I've only seen two ways to stimulate T cells:
(1) coat surface with anti-DC3 and anti-CD28
(2) use bound (or beaded) anti-CD3 but free anti-CD28
Why isn't the CD28 ever free in suspension? Or have I just not crossed those protocols?
Dear Nate Kove
in general agonistic anti-CD28 antibodies are very high affinity and are therefore used in solution, whereas anti-CD3(epsilon) antibodies are medium affinity and crosslinking is facilitated by plate binding or via beads, which are coated with many antibodies. In addition, the density/ amount of surface CD28 and surface CD3 differs, even though I don't I have the exact stochiometry in mind.
We used plate bound anti-CD3 (145-2C11) and anti-CD28 (37.51) with good results as laid out here.
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
I also attached a small review on different anti-CD28 antibodies (made in mouse and their properties in different experimental models) and a side by side comparison of commonly used anti-CD3 antibodies (for murine T cell stimulation).
Article CD28-Specific Immunomodulating Antibodies: What Can Be Learn...
https://academic.oup.com/intimm/article/20/10/1247/699489
All the best & kind regards,
Michael
Dear Nate Kove
in general agonistic anti-CD28 antibodies are very high affinity and are therefore used in solution, whereas anti-CD3(epsilon) antibodies are medium affinity and crosslinking is facilitated by plate binding or via beads, which are coated with many antibodies. In addition, the density/ amount of surface CD28 and surface CD3 differs, even though I don't I have the exact stochiometry in mind.
We used plate bound anti-CD3 (145-2C11) and anti-CD28 (37.51) with good results as laid out here.
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
I also attached a small review on different anti-CD28 antibodies (made in mouse and their properties in different experimental models) and a side by side comparison of commonly used anti-CD3 antibodies (for murine T cell stimulation).
Article CD28-Specific Immunomodulating Antibodies: What Can Be Learn...
https://academic.oup.com/intimm/article/20/10/1247/699489
All the best & kind regards,
Michael
Hi Nate Kove .. that is because, the functional activation of T cells in vitro can only be achieved via immobilized CD3 Abs. Triggering of the TCR/CD3 by soluble CD3 Abs, irrespective of their affinity, will give only transient stimulation and induce anergy or unresponsive state in T cells.
Because the occupancy of TCR/CD3 complex is high with immobilized CD3 Abs such that it can override the quantitative support of CD28-mediated signalling in in vitro conditions. Therefore the co-stimulatory signalling via CD28 Abs often becomes redundant in in vitro stimulation of T cells. Therefore, it does not matter how you provide co-stimulation when the CD3 Abs are immobilized on the beads or in the plastic wells.
Cheers
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