I am trying to compare different groups of mice for T cell proliferation responses. I use unsorted splenocytes and stimulate them with different concentrations of antiCD3/CD28 antibodies and ConA. I see some difference in proliferation response, between the groups, with CD3/CD28 but no difference with ConA. Could someone help in interpreting this in terms of mechanism?
Samra
These two types of stimulation use very different pathways. More importantly, Anti-CD3 is specific for T-cells whereas ConA stimulates many different cells and promote aggregation of some cell types (e.g.red blood cells).
Therefore, you can't use unsorted spleen cells to study T cell proliferation responses to ConA. You won't be able to differentiate the direct effects of ConA on T-cells from the undirect effects via none-CD3+ spleen cells. You have to use purified CD3+ T-cells.
Once you do this, the following paper may be of help:
Differential pathways for calcium influx activated by concanavalin A and CD3 stimulation in Jurkat T cells. Pang B, Shin DH, Park KS, Huh YJ, Woo J, Zhang YH, Kang TM, Lee KY, Kim SJ. Pflugers Arch. 2012 Feb;463(2):309-18.
Samra
These two types of stimulation use very different pathways. More importantly, Anti-CD3 is specific for T-cells whereas ConA stimulates many different cells and promote aggregation of some cell types (e.g.red blood cells).
Therefore, you can't use unsorted spleen cells to study T cell proliferation responses to ConA. You won't be able to differentiate the direct effects of ConA on T-cells from the undirect effects via none-CD3+ spleen cells. You have to use purified CD3+ T-cells.
Once you do this, the following paper may be of help:
Differential pathways for calcium influx activated by concanavalin A and CD3 stimulation in Jurkat T cells. Pang B, Shin DH, Park KS, Huh YJ, Woo J, Zhang YH, Kang TM, Lee KY, Kim SJ. Pflugers Arch. 2012 Feb;463(2):309-18.
I agree with Eric Brandt. Using CD3/CD28 ligation is a better way of inducing polyclonal T cell proliferation.
The exact mechanism of Con A activation has not resolved, it is a lectin and likely to bind pathogen receognition receptors on innate immune cells (an example of a classical lectin receptor is the mannose receptor, which is expressed on the surface of monocytes).
In contrast, anti-CD3 and anti-CD28 specifically activate T cells - mimicking T cell receptor ligation with antigen, in the context of MHC. CD3 is the T cell co-receptor, in which the intracellular signalling function resides. CD28 is a co-stimulatory molecule, which binds to CD80 or CD86 on the surface of antigen presenting cells (both these molecules are upregulated by danger signals).
Together this provides a signal to the T cells, similar to antigen and co-stimulation, with danger signal - all 3 are key to immunological synapse.
Con A can indirectly influence this process by effects on monocytes - upregulation of CD80/86.
Totally different pathways. In fact, if you follow the cytokine secretion of your T cells over time, you will see that the strength of response is quite different, and the levels of cytokines (and type) will differ.
Hi Bruno, thanks for your input. I measure IL-2 for both CD3/CD28 and ConA stimulated splenocyte cultures. Will you please elaborate if it's not right to do so? Please provide reference so that I can read it in detail. Thanks
If you are stimulating naive T cells, IL-2 is not the best (at least in CD8+ T cells). TNF is expressed rapidly after CD3/28 stimulation (within an hour) and IFNg a few hours later. IL-2 expression is turned on after TNF and IFNg. I've also found that if you stimulate with CD4 and/or CD8 on top of CD3/28 cytokine production is enhanced.
If you're stimulating effector T cells the cytokine hierarchy is usually IFNg>TNF>IL-2 at least in CD8 T cells. So I'd recommend looking at all three if you can.
Hi Samra!
I have no right to say if that is right or wrong. In my hands, stimulation of lymphocytes with aCD3/28 and ConA can give you different results, depending on what culture conditions you are using. You can't forget for a fact that these are different stimulations which use significantly different cellular pathways. In that line, you will be facing different gene expressing profiles and consequently different strengths and quality in cytokine profiles.
I cannot give you a reference because the data I have are bits and peaces of unpublished observations (so far).
Good luck!
Hi Smara
I agree with Eric that for comparing differential proliferation response of T cells in diferent strains of mie one should use purified CD3+ population which will help you nullifying the associated millieu in unsorted lymphocytes. But even if sorting is not possible then also you will observe difference in proliferation response. Try using lower concentration of ConA compared to what you are using now and hopefully you will se the differential response. Also try using CFSE dye dilution assay for proliferation to enhance the minor differences.
Good Luck!
Thanks everyone for your nice input. I appreciate your contribution and I will definitely consider your suggestions.
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