I would like to perform MLR reactions with DCs and autologous naive T cells, in both human and murine models. In autologous MLRs with antigen-loaded DCs, do I need to put some anti-CD3/CD28 and do I need to irradiate the DC?
From what I've read and already tried, I think it is not useful with human moDC, but is better with mouse BMDC, am I right? And is there any alternative protocol instead of irradiation (for example glutaraldehyde fixation, does anyone use this method?).
You should irradiate the antigen presenting cell (DC). You can use other methods of fixation, but this will likely reduce the sensitivity.
You should not add anti CD3/CD28 -- this will stimulate the T cells non-specifically.
Yes you must irradiate your stimulators (2500-3000 rad) if they are going to be PBMCs. Otherwise will not be able to distinguish between the immune response in the responders and the stimulators.
Good luck.
Said.
Ditto APC need to be inactivated. fixing kills them and they are way less effective-probably because they cann rearrange thier membranes into synapses with the TCR.
This is very experiment dependent. Irradiation of "feeders" or "stimulators" is OK if you are truly doing an MLR, which is looking at the response of the "responders" against the feeder cells.
However, if you are looking for antigen-specific responses, the situation changes. If you are attempting to process and present whole protein, you cannot irradiate as the DCs and other APCs will not properly cut up the protein into antigenic peptides. If you are merely adding a soluble peptide/peptide pool of known antigenic epitopes, you can safely irradiate. This activation is driven by the affinity binding/loading of soluble peptides onto MHC class I and II complexes, so you don't really need a living DC. In this scenario, you really only need the DCs/APCs for their surface MHC complexes.
When you use dc as antigen presenting cells , it does not need irradiation. If anticd3 and cd28 are uses for stimulation you will miss the antigen specific t cell response. Lalitha kabilan.
Thanks to you all for your answers and your help. I use infected DCs @24h post-infection. I agree that for moDCs irradiation is not useful because they do not proliferate, my doubt was rather for BMDCs.
That is definitely a good techical question for T cell immunologists. Personally, I have tried inactivating APC for T cell stimulation either by irradiation or using Mitomycin C (a suitable alternativs, see. Ramos et al., 2008). In mouse MLR with allogeneic bone marrow-derived DC, we routinely irradiate DC, but also check for activation marker expression CD80/86/MHCII after differentiation.
In my experience human monocyte DCs do not have to be irradiated for an MLR. Immature dendritic cells loaded with antigen/stimulant and then directly put on with autologus T cells serve best. Human monocyte DCs do not proliferate so they are a safe bet as antigen presenters to be used without irradiation in an MLR.
With the mouse BMDCs it depends on your experiment. It is advisable to irradiate the APCs at 2000-3000 rads or treat them with mitomycin C before the stimulation. Do not use anti CD3 or CD28 as this will give non specific proliferation and not antigen specific.
I agree with Antonio. Dendritic cells do not proliferate, so no need to irradiate. Irradiation is necessary only if PBMCs/monocytes or B cells are used as stimulators.
However, it is likely that your DC population is not pure - it is not possible to get 100% monocytes to differentiate to DCs; there may be B cell impurity if the starting population is bone marrow cells. In this case, proliferation of monocytes or B cells may skew your results in the mixed cell culture. I would therefore suggest treating the DCs with Mitomycin C, but only after antigen-loading and maturation.
Do not add anti-CD3/CD28. Very low dose (=/
I agree with Prahlad. You should never assume you have a 100% pure population of DC so his suggestion is a good one because after antigen-loading and expression of antigen by DC, cross-linking the DNA of the cell should have no effect on their ability to present peptide antigens. In addition, you can use a low dose of Mito-C, which will insure that any contaminating T or B-cells are unable to proliferate.
i am not sure why you would bother using MLR if your going to the trouble to drive DC from progenitors? If its is CD4 help your worried about, why not use transwell systems? Anyhow, MLR irradiation is used when mixing PBMC for your MLR's, because you do not want stimulators to proliferate. No need to irradiate your DC.
I assume these responses are baeed on non-FACs based measurements of proliferation for which I would irradiate The APC cell. However, w a FACs baded method, I can gate on CFSE labeled (or EDU or BrdU) Teff cells and strictly measure the effect on CD4 (or other cells) proliferation and therefore should not need to irradiate the APCs.
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