Comparison of two commonly used methods for stimulating T cells
- 149 Downloads
Although several methods have been reported and used for in vitro T cell amplification, there are no consistent reports on the optimal stimulation conditions and the characterization of these stimulated T cells. The current study aimed to determine the optimal conditions for efficient T cell amplification by two commonly used methods involving CD3/CD28 antibody and phytohemagglutinin (PHA), respectively.
Orthogonal design and CCK8 assay showed that 5 μg/mL CD3, 5 μg/mL CD28, and 100 ng/mL IL2 for the first method and 50 μg/mL PHA for the second method was optimal for T cell stimulation. Flow cytometry demonstrated that the percentage of CD8+ in the stimulated groups significantly increased, while the percentage of CD4+/CD8+ was significantly decreased compared with the unstimulated group. The percentage of CD4+ showed no significant difference among the three groups. Notably, there was no significant difference between the two stimulated groups. In addition, the percentage of apoptotic cells was significantly increased in the stimulated groups compared with the unstimulated group, but showed no remarkable difference between the PHA and CD3/CD28 stimulation groups. Glycolysis analysis showed that the glycolytic capacity and glycolytic reserve were both significantly increased in the PHA and CD3/CD28 groups compared with the unstimulated group, with no significant difference noted between the stimulated groups.
Although both stimulation methods showed similar efficacies, we suggest the PHA method might be better considering its easy application and cost-effective nature.
KeywordsApoptosis CD3/CD28 CD4+/CD8+ Glycolysis PHA T lymphocytes
This research was supported by National Nature Science foundation of China (Grant Nos. 81602768 and 81803146), and applied basic research foundation of Shanxi province (Grant No. 201801D221441).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Hombach A et al (2001) Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule. J Immunol 167:6123–6131CrossRefGoogle Scholar
- Jorgensen JL, Reay PA, Ehrich EW, Davis MM (1992) Molecular components of T-cell recognition. Annu Rev Immunol 10:835–873. https://doi.org/10.1146/annurev.iy.10.040192.004155 CrossRefPubMedGoogle Scholar
- Pandit H, Thakur G, Koippallil Gopalakrishnan AR, Dodagatta-Marri E, Patil A, Kishore U, Madan T (2016) Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells. Immunobiology 221:310–322. https://doi.org/10.1016/j.imbio.2015.10.004 CrossRefPubMedGoogle Scholar
- Skendros P, Sarantopoulos A, Tselios K, Boura P (2008) Chronic brucellosis patients retain low frequency of CD4+ T-lymphocytes expressing CD25 and CD28 after Escherichia coli LPS stimulation of PHA-cultured PBMCs. Clin Dev Immunol 2008:327346. https://doi.org/10.1155/2008/327346 CrossRefPubMedGoogle Scholar
- Toth R, Szegezdi E, Molnar G, Lord JM, Fesus L, Szondy Z (1999) Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively. Eur J Immunol 29:383–393. https://doi.org/10.1002/(SICI)1521-4141(199902)29:02%3c383:AID-IMMU383%3e3.0.CO;2-A CrossRefPubMedGoogle Scholar
- Wang H, Daniel V, Sadeghi M, Opelz G (2013) Differences in the induction of induced human CD4(+) CD25(+) FoxP3(+) T-regulatory cells and CD3(+) CD8(+) CD28(-) T-suppressor cells subset phenotypes in vitro: comparison of phorbol 12-myristate 13-acetate/ionomycin and phytohemagglutinin stimulation. Transpl Proc 45:1822–1831. https://doi.org/10.1016/j.transproceed.2012.10.061 CrossRefGoogle Scholar