Hello,
For a project, I need to use frozen PBMC with rare T cells (hopefully) and expand those T cells with synthetic peptides. After the expansion, I will confirm the activation of T cells with ELISA using the supernatant (it won't be an antigen specific confirmation, I just need to know if there is activation), and I will further isolate CD8+ T cells from the bulk for other experiments. I am new to immunology, so here are my questions.
1) I have 15 peptides (some known, some Neoantigens). Would they interfere with each other If I present them to PBMC as one pool? What are the concentrations that you normally use?
2) Which protocol(s) are you using for this expansion? I found multiple ones with different cytokines. It looks like IL-2 is an absolute must. But I also see IL-7, IL-15 and IL-21 sometimes. What is the most efficient cocktail and culture conditions?
Many thanks for the answers!
Regards,
Ilayda
Hi
Mixing several antigens and presenting them to T cells would lead to non-specific activation (If there is any activation that may occur). It is very unlikely that such peptides may interact with each other.
follow this protocol which I have implemented in my PhD:
Restimulation and the selection of well growing cells
Principle:
Incubating the T-cells with irradiated allogeneic PBMCs stimulates proliferation.
This proliferative response is augmented by the presence of IL-2 and PHA. IL-2
is a cytokine that is well known as a T-cell stimulator (Liao et al., 2013). Likewise,
PHA is known to induce T-cell mitosis (Movafagh et al., 2011). Using allogeneic
PBMCs would provide HLA mismatch environment which adds to the strength of
the stimulatory reaction in addition to their role as feeder cells.
Procedure:
The serial dilution plates were restimulated by using 45 Gy irradiated, allogeneic
PBMCs at a concentration of (5x10 4 cell/mL), IL-2 (200 U/mL) and PHA (10
µg/mL). The cultures were incubated at 37 °C and 5% CO 2 for two weeks. They
were fed every 2 days with culture medium (25 μL /well) supplemented with IL-2
(60 U/mL). Plates were assessed regularly to check for the outgrowth of TCCs.
Any well containing a large cell pellet (about 2mm) was transferred into new 96-
U bottomed plates and expanded into 2 wells and later into 4 wells.
The usual way I used to expand T cells is also to plate PBMCs on irradiated feeder cells that you 'pulse' with the desired peptides, in presence of IL-2. IL-7 is recommanded if the T cells you want to expand are naive. I used 50UI/ml of rhIL-2 and 10 ng/ml ofrh IL7. With IL-2 you should never decrease the dose you will give to T cells when you change medium.
If you use PHA, you will expand all T cells regardless of their antigen specificity. This can be a good strategy to increase the total population of your T cells before selecting antigen-specific cells by FACS or assessing their response to your peptides by ELISpot or ELISA or cytotoxicity assay. You can begin with 1 ug/ml of each peptides that you provide each time you change medium (every 3 days).
Another consideration for your feeders cells. Usually they are PBMCs but you can use transformed B cell lines. The time of irradiation required will be different. If your peptides are not the right length to be directly presented by MHC class I and need to be processed then you need to give it to your feeders cells before irradiating them for some hours, so that they process it.
I believe there are alternative methods that do not require Feeder cells such as in this discussion
https://www.researchgate.net/post/How_do_you_culture_antigen_specific_T_cells
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