Five to 6 weeks female NOD/ShiJic-scidJcl mice (NOD-SCID) (Japan CLEA) were used for efficacy studies. A-549 human alveolar lung adenocarcinoma cells were harvested from cell culture dishes by trypsinization, washed with PBS and prepared as cell suspension. Human PBMCs (Cellular Technology Limited) were cultured in vitro in the presence of Dynabeads human T-activator (CD3/CD28) (Veritas Labettor B.V. Groningen, Netherlands EU) for 4 days before inoculation in mice with A-549 cells. Cell suspensions of 5 × 106 A-549 cells were mixed with equal number of activated hPBMCs (the ratio of implanted activated hPBMCs to A-549 cancer cells was 1:1). Mixed cell suspensions were subcutaneously transplanted in right flank of NOD-SCID mice (day 0) and intravenous treatments of indicated tribodies at the indicated dosage were performed at day 0, 2, 4, 6, 8, 10 (total 6 treatment) after the cell transplantation. Tumor growth was measured with calipers in 2 perpendicular dimensions, and tumor volume (mm3) was calculated by using the formula (width2 x length) x π/6. Tumor volumes were expressed as mean ± standard deviation (SD). Statistical significances of tumor size differences on the final day of each study were evaluated by Dunnett test (vs vehicle treatment group), and P < 0.05 was mentioned as significant difference.
Human pbmcs
Manufactured by Cellular Technology
Sourced in United States
Human PBMCs are a type of laboratory cell product derived from human peripheral blood. They consist of a mixed population of mononuclear cells, including lymphocytes and monocytes. PBMCs are commonly used in various in vitro research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 28 beads, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Complete rpmi 1640 medium, by Merck Group (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Streptavidin coated microbeads, by Miltenyi Biotec (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Interleukin 6 (il 6), by Merck Group (1 mentions)
24 well transwell plate, by Corning (1 mentions)
Easysep mouse t cell isolation kit, by STEMCELL (1 mentions)
A23187, by Merck Group (1 mentions)
Elisa kit, by R&D Systems (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Human na ve cd4 isolation kit, by Miltenyi Biotec (1 mentions)
Il 12p70, by Thermo Fisher Scientific (1 mentions)
Il 22, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Vero e6 cells, by Welgene (1 mentions)
6 protocols using human pbmcs
1
Efficacy of Tribodies in Lung Tumor Model
2
Isolation and Differentiation of MDSCs from PBMCs
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Human PBMCs were purchased from Cellular Technology Ltd (CTL). For CD33+ PBMC isolation, cells were labeled with biotinylated anti‐CD33 antibody (Biolegend), incubated with streptavidin‐coated microbeads (Miltenyi Biotec) and separated on an MACS columns (Miltenyi Biotec). CD33+ PBMCs were differentiated into MDSCs in the lower chambers of a 24‐well transwell plate (Corning) in complete RPMI‐1640 medium supplemented with IL‐6 (10 ng/mL, Sigma‐Aldrich) and GM‐CSF (10 ng/mL, PeproTech) for 7 days as previously described.23
3
Influenza Virus T Cell Epitopes
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Eleven de-identified human PBMCs purchased from Cellular Technology Limited were chosen on the basis of positive reactivity (as measured by IFN-γ ELISpot performed by Cellular Technology Limited) to peptides of common influenza virus T cell epitopes, described in Table 1 . Patient demographic data delineated in Table 2 . The country of origin of the human PBMC samples was the United States of America with ten of the eleven samples being collected between May 2006 and September 2012; a single patient in our study donated PBMCs early in February 2013.
4
Evaluating DS-2741a Immunomodulatory Effects
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Human PBMCs were purchased from Cellular Technology Ltd. (OH, USA). Mouse CD3+ T cells were isolated from mouse spleen by using EasySep mouse T‐cell isolation kit (STEMCELL, Vancouver, Canada) according to the manufacturer's protocol.
Jurkat cells, Human PBMCs, or CD3+ T cells of hu‐ORAI1 KI mouse were cultured in 96‐wells microtiter plates containing the indicated concentrations of DS‐2741a or the isotype control IgG1 (Eureka Therapeutics, Inc) diluted in RPMI culture media, and were incubated for 1 hour at 37°C in a humidified incubator containing 5% CO2. The cells were stimulated by 61.7 nmol/L PMA (Phorbol 12‐myristate 13‐acetate, SIGMA) and 141 nmol/L A23187 (SIGMA) or anti‐CD3/28 beads (Thermo Fisher Scientific K.K.) at a bead‐to‐cell ratio of 1:1 for 18 to 24 hours at 37°C in a humidified incubator containing 5% CO2. The supernatants were then collected, and IL‐2 concentration was determined using an ELISA kit (R&D systems, Minneapolis, MN, USA).
Jurkat cells, Human PBMCs, or CD3+ T cells of hu‐ORAI1 KI mouse were cultured in 96‐wells microtiter plates containing the indicated concentrations of DS‐2741a or the isotype control IgG1 (Eureka Therapeutics, Inc) diluted in RPMI culture media, and were incubated for 1 hour at 37°C in a humidified incubator containing 5% CO2. The cells were stimulated by 61.7 nmol/L PMA (Phorbol 12‐myristate 13‐acetate, SIGMA) and 141 nmol/L A23187 (SIGMA) or anti‐CD3/28 beads (Thermo Fisher Scientific K.K.) at a bead‐to‐cell ratio of 1:1 for 18 to 24 hours at 37°C in a humidified incubator containing 5% CO2. The supernatants were then collected, and IL‐2 concentration was determined using an ELISA kit (R&D systems, Minneapolis, MN, USA).
5
T cell Differentiation and Cytokine Inhibition
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Human PBMCs were purchased from Cellular Technology Ltd.. Naïve CD4+ T cells were isolated from PBMCs using a human Naïve CD4 isolation kit (Miltenyi Biotec K.K., Tokyo, Japan) according to the manufacturer's instructions. To initiate differentiation into Th1, Th2, Th22, and Treg cells, the naïve CD4+ T cells (5 × 104/well) were treated with anti‐CD3/28 beads (Thermo Fisher Scientific K.K.) and relevant cytokines (listed below) for 5 days. DS‐2741a or FK‐506 was added 1 hour before the differentiation procedure commenced.
Th1: 5 ng/mL IL‐2 (Peprotech Inc., NJ, USA) and 10 ng/mL IL‐12 p70 (Peprotech Inc.)
Th2: 10 ng/mL IL‐4 (Peprotech Inc.)
Th22: 100 ng/mL TNFα (R&D systems) and 20 ng/mL IL‐6 (Peprotech Inc.)
Treg: 20 ng/mL IL‐2 and 10 ng/mL TGFβ (R&D systems).
To evaluate the inhibitory effects on cytokine production using DS‐2741a or FK‐506, the levels of several cytokines (IFNγ, IL‐13, IL‐31, and IL‐22) were measured using ELISA after treatment with 61.7 nmol/L PMA and 141 nmol/L A23187 for 24 hours (IFNγ, PerkinElmer, Inc. MA, USA; IL‐13 and IL‐31, Thermo Fisher Scientific K.K.; IL‐22, R&D systems). For the evaluation of Treg differentiation, the number of Foxp3+CD4+ cells was determined by flow cytometry.
Th1: 5 ng/mL IL‐2 (Peprotech Inc., NJ, USA) and 10 ng/mL IL‐12 p70 (Peprotech Inc.)
Th2: 10 ng/mL IL‐4 (Peprotech Inc.)
Th22: 100 ng/mL TNFα (R&D systems) and 20 ng/mL IL‐6 (Peprotech Inc.)
Treg: 20 ng/mL IL‐2 and 10 ng/mL TGFβ (R&D systems).
To evaluate the inhibitory effects on cytokine production using DS‐2741a or FK‐506, the levels of several cytokines (IFNγ, IL‐13, IL‐31, and IL‐22) were measured using ELISA after treatment with 61.7 nmol/L PMA and 141 nmol/L A23187 for 24 hours (IFNγ, PerkinElmer, Inc. MA, USA; IL‐13 and IL‐31, Thermo Fisher Scientific K.K.; IL‐22, R&D systems). For the evaluation of Treg differentiation, the number of Foxp3+CD4+ cells was determined by flow cytometry.
6
Macrophage Polarization and Immune Cell Culture
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Human monocytic leukemic cell line THP-1 (Korean Collection for Type Cultures, KCTC; Jeongeup-si, Jeollabuk-do, Korea) was cultured in RPMI1640 medium (Welgene, Republic of Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin/streptomycin (P/S), and 0.5 nM 2-mecaptoethanol. THP-1 monocytes were differentiated into Mɸs by treating with 25 nM PMA for 48 h prior to polarization. Human CD14+ monocytes (PromoCell, Heidelberg, Germany) were cultured with 50 ng/mL of GM-CSF or M-CSF for seven days to achieve differentiation. All Mɸs were polarized in vitro with either LPS (20 pg/mL; Invitrogen, USA) and IFN-γ (20 ng/mL) or IL-4 (20 ng/mL) and IL-13 (20 ng/mL) (Peprotech, USA) for 24 h. Cells were washed thrice with phosphate-buffered saline (PBS) to remove any remaining cytokines. Human PBMCs were purchased from Cellular Technology Limited (CTL; OH, USA). Vero E6 cells were purchased from KCTC and maintained in minimal essential medium (MEM; Welgene, Daejeon, Korea) supplemented with 10% FBS and 1% P/S.
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