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Gm csf and il 4

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GM-CSF and IL-4 are recombinant cytokines used in cell culture applications. GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) is a protein that regulates the production and function of two types of white blood cells, granulocytes and monocytes. IL-4 (Interleukin-4) is a cytokine that plays a role in the differentiation of naive helper T cells into Th2 cells. These products are intended for research use only and their specific applications should be evaluated by the user.

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Lab products found in correlation

Interleukin 4 (il 4), by Thermo Fisher Scientific (5 mentions) Gm csf, by Thermo Fisher Scientific (5 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Soluble ova protein, by InvivoGen (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Cck 8 kit, by Dojindo Laboratories (2 mentions) Il 1b, by Thermo Fisher Scientific (1 mentions) X vivo 15 media, by Lonza (1 mentions) Tnf α, by Thermo Fisher Scientific (1 mentions) Human ab serum, by Merck Group (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Cd4 t cell purification kit, by BD (1 mentions) E coli 055 b5, by Merck Group (1 mentions) Magnetic bead, by Miltenyi Biotec (1 mentions) Mem neaa, by Thermo Fisher Scientific (1 mentions) Cell counting kit 8 (cck8), by Thermo Fisher Scientific (1 mentions)

15 protocols using gm csf and il 4

1

Inhibition of T Cell Proliferation by Anti-CD28 Domain Antibodies

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Example 11

This example demonstrates that anti-CD28 domain antibodies inhibit T cell proliferation initiated by different antigen presenting cells.

T cells were prepared from E+ fractions of PBMC rosetted with sheep red blood cells (Colorado Serum Company). Dendritic cells (DCs) were generated by adherence of monocytes from E− fractions of PBMC to plastic and culture with GM-CSF and IL-4 (Invitrogen) for 7 days, followed by the addition of LPS (Sigma, 1 μg/ml) for 24 hours to induce maturation. Monocytes were prepared from E− fractions of PBMC by elutriation. The lymphoblastoid cell line (PM-LCL) is an EBV-transformed B-cell line from a normal donor. The various APCs were combined with allogeneic T cells at a ratio of 1:50. Anti-CD28 domain antibodies were titrated in half log dilutions for a nine point dose response curve to evaluate their inhibition of proliferation, which was measured by 3[H]-thymidine incorporation on day 5. EC50 values were generated from inhibition curves of each treatment. Results are shown in Table 10.

TABLE 10
dAbDCsLCL B cellsMonocytes
239-891 (n = 2)0.2, 0.20.3, 0.10.2, 1.4
239-891-D70C P30L PEG0.5 ± 0.10.8 ± 0.32.3 ± 0.8
239-891-D70C P40B PEG1.2 ± 0.12.4 ± 0.410 ± 4 

US10919965B2. Compositions monovalent for CD28 binding and methods of use (2021-02-16). Bristol-Myers Squibb Company [US], Domantis Limited [GB]. Inventors: Murray McKinnon [US], Steven G. Nadler [US], Suzanne J. Suchard [US], Brendan Classon [US], Steve Holmes [GB], Olga Ignatovich [GB], Christopher Plummer [GB], Steve Grant [GB].
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2

Inhibition of T Cell Proliferation by Anti-CD28 Domain Antibodies

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Example 11

This example demonstrates that anti-CD28 domain antibodies inhibit T cell proliferation initiated by different antigen presenting cells.

T cells were prepared from E+ fractions of PBMC rosetted with sheep red blood cells (Colorado Serum Company). Dendritic cells (DCs) were generated by adherence of monocytes from E− fractions of PBMC to plastic and culture with GM-CSF and IL-4 (Invitrogen) for 7 days, followed by the addition of LPS (Sigma, 1 μg/ml) for 24 hours to induce maturation. Monocytes were prepared from E− fractions of PBMC by elutriation. The lymphoblastoid cell line (PM-LCL) is an EBV-transformed B-cell line from a normal donor. The various APCs were combined with allogeneic T cells at a ratio of 1:50. Anti-CD28 domain antibodies were titrated in half log dilutions for a nine point dose response curve to evaluate their inhibition of proliferation, which was measured by 3[H]-thymidine incorporation on day 5. EC50 values were generated from inhibition curves of each treatment. Results are shown in Table 10.

TABLE 10
dAbDCsLCL B cellsMonocytes
239-891 (n = 2)0.2, 0.20.3, 0.10.2, 1.4
239-891-D70C P30L PEG0.5 ± 0.10.8 ± 0.32.3 ± 0.8
239-891-D70C P40B PEG1.2 ± 0.12.4 ± 0.410 ± 4 

US09908937B2. Compositions monovalent for CD28 binding and methods of use (2018-03-06). Bristol-Myers Squibb Company [US], Domantis Limited [GB]. Inventors: Murray McKinnon [US], Steven G. Nadler [US], Suzanne J. Suchard [US], Brendan Classon [US], Steve Holmes [GB], Olga Ignatovich [GB], Christopher Plummer [GB], Steve Grant [GB].
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3

CMV-Specific T Cell Response Assay

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Example 7

CD14+ monocytes and CD3+ T cells were obtained from peripheral blood mononuclear (PBMC) isolated from CMV+ human donors (AllCells, Alameda, Calif.) using MACS Cell Separation kits (Miltenyi Biotec). CD14+ monocytes were differentiated into immature dendritic cells (DC) by culturing cells at 106 cells/ml for 7 days in presence of GM-CSF and IL-4 (Peprotech) in X-Vivo 15 media (Lonza) containing 2% human AB serum (Sigma-Aldrich), penicillin-streptomycin (Corning Mediatech) and GlutaMAX (Life Technologies). Following differentiation, DCs were matured by culturing in X-Vivo 15+2% human AB serum media at 106 cells/ml for 2 days in the presence of GM-CSF, IL-4, TNF-α, IL-1b, IL-6 (Peprotech) and prostaglandin ε2 (Sigma-Aldrich). To set-up the CMV recall assay, mature DCs were collected, washed and 10,000 DCs and 100,000 pan CD3+ T cells were plated per well in a 96-well U-bottom plate in a total volume of 100 μl media containing peptide pools for the CMV IE-1 and CMV pp65 protein (Miltenyi Biotec). IgG antibodies (50 ul) were added starting at a final concentration of 133 nM with 5-fold serial dilutions. Cells were co-cultured with peptides and antibodies for 5-6 days. Conditioned media was collected and tested for human IFN-g levels by ELISA (BD Biosciences).

US11014983B2. Anti-Tim-3 antibodies, compositions comprising anti-Tim-3 antibodies and methods of making and using anti-Tim-3 antibodies (2021-05-25). SUTRO BIOPHARMA, INC. [US]. Inventors: Ryan Stafford [US], Alice Yam [US], John Lee [US], Junhao Yang [US], Aaron Sato [US].
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4

Anti-PD-1 Antibody Enhances T Cell Activation

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Example 22

In this example, effect of blockade of PD-1 pathway by anti-PD-1 antibody 5C4 on T cell activation was examined. Purified human CD4+ T cells (Dynal CD4 T cell purification kit) were activated with 1 μg/ml soluble anti-CD3 antibody (BD) in the presence of autologous monocytes or monocyte-derived dendritic cells (DCs). Monocytes were purified using Miltenyi CD14 monocyte purification kit, and DCs was generated in vitro after culture of monocytes with GM-CSF and IL-4 (PeproTech) for 7 days. After three days of activation in the presence or absence of titrated anti-PD-1 antibody or irrelevant isotype control mAb, culture supernatants were harvested for ELISA analysis of IFNγ secretion while tritiated thymidine was added during the final 18 hours of the assay in order to measure T cell proliferation. The results shown in FIGS. 52A and 52B demonstrate that PD-1 blockade by anti-PD-1 antibody resulted in enhanced T cell proliferation and IFN-γ secretion. Synergic effect by anti-PD-1 antibody and anti-CTLA-4 antibody on T cell activation (specifically on IFN-γ secretion) in the presence of monocytes was also observed.

US10441655B2. Monoclonal antibodies to programmed death 1 (PD-1) (2019-10-15). E.R. SQUIBB & SONS, L.L.C. [US], Ono Pharmaceutical Co., LTD. [JP]. Inventors: Alan J. Korman [US], Mohan Srinivasan [US], Changyu Wang [US], Mark J. Selby [US], Bingliang Chen [US], Josephine M. Cardarelli [US], Haichun Huang [US].
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5

Generating OVA-loaded DCs for Thymic Co-culture

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Bone marrow was flushed from femurs and tibias of mice, followed by RBC lysis using ACK buffer. Cells were resuspended in 10 ng/ml GM-CSF and IL-4 (Peprotech), plated at 0.5 × 106 /ml and cultured for 7 d. A half media change was performed on d2, and full media change with GM-CSF + IL-4 was performed on d5. Semi-adherent cells were isolated on d7, washed, and loaded with soluble OVA protein (1 mg/ml, Invivogen) for 30 min at 37 °C. Cells were washed 3x with HBSS, and overlaid onto thymic slices 4 h after addition of OT-II thymocytes. Cells were allowed to migrate into thymic slices for at least 4h prior to washing off excess. Bone marrow from Il2−/− mice was harvested when mice were < 4 weeks of age, before the onset of pathology.
Weist B.M., Kurd N., Boussier J., Chan S.W, & Robey E.A. (2015). Thymic regulatory T cell niche size is dictated by limiting interleukin 2 from antigen-bearing dendritic cells and feedback competition. Nature immunology, 16(6), 635-641.
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6

Generation of immature and mature dendritic cells from human PBMCs

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DC was generated from peripheral blood mononuclear cells (PBMC) isolated from buffy coats obtained from anonymous healthy blood donors (n = 3). Written informed consent was obtained from blood donors at the Department of Clinical Immunology, University Hospital Rigshospitalet, Copenhagen and used without the possibility to identify case specific information. The ethical committee, Region H, The Capital Region of Denmark, approved the use of these buffy coats for research. The research was carried out in accordance with the approved guidelines. The mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation and subsequently CD14+ monocytes were isolated from PBMCs by positive selection using magnetic beads (Miltenyi Biotec, Germany), according to the manufacturer’s instructions. Monocytes were cultured at 37 °C in 5% CO2 in media supplemented with 10% fetal bovine serum (FBS, Gibco) GM-CSF and IL-4 (50 ng/ml both, PeproTech) for 7 days to generate im-DC and m-DC, after LPS (100 ng/ml, E.coli 055:B5, Sigma-Aldrich) stimulation for the last 24h.
Nastasi C., Candela M., Bonefeld C.M., Geisler C., Hansen M., Krejsgaard T., Biagi E., Andersen M.H., Brigidi P., Ødum N., Litman T, & Woetmann A. (2015). The effect of short-chain fatty acids on human monocyte-derived dendritic cells. Scientific Reports, 5, 16148.
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7

In Vitro and In Vivo Immunomodulation

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DOTAP and CHOL were purchased from Avanti Co., Ltd. (AL, USA; purity ≥ 98%). Dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS) were provided by Sigma-Aldrich (MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, DMEM-F12, penicillin-streptomycin, fetal bovine serum (FBS), MEM NEAA, HEPES, sodium pyruvate, trypsin, and phosphate-buffered saline (PBS) were provided by Thermo Fisher Scientific (Waltham, MA, USA). The APC-CD40, APC-CD80, APC-CD86, FITC-CD11c, PE-MHCII, and FITC-CD8 antibodies were purchased from BD Bioscience Co., Ltd. (USA). GM-CSF and IL-4 were obtained from Pepro Tech (USA). CD8 immunomagnetic bead kits were purchased from Intervention (USA). The Cell Counting Kit-8 from Gibco (USA) and Cyto Tox 96 Non-Radioactive Cytotoxicity Assay Kit was purchased from Promega (Japan).
Hepa1-6 cells were purchased from the Institute of Chinese Academy of Sciences and cultured in a humidified atmosphere of 5% CO2 at 37°C. The cells were cultured in DMEM supplemented with 10% FBS and 100 μg/mL penicillin-streptomycin. C57BL/6 mice were purchased from Jinan Pengyue Laboratories. All animal studies were approved by the Ethics Committee of Liaocheng University. All animal procedures were performed in accordance with the guidelines of the Committee on Animal of Liaocheng University.
Zhang Y., Xie F., Yin Y., Zhang Q., Jin H., Wu Y., Pang L., Li J, & Gao J. (2021). Immunotherapy of Tumor RNA-Loaded Lipid Nanoparticles Against Hepatocellular Carcinoma. International Journal of Nanomedicine, 16, 1553-1564.
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8

Generating OVA-loaded DCs for Thymic Co-culture

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Bone marrow was flushed from femurs and tibias of mice, followed by RBC lysis using ACK buffer. Cells were resuspended in 10 ng/ml GM-CSF and IL-4 (Peprotech), plated at 0.5 × 106 /ml and cultured for 7 d. A half media change was performed on d2, and full media change with GM-CSF + IL-4 was performed on d5. Semi-adherent cells were isolated on d7, washed, and loaded with soluble OVA protein (1 mg/ml, Invivogen) for 30 min at 37 °C. Cells were washed 3x with HBSS, and overlaid onto thymic slices 4 h after addition of OT-II thymocytes. Cells were allowed to migrate into thymic slices for at least 4h prior to washing off excess. Bone marrow from Il2−/− mice was harvested when mice were < 4 weeks of age, before the onset of pathology.
Weist B.M., Kurd N., Boussier J., Chan S.W, & Robey E.A. (2015). Thymic regulatory T cell niche size is dictated by limiting interleukin 2 from antigen-bearing dendritic cells and feedback competition. Nature immunology, 16(6), 635-641.
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9

Isolation and Culture of Murine Immune Cells

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Single cell suspensions were prepared from livers, spleens, pooled peripheral lymph nodes, and/or kidneys of infected or immunized hCD1Tg mice. Lymphocytes were isolated from livers and kidneys using a 37.5% Percoll gradient centrifugation. At times, αβ T cells were enriched through depletion of B220+, CD11c+, F4/80+, Ly-6G+, MHC-II+, and TCRγδ+ T cells using biotin-conjugated antibodies (BioLegend, San Diego, CA) and the Dynabeads depletion kit (ThermoFisher Scientific, Waltham, MA). BMDCs were derived from mouse bone marrow progenitors using GM-CSF and IL-4 (PeproTech) as previously described [74 (link)]. BMM were derived from bone marrow progenitors using complete DMEM supplemented with 30% L929-conditioned medium or 20ng/mL recombinant MCSF (PeproTech).
Visvabharathy L., Genardi S., Cao L., He Y., Alonzo F I.I.I., Berdyshev E, & Wang C.R. (2020). Group 1 CD1-restricted T cells contribute to control of systemic Staphylococcus aureus infection. PLoS Pathogens, 16(4), e1008443.
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10

Generating Bone Marrow-Derived Dendritic Cells

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Female C57BL/6 mice (6–10 weeks old) were obtained from The Jackson Laboratory.
All mice were treated according to protocols approved by the University of
Alberta Animal Care and Use Committee. Bone marrow–derived DCs (BmDC) were
generated using a standard protocol with little modification.23 Briefly, bone marrow was flushed dispersed and collected from femurs and
tibias of Female C57BL/6 mice, passed through a 70 µm nylon mesh, and suspended
in bone marrow–derived DC-complete media (RPMI 1640 containing 5 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 U Pen/Strep, 2 mM
glutamine, 50 µM 2-ME, 50 mM gentamycin sulfate, and 10% fetal bovine serum
(FBS)) in the presence of granulocyte-macrophage colony-stimulating factor
(GM-CSF) and IL-4 (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) and cultured in
tissue culture dishes (Thermo Fisher, Carlsbad, CA, USA) in a humidified
atmosphere of 5% CO2 in air at 37oC. All media components,
except for GM-CSF and IL-4, were obtained from Gibco (Carlsbad, CA, USA). During
culture, half of the media was replaced on days 3 and 6. On day 8, BmDC were
harvested, and their morphology was confirmed by optical microscopical analysis
(Supplementary Figure 1).
Arizmendi N., Hou C., Guo F., Li Y, & Kulka M. (2018). Bicyclic eremophilane-type petasite sesquiterpenes potentiate peroxisome proliferator–activated receptor γ activator–mediated inhibition of dendritic cells. International Journal of Immunopathology and Pharmacology, 32, 2058738418787739.
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