Hi! I am new to DC and T-cell co-culturing, I am interested in inhibiting a protein in DCs and seeing its effect on antigen presentation and activation of T cells. I plan to look at antigen-MHCII complex formation in DCs, but am also interested in looking at the ability for the inhibited DCs to activate T-cells, by measuring IL-2 production.
My main two questions are:
i) I need to use pharmacological inhibition to block antigen presentation, and am worried that this drug may affect T-cell activation. Do you have any advice or protocols/literature where DC function is blocked during co-culture. I came across a suggestion to fix DCs after peptide loading/pharmacological inhibition, then co-culture with T-cells and measure Il-2 production (but I imagine this may affect proper immune synapse formation).
ii) Do DCs contribute to the production IL-2 during co-culture, thus affecting IL-2 T-cell activation assay?
Any help or advice would be greatly appreciated! Many thanks!
This type of work has been conducted in the past: the objective was then to determine the role of DCs in the interaction between T cells and B cells. The ability of maturation (secretion of B cells), proliferation (secretion of cytokines by T cells), processing of antigens (capture and presentation via MHC I / II), and migration (mip3a / mip3b) were studied. To do this antibodies were used to block the interaction between the DC (or) DCs and the T cell (or B cell). But each interaction pathway corresponded to an activating or blocking antibody. For example: Blocking / Activation of DCs: anti CD 40, 83, 201, 205, 206, 209, Dora, DCIR ... MHCI, MHCII, TLR ... other ligands: TLRs Blocking / activation of T cells: anti CD 2, 3, 28, 154 ... other ligands: TLRs, NOD ... Blocking / B cells: anti CD 10, 20, 38, 40, 44, 71 ... MHC I, IgD / IgM ... other ligands: TLRs But for each signaling pathway there is a specific readout: proliferation, secretion of cytokines IL2,4,6,7,8,10,12,17a ..., apoptosis, isotypic switching to Ig A, E, G .. . migration ...
But all this depends on the dendritic cell studied: DCs 34+ GM / TNF are the most efficient, but GM / IL4 monocytes are the most used. Blood DCs are the most accurate but the most delicate to obtain and interpret without making mistakes. In fact T cells can interact with most DCs but the observed results will be very different from one CD to another. The readout and blocking pathway will be very different. It is therefore necessary to define the DC cells to be used and the corresponding T cells, to be able to choose a blocking pathway and especially to select the readout adapted to your question.
Thanks for the valuable information Jean-Jacques!
Just a bit more information to clarify what I am interested in doing.
We are using GM monocyte derived DCs and a complementary T-cell line to our MHCII complex of interest. We want to use a drug that blocks our protein of interest and measure (1) MHCII complex formation/ Ag presentation in DCs (2) T-cell stimulation during co-culturing (here we are worried that the drug may affect T-cells as well).
I'm not sure that activated monocytes GM be able to capture the antigen and then present conveniently via the MHC II. In reality, monocytes-GM are not dendritic cells: too much scavenger receptor, no functional MCH II. The GM + IL4 monocytes are already more efficiently able to capture, but present the Ag rather badly. The best is the DCs from CD34 + / GM + TNFa, because they capture everything and present everything efficiently. An example with an antibody (405H1) showing the reliable capture and presentation of an M1 peptide by DCs GM / TNF. But here it is the MHC I. This remains a very good model for the readout.
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