Hi, everyone!
I tried to do mouse MDSC and T cell coculture assay, but failed. I have some questions.
1.Bone marrow derived MDSC can inhibit non-specific activated CD8+ T cell or not? (CD3 and CD28 activate)
2.I tried to isolate MDSC from 4T1 tumor using BD INFLUX. But the percentage of MDSC in tumor is too low to get enough cells, only 2-3%.
Which tumor model do you use, and how do you isolate MDSC from tumor.
Are there any flow cytometry better and faster than BD INFLUX?
Thank you!
Hi Xin,
First off MDSC assays can be tricky based on the basic phenotypes you might be using for monocytic (CD11b+ Ly6c+ Ly6g- or CD11b+ Gr-1 int) vs granulocytic (CD11b+ Ly6c+ Ly6g+ or CD11b+ Gr-1 high) MDSCs that are also the same markers you would use to identify monocytes and neutrophils, respectively. Because of this, you should be aware endogenous MDSCs are a mixed population of all these myeloid cells and you’re really looking at the net effect of the population as a whole.
1) Yes, CD3/CD28 agonism-induced T cells can be suppressed by MDSCs but the extent depends on your readout. These cells suppress but do not completely impair T cell proliferation/function, so you would probably not notice big difference in proliferation by Ki-67 compared to something like CSFE dilution. Likewise, a subsequent T cell killing assay would be more sensitive assay compared to an IFNy stain.
2) Recovery of immune cells can be difficult in the 4T1 model if you allow your tumor to progress to the point where most of the tumor is dead fibrotic tissue. If you’re seeing ulcerations on your mouse, try using an earlier time point for recovery from a 4T1 tumor. MDSCs might actually be that rare if you’re recovering them from the primary tumor; however, in theory they are a systemic population that you can recover from alternative organs (e.g., spleen). This would most definitely affect the ratios of MDSCs vs monocytes/neutrophils this population would represent.
3) In the past I have used Gr-1 column enrichment from splenocytes recovered from a 4T1-bearing mouse. These populations did show suppression but have to be interpreted with the aforementioned caveats. Alternatively, you might try negative selection for Ly6c followed by positive selection of Ly6g (or FACS sorting) if you want to parse out monocytic vs granulocytic suppression.
Hope this helps and good luck with your experiments.
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