Anti cd28 cd49d antibodies

Manufactured by BD

Anti-CD28/CD49d antibodies are laboratory reagents used to activate and stimulate T cells. These antibodies bind to the CD28 and CD49d receptors on the surface of T cells, providing co-stimulatory signals that enhance T cell proliferation and cytokine production.

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Lab products found in correlation

Ifn γ apc, by BioLegend (2 mentions) Tnf α pe, by BD (2 mentions) Cytofix cytoperm, by BD (2 mentions) Golgiplug, by BD (2 mentions) Trypan blue, by Thermo Fisher Scientific (1 mentions) Cd40l, by BD (1 mentions) Staphylococcus enterotoxin b, by Merck Group (1 mentions) Cd4 percp cy5, by BD (1 mentions) Cd3 apc h7, by BD (1 mentions) Cd8 pe cy7, by Beckman Coulter (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Canto 2 flow cytometer, by BD (1 mentions) Penicillin, by Lonza (1 mentions) Streptomycin, by Lonza (1 mentions)

7 protocols using anti cd28 cd49d antibodies

Intracellular Cytokine Staining of T Cells

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For intracellular cytokine staining, T cells were rested with IL-2 (100 U/mL) overnight and then stimulated with PepMixes encompassing viral or control proteins (JPT) at 0.5 nmol/μL, along with anti-CD49d/CD28 antibodies (BD Biosciences) at 1:1,000 for co-stimulation and Golgi stop at 1 μL/mL. SEB and Actin were utilized as controls. T cells were stained with cell surface markers (CD3, CD4, and CD8), followed by permeabilization with Cytofix/Cytoperm (BD Biosciences), washing, and staining with IFN-γ-APC (BioLegend), IL-2-fluorescein isothiocyanate (FITC), and TNF-α-PE (BD Biosciences).

Dave H., Luo M., Blaney J.W., Patel S., Barese C., Cruz C.R., Shpall E.J., Bollard C.M, & Hanley P.J. (2017). Toward a Rapid Production of Multivirus-Specific T Cells Targeting BKV, Adenovirus, CMV, and EBV from Umbilical Cord Blood. Molecular Therapy. Methods & Clinical Development, 5, 13-21.

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Stimulation and Profiling of T-cells

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T-cells were rested with IL-2 (100U/ml) overnight and then stimulated with pepmixes encompassing viral or control proteins (JPT) at 0.5 nmol/ul, along with anti-CD49d/CD28 antibodies (BD) at 1:1000 for co-stimulation and Brefeldin A at 1ul/ml. SEB and Actin were utilized as controls. T-cells were stained with cell surface markers (CD3, CD4, CD8), followed by permeabilization with Cytofix/Cytoperm (BD FastImmune), washing, and staining with IFN-γ-APC (Biolegend, San Diego, CA), IL-2-FITC, and TNFα-PE (BD).

McLaughlin L.P., Lang H., Williams E., Wright K.E., Powell A., Cruz C.R., Colberg-Poley A.M., Barese C., Hanley P.J., Bollard C.M, & Keller M.D. (2016). Human Parainfluenza Virus-3 can be Targeted by Rapidly ex vivo Expanded T-Lymphocytes. Cytotherapy, 18(12), 1515-1524.

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Screening TCR-transduced PBMCs for Antigen Reactivity

Cited 2 times

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To measure antigen-specific reactivity of recovered TCR sequences, TCRs were expressed and screened in Jurkat-NFAT-GFP cells as described previously (34 (link)). Paired TCR alpha and beta chains of interest were cloned into a retroviral pMSGV construct as previously described (17 (link)). PBMCs for retroviral transduction were processed and cultured according to our rececent publication(19 (link), 34 (link)). To assess function of the transduced TCRs in human PBMCs, TCR expressing cells were mixed with K562-A2 cells at a ratio of 1:2 (Effector:Target) in the RPMI media and supplemented with 1 μg/ml of anti-CD28/CD49d antibodies (BD Biosciences, 347690) and 1 μg/ml of cognate peptides. For PBMCs, supernatants were collected after 48 hours and analyzed by ELISA (BD Biosciences) to estimate IFN-γ concentration. PBMCs transduced by the vector without a TCR was used as a negative control.

Koo D., Mao Z., Dimatteo R., Tsubamoto N., Noguchi M., McLaughlin J., Tran W., Lee S., Cheng D., de Rutte J., Sojo G.B., Witte O.N, & Di Carlo D. (2023). Defining T cell receptor repertoires using nanovial-based affinity and functional screening. bioRxiv.

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Stimulation and Immunophenotyping of PBMCs

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PBMC were suspended in culture medium (complete medium) for viability test with trypan blue (Gibco) and rested in an incubator overnight at 37°C, with 5% CO2. After counting, cells were suspended in culture medium and stimulated with 4μg/ml of GMZ2 vaccine antigen (Henogen S.A. Belgium) for 18h for the T cell panel using 1μg/ml of Staphylococcus Enterotoxin B (Sigma-Aldrich) as positive control, and 6h for the B cell panel using 10μg/ml CPG (Invivogen) + 50ng PMA (Thermo Fisher) + 1μg IONO (Thermo Fisher) as positive control. Unstimulated cells as negative control were incubated in culture medium alone. For the T and B cell panels, cells were cultured in the presence of anti-CD28/CD49d antibodies (BD Biosciences) and CD40L (BD Biosciences) respectively at a concentration of 1μg/ml. In order to assess Forkhead box P3 (Foxp3) expression and IL-10 production, 1μg/ml of Golgi Plug (BD Biosciences) was added two hours after PBMC stimulation. Each plate used for cell stimulation included PBMC isolated both on D0 and on D84.

Nouatin O., Ngoa U.A., Ibáñez J., Dejon-Agobe J.C., Mordmüller B., Edoa J.R., Mougeni F., Brückner S., Hounkpatin A.B., Esen M., Theisen M., Moutairou K., Hoffman S.L., Issifou S., Luty A.J., Loembe M.M., Agnandji S.T., Lell B., Kremsner P.G, & Adegnika A.A. (2020). Effect of immune regulatory pathways after immunization with GMZ2 malaria vaccine candidate in healthy lifelong malaria-exposed adults. Vaccine, 38(27), 4263-4272.

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Phospho-Signaling Pathway Analysis in Activated PBMCs

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Peripheral blood mononuclear cells (PBMCs) were separated on Ficoll-Paque (GE Healthcare) according to the manufacturer's instructions. PBMCs were reconstituted in a culture medium consisting of RPMI 1640 with 25 mM HEPES, L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Lonza, Basel, Switzerland) to a final concentration of 2 million cells per milliliter. After a 1-h rest at 37°C in a 5% CO₂ atmosphere, the cells were stimulated on 96-well plate containing coated anti-CD3 (10 μg/ml, Exbio Praha) and free costimulatory anti-CD28/CD49d antibodies (1 μg/ml, BD Biosciences) for 5, 15, and 30 min. The cells were fixed with 4% formaldehyde for 10 min and permeabilized with ice-cold methanol for 30 min. The following fluorochrome conjugates were used for cytometric detection: phospho-Akt (Ser473)-Alexa Fluor 488, phospho-S6 (Ser235/236)-Pacific Blue (Cell Signaling Technologies), phospho-mTOR (Ser2448)-PE (eBioscience, Thermo Fisher), CD45-Pacific Orange, CD45RA-APC (Exbio), CD8-PE-Cy7 (Beckman Coulter), CD4-PerCP-Cy5.5, and CD3-APC-H7 (BD Biosciences). The samples were acquired on Canto II flow cytometer and analyzed using FlowJo software (BD Biosciences).

Polaskova K., Merta T., Martincekova A., Zapletalova D., Kyr M., Mazanek P., Krenova Z., Mudry P., Jezova M., Tuma J., Skotakova J., Cervinkova I., Valik D., Zdrazilova-Dubska L., Noskova H., Pal K., Slaby O., Fabian P., Kozakova S., Neradil J., Veselska R., Kanderova V., Hrusak O., Freiberger T., Klement G.L, & Sterba J. (2020). Comprehensive Molecular Profiling for Relapsed/Refractory Pediatric Burkitt Lymphomas—Retrospective Analysis of Three Real-Life Clinical Cases—Addressing Issues on Randomization and Customization at the Bedside. Frontiers in Oncology, 9, 1531.

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PBMC Stimulation and Cytokine Assay

Cited 1 time

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The different steps of PBMC isolation after sample collection, and of PBMC stimulation are described in detail elsewhere [26 (link)]. Briefly, after counting, cells were suspended in the culture medium and stimulated for 18 h with either the vaccine antigen (GMZ2, 4 µg/ml, Henogen S.A. Belgium) or staphylococcal enterotoxin B (SEB, 1 µg/ml, Sigma-Aldrich) as positive control. Unstimulated cells as negative control were incubated in culture medium alone. The culture was done in the presence of anti-CD28/CD49d antibodies (BD Biosciences). Two hours after PBMC stimulation, 1 µg/ml of Golgi Plug (BD Biosciences) was added. Each plate used for stimulation included PBMC isolated both on D0 and on D84 from the same participant. The working conditions remained stable for all samples.

Nouatin O., Ibáñez J., Fendel R., Ngoa U.A., Lorenz F.R., Dejon-Agobé J.C., Edoa J.R., Flügge J., Brückner S., Esen M., Theisen M., Hoffman S.L., Moutairou K., Luty A.J., Lell B., Kremsner P.G., Adegnika A.A, & Mordmüller B. (2022). Cellular and antibody response in GMZ2-vaccinated Gabonese volunteers in a controlled human malaria infection trial. Malaria Journal, 21, 191.

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Peptide-Specific T Cell Coculture

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For peptide pulsing coculture experiments, target cells were mixed with TCR-engineered PBMCs at a ratio of 1:2 (T:E) in the media desired by target cells and supplemented with 1 µg/mL of anti-CD28/CD49d antibodies (BD Biosciences). For cell lines expressing full-length PAP, target cells were first treated with 2 ng/mL IFNγ and 3 ng/mL TNFα for 8 h to 10 h. Target cells and PBMCs were then mixed at a ratio of 1:16 (T:E) for coculture analysis. Supernatants were collected after 48 h and analyzed by ELISA (BD Biosciences) to estimate IFNγ concentration.

Mao Z., Nesterenko P.A., McLaughlin J., Deng W., Burton Sojo G., Cheng D., Noguchi M., Chour W., DeLucia D.C., Finton K.A., Qin Y., Obusan M.B., Tran W., Wang L., Bangayan N.J., Ta L., Chen C.C., Seet C.S., Crooks G.M., Phillips J.W., Heath J.R., Strong R.K., Lee J.K., Wohlschlegel J.A, & Witte O.N. (2022). Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation. Proceedings of the National Academy of Sciences of the United States of America, 119(31), e2203410119.

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