If you are only interested in MHC levels you could look at presentation of peptide antigen to a T cell clone. You need to know the HLA genotype that is expressed by your donors. Otherwise you could try superantigen SEB presentation to autologous T cells.

Dear Kristiina, MHC molecules are not expressed on sell surface without antigenic peptides derived from self or foreign proteins. So, you can incubate macrophages with fluorescently labeled antibodies specific to MHC class II molecules for subsequent estimation of fluorescence intensity.

A functional assay usually means you should use a test antigen, what about tetanus toxoid , most everybody will respond to that. Incubate APC plus TT in the presence of autologous T cells and measure proliferation. Otherwise  anti HLA Class II abs and flow cytometry would be a method.

Thank you so much for the suggestions!

I´m interested in the macrophage / T cell -coculture experiments using SEB or TT as the antigen. However, as the T cells need to be autologous I would have to culture them separately for the 7 days that the human monocyte-to-macrophage differentiation takes. Is this feasible or should the T cells be used freshly after isolation from blood? Sorry for the basic questions; haven´t been working with T cells before.

Also, I would be very grateful if you know some good example papers describing in detail this type of antigen presentation assay to autologous T cells.

Best regards,

Kristiina

fresh PBMC are always best. When we do mixed lymphocyte culture using as apc's  monocyte adherent cells versus  monocytederived  dendritic cells ( obtained after 6 days of culture in GMCSF plus IL-4)  there is a big difference in the proliferative response of the allogeneic T cells, this difference is in part due to the greater MHC class II expression on the DC...is this an option for you?  this way you just need two human donors , rather than freezing cells.   I sometimes end up bleeding myself ( or other people that can't get away so easily) for the autologous cultures. 

Hi Kristiina,

I agree with Paul, fresh PBMCs are always best. What you can do is, after PBMCs isolation on day 1 (besides setting up the monocyte-to-macrophage differentiation) you can in parallel put an aliquot of PBMCs (containing T cells plus several APCs) + TT for ~4 days. That will help you expand the population of TT-specific T cells. On ~day 4 you ficoll this T cell culture (activated T cells/lymphoblasts are less dense, while non-activated cells are more dense), recover the lighter T cells and put them back in culture, now with IL-2 to help expand those T cells. Important: IL-2 has to be removed from T cell media ~24/36h before your final assay with the Macrophages.

Thus, by day 7 you'll have your autologous T cells enriched in TT-reactive cells (which you can enrich further for CD4+ T cells by negative selection) on one side, and your macrophages on the other, to test the MHC II-mediated Ag presentation (you'll have to test the optimal T cell/Macrophage numbers). Besides the usual conditions/controls you would consider (e.g. with/without TT, Yes/No T cells, Yes/No APCs) I recommend you to also set some positive control for T cells (could be anti-CD3/anti-CD28 beads, for example). Readout you could use is IFN-g production from T-cells, either in supernatant (ELISA), by IFN-g ELISPOT, intracellular flow cytometry, etc. 

Good luck,

Ricardo

Thank you again for all the information and thank you Ricardo for the detailed protocol, it seems like a good option. I would need to apply for an ethical permit to be able to draw blood from volunteers at our lab, as 2 blood samples required on consecutive weeks from the same person can´t be done via the Red Cross Blood Service buffy coats we regularly use.

Hi guys,

Thank you Kritiina for asking this question it is exactly waht I was looking for!

Except I am using Bone Marrow to derive macrophages so I was wondering if there is any detailed protocol to do this? like at what concentration should I use the TT?

Thank you all

Sanda