It depends on your leukemia model. You should test beforehand how much times does it takes for your mice to die given your model. In my experience (paper attached and thesis on my profile) we were injecting the tumor cells on the day of the marrow transplant, and depending on the model all the untreated mice died between 10 and 20 days, but in another model even before. So you should be sure to characterize well your model, and on that base you can decide the time points. Remember that if you inject the T cells from a mismatched host you will have GvT (and therefore a much longer survival, sometimes more than 50 days), but you will also have GvHD (so try to avoid it or calculate that mice with GvHD will not last very much).

Article Pharmacologic inhibition of JAK1/JAK2 signaling reduces Expe...

Hi Davide,

Thanks for the reply and i am reading through the article very carefully. Although i am taking a different approach, let me know what you think of it. I have never  done the GVL experiment before, so i  need an expert opinion about my method.

The chimeras that we have prepared are mixed and stable for 6 weeks now. We observed no GVHD. Now i want to challenge the chimeras with the host leukemia cell line, in order to verify if the newly acquired acquired chimerism by T cells (which are half allogeneic, H2b+H2d) make them reject (kill) the host leukemia (H2d).

I am not planning on doing the survival of the mice, i want to inject a big load of CFSE stained leukemia (10 million cells) to the chimeras and terminate them at day 3 or 5 and observe the less CFSE stained cells in treatment compared to control.

Do you think 3 or 5 days is is optimal to see a difference in rejection or tolerance to the leukemia?

I am planning to do this experiment just on a B6 and Balb/c mice before i go to chimeras, but even here i would like to know what day should i choose to do FACS after injecting leukemia?

If you inject 10 million leukemia cells in your mice it´s most likely that they will die in less then a week. I think you should have some more informations about your leukemia cells in general. If you have stable chimeras without GvHD it´s most likely that your T cells are already tolerant to the H2b/H2d antigens, and therefore I don´t see why they should attack the leukemia cells. If the leukemia cells in your case are lacking co-stimulus proteins/MHC etc, then the reaction will be carried out by the NK. If  the leukemia cells are expressing another molecule that will distinguish them from the chimeras, then the adaptive immunity will need at least one week. Even GvHD mice will develop the GvHD in a week more or less, and that´s a full BM transpant. So, since you are just taking blood samples, you can for sure try at day 3 and day 5, and it can also be that you will find a reduction of your leukemia cell title, but I would expect something later. In any case, think carefully about your negative controls.

Thank you Davide. The chimeras that we developed are through Hematopoeitic stem cell transplantation and we used a semi-lethal model so they became stable and exhibit mixed chimerism.

In invitro the T cells from the chimeras exhibited good amount of CD8-cytotoxicity against the WEHI3 leukemic cells compared to just BALB/c T cells, which was our objective from the beginning, and i just want to see if the anti-luekemic activity is replicated in vivo conditions.

I will read more into the WEHI3, but i was hoping that the minor antigen differences with leukemic cells should be enough to activate the CD8 chimeric cells against them.

As you suggested i will check the blood on day 3 and 5. but do you think CFSE will be stable if i go beyond that day?