We have treated tumor bearing animals and had complete clearance of their tumors. We then challenged these animals with fresh tumor cells and they were protected. We would now like to determine if there is a CD8+ T-cell response against the tumors. There is no known tumor antigen so we would like to isolate the CD8+ cells and stimulate them in vitro with irradiated tumor cells. These stimulated cells will then be used to do proliferation, CTL, and IFN-gamma assay.
Could someone provide me with a protocol for the stimulation of the T-cell?
Thanks!
In the paper Responses of human T cells to peptides flanking the tandem repeat and overlapping the signal sequence of MUC1 available on my profile there is a protocol for stimulation with irradiated PBMCs. You would need to adjust the RADS for the tumour cells though, as they need higher irradiation. Hope that helps.
You can use mixed lymphocyte tumor culture (MLTC). 4-8 x 10(5) splenocytes from immune mice should be mixed with 4-8 x 10(4) irradiated or heat treated (45 degrees, 1h) tumor cells in complete RPMI-1640 medium (RPMI+10%FBS+L-glutamine+2-ME) in wells of 96-well U-shape plate (for 24-well plates you can use 4-8 x 10(6) splenocytes and 4-8 x 10(5) irradiated or heat treated (45 degrees, 1h) stimulating tumor cells). Four days later, you can see propagation of memory cells, which you can characterize by flow cytometry, to measure 3H-thymidine incorporation or CTL activity. IFN gamma you should determine much earlier, ~ in 12 h after stimulation of immune splenocytes.
With your purified CD8+ T cells, you will probably want to use a variety of E:T ratios. The important thing to remember is that a small % of CD8's are going to be specific to your tumor antigens. For example, I would think you would want a high amount of targets (per effector) to ensure that the majority of your reactive CD8's are activated for your proliferation and IFNG assays. However, for your killing assay, I would think that you would want a high amount of effector CD8's (per target) to ensure that your targets are eliminated at a detectable level. Good luck.
plenty papers in the earlier years of CTL generation using tumour cells. It will have a lot of non-specific response as tumour cell have a lot of antigen especially self antigen. Nowadays, efforts are made to look at tumour specific/associated antigen first or screen vast antigen firs and then use proteins, long peptide etc to generate CTL if you just want tumour specific CTL.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning