × 106 cells/ml, seeded in the 48-well plate, and stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml. Cells were also added with different concentration of PTX and TPT. After 48 hours, the cultured supernatant was collected for cytokine measurement.
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3H-thymidine incorporation assay
Purified CD4 T cells were adjusted to the concentration of 1 × 106 cells/ml, seeded in the 96-well round-bottom plate (Nunc, Roskilde, Dermark), and stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml. Cells were also added with different concentration of PTX and TPT. After 72 hours, cells were prepared for thymidine incorporation assay.
Regulatory T cell induction
Purified CD4 T cells were adjusted to the concentration of 2 × 106 cells/ml, seeded in the 24-well plate, and stimulated with plate-coated anti-CD3 and anti-CD28 at 2μg/ml. IL-2 was added at 100U/ml in each well. TGF-β was added at 10ng/ml as positive control. After 72 hour of cultured, these cells were harvested to analyze the surface marker expression, and the cultured supernatant was collected for cytokines measurement.
Drug treatment on bone marrow-derived dendritic cells
The DCs, which were already cultured for 6 days, were adjusted to the concentration of 1 × 106cells/ml, and also treated with different concentration of PTX and TPT. The 100 ng/ml of LPS were used to stimulate DCs. After 48 hours, the DCs
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were harvested to analyze surface marker (CD11c, CD80, CD86, MHC class II) expression, and the cultured supernatant was collected for cytokines (IL-10, IL-12p40, IL-12p70) measurement.
Proliferation assay
The cultured CD4 T cells were pulsed with 1.0 μCi/ml of 3H-thymidine (Amersco, USA) and further incubated in 37℃ and 5% CO2 incubator for 16~18 hours. After incubation, these cells were harvested to glass fiber filter (Packard Instrument Co., Meriden, CT) with semi-automated device (Harvester, Packard, Filtermate 196). 3H- thymidine incorporation was quantitated by using Direct Beta Counter (Packard Instrument Co., Meriden, CT). The results were presented in counts per minute (CPM).
Measurement of cytokine production
Supernatants were collected from cell culture well to measure cytokine concentration by ELISA. The protocol was followed by Duoset, R&D Systems ( Minneapolis, MN, USA). First, ELISA plates (Nunc, Roskilde, Denmark) were coated with diluted capture antibody, 100 μL per well, at room temperature overnight . The next day, the plate were washed three times and blocked with blocking buffer. After washing, sample and standards were added at 100 μL per well and incubated 2 hours at
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room temperature. Plates were washed and incubated with 100μL per well of diluted detection antibody. After two hours at room temperature, diluted streptavidin-HRP were added (100 μL per well) and then 1:1 mixture of H2O2 and Tetramethylbenzidine were used as the substrate. At last, H2SO4 were used as stop solution (50 μL per well). The OD450 and OD540 were read by using a microplate reader (Anthosreaser 2010, Austria), and the cytokine levels were determined by appropriate cytokine-specific standard curve.
Flow cytometry analysis
CD25 & Foxp3 expression of T cell
The cultured CD4 T cells were harvested and washed twice with FACS buffer, and then they were stained with anti-CD25 antibody (1μL /106 cells) at 4℃ for 30 minutes.
After washing to remove extra antibodies with FACS buffer, cells were resuspended with 1X FOXP3 Fix/Perm solution 1ml, and incubated at room temperature in the dark for 20 minutes. After removing the supernatant, the cells were washed once with FACS buffer and also with 1X FOXP3 Perm solution, then re-suspended with 1X FOXP3 Perm solution 1ml per tube, and incubated at room temperature in the dark for 15 minutes. Afterward, the supernatants were removed and added appropriate amount of anti-Foxp3 antibody (20μL /106 cells) and incubate at room temperature in the dark for
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30 minutes. After washing twice with FACS buffer, cells were re-suspended in appropriate volume of FACS buffer for analyzing on FACSort cell analyzer (Becton-Dickinson).
MHC class II & CD11c & CD80 & CD86 expression of DC
Cultured DCs were harvested on day 8. In order to remove the culture medium, cells were washed twice with FACS buffer. Antibodies (anti-MHC class II, anti-CD11c, anti-CD80, anti-CD86, 1μL/106 cells) were added for staining. After incubated at 4℃
for 30 minutes, cells were further washed twice with FACS buffer and re-suspended in appropriate volume with FACS buffer for analyzing on FACSort cell analyzer (Becton-Dickinson).
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CHAPTER III
RESULTS
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Part I. Pentoxifylline (PTX)