Hello Lily Watson

Yes, Jurkat cells can be activated by anti-CD3 and anti-CD28 antibodies. But even though they exhibit some signaling pathways similar to the primary human T cells, there are notable differences in their activation mechanisms and signaling responses.

Ø When we treat Jurkat cells with anti-CD3 or anti-CD28 antibodies, do they trigger the same signaling pathways as in regular human T cells, like IL-2 production?

Jurkat cells and primary human T cells do activate similar signaling pathways when stimulated with anti-CD3 and anti-CD28 antibodies, including those involved in IL-2 production. However, there might be some differences in the intensity or nuances of these pathways, especially when it comes to CD28 signaling. While both cell types activate NF-κB via CD28, Jurkat cells and primary T cells may rely on slightly different signaling modules downstream of CD28 for NF-κB activation. Primary T cells might rely more heavily on adaptor proteins like ADAP for NF-κB activation by anti-CD3, while Jurkat cells may have a more direct route. Both cell types can produce IL-2 in response to combined CD3 and CD28 stimulation, though the absolute levels and kinetics of IL-2 production can differ between Jurkat cells and primary T cells.

Ø Do Jurkat cells respond to these antibodies in the same way as primary T cells, or are there differences in how the signals get transmitted or how the cells behave?

While both cell types are T lymphocytes and share the same core receptor machinery (CD3 and CD28), their specific responses to activation stimuli can be different. Jurkat cells, as a cell line, may have undergone genetic changes or adaptations that alter their signaling pathways and functional outcomes compared to primary T cells. For instance, as far as signaling pathway is concerned, while both cell types utilize the same receptors (CD3 and CD28) for activation, the downstream signaling cascades can differ. For example, the activation of MAPK pathways like ERK phosphorylation can be differentially regulated in Jurkat cells compared to primary T cells. Also, as far as proliferation is concerned, while both cell types can proliferate upon stimulation, the extent and kinetics of proliferation might vary. Jurkat cells have been shown to exhibit specific patterns of cell cycle progression (S and G2 phase) upon stimulation.

Ø Except for using antibodies, are there any special conditions or other tweaks needed to get Jurkat cells activated?

Yes. The ratio of anti-CD3 to anti-CD28 antibodies can influence the strength of T cell activation. I came across one study (see the article attached below) that showed a 2:1 mass ratio of CD3 to CD28 antibodies on magnetic beads led to the most significant T cell expansion. So, carefully titrating the antibody concentrations can optimize the activation process.

Article Enhancing the activation of T cells through anti-CD3/CD28 ma...

Another factor is the substrate stiffness. T cell activation can be influenced by the stiffness of the substrate the cells are cultured on. This mechano sensitivity can affect cytoskeletal forces and signal transduction. You may want to refer to the article attached below which showed that Jurkat cells exerted different forces on substrates of varying stiffness, which correlated with the efficacy of signal transduction.

Article Cytoskeletal forces during signaling activation in Jurkat T-cells

Protocol for activation of Jurkat cells using anti-CD3 and anti-CD28

1a. First and foremost, prepare the antibody solutions. Prepare a solution of anti-CD3 antibody (a concentration range of 1-3 µg/ml) and anti-CD28 antibody (a concentration range of 3-5 µg/ml) in culture medium.

1b. If you are using beads for activation, coat them with anti-CD3 and anti-CD28 antibodies. This can be done by incubating the beads with a solution of the antibodies in PBS at 4°C overnight.

2. Seed Jurkat cells in a suitable culture vessel, such as a well plate at a concentration of 2 x 10^6 cells/ml or 1 x 10^6 cells/ml depending on your requirements.

3a. Add the prepared antibody solution from step 1 to the cells in the culture well plate.

3b. If you are using beads, then add the antibody-coated beads to Jurkat cells in a 1:2 ratio (cells to beads). Please note that when using beads, the ratio of beads to cells can also influence the activation level.

4. Incubate the cells at 37°C in a humidified incubator with 5% CO2. The incubation time is generally 45 minutes to several hours. If you wish to keep it for longer incubation (say, 24-72 hours), then you may need to add IL-2 for optimal cell expansion. I would suggest you adjust the incubation time based on your experimental needs. Shorter incubation (like 45 minutes) is suitable for studying early signaling events, while longer incubation (such as 24-72 hours) may be needed for cell proliferation and cytokine production.

5. After the desired incubation time, stop the reaction by adding ice-cold PBS to the cells and wash them and later centrifuge at 300g for 5 minutes.

6. Resuspend the cells in PBS or as per the desired buffer for further analysis.

Best,

Lily Watson ,

As a tumor cell line, Jurkat cells cannot directly simulate the physiological state of primary T cells, so the effect of using T cell antibodies to activate Jurkat cells may be limited.

According to literature reports (PMID: 17706604), antibodies that activate human T cells in vitro (such as anti-CD3, anti-CD28) can cause Jurkat cells to produce responses similar to T cell activation.

In addition, overexpression of FOXP3 causes Jurkat cells to exhibit phenotypic characteristics of regulatory T cells (Treg). Therefore, it is necessary to avoid overexpression of FOXP3, otherwise it will not only fail to activate Jurkat cells, but will inhibit their activation.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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