PHA and PMA/Iono act on cells in a quite different manner.
PHA (Phytohaemaglutinin) is a lectin, binds to the sugars on glycosylated surface proteins, including the TCR, and thereby crosslinks them. So with PHA you get TCR crosslinking and signal 1 (and possibly also signal 2 via crosslinking of co-stimulatory molecules) required for T cell activation. These processes happen on the cell surface and might therefore involve a variety of signalling pathways.
PMA on the other hand is a small organic compound which diffuses through the cell membrane into the cytoplasm, where it directly activates Protein Kinase C (PKC) omitting the ‘need’ of surface receptor stimulation. Ionomycin, a calcium ionophor, is used in addition to trigger calcium release which you need for NFAT signalling.
In addition, LPS is a TLR-4 and CD14 ligand. The main receptor for bacterial LPS seems to be CD14, so if you are using that to stimulate T cells, make sure you are just using T cells. It may also be important to note that soluble CD14 is present in the circulation and has been shown to inhibit T cell activation. TLR-4 activation has been shown to stimulate autophagy by macrophages, and to affect a pro-inflammatory response in T cells. The TLRs are present on many cell types but seem to correlate to innate immune pathways. As such, using a TLR ligand alone to stimulate T cells will probably give you a more innate, pro-inflammatory cytokine response profile than using a more broad T cell activating agent, like anti-CD3/28, PMA/Iono or PHA. Autophagy stimulated by TLR-4 activation may also lead to erratic, inconsistent cytokine staining.
Souce: NovoPro 2018-03-12