Has anyone measured surface expression of PDL-1 on EL4 cells?
And while I am it: do they produce TGF-beta?
Just wondering what kind of immunosuppressive "arsenal" EL4 cells have to fight off lymphocytes.
Still, I do not know exactly. Immune selection resulting in tumor escape really forms specific phenotype of EL4 cells. We see both the loss of MHC class I molecules, and significant percent of CD4+ tumor cells. It may be, this phenotype reproduces the one of Tregs. Hope to know more soon.
I am planning to stain the EL4 I have in culture for PDL-1, CD80 and CD86. I will try to post the results here.
In our in vivo studies we routinely use EL4 expressing the flu NP antigen, then try to eradicate the tumour with T cells expressing the F5 TCR. In some animals the tumors escape T cell control and, once isolated, we find they have reduced (but not absent) class I expression but have completely lost the NP antigen.
It would be interesting to compare the PDL-1, CD80 and CD86 expression before and after passage in animals.
Thanks, Pedro! We use B10.D2(R101) (KdIdDb) mice with transgenic expression of 1D1 TCR beta chain. Immunization of these mice with lymphoma EL4 cells results in long-lasting compromised immune response resulting in tumor escape and death of animals 1-2 months later. In wild type and TCR transgenic mice this response is targeted to H-2Kb molecule expressed by EL4 cells (in wild type we see full rejection of tumor cells, whereas transgenic mice die). In 12-15 days after immunization of transgenic mice we see increase in PD-1 expression on CD8+ T cells and increased percent of KLRG1+CD8+ T cells as compared with wild type. To the end of this period we see formation of specific phenotype of EL4 cells (loss of H-2Kb, increased percent of CD4+ cells, increased adherence to plastic surface, tendency to grow by small groups in vitro). I think, it would be very important to determine cellular source of PDL-1 (it may be, not only EL4 cells?). I have some doubts to detect expression of CD80 and CD86 on T lymphoma (but who knows?)
I stained both EL4 and the derived EL4-NP (express the flu NP antigen).
Looks like 8% of EL4-NP that express PDL-1 and also CD80 and CD86.
I wonder why this population is not present in the parent line.
What do you make of it?
Thank you very much, Pedro! Nice results! Appearance of PDL1+ cells is convincing. CD80 and CD86 are less evident, but may be significant. Did you see any difference in FSC-SSC plots and in 7-AAD (or PI) uptake?
Yeah, the CD80 and CD86 isn't very convincing. These cells do provide good initial stimulation to NP-specific T cells but the T cells fail to proliferate massively, perhaps due to lack of co-stimulation in vitro.
The PDL-1+ EL4-NP cells were larger cells, sitting on a higher FSC and SSC.
I wonder if this population is a contaminant (that still expresses NP though, as it is selected with G418).
It is very hard to find a cell line that does not express PD-L1 in my experience.
That does not make good sense though. Have you got another Flu NP line? For stimulating T cell line, you may have to use fewer APC (sorry for assuming you used too many as most people do), 1 in 10 (APC:T cell) or lower if your line is not of high purity.
Weisan Chen,
these are not professional APCs so we tend to use quite a lot (2 or 3 EL4-NP for every T cell expressing the F5 TCR). I am planning to titrate this to very low numbers so I will report if I see a difference.
I have now transduced EL4 with NP using a different vector and will look at the behaviour of several clones. As I posted earlier, the parent EL4 had no PD-L1 expression. Lets hope it stays homogeneous.
Yes, I was referring your EL-4/NP as APC. Check whether some of the T cells express B220 when they appear unhappy. We published a method paper recently about depleting B220 cells to keep healthier T cell lines
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