To measure cell proliferation of immune response commonly are used compound to stimulate cellular mitogen capacity of immune cells. In paper usualy are described these mitogenic compounds being Concavalina A, Pokeweed mitogen and phytohemaglutinin. This compounds stimulate different cells? How can I interpret the results for different compounds?
Dear Camila,
Here is a little bit of information:
Phytohaemagglutinin (PHA) is a lectin that has carbohydrate-binding specificity for a complex oligosaccharide containing galactose, N-acetylglucosamine, and mannose. It agglutinates most mammalian red blood cell types. PHA has multiple effects on cell metabolism; it induces mitosis triggering cell division in T lymphocytes, and affects the cell membrane in regard to transport and permeability to proteins.
Concanavalin A (ConA) is also a lectin and binds mainly internal and nonreducing terminal α-D-mannosyl and α-D-glucosyl groups. ConA is known to stimulate mouse T-cell subsets (four functionally distinct T cell populations) and at least one subset of human T cells (Dwyer JM, Johnson C (1981). "The use of concanavalin A to study the immunoregulation of human T cells". Clin Exp Immunol 46 (2): 237–49. PMC 1536405. PMID 6461456.)
Pokeweed mitogen is a lectin from the pokeweed (Phytolacca americana). It is is mitogenic for both T and B cells.
Among these three, the pokeweed mitogen is probably the safest choice for stimulating lymphocytes from various species.
An alternative stimulator is PMA+I: phorbol 12-myristate 13-acetate (PMA) which activates ctivate the signal transduction enzyme protein kinase C; combined with Calcium Ionophore which increases intracellular Ca2+ in intact cells.
My personal favourite for T cell stimulation is SEB (Staphylococcal enterotoxin B) which covalently binds MHC and TCR in a variety iof species and triggers proliferation and cytokine responses in a way very similar to "true" TCR triggering.
Another option close to "real life" are peptide cocktails (from recall antigens).
Cheers,
Axel
Nothing to add to the previous explanation, except that I prefer PHA instead of PMA, because it ist more "physiological". SEB is certainly very potent. Another "physiological" option for T cells would be anti CD3/CD28.
Best regards.
W. Solbach
Camila,
you already have a lot of information. PWM is a T-dependen B mitogen, and ver weak T-mitogen. Both PHA and ConA (among lectins) are better to test T-cells
The most powerful stimulus is PMA+Ionomycin, but that combination is the less physiological.
You can also cant test cytokines in the mitogen cocktails, and also antigens (as candida, PPD, etc...) thus it depends if you want to asses the general function test or a more specific response.
In monoclonals, apart from anti-CD3, you could also use some clones of anti-CD2 (alone or together with other stimuli)
And I agree with the colleagues that the more closed to "real stimulation" are either cd3+cd28 (my favourite) or SEB (superantigens -thanks god- are not normally triggering our cells).
attached is an article by a Research Gate's colleague working on mitogens
Great and clear answers.
An additional question:
Some patients (especially children) show a low or absent response to PHA and ConA, while PWM works perfectly. The lack of IL-2 synthesis (a key aspect for the progress of these stimulations to complete proliferation) seems to be a main reason for this fact, while PWM is able to induce a normal IL-2 secretion. While IL-2 alone is unable to induce direct proliferation, how do you interpret when the combination of IL-2 + PHA or ConA is able to recover a complete proliferation?
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