Anti cd28 mab

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Anti-CD28 mAb is a monoclonal antibody that targets the CD28 receptor on T cells. It is a laboratory tool used in immunology research to study T cell activation and function.

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18 protocols using anti cd28 mab

HBV-specific T cell response assay

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Spleens, mesenteric lymph nodes and liver were isolated from individual animals and 0.5×10⁶ human spleen plus mesenteric lymph node or liver leukocytes were stimulated for 20 hours with phytohemagglutinin (PHA) or HBV antigens [HLA2 core (18–27), envelope (183–191, 185–194, 172–181), polymerase (573–581) peptides (ProImmune) +/− recombinant HBc and HBs (ProSpec) at 10 ug/ml each]+1 ug/ml anti-CD28 mAb (Invitrogen) in IMDM+10% FBS media (Gibco). The cells were then cultured for 14 days with fresh media replaced every two days (IMDM, 10% FBS, 10 U/mL human IL-2 and 125 ng/mL IL-7; human T cell expansion was examined by FACs (Guava, Millipore) using CD45, CD3 and live cell marker. Additionally, cells were stain for HBV core (18–27) or HBV envelope (183–191) pentamer+ human CD8+ T cells (ProImmune). PHA expanded cells were re-stimulated with Phorbol 12-myristate 13-acetate (PMA) plus ionomycin for approximately 6 hours and cytokine secretion was block with brefeldin A; intracellular cytokine levels were examined using FACs.

Bility M.T., Cheng L., Zhang Z., Luan Y., Li F., Chi L., Zhang L., Tu Z., Gao Y., Fu Y., Niu J., Wang F, & Su L. (2014). Hepatitis B Virus Infection and Immunopathogenesis in a Humanized Mouse Model: Induction of Human-Specific Liver Fibrosis and M2-Like Macrophages. PLoS Pathogens, 10(3), e1004032.

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Platelet-T Cell Interaction in Pediatric Immune Response

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Separated T cell activation was induced by coated anti-CD3 monoclonal antibody (MAb) (1 μg/mL, 500 μL/well; 3 h, 37 °C) and soluble 1 μg/mL anti-CD28 MAb (all from Invitrogen, Carlsbad, CA, USA). Platelets and CD4⁺ T cells were cultured alone or in combination in RPMI-1640 medium (Biological Industries, Kibbutz Beit Haemek, Israel) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Sangon Biotech, Shanghai, China) at naive CD4⁺ T cell–platelet ratios of 1:0, 1:20, 1:40, 1:80, and 1:200. Platelets come from healthy children, mild children, and severe children. Cells were cultured at 37 °C with 5% CO₂ for up to 3 days.

Li Q., Wang Y., Xue W., Bian Z., Gao Y., Zeng Y., Tang L., Tang T., Tian Y, & Guo W. (2020). Immunomodulatory effects of platelets on the severity of hand, foot, and mouth disease infected with enterovirus 71. Pediatric Research, 89(4), 814-822.

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CFSE-Based T Cell Proliferation Assay

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Splenic T cells were isolated from OT-II mice and purified by nylon wool. 0.2×10⁶ CFSE labeled T cells (1.5 μM, Molecular Probes, Eugene, OR, USA) were cultured with WT DCs (1:10, DC/RA-DC:T) and OVA protein (100 μg/mL; Worthington Biochemical Corporation, Lakewood, NJ, USA). For antigen pulsed experiments, DCs and RA-DCs were incubated with OVA protein (400 μg/mL, 18 hrs; Worthington Biochemical Corporation, Lakewood, NJ, USA) or ovalbumin peptide OVA_323–339 (0.6 μM, 3hrs). In most conditions, additional DCs or RA-DCs were added into co-cultures (1:10, DC/RA-DC:T). Cultures were maintained in 10% FBS complete RPMI-1640 medium for 3 days at 37°C, 5% CO₂. T cell proliferative response was determined by CFSE dilution assay and analyzed by flow cytometry. For anti-CD3/CD28 studies, T cells were added to an anti-CD3 mAb coated plate (1 μg/mL, overnight; eBiosciences, San Diego, CA, USA) followed by addition of anti-CD28 mAb (1μg/mL; eBiosciences). To inhibit Arg-1 or iNOS activity, N^w-hydroxy nor-L-arginine (nor-NOHA) or N^G-monomethyl-L-arginine, monoacetate salt (L-NMMA) (Caliobiochem, San Diego, CA, USA) were added at the beginning of co-culture to achieve a final concentration of 300 μM. L-arginine (Sigma Aldrich) was added in excess at the time of co-culture to achieve a final concentration of 3 mM.

Bhatt S., Qin J., Bennett C., Qian S., Fung J.J., Hamilton T.A, & Lu L. (2014). All-trans retinoic acid induces Arg-1 and iNOS producing dendritic cells with T cell inhibitory function. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5098-5108.

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PBMC Activation and Expansion for ATC Generation

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Peripheral blood mononuclear cells (PBMCs) were separated immediately by Ficoll-Hypaque density gradient centrifugation. Blood was obtained from healthy donors which supplied by the Beijing Blood Bank. PBMCs were cultured at 1×10⁶/ ml in RPMI-1640 medium supplemented with 10% FBS and 5 μg/ml anti-CD3 mAb (eBioscience, San Diego, CA, USA) combined with 5 μg/ml anti-CD28 mAb (eBioscience) in the presence of 100 IU/ml recombinant human IL-2 (Peprotech, Rocky Hill, NJ, USA). IL-2 was added every 2 or 3 days to stimulate ATC cells and the cells were cultured for 14 days. On day 14, ATC expansion products of healthy donors were on 98.99% CD3+ cells (2.66% CD3+CD4+ cells, 85.95% CD3+CD8+ T cells, and 16.16% CD3+CD56+T cells), the cells were used immediately or cryopreserved for further use.

Ma W., Ma J., Lei T., Zhao M, & Zhang M. (2019). Targeting immunotherapy for bladder cancer by using anti-CD3 × CD155 bispecific antibody. Journal of Cancer, 10(21), 5153-5161.

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Isolation and Differentiation of CD4+ T Cells

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Peripheral blood cells were collected and isolated with Ficoll-Paque^TM (GE healthcare, Uppsala, Sweden) as described previously.19 (link) Briefly, peripheral blood (3 mL) diluted with sterile PBS (3 mL) was added to equal volume Ficoll. The tubes were vortexed for 30 minutes under 2,000 rpm at 20°C. PBMCs were collected and incubated with CD4⁺ microbeads for 30 minutes at 4°C. CD4⁺ T-cell clones were expanded after being stimulated with plate-bound anti-CD3 mAb (5 mg/mL), and anti-CD28 mAb (2 mg/mL, eBioscience, San Diego, CA), followed by being stimulated with recombined human (rh) TGFβ (20 ng/mL), rh IL-6 (30 ng/mL), anti-IFNγ (20 µg/mL), and anti-IL-4 (20 µg/mL) with PBS or TWP (1.25 mg/mL) for 72 hours. Differentiated CD4⁺ T-cells were collected for qRT-PCR. CD4⁺ T-cells were stimulated with mentioned cocktails for 5 days followed by being stimulated with PMA (50 ng/mL, Sigma-Aldrich, St Louis, MO, USA), ionomycin (750 ng/mL, Sigma-Aldrich), and Brefeldin A (3 μg/mL, eBioscience) for 5 hours. Differentiated CD4⁺ T-cells were collected for cell membrane and intracellular staining, eg, CD25, IFNγ, IL-17A, and FOXP3 for flow cytometer analysis.

Zhang C., Ju J., Wu X., Yang J., Yang Q., Liu C., Chen L, & Sun X. (2021). Tripterygium wilfordii Polyglycoside Ameliorated TNBS-Induced Colitis in Rats via Regulating Th17/Treg Balance in Intestinal Mucosa. Journal of Inflammation Research, 14, 1243-1255.

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Generation of Chimeric Antigen Receptor-Expressing CIK Cells

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Human PBMCs were isolated from blood samples of healthy donors using density gradient centrifugation with Ficoll-Hypaque (TAKARA). To generate CIK cells, PBMCs were initially cultured at a density of 2 × 10⁶ cells/ml in serum-free X-Vivo15 medium (Lonza) supplemented with IFN-γ (1,000 U/ml, Peprotech). After 24 h, anti-CD3 mAb (OKT3, 100 ng/mL, eBioscience), anti-CD28 mAb (100 ng/mL, eBioscience) and IL-2 (500 U/ml, Peprotech) were added. After an additional 48 h of cultivation, 5 × 10⁵ CIK cells were mixed with 400 ng of concentrated lentiviral particles, seeded into a 24-well cell culture plate and centrifuged at 1,000 g for 1 h in the presence of protamine sulfate (8 μg/ml, Sigma). Then, the plate was incubated for 18 h at 37 °C under 5% CO₂ before the supernatant was replaced with fresh complete medium containing 500 U/ml of IL-2. After 72 h, the GFP-positive cells were sorted using a BD FACSAria^TM IIu flow cytometer. An anti-myc-tag-Alexa Fluor^® 647 antibody (clone#9B11, CST) was used to identify CAR expression on the surface of CIK cells. CAR-expressing CIK cells and NT-CIK cells were propagated at a density of 2 × 10⁶ cells/ml for 2~3 weeks, and cultures were supplemented with fresh medium every 2~3 days. Phenotypic analysis was performed weekly using FACS.

Guo X., Zheng H., Luo W., Zhang Q., Liu J, & Yao K. (2017). 5T4-specific chimeric antigen receptor modification promotes the immune efficacy of cytokine-induced killer cells against nasopharyngeal carcinoma stem cell-like cells. Scientific Reports, 7, 4859.

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Comprehensive T Cell Immunophenotyping

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After careful thawing in 20% FCS/80% IMDM, cells were incubated with FcR Blocking Reagent (Miltenyi Biotec) for 10 min at room temperature. Surface staining of cells was done in PBS containing 0.5% FCS for 15 min at room temperature, except for CCR7, which was stained for 15 min at 37°C. To exclude dead cells from the analysis, Near IR (Life Technologies) was used as a Live/Dead marker. For intracellular staining of transcription factors, ki67 and Granzyme B, cells were fixed and permeabilized after surface staining with the Transcription factor buffer set (BD Pharmingen) following manufacturer's instructions. To measure cytokine production, cells were washed with culture medium and stimulated with 20 ng/ml Phorbol 12‐myristate 13‐acetate (PMA; Sigma) and 1 μM Ionomycin (IM; Sigma) in the presence of Brefeldin A Solution (eBioscience) for 4h at 37°C. Anti‐CD3 mAb (Pelicluster; 0.1 μg/ml) and anti‐CD28 mAb (eBioscience; 0.1 μg/ml) were used to mimic antigen‐specific T cell stimulation and co‐stimulation, respectively. Cells were stimulated with these antibodies in IMDM overnight at 37°C. Data were acquired on an LSR Fortessa cytometer (BD Biosciences), and analyzed using the FlowJo software (version 10; Tree Star). Data were analyzed and reported according to chapter VII “Data handling, evaluation, storage & repositories” of the EJI Guidelines for the use of flow cytometry [37].

Opstelten R., Suwandi J.S., Slot M.C., Morgana F., Scott A.M., Laban S., Nikolic T., Turksma A.W., Kroeze A., Voermans C., Zwaginga J., Roep B.O, & Amsen D. (2021). GPA33 is expressed on multiple human blood cell types and distinguishes CD4+ central memory T cells with and without effector function. European Journal of Immunology, 51(6), 1377-1389.

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T Cell Proliferation Assay for Nf1 Mice

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Following the manufacturer’s recommendations, CD3⁺ T cells were purified from the lymph nodes of Nf1^+/+ and Nf1^+/− mice (age 3–6 months) using pan-T-cell isolation kit (Miltenyi Biotec, Auburn, CA). Purified CD3⁺ T cells (2 × 10⁵/well) were cultured for 2 days in 96-well plates (in triplicate) precoated with indicated doses of anti-CD3 Ab (eBioscience; cat. no. 16-0031-86) alone or anti-CD28 mAb (eBioscience; cat. no. 16-0281-85). After pulsing with ³H-thymidine (1 μCi/well [0.037 MBq/well]) for 20–22 h, cells were collected and evaluated for ³H radioactivity. For each experimental group, the T cell proliferation assay was performed in two pooled biological and three technical replicates.

Brosseau J.P., Liao C.P., Wang Y., Ramani V., Vandergriff T., Lee M., Patel A., Ariizumi K, & Le L.Q. (2018). NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation. Nature Communications, 9, 5014.

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Immunomodulatory Agents in T-cell Activation

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Concanavalin A (ConA) were purchased from Sigma–Aldrich. Anti-CD3ε mAb was purchased from R&D Systems (United States). Anti-CD28 mAb was from eBioscience (United States). ATP, ADP, and adenosine were from Sigma (United States). PPADS, BDBD, MRS2578, SCH442416, PSB36, MRS3777, A438079, CGS15943, PSB603, and MRS2111 were from Tocris Bioscience (United Kingdom). Suramin and oxATP were purchased from Sigma–Aldrich. NF449 was from Abcam (United Kingdom). adenosine 5′-[γ-thio]triphosphate tetralithium salt (ATP-γ-S), α,β-methyleneadenosine 5′-triphosphate lithium salt (α,β-Me-ATP), BzATP, α,β-methyleneadenosine 5′-diphosphate sodium salt (α,β-Me-ADP), and 2-methylthioadenosine diphosphate trisodium salt (2-MeS-ADP) were purchased from Sigma–Aldrich. Anti-ERK1/2 mAb and anti-phospho-ERK 1/2 (Thr²⁰²/Tyr²⁰⁴) mAb and other secondary Abs were obtained from Cell Signaling Technology (United States). All other chemicals used were of the highest purity available.

Shinohara Y, & Tsukimoto M. (2018). Adenine Nucleotides Attenuate Murine T Cell Activation Induced by Concanavalin A or T Cell Receptor Stimulation. Frontiers in Pharmacology, 8, 986.

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CD4+ T Cell Proliferation Assay

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CD4⁺ T cells were cultured in 24-well plates (1 × 10⁶/ml), stimulated with recombinant human IL-2 (10 units/ml, eBioscience) in RPMI 1640. In the IL-2 superimposed TCR stimulation group, anti-CD3 mAb (1 μg/ml, eBioscience, CA, United States) and anti-CD28 mAb (1 μg/ml, eBioscience) were added at the same time as IL-2. After 24, 48, 72, and 96 h, respectively, the stimulated CD4⁺ T cells were seeded into 96-well plates. Then, 10 μl of Cell Counting Kit-8 (CCK-8) reagent was added to each well and incubated at 37°C in the presence of 5% CO₂ for 1 h in the incubator. Absorbance was measured at 450 nm on a microplate reader.

Hua J., Chen Y., Fu B., Chen X., Xu X.J., Yang S.H., Chen C, & Xu Y.J. (2020). Downregulation of p53 by Insufficient CTCF in CD4+ T Cells Is an Important Factor Inducing Acute Graft-Versus-Host Disease. Frontiers in Immunology, 11, 568637.

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