The canonical activation of a T-cell is by way of an antigen presenting cell (APC). This happens by the APC presenting an antigen to the T-cell by way of the T-cell receptor (or CD3) and at the same time co-stimulating by activating the CD28 ligand on the T-cell. Thus, when mature T-cells are expanded in vitro, stimulating antibodies targeted against CD3 and CD28 are supplemented.
In contrast, a quick view of the literature will demonstrate that some investigators choose NOT to use anti-CD28 when testing a drug or compound on T-cells. Are there any times in vivo that a T-cell would be activated WITHOUT CD28 stimulation? Is this a legitimate way of testing a drug or compound on T-cells? Do people choose not to use anti-CD28 because it masks the small effects their drug may induce on T-cells?
The main reason I ask is we are currently growing mouse T-cells in vitro and have been treating with various compounds of interest to our lab. If we use CD28 stimulation, we will obtain a positive response from the T-cells. If we omit CD28 stimulation, we obtain a negative response. So which is the "correct" answer? Which one is more likely happening in vivo? Last, how can we interpret literature reports when they only perform one form of stimulation?
anti-CD3/CD28 is artificial, much like using PMA/ionomycin. The cells are not activated by antigen presentation of peptides by APCs.
Using peptide+APCs+IL2 is an attempt at trying to emulate in-vivo priming. Yes, using CD3/28 or PMA/I will give you a very strong response, but it doesn't mean anything other than "the cell can produce cytokines". The answers provided here pretty much summarize this.
When looking at the literature, this should actually be something to look for. It is easier to induce a cytokine production with artificial methods, and more significant/relevant to show this with peptide/APC stimulation.
If you have antigen specific cells from either transgenic mice or hybridomas (eg, CD8 SIINFEKL/OT-I or p14, CD4 OT-II or SMARTA) then using the appropriate peptides along with transgenic cells will be a more meaningful experiment. Doing CD3/CD28 stimulation alongside this is useful to see differences, as some have already pointed out.
Dear Adam,
1) Treatment with anti-CD3 plus anti-CD28 is too artificial and allows to answer restricted number of questions, for example, to study some aspects of signaling in purified T cells. Obviously, the subjects of interest are immunoregulation and immune response, which are integral results of interactions of T cells with real APCs. Stimulation with anti-CD3 only at some extent keeps APCs as an active players in experimental system, whereas additional stimulation with anti-CD28 fully obviate them. Normal interactions with APC and feedback inhibition of response via CTLA-4, as well as consecutive production of regulatory cytokines become impossible. Normal immunoregulation and any mean to estimate impact of tested compound on it become elusive.
2) Example of immune response without CD28 stimulation exists, for example, “costimulation blockade resistant allograft rejection”. It seems, responses of CD8+ cells may be less dependent on the B7/CD28 as compared with CD4 T cells.
Dear Adam,
I was wondering if your anti-CD3 stimulation was done with previous pre-bound of the antibody on the plate. This is necessary if you are working with purified T cell populations to get full T cell activation (if you are working with cell suspensions that have APCs, anti-CD3 pre-bound is useless since they will "hold" the MAb by their Fc receptors). However, if you put together anti-CD28, this pre-bound is not needed.
Thank you both for your responses. I am indeed using anti-CD3 pre-bound to the plate, and anti-CD28 in soluble form in the media. Additionally, I have no APC's (or should not have any) in my culture as I use antibody negative selection to isolate pan-T-cells from spleens of mice.
Another interesting fact: people who analyze the conversion of CD3+FoxP3- into FoxP3+ Treg usually don't take CD28 Ab because it interferes with the conversion... the signal is too strong. Using irradiated APCs instead is much more physiological
If you are using highly purified T-cells you are providing a TCR-trigger (anti-CD3) and a costimulatory signal (anti-CD28). In any case it is artificial and the value depends on the question you ask. In vivo, you may have even more costimulatory and inhibitory signals, so that is not a valid comparison. If you want to ask, how your immunmodulatory compound influences the activation threshold, both types of stimulation in parallel would make most sense. You might also want to titrate your CD3-antibody, as both, your costimulatory molecule and you drug may possibly increase or decrease your activation threshold (e.g. conc. of anti-CD3-antibody that leads to half-maximal activation). Comparison of the two conditions may tell you, whether your compound interferes with costimulation rather than TCR-triggering.
Matthias
Hi Adam
There is also the consideration of which T cells you want to stimulate. Highly differentiated T cells like those driven by chronic viral infections lose CD28 expression and use other signals for maintenance.
anti-CD3/CD28 is artificial, much like using PMA/ionomycin. The cells are not activated by antigen presentation of peptides by APCs.
Using peptide+APCs+IL2 is an attempt at trying to emulate in-vivo priming. Yes, using CD3/28 or PMA/I will give you a very strong response, but it doesn't mean anything other than "the cell can produce cytokines". The answers provided here pretty much summarize this.
When looking at the literature, this should actually be something to look for. It is easier to induce a cytokine production with artificial methods, and more significant/relevant to show this with peptide/APC stimulation.
If you have antigen specific cells from either transgenic mice or hybridomas (eg, CD8 SIINFEKL/OT-I or p14, CD4 OT-II or SMARTA) then using the appropriate peptides along with transgenic cells will be a more meaningful experiment. Doing CD3/CD28 stimulation alongside this is useful to see differences, as some have already pointed out.
Adam, T cell stimulation will always require cells presenting antigen and providing co-stimulation, if you're looking at antigen-specific responses. With very strong T cell activity, the requirement for co-stimulation is low, and perhaps not needed at some point. For your experiments, perhaps you gain some info by titrating how much CD28 you need to get a response from the T cells. This may help you differentiate between strong and weak treatments for the T cells in your system.
Hi Adam,
I did several proliferative studies using plate pre-bound with anti-CD3 and anti-CD28 in purified T cells. I think that is the closest model of T cell activation if you are not using APC.
Good luck in your experiments
Hi Adam
anti-CD3 stimulation of T cells induce defective activation of T cells that leads to apoptosis when the T cells see the antigen again (anti-CD3 re-stimulation), after a resting period in culture without anti-CD3 but IL-2. This process can pretend the in vivo induction of tolerance of anergy of T cells in the absence of inflammation and dendritic cell activation. anti-CD3 and anti-CD28 stimulation in purified T cells prevents apoptosis of T cells in repeated rounds of re-stimulation.
Hello, Adam,
If you found some references without anti-CD28, it's possible that they just stimulated the whole splenocytes. In your system, purified T cells were used, you need anti-CD28. 2C11 should be coated, but anti-CD28 could be soluble or coated. if you work on the whole splenocyte, soluble anti-CD3 works fine, because B cells and other cells with FcR will provide the cross-linking of anti-CD3.
Thank you all for your insight. This information will be very helpful in designing my next set of experiments.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology