I want to perform an in vitro Antigen presentation assay using NSCLC and OT1 CD8 T cells. First I want to know if it is better to pulse the NSCLC cells with OVA257-264 peptide or with the complete OVA protein? And regarding the procedure, is it done by just incubating the cells with the peptide or the protein for couple of hours at 37 °C (incubator)?
Also should I activate the OT1 T cells (culture for 2 days culture with IL-2, antiCD3 and antiCD28) before their co-culture with OVA-NSCLC?? since most of the in vitro Ag presenting assays used DC as an APC which activate the T cells by themselves due to their CDs.
Hi, you cannot pre activate the OTI T cells before the coculture (this will not depict an antigen specific activation). If your tumor cells are able to present the ova peptide, then the OTI T cells (added after a few hours after pulsing the NSCLC cells) will anyways get activated. DC are potent in antigen processing and presentation and hence whole ovalbumin protein can be used. However I am not sure if tumor cells are as potent as DC in processing the whole protein. Probably you can pulse ova peptide and whole protein in parallel to check this out. All the best!
You should use peptide since whole protein will require cross-presentation (exogenous antigen in Class I pathway). Silly question, but what is the origin of your NSCLC?
Thanks for the answer.
Actually the preactivation is usually done using the same peptide but this process is important to test T cell-mediated cytotoxicity. For in vitro antigen assay and after reading several papers, I noticed that no need to activate the T cells. It is enough for the T cells to recognize the antigen in order to be activated even in the absence of the costimulatory molecules (presented on DCs).
I still do not know how to pulse the tumor cells with the peptide, I need to know if it can work with the incubation of tumor cells with the peptide for couple of hours or I need to transfect the tumor cells with the peptide?
Hi Greg.
The cells are mouse NSCLC.
I do not know if that what do you mean by your question.
Hi Taghreed, if you do not know how efficient your Tumor cells are in the processing of ova and still unclear of how much time is required for pulsing, try one or 2 experiments by pulsing the Tumor cells with DQ-ovalbumin (BODIPY FL conjugated, invitrogen) and in parallel pulse DC with the same. Do this in different time Points , 0, 15 min, 30 min, and 1 hour , 2 hrs at 37oC and then take the samples on ice. Then you can visualize the percentage of processed ova using FACS. The Advantage of DQ-Ova is that, when the ovalbumin is processed inside the organelles (that have an acidic pH) of the Tumor cells /DC it emits Flourescence (FITC) that could be detected by FACS. Unprocessed ova can be detected in PE channel. However, there are a series of BODIPY conjugates and u can selected the flourescence as per your choice. Best!
Taghreed
You answered my question. I was just confirming that your cell line expressed Kb. I would recommend just adding the OVA peptide (SIINFEKL) at 1 or 10µg/mL (or omit as negative control) to co-cultures of your NSCLC + OT-I and measuring CFSE dilution by the OT-I cells to confirm presentation.
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